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Analysis of the ex-vivo transformation of semen, saliva and urine as they dry out using ATR-FTIR spectroscopy and chemometric approach

The ex-vivo biochemical changes of different body fluids also referred as aging of fluids are potential marker for the estimation of Time since deposition. Infrared spectroscopy has great potential to reveal the biochemical changes in these fluids as previously reported by several researchers. The present study is focused to analyze the spectral changes in the ATR-FTIR spectra of three body fluids, commonly encountered in violent crimes i.e., semen, saliva, and urine as they dry out. The whole analytical timeline is divided into relatively slow phase I due to the major contribution of water and faster Phase II due to significant evaporation of water. Two spectral regions i.e., 3200-3400 cm −1 and 1600-1000 cm −1 are the major contributors to the spectra of these fluids. Several peaks in the spectral region between 1600 and 1000 cm −1 showed highly significant regression equation with a higher coefficient of determination values in Phase II in contrary to the slow passing Phase I. Principal component and Partial Least Square Regression analysis are the two chemometric tool used to estimate the time since deposition of the aforesaid fluids as they dry out. Additionally, this study potentially estimates the time since deposition of an offense from the aging of the body fluids at the early stages after its occurrence as well as works as the precursor for further studies on an extended timeframe.

analysis_of_the_ex-vivo_transformation_of_semen,_saliva_and_urine_as_they_dry_out_using_atr-ftir_spe

<!>Body fluid<!>Semen<!>Statistical results.<!>Materials and methods

<p>Body fluids are recurrently confronted as major evidence in violent crimes 1 . The potential application of different body fluids typically ranging from their identification to the successful extraction of DNA and its profiling 2 . All of these fluids experience instant biochemical change as they come out from the body. This change can be referred as aging [1][2][3][4] . Some of these changes are rapid and the remaining are gradual 3 , but both the changes are significant to reveal one of the most important aspects of forensic examination; 'time since the deposition' of a crime 4 . The estimation of 'time since deposition (TSD) of a body fluid at the crime scene potentially solves the problem of situating the time of an offense 3 . The TSD of body fluids additionally counter a wide range of issues regarding the crime scene investigation 3,4 . Most of the TSD studies are based on bloodstain aging [2][3][4][5][6][7] except for a solitary publication on semen very recently 8 . All these studies except one 9 investigated the TSD for an extended period, escaping the initial changes in the fluid. With the inception of the TSD estimation study in the early twentieth century, various researchers have investigated this phenomenon on bloodstains 3,4 . Since then the techniques used for TSD estimation went through a drastic evolutionary transformation. The analytical technology gradually transformed into nondestructive from destructive methods 3,4,10 . A variety of techniques including Gas chromatography 11 , Liquid chromatography [12][13][14] , Oxygen electrode 3,15 , microRNA based assay 3,[16][17][18][19] , Color transformation chart 20 , Electron paramagnetic resonance [21][22][23] , Reflectance spectroscopy in Ultraviolet, Visible and Infrared (IR) region [24][25][26] , smartphone imaging 27 , IR absorption 1,3,4,8,9,28,29 , Raman scattering 4,6,7,11,30 and fluorescence spectroscopy is explored around the world for the TSD estimation of blood (peripheral and menstrual) 31 and semen 8 . As an age prediction tool, most of these methods realistically depict promising results. Yet, few methodological limitations hinder the universal acceptability of one or more of these techniques as the Table 1. Strong, medium and weak peaks observed in all the three body fluids during their ATR-FTIR spectral analysis with their vibrational modes and spectral assignments. 'Bold' marks denote the peaks changed during the transition from phase I to phase II and 'Bold' + 'Italic' mark denotes the age-linked peaks.</p><!><p>Wave number (cm −1 ) (approx.) Spectral assignment</p><!><p>O-H stretching (Phase I) and Symmetric N-H stretching of Amide A (Phase II) 37,38,39 spectra in the present study showed similarity with previously reported spectra by several researchers. Although, some minor spectral bands are not visible. It would probably occur due to the spectral acquisition timing, as in this study, all the spectra were collected at the earliest stage of the ex-vivo degradation process up to their drying. The minor bands may have occurred at the later stages in the ex-vivo environment 8,[39][40][41][42] . The spectra in other reported articles were collected at least 4-6 h from the time of deposition 39,40 . Although several peaks were identified in the spectra of each fluid during the study, only a few (age-linked peaks) showed linear changes in their absorption intensity with time. At the initial stage, a similar phenomenon has been observed in the drying of every body fluid. For the first several minutes, only two strong absorption peaks were visible (Fig. 1a-f). These two peaks at 3270-3273 cm −1 (O-H stretching) and 1637 cm −1 (approx.) (scissoring of two H atoms bonded with O molecule) appeared due to the high amount of water in all the fresh fluid samples [43][44][45] . Similar results were obtained in the study by Zhang et al., on blood 9 . After a certain amount of time multiple significant peaks corresponding to the biochemical profile of the fluids are revealed throughout the fingerprint region of the IR spectra (Fig. 2a-f). Following the trend in each fluid, the whole drying time was divided into two phases. www.nature.com/scientificreports/ Except for the concentrated samples of fluids, diluted samples of 2:3 ratio (40%) were also prepared to investigate the changes in the drying of the fluids. The dilution was kept constant for all the fluids and a similar extended drying time had been recorded. Multiple dilutions can also alter the drying time as these factors can be studied in future studies on these fluids separately with other factors as experimented by Zhang et al., on blood 9 . The dilution of the fluids showed an extended (2-4 min) phase I due to the excess amount of water in the diluted sample. The longest phase I observed in the Semen samples and the shortest in the saliva samples. Phase II was relatively similar in the spectra of both raw and diluted samples. The duration of phase II of three body fluids was relatively the same (10-12 min). Table 2 demonstrates the minimum, maximum, and mean values of both phases. The difference in the drying time of all three fluids is potentially a result of the qualitative and quantitative variability in their biochemical components.</p><p>Few researchers reported the correlation between the evaporation of distilled water and time 48,49 . Except for similar height, the peak corresponding to O-H stretching was broader than the peak due to H-O-H scissoring. Except for urine, the absorption intensity of these two peaks showed insignificant change throughout phase I in the spectra of semen and saliva (Figs. 1a-f, 3). On the contrary, Zhang et al. 9 found a different result for blood as the peak at 3308 cm −1 showed very weak but linear absorption change during the early stage. Only the spectra of urine (100% and 40%), showed analogous results with the study by Zhang et al. 9 as the peak at 3273 cm −1 showed a significant decline in the mean absorbance with time during phase I (Figs. 2c,f, 3c). www.nature.com/scientificreports/ Phase II is the fast declination stage where a significant amount of water evaporates rapidly and reveals the other peaks and their intensity changes with time in each body fluid. The peaks at 3271 cm −1 (Amide A) and 1637 cm −1 (Amide I) showed no shift during the whole drying (phases I and II) process of semen stain but the former one sharpens with time and rapidly declined during phase II (Figs. 1a,d, 2a,d) as the peak in Phase II appeared due to the N-H stretching of Amide instead of O-H stretching of water 8,[43][44][45] . In the phase II drying of semen droplets, one strong (1546 cm −1 : Amide II), two medium (1446 cm −1 : methylene; CH 2 and CH 3 and 1066 cm −1 : Glycosylated proteins: probably prostate-specific antigen) and three weak (2968 cm −1 : CH 3 stretching, 1396 cm −1 : fatty acids and polysaccharides and 1243 cm −1 : Amide III) significant age-linked peaks were observed (Fig. 2a,d). Zha et al. 8 investigated the changes in few similar peaks at marginally different positions i.e., 1539 cm −1 (Amide II), 1448 cm −1 (Methylene: CH 2 and CH 3 ), 1392 cm −1 (Fatty acids and polysaccharides), 1059 cm −1 (Prostate-specific antigen). Few more researchers reported the IR spectra of semen in several body fluid identification research articles [38][39][40] . Phase II spectra of saliva showed the shift of strong peaks at 3273 cm −1 (O-H stretching) to 3286 cm −1 (amide A) and 1637 cm −1 (H-O-H scissoring) to 1645 cm −1 (amide I) that indicated the initiation of this phase (Fig. 2b,e). The shifted peak of amide A sharpens following the trend of semen samples. Among others, one strong and sharp (1546 cm −1 : amide II), four weak (1448 and 1403 cm −1 : Methylene, 1078 cm −1 , and 1043 cm −1 : glycosylated proteins) significant age-linked peaks were found (Fig. 2b,e). Including the peak corresponding to amide II, two peaks at 1448 cm −1 and 1078 cm −1 are similar to the peaks at 1446 and 1066 cm −1 in the spectra of semen and placed at marginally different positions. But the intensity of the peak corresponding to glycosylated protein is relatively weaker in the spectra of saliva. The peaks in phase I spectra of urine bifurcated in phase II. The peak at 3273 cm −1 divided into 3346 cm −1 and 3205 cm −1 and 1637 cm −1 divided into 1623 and 1658 cm −1 (Fig. 2c,f). One strong (1658 cm −1 : amide I), one medium (3346 cm −1 : H-O-H stretching), and 2 weak (1156 cm −1 : urea and 1081 cm −1 : Glycosylated proteins) peaks were observed in the phase II spectra of urine samples (Fig. 2c,f) that significantly changes during the drying process. The peak at 1081 cm −1 is similar to the peaks at 1066 cm −1 and 1078 cm −1 of semen and saliva, respectively. Due to the presence of a significant quantity of prostate-specific antigens in semen, the peak corresponding to glycosylated protein is stronger in its spectra than saliva and urine [37][38][39] . In several previous literatures on the IR signature of saliva and urine, the above-mentioned peaks were reported by researchers [37][38][39][40][41][42] . Amide A, I, II, and glycosylated proteins are the common biochemical components found in all the 3 body fluids. Elkins, Orphanou, and Takamura et al. [37][38][39] previously reported the presence of the common biochemicals in all these body fluids in their article on body fluid identification by ATR-FTIR. In Phase II, all the age-linked peaks of each fluid showed a linear relationship between the mean absorbance at each time point and TSD (Fig. 2a-f).</p><!><p>The regression equation of a line is the representation of a prediction model. Table 3 depicts the slopes and intercepts calculated for all the age-linked peaks of three body fluids with a 95% level of significance.</p><p>Body fluids show significant ex-vivo degradation when exposed for a relatively longer period. While early changes are very limited as only a few relevant peaks due to aging are visible. Hence, the TSD estimation was www.nature.com/scientificreports/ performed only on the age-linked peaks of three body fluids. Among several strong and medium peaks 6 (semen), 5 (saliva), and 4 (urine) peaks were selected to calculate the TSD of the body fluids. Irrespective of the variation in the numbers of age-linked peaks for the three body fluids, it was evident that the regression models successfully estimated the TSD with very high accuracy. Both Principal Component Regression (PCR) Analysis and Partial Least Square Regression (PLSR) are strong chemometric tools for the estimation of TSD of body fluids as reported in previous studies 1,[4][5][6][7][8][9][10][11] . The calculated R 2 values for the calibration and prediction of both models are more than 0.9. While the Root Mean Square Error of Cross-Validation and Prediction (RMSECV and RMSEP) values in both the models for all the three fluids except diluted urine, showed low values (Table 4a,b). Although the initial changes in the IR spectra of body fluids are relatively less distinguishable in comparison to the samples exposed for a longer period, the High R 2 and low RMSE values indicate a good prediction of TSD during this timeframe.</p><p>In diluted urine samples, the RMSECV (PCRA: 0.7659; PLSR: 0.7622) and RMSEP (PCRA: 0.7383; PLSR: 0.7327) (Table 4a,b) in both the models are relatively higher, that potentially interfere in the accurate age estimation. While, diluted semen also showed higher RMSECV (0.7167) and RMSEP (0.6840) in the PCRA regression model, while PLSR predicts the age for the same condition with significantly higher accuracy with RMSECV and RMSEP values of 0.1897 and 0.1546, (Table 4a,b) respectively. The lower RMSE value also depicts that there is very minute inter-donor variation and low standard deviation values (0.00002-0.00004) of the spectral data obtained from the repeated sampling showed minimal intra-donor variation. Additionally, the RPD (Residual Predictive Deviation) values for each model were also calculated and it has been found that all the values are above 3 which indicates an excellent prediction model accuracy (Fig. 4) 50,51 . Comparatively, PLSR showed better efficiency of prediction than PCRA as for every fluid it records relatively higher R 2 and lower RMSE values than in PCRA. Figures 4 and 5 depicts the PCR and PLS plots of actual vs predicted regression lines for age-linked peaks of three body fluids. Finally, one-way ANOVA has also been applied to the age-linked peaks separately. The F-statistic for all the age-linked peaks showed a significant difference between the time intervals (Table 5).</p><p>In the present study three forensically significant body fluids other than blood i.e., semen, saliva, and urine were considered to explore the instantaneous changes in their ATR-FTIR spectra up to their drying. This study revealed that all the body fluids undergo significant water loss at the initial stage of their ex-vivo degradation. This rapid loss of water significantly divided the drying process into two phases as the first phase consists of slow evaporation with minimal spectral change with a major contribution of water and the second phase depicts relatively faster evaporation. These two phases can be distinguishable from the ATR-FTIR spectra of each fluid which is a significant marker for estimating the time since deposition of the fluid(s). This study also revealed the spectral regions of interest for the TSD estimation of these fluids as saliva and urine are not explored previously for this purpose. Additionally, if a body fluid is accidentally diluted during the deposition on a wet nonporous substrate during the earliest phase post-deposition, the accuracy of the estimation of TSD can be significantly altered as the water evaporation potentially take more time. In the practical scenario, fresh body fluid samples in liquid and semi-liquid (fluid samples with loss of a certain quantity of water since deposition) conditions from any non-porous surface (e.g., glass, metal, tile, etc.) can be easily collected through a pipette-like apparatus. We can acquire the spectra of the freshly collected fluid with a portable IR instrument containing an ATR-FTIR crystal face exclusively dedicated to forensic crime scene investigation purposes. Although the drying process of body fluids is different on porous substrates like, cloth fabrics, carpets, etc. Hence, further experiments on this subject can be framed based on several factors like, different concentrations, quantities, and interference of porous substrates. Despite, relatively low changes in the IR absorption, chemometric tools like; PCR and PLSR successfully estimate the TSD for each fluid during the initial spectral changes with a very low RMSECV and high R 2 values. Hence, the results (spectral and statistical) of this study potentially be used as a reference for the further TSD studies on these three fluids with a longer timeframe including different factors.</p><!><p>Sampling. Samples of saliva, semen, and urine were collected from randomly selected eight healthy volunteers (28-40 years). All the methods of this study were carried out in accordance with the World Medical Association Declaration of Helsinki. The experimental protocols were approved by the Ethical Committee of Maharani Laxmi Bai Medical College (4647/IEC/2020/SC-1), Bundelkhand University, Jhansi, Uttar Pradesh, India. All the donors were informed about the nature and procedure of the work and written informed consent was taken from each donor. Each body fluid from a donor was collected separately in a glass test tube(s) just before the spectral analysis to avoid any loss of time after its release from the human body. All the fluids were collected by voluntary secretion, ejaculation, and excretion process without using any invasive technique. Spectra of fluids were obtained in two different concentrations i.e., 100% and 40% (2:3) to investigate the effect of water content on the drying time. The dilution concentration was randomly selected for this study to investigate the difference in drying time between concentrated and dilute fluid samples. 40% solutions of all the fluids were prepared by mixing 2 parts of the fluid and 3 parts of distilled water in a test tube instantly after the collection of the fluid. For experimental purposes, saliva and urine samples were repeatedly taken for three consecutive days, and semen was collected three times with an interval of 3 days to observe any intra-variations of their composition. All the repeated samples were collected from the same individuals.</p><p>Collection and pretreatment of FTIR spectra. IR spectra of all the body fluid samples were collected by a 'Spectrum Two' FTIR spectrophotometer manufactured by Perkin Elmer corporation, equipped with a 2 mm diameter diamond crystal ATR accessory and spectrum software (Version 10.0). The spectrum software is used for the collection of spectra. The crystal face was cleaned with a 70% methanol solution before drop the fluid sample for each spectral measurement. One drop (approximately 50 µL) of each body fluid was separately added onto the crystal from a constant height of 4 cm. The crystal was used as the drying surface to reduce any loss of water from the fluids during the transfer of the samples from the substrates. Throughout the whole study, the volumes of fluids were kept relatively constant. The experiment was performed in the month of January. The approximate temperature and relative humidity during the complete spectral collection varied between 13 and 17 °C and 56-75%, respectively. As the ambient conditions during the study did not vary significantly, the effect of variable temperature and humidity were not considered in this study. The spectra of each fluid were collected with an interval of two minutes immediately after placing the droplet on the crystal within the range of 4000 cm −1 to 500 cm −1 with 12 scans and a resolution of 4/cm. In this study, the term 'drying out' is the time point at which there was a negligible change between the three consecutive scans taken at a 2-min interval 26 . At this point, the droplet transformed into a dried stain. Before analyzing the obtained data, all the spectra of body fluids were preprocessed by using Unscrambler X software with several spectral corrections i.e., baseline offset, spectral smoothing with Savitzky-Golay algorithm including 13 smoothing points and 3 polynomial orders in a symmetric kernel and range normalization 52,53 .</p><p>Statistical and chemometric analysis. The mean values of the absorbance of all the significant peaks and the drying time were calculated. Peak identification, fitting, and statistical analysis were carried out by using Origin Pro 2016 and Microsoft excel 2019 software. The correlation coefficient (R) and coefficient of determination (R 2 ) between the TSD and changes in the absorbance values for each body fluid was established with a fitting correlation equation. All the equations showed a 'P' value of < 0.05 which is statistically significant. PCR and PLSR are the two most frequently used tools for prediction model creation and estimation studies. Both of these tools decompose the multiple X variables with respect to the values of Y variables and generate single values for each sample and establish a correlation between X and Y.1 In the present study, the time has been considered as Y and the different age-linked peaks with absorbance values are considered as X. All the chemometric analyses were performed by using 'Unscrambler X' (CAMO Analytics) software. RMSECV and RMSEP were calculated to check the consistency and predictive ability of the regression model. Higher R 2 values and lower RMSE values are indicative of a good prediction model. Full cross-validation was performed for each fluid. Eight samples randomly for each fluid were selected for the model creation and two randomly left out samples were applied for external validation purposes.</p>

Scientific Reports - Nature

Thermochemistry and Kinetics of the Thermal Degradation of 2-Methoxyethanol as Possible Biofuel Additives

Oxygenated organic compounds derived from biomass (biofuel) are a promising alternative renewable energy resource. Alcohols are widely used as biofuels, but studies on bifunctional alcohols are still limited. This work investigates the unimolecular thermal degradation of 2-methoxyethanol (2ME) using DFT/BMK and ab initio (CBS-QB3 and G3) methods. Enthalpies of the formation of 2ME and its decomposition species have been calculated. Conventional transition state theory has been used to estimate the rate constant of the pyrolysis of 2ME over a temperature range of 298-2000 K. Production of methoxyethene via 1,3-H atom transfer represents the most kinetically favored path in the course of 2ME pyrolysis at room temperature and requires less energy than the weakest C α − C β simple bond fission. Thermodynamically, the most preferred channel is methane and glycoladhyde formation. A ninefold frequency factor gives a superiority of the C α − C β bond breaking over the C γ − o β bond fission despite comparable activation energies of these two processes.Limited energy reserves and global environmental impact of fossil fuel burning became a crucial issue pushing to searching for alternative renewable sources of energy [1][2][3][4][5][6] . Biofuels represent a promising alternative renewable source of energy. Biofuels appear in the energy map of many industrial countries 7,8 . Therefore, a revolution occurred in the forums of the production of biofuels from different biomasses.Among biofuels, the most popular bioethanol suffers from some drawbacks such as low internal energy, water absorption, very high ignition temperature, lower combustion efficiency, and high vapor pressure causing massive emissions to the atmosphere 8-10 giving rise to adverse effects on the human health 11 . In order to avoid most of the above issues, bigger oxygenated materials are preferred. For instance, 2-methoxyethanol (2ME) with bifunctional groups namely etheric (O) and hydroxyl group (OH) is proposed as a model for sizeable molecular biodiesel additive hydroxyethers 12 since it can mimic the behavior of the latter in the combustion process. Furthermore, 2ME is an excellent indirect biofuel candidate due to its original synthesis from small bioalcohols like methanol and ethanol. Besides, it can be obtained by modifying ethylene glycol (EG) itself. Ethylene glycol had recently become available from different biomass categories using various procedures with high yield [13][14][15][16][17][18][19] as a biofuel, but there still some concerns related to its low carbon content, low melting point (−13 °C), high viscosity, high toxicity, and high hydrophilic nature 20 . Those issues can be avoided by using alone in the current engine infrastructure. 2ME could function as a biofuel that might be better than ethanol, ethylene glycol regarding lower vapor pressure, higher boiling point, and high energy content. It also shows high miscibility with oils and gasoline besides the expected enhanced ignition behavior due to its high oxygen content (42.1% per mol). These represent some essential useful properties for 2ME as a good biofuel candidate.2ME has a wide range for applications in industrial and pharmaceutical proposes. For instance, it is used in inks, resins, dyes, paints, metal coatings, phenolic varnishes, detergents, cosmetics, cleaners' products, protective

thermochemistry_and_kinetics_of_the_thermal_degradation_of_2-methoxyethanol_as_possible_biofuel_addi

<!>Results and Discussion<!>Bond dissociation energy.<!>Enthalpies of Formation.<!>Complex fission reactions<!>Species<!>Rate constant calculation.<!>Plotting ln k TST (T) versus

<p>X HF CBS HF bx where X = {T, Q, 5} for aug-cc-pVTZ, aug-cc-pVQZ, and aug-cc-pV5Z, respectively 36 . The extrapolation of the MP2 was obtained by the two-parameter polynomial equation:</p><p>where X = {Q, 5} for aug-cc-pVQZ and aug-cc-pV5Z, respectively. The transition states for different reactions of 2ME pyrolysis have been located with the aid of the eigenvector-following (EF) optimization technique as implemented in the Gaussian programs. Vibrational analyses have been conducted at BMK/6-31+G(d, p) to characterize the nature of the obtained stationary points whether they are minima or transition states with real frequencies or one imaginary frequency, respectively, and for the zero-point vibrational as well as the thermal corrections of energies at 298 K. Vibrational modes have been analyzed using the Chemcraft program 37 . For further confirmation of correct transition states that connect desired reactants and products, minimum energy paths (MEP) have been computed through intrinsic reaction coordinates (IRC) 38,39 . All electronic structures calculations have been conducted using the Gaussian 09 W suite of programs 40 .</p><p>The atomization energy approach has been exploited to estimate the gas phase enthalpies of formations for 2ME and its released species at the standard state of temperature and pressure, as it is deduced from the well-known 41 enthalpies of formation of the separated atoms. For any molecule M containing X numbers of isolated atoms, the gas phase enthalpy of formation is obtained from where E e (M) and E e (X i ) are the theoretically calculated electronic energy of molecule M, and the i th atom X at the same level of theory, respectively. ZPVE is the zero-point vibrational energy of the molecule. [H 298 (M) − H 0 (M)] and [H 298 (X i ) − H 0 (X i )] are thermal corrections to the enthalpy for the molecule M and the separated atoms X, respectively. The individual atomic enthalpies ΔH °f(X i ) are extracted from the NIST WebBook 41 . Kinetic parameters for different channels of 2ME pyrolysis have been estimated over a wide range of temperatures using the Kisthelp package program 42 , where the classical transition state theory (TST) 43 is coupled with Eckart tunneling correction 44 to compute rate constants (k) for H-atom transfer reactions of 2ME pyrolysis over the applied range of temperatures (298-2000 K). The rate constant reads:</p><p>where h, k B , and R symbols are Planck, Boltzmann, and universal ideal gas constants, respectively, and χ T ( ) is the Eckart tunneling correction. T is the system's temperature in Kelvin, σ is reaction path degeneracy, p° is the standard pressure (1 atm), and Δ ° †G T ( ) is the standard Gibbs free energy of activation for reaction. Δn takes two value either zero in the case of unimolecular decomposition or 1 in the case of the bimolecular oxidation. The more accurate correction term Eckart tunneling correction χ(T) which obtained through the numerical integrating probability of transmission ρ(E) over Boltzmann distribution of energies. The asymmetric Eckart tunneling correction gives reliable results at low temperatures and previously demonstrated in many previous publications [45][46][47] .</p><p>The transmission probability coefficient χ(T) can deduce from the following equation:</p><p>where ρ(E) is the probability of transmission through the one-dimensional barrier at energy E. ΔH f 0K is the zero point correlated energy barriers in the forward direction.</p><p>Equilibrium relation (K eq = k forward /k reverse ) has been used to calculate the rate constant of simple fission reactions. At first, the equilibrium constant (K eq ) was calculated automatically by the assistance of the Kisthelp program 42 then the previous experimental well-known association rate constants [48][49][50] have been used as values for k reverse to get the forward rate constants (k forward ) for the selected simple bond fission reaction.</p><p>All complex fission reactions barrier heights have been investigated using the more accurate ab initio CBS-QB3, G3, and BMK/6-31+G (d, p). The last level of theory has been proven to have a significant efficiency for the structure optimization in previous works 51,52 .</p><!><p>Methoxyethanol conformers. 2ME has 12 conformers. Three of them are illustrated in Fig. 1, and the rest of the optimized structures and energies are presented in the Supporting Information (SI). Several studies on 2ME conformers highlighted the effect of the intramolecular hydrogen bond (IHB) between the alcoholic hydrogen and etheric oxygen on molecular properties [53][54][55][56] . Our findings at CBS-QB3, G3, and BMK/6-31+G(d, p) are in mutual harmony, see Fig. 2. The most stable 2ME conformers adopt tGg − and gGg − structures with IHB [54][55][56] . However, tGg − is 1.6 kcal/mol more stable than gGg − . On the other hand, the least stable conformer (gGt), among the studied conformers, is 4.38 kcal/mol higher than tGg − at the CBS-QB3 level of theory.</p><p>Energy of 2ME conformations: Extrapolation to CCSD(T)/CBS level using FPA. Tables 1 and 2 collect the results of FPA for the most and least stable 2ME conformers, while Table 3 shows a comparison of FPA results at MP2/CBS and CCSD(T)/CBS with our obtained values using ab initio methods (CBS-QB3 and G3) and the DFT/BMK/6-31+G(d,p). The CCSD(T)/CBS energies are 1.43 ± 0.15, 2.47 ± 0.19, 4.11 ± 0.04, and 4.25 ± 0.04 for the conformers gGg-, tTt, gTg, and gGt, respectively. The uncertainty term is obtained using δ [CCSD(T)] ± ∆E CCSD(T) − ∆E CCSD . Comparing the obtained results of the FPA with that of our 2ME conformation analysis using ab initio composite methods and BMK/6-31+G(d,p) level shows harmony as appeared in Table 3, while the convergence of the quantum chemical electron correlations methods at the aug-cc-pVTZ basis set is sketched in Fig. 3.</p><!><p>In order to assess the strengths of different bonds in 2ME, their bond dissociation energies have been calculated. Figure 4 displays the bond dissociation energies of 2ME using the CBS-QB3 composite method. The results indicate that the C ɤ −O β and C α −C β are the weakest bonds with bond dissociation energies of 86.2 and 86.7 kcal/mol, respectively. The alcoholic O α -H bond is the strongest one which is close to our previous results (104.5-106.3 kcal/mol) obtained for C1-C4 alcohols 52,57 . The C α -H and C β -H hydrogen atoms are the most acidic and are expected to be abstracted easier in the presence of oxidizing agents as compared to the other hydrogen atoms which agreed with similar bifunctional compound 58 .</p><!><p>Enthalpies of formation for 2ME and its released compounds through combustion have been calculated using atomization approach (at CBS-QB3) and isodesmic equations procedures (at BMK/6-31+G (d, p)). The obtained results are collected in Table 4 using experimental enthalpies of formation values of Table 5. The results have been compared with one another and with available experimental data. The comparison shows impressive agreement with a maximum deviation of ±2 kcal/mol which gives confidence in the future experimental determination of unknown species.</p><p>The current study concentrates on 2ME pyrolysis. The decomposition mechanism can be expanded into nine complex fissions (barrier reactions) and eight simple bond scission reactions (barrierless reactions). Table 1. The valence focal-point analysis (FPA) of energy differences (kcal/mol) of the most stable 2ME conformers (a) gGg-and (b) tTt. Conformer geometries have been optimized at B3LYP/augcc-pVTZ level. aCCD = aug-cc pVDZ; aCCT = aug-cc-pVTZ; aCCQ = aug-cc-pVQZ; aCC5 = augcc-pV5Z; CBS = complete basis set. The symbol δ denotes the increment in the relative energy concerning the previous level of theory, as given by the competing higher-order correlation series:</p><!><p>Values listed in brackets are taken for extrapolation. Equations ( 1) and ( 2) have been used for extrapolation of HF and MP2 energies to complete the basis set, respectively. Final values (in bold) include core correction. Simple bond fission reactions</p><p>Complex fission reactions are those reactions proceeding by H-atom transfers via cyclic transition state, while simple bonds fission are those occurring by homolytic cleavage of the chemical bonds. We will concentrate here on that formed due to complex ones. Among nine unimolecular complex reactions, the formation of methoxyethene, methoxy methylcarbene, and oxetane occurs by dehydration (R1, R3, and R8), while 2-methoxy acetaldehyde is formed via hydrogen molecule elimination (R6) reactions. Reaction R5 proceeds via three-membered ring transition state producing ethylene glycol and triplet methylene. The other complex fission reactions R2, R4, R7, and R9 are accomplished by 1,3-H atom transfer reactions via four-membered ring transition state to produce www.nature.com/scientificreports www.nature.com/scientificreports/ methanol and vinyl alcohol, formaldehyde and ethanol, formaldehyde and dimethyl ether, and methane and glycolaldehyde, respectively.</p><p>The optimized structure of 2ME and proposed transition states leads to the formation of methoxyethene (TS1), vinyl alcohol (TS2), methoxymethyl carbene (TS3), ethanol (TS4), ethylene glycol (TS5), 2-methoxy acetaldehyde (TS6), dimethyl ether (TS7), oxetane (TS8), and glycolaldehyde (TS9) given in Fig. 5.</p><p>Detailed optimized structures of products and bonds lengths variations versus IRC of complex fission reactions are given in the SI (Figs 1S-9S), while the potential energy diagrams of 2ME pyrolysis at the G3 and CBS-QB3 methods are shown in Fig. 6 and the results at BMK/6-31+G(d,p) are listed in Table 8S. The barrier heights and reaction energies of the main favorable routes at CBS-QB3 and G3 methods are tested against the W1 68 , e ref. 69 , f ref. 70 , g ref. 71 , h ref. 72 , i ref. 73 , j ref. 74 , k ref. 75 , l ref. 76 , m ref. 77 , n ref. 78 , o ref. 79 , p ref. 80 , q ref. 81 , r ref. 82 , s ref. 83 . Energies and IRC analysis. Among complex reactions, two reactions (R3 and R5) proceed via three-membered ring transition state, while the rest is passing over the four-membered ring. Almost all reactions are endothermic so that structures of transition states are close to those of products more than reactants according to Hammond postulate 59 . As a result of the high oxygen content in 2ME (42.1% per molecular weight), a theoretical study on 2ME combustion is essential since many oxygenates like ether, alcohols, and carbonyl compounds can be released to the atmosphere during its ignition.</p><!><p>Formation of ethers compounds. Decomposition of 2ME resembles a platform of many ether categories like methoxyethene, methoxymethyl carbene, dimethyl ether (DME), oxetane, and 2-methoxy acetaldehyde. Methoxyethene formation is the preferable kinetic channel on the potential energy diagram of 2ME decomposition with a barrier height and reaction energy of 72.2 and 6.5 kcal/mol, respectively. The reaction can be accomplished via intra-molecular H-atom abstraction from C β by the alcoholic OH (1,2-water elimination) via the four-membered ring transition state TS1. The selected transition state involves inter-rotational of gauche dihedral angle (OCCO) and anti-gauche dihedral angle (CCOH) to be −108° and 102°, respectively. The IRCs of the methoxyethene formation appear in Fig. 1S. Figure 1S shows a fast rapture for the strong C1-O1 bond (BDE = 97.9 kcal/mol) rather than the weakest C2-H3 bond (BDE = 96.7 kcal/mol). The reason is attributed to the high-frequency factor of the C1-O1 bond compared to that of the C-H bonds (see SI). The broken C2-H3 bond at s = −1 amu 1/2 bohr is associated with the O1-H3 bond formation, where the two curves cross each other at the transition state (s = −0.2 amu 1/2 bohr). Formation of the C1-C2 double bond occurs gently during the reaction.</p><p>Methoxymethyl carbene is an unstable compound that is obtained by 1,1-water elimination of C α via TS3. The reaction requires a preliminary structure conversion from the most stable conformer tGg-to the tGt structure through multi-steps with final reaction energy of 2.9 kcal/mol. The reaction proceeds via a three-membered ring transition state with barrier energy of 82.5 kcal/mol. The transformation process of 2ME to methoxymethyl carbene is displayed in Fig. 2S. Figure 2S shows a superior rapture for the C1-O1 bond than the C1-H1 one. The disintegration of the C1-O1 bond begins at s = −1.2 amu 1/2 bohr, while the formation of the O1-H1 bond progresses simultaneously with cracking of the C1-H1 bond. The two curves cross each other at s = −0.5 amu 1/2 bohr. The slight decrease in values of the C1-O1 bond length after the cracking is a clue for the formation of an intermediate compound with an H-bond linking separated atoms near each other.</p><p>Oxetane production has the highest barrier energy value among water elimination reactions from 2ME with a barrier height of 98.3 kcal/mol. The high energy barrier can be attributed to the formation of a highly strained four-membered cyclic product. The reaction proceeds by the alcoholic abstraction of the C ɤ hydrogen (1,4-water elimination) with the four-membered ring transition state TS8. The barrier height and the reaction energy of 98.4 and 12.9 kcal/mol are in line with the work in ref. 60 where the barrier height and the reaction energy were 96.0 and 15.7 kcal/mol, at the same level of theory, for the same investigated channel of 1,4-dehydration of n-butanol. Table 6 shows a comparison between 2ME and n-butanol with respect to 1,1-, 1,2-, and 1,4-H 2 O elimination reactions. Oxetane is formed over multi-conversion processes as the most stable tGg-converts to tGt then to g-Gt conformer by a rotational barrier of 0.6 kcal/mol and reaction energy of 0.4 kcal/mol relative to tGt conformer (2.7 and 1.5 kcal/mol, respectively in case of n-butanol 60 ). Figure 3S illustrates a fast cleavage of the strong C1-O1 bond relative to the weakest C3-H7 bond which occurs at s = 1.5 amu 1/2 bohr. The formation of the O1-H7 bond starts at s = −0.1 amu 1/2 bohr. The two curves of C3-H7 and O1-H7 bonds cross each other at s = 0.9 amu 1/2 bohr, while the formation of the single σ covalent bond C1-C3 occurs gradually during the reaction.</p><p>DME is produced via TS7. The alcoholic H-atom migrates to C β resulting in DME and formaldehyde. The alcoholic H-atom rotates from the gauche dihedral angle of 51° to 0° for facilitating the conversion process. The change of bond lengths for the formation of DME is shown in Fig. 4S. The Figure shows that the weakest C1-C2 bond (BDE = 86.7 kcal/mol) dissociates earlier (at s = −2 amu 1/2 bohr) than the strong alcoholic O1-H8 bond (BDE = 108.1 kcal/mol) rapture at s = −0.8 amu 1/2 bohr. The C2-H8 bond is formed at s = 1 amu 1/2 bohr and the www.nature.com/scientificreports www.nature.com/scientificreports/ carbonyl C1-O1 bond of formaldehyde is formed smoothly during the reaction. The curves of the O1-H8 and C2-H8 bonds cross each other at the transition state.</p><p>2-Methoxyacetaldehyde is a direct result for the 1,2-H 2 elimination from 2ME. The reaction proceeds via the TS6 with a barrier height and reaction energy of 88.9 and 20.9 kcal/mol, respectively. Figure 5S shows a variation of selected bonds lengths during the formation of 2-methoxy acetaldehyde. It is clear that breaking the weak C1-H1 bond (BDE = 96.2 kcal/mol) occurs first and then the alcoholic O1-H8 bond (BDE = 108.1 kcal/mol), while the carbonyl C1-O1 double bond formation progresses smoothly during the reaction.</p><p>Formation of alcohols and carbonyl compounds. Many alcohols such as methanol, vinyl alcohol, ethanol, glycolaldehyde, and ethylene glycol are released through the combustion of 2ME. Vinyl alcohol production occurs via TS2. It is the 2 nd kinetically preferable pathway with a barrier height difference of 0.6 kcal/mol relative to the most stable methoxyethene transition state TS1. The less stable vinyl alcohol (enol) transforms into the most stable acetaldehyde (keto) (TS11) via the 1,3-intramolecular H atom transfer. The reaction barrier is 55.9 kcal/mol and the reaction energy is 11.54 kcal/mol relative to the vinyl alcohol that agrees with our past recorded data 57 and with alkenol -alkanal conversion using CBS composite methods 61,62 .</p><p>According to Fig. 6S, the weakest O2-C2 bond (BDE = 86.7 kcal/mol) is broken first (at s = 2 amu 1/2 bohr) then the C1-H1 bond (BDE = 96.2 kcal/mol) stretches slowly until rapture at s = 0.9 amu 1/2 bohr. Fission of the C1-H1 bond and the formation of the alcoholic O2-H8 bond occur at the same time and the two curves cross each other at s = 0.3 amu 1/2 bohr, while the formation of the enolic double bond C1-C2 occurs step by step during the conversion process.</p><p>EG production is the highest endothermic route among all H-atom transfer channels with reaction energy of 90.6 kcal/mol. The reaction proceeds by 1,2-H-atom transfer via TS5 as one of the C γ hydrogen migrates to the O β via a strained three-membered ring transition state. The high recorded reaction energy may be related to the formation of the less stable triplet methylene. The investigation related to the IRC in Fig. 7S indicates a fast breakage of the O2-C3 bond (BDE = 86.1 kcal/mol) at s = 2 amu 1/2 bohr, while the C3-H6 bond stretches and breaks at s = 1.5 amu 1/2 bohr with the formation of the O2-H6 bond. The two curves interrupted at s = 0.8 amu 1/2 bohr. Similar to the methoxymethyl carbene, the variational of the O2-C3 bond length is a clue for the formation of the H-bond which makes the two separated atoms close to each other after the product formation.</p><p>Ethanol is produced via TS4 with an energy barrier of 86.2 kcal/mol and reaction energy of 6.7 kcal/mol. The reaction occurs by shifting one of the C γ hydrogens to the C β passing over the etheric oxygen O β . Figure 8S reveals that the O2-C2 bond breaks before the C3-H7 bond, which agrees with the bond dissociation values of the two bonds, while the O2-C3 double bond forms slowly during the reaction. Thermodynamically, ethanol formation is preferable than methoxyethene production by 0.2 kcal/mol.</p><p>Glycolaldehyde is also a bifunctional compound that has alcohol and aldehyde groups. It is formed through TS9 which is the highest energy barrier among all complex channels (100.4 kcal/mol). However, it is the preferable thermodynamic pathway with reaction energy of −1.2 kcal/mol. Figure 9S in the SI shows the earlier rapture of the least energy O2-C3 bond (BDE = 86.2 kcal/mol), while the C2-H4 bond (BDE = 96.7 kcal/mol) stretches gently till it gets broken at s = −0.6 amu 1/2 bohr. Formation of the C3-H4 bond occurs at s = 1.5 amu 1/2 bohr. The two curves of C3-H4 and C2-H4 bonds cross each other at s = 0.7 amu 1/2 bohr, while the O2-C2 double bond is formed gently during the reaction. For liner relations between ln k vs. 1000/ T for reactions R10, R11, and R12, the activation energy and pre-exponential factor can be derived from the two-parameter Arrhenius equation:</p><!><p>Taking the Natural Logarithm of the two sides</p><!><p>T 1000 shows a straight line with a frequency factor A (s −1 ) = e Intercept and an activation energy Δ = × . † E (cal/mol) slope 1 987. In Fig. 7, the tunneling correction calculated by Eckert method plays a vital role for the curvature of the relation between ln k vs. 1000/T for R1 and R2 reactions at T ≤ 500 K. Therefore, these reactions can fit the In the case of three-parameter Arrhenius equation, the following equation is used: The equation converts to the general form We will get another two equations of the three variables A, n, and Δ † E . The algebraic solution of the three Eqs (3), ( 4) and ( 5) gives values of A, n, and Δ † E . Arrhenius equations for the calculated rate constant (s −1 ) for main channels R1, R2, R10, R11, and R12 in the temperature range 298-2000 K can be summarized as follow:</p>

Scientific Reports - Nature

Small Molecule Probes of Cellular Pathways and Networks

Small molecules are important not only as therapeutics to treat disease, but also as chemical tools to probe complex biological processes. The discovery of novel bioactive small molecules has largely been catalyzed by screening diverse chemical libraries for alterations in specific activities in pure proteins assays or in generating cell-based phenotypes. New approaches are needed to close the vast gap between the ability to either study single proteins or whole cellular processes. This review focuses on the growing number of studies aimed at understanding in more detail how small molecules perturb particular signaling pathways and larger networks to yield distinct cellular phenotypes. This type of pathway-level analysis and phenotypic profiling provides valuable insight into mechanistic action of small molecules, can reveal off-target effects, and improve our understanding of how proteins within a pathway regulate signaling.

small_molecule_probes_of_cellular_pathways_and_networks

Introduction<!>Pathway-directed small molecule screening<!>Systems chemical biology and chemical profiling<!>Cancer Cell Profiling<!>Whole organism/IN VIVO chemical screening<!>Outlook<!>Small molecule probes of pathways and networks<!>A pathway screen results in small molecules that target the Rho pathway in cytokinesis

<p>Small molecules are essential as drugs in modern medicine and are valuable as probes of biological mechanism in chemical biology. Significant screening efforts to date have focused on the discovery of small molecules that target specific proteins or that confer interesting cellular phenotypes (Figure 1). Between these two spectra, there is a critical need to expand mechanistic studies into how small molecules modulate signaling pathways and larger networks to generate complex phenotypes (Figure 1). Recent advances in high-content screening methods have greatly enhanced the ability to rapidly acquire large volumes of phenotypic data following the treatment of cells with bioactive small molecules, making it possible to maximize this emerging capability (Figure 1). In this review, we discuss how pathway-level screening and analysis methods have expanded the number of pathways that can be probed with small molecules and that have allowed for a deeper understanding of how compounds alter cellular processes controlled by multiple pathways. This information has provided valuable insight into how small molecules perturb larger signaling networks and has enabled more thorough investigations into mechanisms of action, which is critical for both for drug discovery and chemical probe development.</p><p>As probes, small molecules are useful and versatile tools to study complex cellular processes requiring integrated inputs from multiple pathways. Small molecules act quickly and often reversibly upon washout, allowing a high degree of spatial and temporal modulation over protein function. Many compounds have been used successfully to probe basic cellular functions. In a historic example, the natural product cytochalasin was shown to disrupt actin filaments and it has long been used to study the role of actin in different processes of the cell including migration, ruffling and division (1, 2). Given their therapeutic potential and possible utility as research tools, it is important to develop methods to characterize in more detail the mechanism of action of existing compounds and discover new bioactive small molecules.</p><p>Active small molecules can be discovered by screening chemical libraries for alterations of specific activities in pure protein assays or for desired phenotypes in cell-based assays. Many excellent studies have been directed toward developing biochemical screening assays using purified proteins to identify small molecule inhibitors. While these in vitro assays can be extremely useful in identifying potent and selective compounds, their limits include: 1) They primarily target very specific and measurable functions of a protein or complex of proteins, either particular enzymatic activities or particular functional interactions between defined proteins, 2) potent and selective in vitro activity is not necessarily predictive of actual activity in cells, for example due to poor cell permeability and 3) the scope of off-target cellular effects due to, for example, similarities between related proteins, e.g. kinases, as well as toxicity is not readily discernible.</p><p>In contrast, small molecules discovered in phenotypic screens can target any protein, regardless of its activity, and must display some specificity to cause a specific phenotype (3). The potential of phenotypic screens for the discovery of useful compounds is high, but challenges remain because cellular phenotypes that serve as readouts in phenotypic screens often involve intricate biological processes coordinated by multiple signaling pathways. This level of complexity can present significant challenges when performing follow-up secondary assays and target identification, creating a bottleneck, which slows the rate of discovery of useful compounds.</p><!><p>To circumvent many of the challenges associated with traditional pure protein or phenotypic screens, several groups have recently developed novel strategies to identify compounds that target specific biological pathways. Pathway-directed screening approaches can simplify target identification and generate a toolbox of small molecules to systematically interrogate a given signaling pathway and identify potential "druggable" targets.</p><p>Screening strategies that combine genetic with chemical methods have been successfully implemented in several model systems, including bacteria, yeast, Dictyostelium and Drosophila. We will see below how some progress has been made in screens in different human cells lines, especially in cancer cells. Most of the new developments in pathway screening strategies, however, have taken advantage of model systems. Reasons for this include more straightforward methods for genetic manipulations and less complex, and less redundant, signaling pathways. For example, as discussed below, we performed a screen in Drosophila cells because only one isoform of our target protein/pathway, Rho, exists, while there are three isoforms in humans, and all three need to be depleted to achieve our desired phenotype (4).</p><p>Using a screening approach in bacteria that utilized both small molecules and antisense RNA technologies, scientists at Merck discovered the antibiotic platensimycin, a selective inhibitor of FabF, a key enzyme involved in bacterial fatty acid synthesis and thus critical for cell viability (5, 6). The authors screened over 250,000 natural product extracts looking for compounds that selectively inhibited the growth of a bacterial strain genetically engineered to express fabF antisense RNA. The antisense RNA-sensitized strain, because of its already reduced FabF levels, became more susceptible to killing by FabF inhibitors, providing a powerful system to screen for novel antibiotics.</p><p>Our lab developed a phenotypic screening approach to discover compounds that specifically target the Rho pathway in cytokinesis (7). Inspired by classical genetic experiments and the Merck study, we developed a screening strategy analogous to a genetic modifier screen, but perturbed cells by small molecules and RNAi instead of genetic mutations. We sensitized Drosophila cells by RNAi treatment, reducing Rho function, and screened for small molecules that modify a Rho-specific RNAi phenotype. We decided to target the pathway rather than Rho GTPase itself because, despite serious efforts by many labs, especially with the oncogenic GTPase Ras, small molecules that inhibit small GTPases have been elusive. GTP affinity in GTPases is much higher than ATP affinity in kinases, making it energetically unfavourable for a small molecule to displace GTP (8). We modestly impaired cytokinesis using partial RNAi depletion of Rho, added small molecules, and identified compounds that suppressed RNAi-induced cytokinesis defects, or aggravated them. We expected to find enhancers and suppressors because the pathway is positively and negatively regulated. An initial analysis resulted in nine compounds that increased the level of binucleated cells in a Rho RNAi background, which we named Rhodblock 1a/b-8. We confirmed that the Rhodblocks truly target the Rho pathway by showing that 8/9 compounds inhibit the phosphorylation of myosin light chain, a key function of the Rho pathway (Figure 2), and identified Rhodblock 6 as an inhibitor of Rho kinase, a key downstream Rho pathway effector. Rhodblocks will be useful tools to dissect Rho signaling and to study cytokinesis regulation.</p><p>An important pre-requisite for this screening strategy involves identifying proteins in the candidate pathway that, when perturbed by RNAi or small molecule inhibition, yield robust and measureable phenotypes that can be adapted to high-throughput screening and automated analysis. One can imagine many potential phenotypes amenable to this analysis and therefore our screening strategy should be widely applicable to generate small molecule modulators to dissect other dynamic signaling pathways.</p><p>In the previous examples, we describe screening strategies that use amplifications of genetic phenotypes by small molecules as a readout. One can also imagine a reverse approach, i.e. using genetic methods to overcome a small molecule phenotype, which would be especially valuable in small molecule target identification. To study how the mitotic spindle coordinates changes in the cell cortex during cytokinesis in Dictyostelium and to identify potential regulatory pathways, Zhou et al. screened a cDNA library for genetic suppressors of nocodazole-induced growth defects, and identified protein 14–3–3 as a hit in their screen (9). In subsequent follow-up work, the authors dissected the role of 14–3–3 in coordinating the activities of microtubules, RacE and myosin II to modulate cortical remodeling and shape changes during cytokinesis. This work is important because it provides mechanistic insight into how signals emanating from the mitotic spindle initiate cortical changes necessary for cytokinesis.</p><p>The work described above largely focuses on how single small molecules in the context of specific genetic perturbations can alter cellular pathways. Combining small molecule treatments to determine synergistic/antagonistic effects can be useful in dissecting connectivity of targets in a given pathway and understanding functional interactions between pathways in disease states (17). For example, Owens et al. systematically combined inhibitors of the sterol biosynthesis pathway and measured effects on Hepatitis C viral replication. The authors determined that sterol biosynthesis inhibitors administered in combination showed synergistic disruptions in HCV viral replication, highlighting the utility/therapeutic potential of combining drug treatments (18).</p><p>In addition to using combined genetic and small molecule perturbations to identify pathway-specific small molecules, an alternate approach involves measuring to what extent small molecules disrupt downstream signaling events of a given pathway, such as transcriptional activation or phosphorylation state changes. For example, several groups have used a Wnt/beta-catenin-mediated luciferase reporter assay to screen for modulators of Wnt signaling (19, 20).</p><p>The Wnt pathway represents an important therapeutic target because it is misregulated in many cancers. Huang et al. identified a small molecule inhibitor of Wnt signaling (XAV939) that stabilized axin and promoted beta-catenin degradation. The targets of XAV939 were discovered by three-channel iTRAQ quantitative chemical proteomics as the poly-ADP-ribosylating enzymes tankyrase 1 and 2. This study identified tankyrases as new therapeutic targets in Wnt signaling. XAV939 is an important chemical tool to study how tankyrases regulate axin homeostasis and beta-catenin degradation.</p><p>Recent work by Hoffman et al. developed a cell-based screening assay to identify small molecule modulators of mTORC1 signaling, which is known to be altered in several disease states including many cancers (21). The authors utilized a novel in cell Western technique to monitor the activation state of the mTORC1 signaling pathway by quantifying the phosphorylation status of a downstream substrate, ribosomal protein S6. The assay is currently being used to screen libraries of small molecules and siRNAs in parallel to enable direct phenotypic comparisons (22), which will provide valuable insights into target protein identification of promising small molecules.</p><!><p>The work described above largely focused on discovering small molecules that target single pathways. Recently, several groups have taken broader systems-type analyses to determine mechanism of action, where multiple pathways are profiled after small molecule treatment. Using diverse profiling approaches, different types of phenotypic compendia can be created, which are useful both as guides for comparative target identification approaches and to better understand mechanistic context of small molecule perturbations.</p><p>Gene expression profiling is commonly used to assess effects of cellular perturbations. Hughes et al. conducted gene expression profiling to functionally characterize 300 genetic mutations and small molecules in yeast (23). Lamb et al. extended this work to mammalian cells and constructed a "Connectivity Map" based on gene-expression profiles and pattern-matching analysis following small molecule treatment. The Connectivity map promises to be a useful resource to group drugs with common mechanisms of action, discover novel mechanisms for uncharacterized compounds, and find small molecules that mimic or suppress a disease state (24).</p><p>In yeast, the drug-induced haploinsufficiency profiling method (HIP) has been an important tool for drug discovery and target identification. In HIP, approximately 6,000 heterozygous deletion strains exhibit a partial reduction in gene dosage and can be screened in parallel. Strains that show a decrease in growth/fitness in the presence of a small molecule represent functionally interacting genes (10, 11). Giaever et al. and Baetz et al. used the HIP approach to screen diverse small molecules against the entire genome-wide collection of heterozygous deletions strains to determine which cellular pathways are perturbed by a given small molecule, providing insight into drug mechanism of action (11, 12). The authors validated the HIP approach by identifying previously known targets of several compounds, including methotrexate and statins, while also uncovering new interactions and mechanisms. For example, Baetz et al. tested dihydromotuporamine C, an anti-cancer compound with uncharacterized targets, and discovered that it disrupted key steps in sphingolipid metabolism.</p><p>Like HIP, the HOP (homozygous profiling) approach uses homozygous (or haploid) deletion strains (13). Parsons et al. used HOP to generate chemical-genetic fitness profiles to characterize approximately 80 compounds and natural product extracts (14). Through clustering analysis, the authors discovered that the breast cancer drug tamoxifen disrupts calcium homeostasis. Several studies have used multicopy suppression profiling to confirm a drug target (15, 16). Using this approach, overexpression of the target protein should confer resistance to the small molecule inhibitor. Hoon et al. combined the utility of HIP, HOP, and multicopy suppresssion approaches into a systems-level integrated platform to study drug mechanism of action using eight reference compounds and 188 compounds of unknown activity (16). The authors validated the platform by identifying the known molecular targets of the phosphatase inhibitors cantharidin and calyculin A and further characterizing their cellular affects, and also demonstrated its utility in finding novel interactions/potential targets.</p><p>To group known compounds of similar mechanisms of action and to suggest targets/mechanisms for new drugs, Perlman et al. developed a high-throughput and quantitative phenotypic profiling method in HeLa cells (25). Using automated fluorescence microscopy and 11 distinct biological probes, the method allowed the extraction of multidimensional phenotypic information at the single-cell level for many compounds (100) over a range of doses (13 threefold dilutions) where a set of 93 descriptors (i.e. measures of size, shape, intensity and ratios of intensities) are measured for nuclear, cytoplasmic and probe regions. Similarly, Tanaka et al. conducted a phenotypic screen of 107 small molecules where changes in cell and organelle morphology were systematically profiled to identify novel bioactive small molecules that generated unique signatures in cancer cell lines (26). From the screen and subsequent affinity-chromatography experiments, the authors identified a hydroxyl-substituted analog of the Src-family kinase inhibitor PP2 as an inhibitor of carbonyl reductase I and show that it can be used to enhance the effectiveness of established anti-cancer drugs. These two studies highlight the utility of using multidimensional phenotypic profiling to discover and characterize interesting new compounds.</p><p>While the work discussed above provided useful snapshots of the effects of different small molecule treatments, it would be very helpful to have an opportunity to observe if functional interactions in cells have been perturbed. MacDonald et al. utilized a method to probe the effects of over one hundred compounds on multiple signaling pathways simultaneously in living cells by imaging of protein-fragment complementation assays (PCAs) (27). PCA technology involves fragments of inactive reporter polypetides fused to proteins that are known to interact upon activation of a signaling pathway. When these pathway proteins interact and come into close proximity with each other, the reporter fragments are able to complex into an active reporter to generate a fluorescent signal at the site of interaction. In this study, PCAs served as sensors of specific protein-protein interactions in a given pathway and thus reported on dynamic changes occurring in that pathway in response to a compound. 49 PCAs acted as readouts for pathway activation or inhibition and reported on the status of multiple pathways involved in cell division, apoptosis, inflammation, DNA damage responses, and nuclear hormone receptor signaling. From these data, the authors assembled a multi-pathway signature profile after compound treatment. These profiles were predictive and provided insight into a drug's mechanism of action and highlighted/identified previously uncharacterized off-target phenotypes. PCAs have also been used to probe more specifically for modulators of Rho family GTPases (Ras) and MAPK signaling pathways (28).</p><!><p>The studies described above highlight the utility of image-based phenotypic and gene-expression profiling to make informed predictions about a drug's mechanism of action and make previously unidentified connections between groups of compounds. Other profiling approaches have been developed to discover compounds that specifically kill cancer cells.</p><p>For example, Dolma et al. performed a high-throughput synthetic lethal screen to look for compounds that kill genetically engineered tumor cell lines (primary fibroblasts expressing hTERT and oncogenic proteins Large T, Small T and HRAS) but not the isogenic parent cell line (29). The readout in the screen utilized calcein AM dye, which is cleaved in living cells by esterases and remains trapped in cells and exhibits green fluorescence. The authors identified nine compounds including five previously known anti-cancer drugs (doxorubicin, daunorubicin, mitoxantrone, camptothecin, and echinomycin). They examined the killing potential of the nine hit compounds in their full complement of engineered tumor cell lines (14 cell lines total) to tease apart the killing mechanism and identified which genetic elements were important for compound activity. They identified three groups of compounds (1) compounds that killed the engineered tumor cells indiscriminately (2) compounds that killed only tumor cells expressing hTERT and inactive RB, and (3) compounds that required oncogenic RAS and ST expression. This is interesting because it enables insight into which compounds might be more effective against specific cancers depending on their particular genetic expression profile.</p><p>Cancer stem cells (as well as most other stem cells) have been difficult to screen with small molecules because they exist in low numbers in tumors and are challenging to maintain in culture. Gupta et al. used hTERT-immortalized mammary epithelial cells treated with E-cadherin RNAi to induce a cancer stem cell-like state (30). The authors used their enriched cells to perform a high-throughput screen to look for compounds that selectively kill E-cadherin RNAi-induced cancer stem cells, but not control cells. They identified salinomycin as a compound that selectively killed cells with breast cancer stem cell-like properties. This is important because cancer stem cells drive cancer progression and contribute to relapse following treatment, so targeting these cells to specifically is a promising potential therapy for breast cancer.</p><p>To identify new inhibitors of Bcr-abl kinase for Chronic Myeloid Leukemia (CML) treatment, Adrian et al. utilized a cytotoxicity screen to look for compounds that selectively killed Bcr-abl transformed cells, but not non-transformed cells (31). Their screen identified the non-ATP-competitive inhibitor GNF-2. GNF-2 was shown to be effective against imatinib-resistant Bcr-abl mutants. Zhang et al. extended these studies to show that combining GNF-5, a more potent analog of GNF-2, with imatinib had additive inhibitory effects in cell-based assays and suppressed imatinib resistance emergence in vitro and in vivo (32). These studies were important because they demonstrated the therapeutic potential of combining allosteric and ATP-competitive kinase inhibitors to suppress resistance to either compound alone.</p><!><p>As discussed in the above papers, profiling in established cell lines has been very informative in identifying cellular pathways affected/perturbed by small molecules. Many diseases, however, are caused by problems in three-dimensional structures that involve connected cells (e.g. an organ) and can therefore not be studied in a cultured cell model. For this reason, whole organism high content chemical screening is becoming an important resource for expanding drug discovery and target identification efforts.</p><p>Since C. elegans was one of the first organisms where RNAi was possible, much insight has been gained into biological signaling networks using this model organism. As we have seen above, combinations of small molecule and RNAi experiments are appealing and therefore C. elegans would have been a natural model for whole organism screening (33). Despite some promising screens having been performed in C. elegans (34, 35), it appears that the general consensus now is that small molecule screens in C. elegans are difficult because small molecules typically do not accumulate in the worms due to significant physical barriers and powerful xenobiotic defenses (36).</p><p>A number of chemical screens have been conducted in zebrafish to probe a diverse array of biological processes. Zebrafish are readily adaptable to screening because the embryo, unlike C. elegans, readily absorbs small molecules. This quality makes zebrafish a nice system to study early development, a highly dynamic process involving the coordinate regulation of multiple signaling cascades. Because the embryo is quite transparent, phenotypes can be visualized in detail. For example, Peterson et al. screened for chemical suppressors of a genetic mutation disrupting aortic blood flow in zebrafish (gridlock mutation) (37). The compound GS4012 rescued the gridlock phenotype and restored blood flow by activating the VEGF pathway. Mathew et al. utilized chemical screening in zebrafish to identify modulators of tissue regeneration (38). The authors identified glucocorticoids as inhibitors of caudal fin regeneration.</p><p>Many screens involve measuring changes in visual phenotypes in response to small molecule treatment. In an exiting new study, Rihel et al. extended these approaches by monitoring animal behavior as a readout for active compounds (39). The authors profiled dynamic changes in zebrafish locomotion during rest and wake periods. Behavioral profiling allowed the authors to cluster drugs of known mechanism of action with less characterized drugs and allowed them to make meaningful predictions as to the targets and mechanisms of these unknown drugs.</p><!><p>In this review, we discussed recent work utilizing pathway-level screening and analysis methods. These approaches have: 1) expanded the number of pathways that can be probed with small molecules, 2) allowed for a more comprehensive understanding of how compounds alter larger networks of pathways by generating phenotypic profiles based on these changes, and 3) enabled compound clustering to shed light on new mechanisms of action, including identifying the target proteins of previously uncharacterized compounds.</p><p>Although much progress has been made in the effort to discover and develop small molecules as therapeutics and probes, a host of significant challenges remain: Which model systems are best to utilize for chemical screening? How do we determine which pathways/processes are best to profile? How do we integrate large profile data sets with secondary assays to make informed choices as to which leads/hits to follow-up for detailed studies? As the field of small molecule discovery moves to address these questions, we expect that significant inroads will be made. A major advance will be a better understanding of what constitutes the "druggable genome".</p><!><p>Although small molecules can be discovered in, and can target, different levels of complexity, they have been mostly used in "bottom-up" approaches starting at the protein level or in "top-down" approaches starting at the phenotype level. This review focuses on the effects of small molecules on cellular pathways and networks. Pathway/network analyses are important because they offer mechanistic insight into how small molecules perturb proteins in signaling pathways and how disruption of those pathways affects whole networks to yield complex observable phenotypes.</p><!><p>A. A small molecule/RNAi modifier screen. Cells with two nuclei are a consequence of failed cytokinesis and the readout in the screen. (Whole cells are cartooned in green, DNA in orange). B. Small molecules from the screen inhibit the accumulation of phospho-myosin at the cleavage furrow, a key function of the Rho pathway. Immunofluorescence images show Drosophila Kc167 cells where phospho-myosin (red), tubulin (green) and DNA (blue) have been visualized. Note the decrease of phospho-myosin at the furrow in compound-treated cells. Adapted from (7).</p>

PubMed Author Manuscript

Occurrence of the potent mutagens 2- nitrobenzanthrone and 3-nitrobenzanthrone in fine airborne particles

Polycyclic aromatic compounds (PACs) are known due to their mutagenic activity. Among them, 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA) are considered as two of the most potent mutagens found in atmospheric particles. In the present study 2-NBA, 3-NBA and selected PAHs and Nitro-PAHs were determined in fine particle samples (PM 2.5) collected in a bus station and an outdoor site. The fuel used by buses was a diesel-biodiesel (96:4) blend and light-duty vehicles run with any ethanol-to-gasoline proportion. The concentrations of 2-NBA and 3-NBA were, on average, under 14.8 µg g −1 and 4.39 µg g −1 , respectively. In order to access the main sources and formation routes of these compounds, we performed ternary correlations and multivariate statistical analyses. The main sources for the studied compounds in the bus station were diesel/biodiesel exhaust followed by floor resuspension. In the coastal site, vehicular emission, photochemical formation and wood combustion were the main sources for 2-NBA and 3-NBA as well as the other PACs. Incremental lifetime cancer risk (ILCR) were calculated for both places, which presented low values, showing low cancer risk incidence although the ILCR values for the bus station were around 2.5 times higher than the ILCR from the coastal site.Cancer is one of the major causes of morbidity and mortality globally. In 2012 new cancer cases accounted for about 14 million new cases, with 8.2 million deaths occurred throughout the world. From that, circa 1.69 million deaths in 2012 resulted from lung cancer. However, only less than one third of them were derived from tobacco smoke 1,2 , what indicates there are other routes contributing to lung cancer incidence. Additionally, it is expected the number of new cancer diagnoses to be risen by about 70% over the next two decades, possibly reaching 21.7 million people, and the prediction of 13 million cancer deaths in 2030 2,3 .Most cancer results from the interaction of genetics and the environment. However, hereditary or genetic factors themselves only respond for less than 10% of all types of cancers 4,5 . The remainder is attributed to environmental factors, and among them physical, chemical or biological toxicants, as well as individual susceptibility 4,6 acting to explain the large cancer incidence worldwide. Human environmental and occupational exposure to atmospheric pollutants may be one of the major causes of lung cancer since the main pathway to atmospheric carcinogenic exposition is through inhalation [6][7][8] . It is well known that energy is the single most important cause of emissions of all main pollutants, and air pollution is an energy problem 9 . Carcinogenic and/or mutagenic compounds occurring in vapor phase and atmospheric aerosols, such as unsubstituted polycyclic aromatic hydrocarbons (PAHs) and their nitrated and oxygenated derivatives (nitro-PAHs and oxy-PAHs, respectively) are of major concern in regard to the potential risk of causing cancer.Nitro-aromatic polycyclic hydrocarbons (nitro-PAHs) are ubiquitous airborne particle contaminants, mainly originated from incomplete combustion or pyrolysis of organic matter 10,11 and/or photochemically-formed in atmosphere 12 . Nitro-PAHs are persistent compounds 13 generally regarded as direct-acting carcinogenic and/or

occurrence_of_the_potent_mutagens_2-_nitrobenzanthrone_and_3-nitrobenzanthrone_in_fine_airborne_part

<!>Results and Discussion<!>Frequency of detection (%)<!>Multivariate Analysis.<!>Cancer risk from inhalation exposure. Estimates of incremental lifetime cancer risk (ILCR) according<!>Concluding Remarks<!>Instrumentation and analysis.

<p>mutagenic agents to humans or animals [13][14][15][16][17] . Even though the nitro-PAH levels are typically one order of magnitude smaller than their unsubstituted congeners in temperate or remote regions, some of their members present high direct-acting mutagenic and/or carcinogenic potency in bacterial and mammalian cells 10,13 . Representatives may be cited, such as mono-and dinitropyrenes, nitrofluoranthenes and nitroketones 18,19 .</p><p>Although the understanding of the exact mechanisms of cancer incidence derived from atmospheric aerosols remains mostly uncertain 7,20 , it may be pointed out the nitroketone species 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA) may be important contributors. They are ubiquously present in atmospheric particle samples as well as it has been reported evidences they contribute to the induction of tumors in animal models [18][19][20][21][22][23] . 3-NBA is a potent bacterial mutagen generally found in diesel and gasoline directly-exhausted particles. The 3-NBA carcinogenicity is comparable to 1,8-dinitropyrene, which is one of the most potent mutagens 19,[21][22][23][24][25][26][27][28][29][30] . Indeed, 3-NBA is likely to form adducts to DNA molecule 19,24,25,28 augmenting its genotoxic potential in living beings. In turn, the isomer 2-NBA is rather an ambient PM contaminant which is likely to be produced from the reaction of its precursor (benzanthrone, BA) with nitrogen oxides or other oxidants under typical atmospheric conditions [24][25][26][27][31][32][33] .</p><p>Despite the fact 2-NBA has been more abundantly found in airborne samples, screening assays studies suggest the genotoxic potency of 2-NBA is significantly lower than 3-NBA [34][35][36] . Both of them are lipophilic substances (K ow = 3.99 for 2-NBA and K ow = 3.90 for 3-NBA) yet they may be considered persistent in the environment 30 and may be transferred from atmosphere to other environmental compartments by wet or dry deposition. Although their inherent relevance, there is limited information about the occurrence of 2-NBA and 3-NBA in aerosol particles.</p><p>2-NBA and 3-NBA have been unevenly and eventually identified in diesel exhaust and ambient air PM worldwide, although they have remained predominantly underdetermined. In part, the reason for finding little information about particle-bound 2-NBA and 3-NBA are their very low concentration levels in atmospheric aerosols (from low ng m −3 to low fg m −3 ), ranging from 0.5-3.5 f mol m −3 (or 0.14-0.96 pg m −3 ) (3-NBA) in Central Tokyo 22 to 6.79 pg m −3 (3-NBA) in other parts of Japan 37 . Even fewer studies have considered 2-NBA. The reported concentration range of 2-NBA in ambient PM is around 49-831 f mol m −3 (or 13.5-229 pg m −3 ) 38 . Their low atmospheric concentrations together to the complex nature of particulate matter demand reliable and efficient sample preparation and analysis methods in order to be able to confidently quantify 2-NBA and 3-NBA in atmospheric samples. Nonetheless, the latest studies regarding 2-NBA and 3-NBA occurrence in atmospheric aerosols and rainwater and possible atmospheric chemistry implications are dated from late 1990s and early and mid 2000s only 22,26,27,30,32,37,39,40 . After that very little has been done in this subject. Consequently, the implications for atmospheric chemistry and their health-related endpoints are underestimated. More studies regarding the 2-NBA and 3-NBA in airborne particles are needed for better understand their role in these fields.</p><p>In the present study, we determined 2-NBA and 3-NBA in order to study the atmospheric occurrence of these species in ambient PM2.5 samples collected from a coastal tropical site in Northeastern Brazil as well as in samples collected in an underground level of a bus station, where buses exhausted mixtures of biodiesel to fossil diesel (B4) combustion during commuting. Together to that we also report some PAH levels in order to help trace atmospheric sources, which may be contributing to the found levels of 2-NBA and 3-NBA in our study. To date, this is the first time 2-NBA and 3-NBA levels are reported in the Southern Hemisphere. Cancer risk and mutagenic risk assessments from inhalation exposure were also calculated. Results are conveniently presented and critically discussed.</p><!><p>Analysis and identification of 2-NBA and 3-NBA. 2-NBA and 3-NBA have been poorly determined in atmospheric aerosols. Their low atmospheric concentrations together to the complex nature of particulate matter demand reliable and efficient sample preparation and analysis methods in order to be able to confidently quantify 2-NBA and 3-NBA in atmospheric samples. In the same way, previous studies regarding 3-NBA and isomers were mostly determined via a derivatization step prior their analysis. Generally, the 2-NBA and 3-NBA derivatization is done through a reduction step which yields 2-aminobenzanthrone and 3-aminobenzanthrone (2-ABA and 3-ABA), respectively. They are further mostly analyzed by HPLC coupled with either fluorescence 22,41,42 , UV 22,39,[41][42][43] , chemiluminescence 37,38,44 and/or mass spectrometer 41 detectors. If we consider possible losses or degradation as well as any artifact formations during 2-NBA and 3-NBA derivatization step associated to their very low atmospheric levels all together also may partially answer for the difficulty of finding them in appreciable levels in the atmospheric environment. Recently, Santos et al. 45 , reported, in a companion paper, a novel miniaturized method for the efficient determination of polycyclic aromatic compounds, and among them 3-NBA, by GC-MS with no derivatization or fractionation steps needed. Details about the sample preparation method is described in the Supplementary Information.</p><p>In the GC-MS system used in the present study 3-NBA is eluted just before 2-NBA, and both of them with retention times between 27.50 min and 27.75 min, as stated in Fig. 1. In this figure, we show two chromatograms of real samples, being Fig. 1a a chromatogram of a real PM2.5 sample from the bus station as well as Fig. 1b being a chromatogram of a real PM2.5 sample from the coastal site considered here. Accordingly, Fig. 2 shows the mass spectra of both 2-NBA and 3-NBA obtained in this study, which are in accordance with Phousongphoung and Arey 27 .</p><p>As stated in Santos et al. 45 , every analyte was monitored in SIM mode by three different m/z ions, with the addition of 2-NBA in the present study. Indeed, when our group published the results from Santos et al. 45 , 2-NBA was not listed as an analyte since we did not have its authentic analytical grade standard at that time. Nonetheless, in the current study we have added the 2-NBA to our mixed analytical standard solution and we could also quantify it in real samples without any modification of the GC-MS method. In order to distinguish 2-NBA from 3-NBA, we monitored three m/z ions, the ion base and two reference ions, in order to approach unequivocal identification. In this way, 2-NBA was identified (and differentiated from 3-NBA) by using m/z 275 (ion base) and m/z 201 and 245 (reference ions), as shown in detail in Fig. 1. In turn, 3-NBA was identified by m/z 275 (ion base) and m/z 215 and 245 (reference ions). Quantification was further done by considering the ion base signal only. Limits of detection (LOD) and limits of quantification (LOQ) were calculated from the calibration curve data. We considered LOD = 3 s/a and LOQ = 10 s/a, where "s" is the standard deviation of the linear coefficient "b" and "a" is the angular coefficient (inclination) from calibration curve (in the format y = ax + b) 45 . LOD and LOQ concentrations values were converted to the minimum absolute mass either detected (LOD) or quantified (LOQ) by the MS in 1 µL standard solution injected in the GC-MS. LOD in terms of absolute mass, were 2.0 pg and 2.4 pg for 2-NBA and 3-NBA, respectively. Limit of quantification (LOQ) were 6.6 pg (2-NBA) and 8.1 pg (3-NBA). Finally, recovery levels were above 95% for both compounds. In this way, we consider our analytical methodology is adequate for studying 2-NBA and 3-NBA in the atmospheric environment.</p><p>Occurrence of 2-NBA and 3-NBA associated to fine particles. Both 2-NBA and 3-NBA were found in PM2.5 samples collected in the bus station and coastal site (Table 1 and Fig. 3). 3-NBA and 2-NBA concentrations (±one standard deviation) were calculated as pg m −3 (picograms per cubic meter) and as mixing ratios, in terms of µg g −1 (micrograms per grams of particles). We decided to do in this way since the former considers the total sampled volume air (or a normalization of the mass of 3-NBA or 2-NBA by total sampled volume air) while the latter consider the compounds masses normalized by the collected PM2.5 masses. We consider the measurement of 3-NBA and 2-NBA in terms of µg g −1 more useful for some of the discussions done here since it better represents the intrinsic or inherent characteristics of the PM considered in relation to 2-NBA and/or 3-NBA and makes sites with different levels of particle mass concentrations (due to different emission rates among sources) more directly and easily comparable. This is also advantageous to use this type of concentration unit when trying to address toxicological responses from those species possibly present in PM. Accordingly, we still present our results in terms of pg m −3 since it is needed when comparing our study with other reports found in the literature (Supplementary Information (SI) Table S1) and for risk assessment calculations (ILCR). In this study 3-NBA concentrations were 431 (±183) pg m −3 in the bus station and 59.0 (±16.6) pg m −3 in the coastal site. In turn, 2-NBA concentration was 200 (±18.8) pg m −3 , although it was found in the coastal site samples only. Indeed, this is consistent with the fact 2-NBA is majorly formed photochemically and, therefore, it would not be found in the bus station samples (since this is a nearly indoor site mainly impacted by direct, freshly emitted vehicular particles). On the other hand, 3-NBA was found in both places, which is mainly derived from fuel burning/vehicular fleet present in these sites.</p><!><p>mass conc. (µg m −3 ) 91. The present study focuses on the determination of 2-NBA and 3-NBA in PM2.5 samples together to a better understanding of their atmospheric and human health implications. Other related and important compounds (such as 1-NPYR, 2-NPYR, 2-NFLT, 3-NFLT, FLT, PYR, BaA, and BaP) are also considered in order to support discussion and possible conclusions in regard to PAH and Nitro-PAH reactivities and source identification. Our findings show the 2-NBA levels is only about 3.4 times higher than the 3-NBA levels (Table 1). However, the few studies reported both 2-NBA and 3-NBA levels showed 2-NBA is ~35-70 higher than 3-NBA (Table S1) 22 . In our study while 2-NBA concentrations are similar to the literature, 3-NBA levels seem to be higher. This may be happening for some reasons. Considering 3-NBA is mostly emitted during vehicle fuel burning, this may be a consequence from the differences in the Brazilian fuel composition with other places in the world. Indeed, the Brazilian gasoline is 22-26% ethanol, and diesel fuel was actually a mix of 4% (v v −1 ) biodiesel into mineral diesel. Yet light-duty vehicles in Brazil has been set from the manufacturer to run with any ethanol-to-gasoline proportion. So, the 3-NBA emission is likely to differ from any other country in the world. How much is the 3-NBA emission rate from different fuel compositions should be better addressed in future studies.</p><p>Another point is that previous 2-NBA or 3-NBA studies were done mainly in temperate regions, with climate conditions completely different from our sampling places. In the tropics, the higher ambient temperature and more effective sun incidence could either favor 2-NBA photochemical production or its degradation through photolysis. All those points stated here could be plausible reasons in our study the ratio 2-NBA/3-NBA is lower than seen in literature. Since there are quite a few reported studies regarding both 2-NBA and 3-NBA these differences may also be due to limited understanding about their role in the atmosphere.</p><p>To our best knowledge, this study is the first to find 2-NBA and 3-NBA in a tropical area around a coast, and also adjacent to a large city (Salvador city and Metropolitan area) as well as to find 3-NBA emitted in the exhaust of fossil diesel-biodiesel mixes (B4) under real conditions. Yet, this is also the first study to investigate these species in fine particles, which brings more direct concern in regard to health-related endpoints, than other reports because they considered larger PM fractions only (such as PM10 and TSP). In this way, our results (Table S1) are not directly comparable with the previous ambient 22,32,38,39,42,43,[46][47][48] , nor chamber studies 26,27 , especially concerned to particle size, different concentrations units reported, the lack of information regarding sampling data and the broad sort of analytical methods used. Some selected PAHs and nitro-PAHs levels are stated in Table 1 and Fig. 3 in order to be able to better discuss possible nitro-PAHs photochemical routes and trace 2-NBA and 3-NBA main sources. 1-NPYR, 2-NPYR, 2-NFLT, 3-NFLT, and 3-NBA concentrations determined within the bus station (Table 1 and Fig. 3) represent the primary emission while the 1-NPYR, 2-NPYR, 2-NFLT, 3-NFLT, 2-NBA and 3-NBA concentrations determined in the coastal area represent both the primary and secondary contributions. They may also depend on the concentrations of PAH precursors in this site.</p><p>Our reasoning in this study in using two different sites, a bus station and a coastal site, was to investigate the emission profiles (which and how much) selected polyaromatics are emitted to atmosphere. In this study, we chose to collect PM2.5 samples in the underground floor of a bus station in order to better understand 3-NBA and related species emissions from diesel/biodiesel burning under real conditions (as opposed to sometimes unrealistic dynamometer studies). On the other hand, in the coastal area, which is affected by different sources including diesel/biodiesel burning, our goal was to evaluate the relative contribution among different sources to the found PAHs and nitro-PAHs levels in PM2.5. Possible differences in the PM2.5 emission rates from the bus station to the coastal site are minimized when using concentrations in terms of µg g −1 , which consider the polyaromatic masses (in µg) normalized by the collected PM2.5 masses (in g). Thereby, the observed differences in the PAHs and nitro-PAHs levels between the sites (Table 1) are mainly derived from different sources relative contributions in each case.</p><p>The concentrations of FLT and PYR observed in the present study, in the bus station (19.6 ± 11.0 and 37.3 ± 19.7 μg g −1 , respectively) are higher than the concentrations of 2-NFLT (2.47 ± 0.93 μg g −1 ) and 3-NFLT (11.8 ± 3.95 μg g −1 ) as well as 1-NPYR (7.81 ± 2.04 μg g −1 ) and 2-NPYR (2.82 ± 0.58 μg g −1 ). However, 2-NFLT and 2-NPYR concentration levels in the bus station are considered estimates only since they were found in 36% of the samples (which is considered a low detection frequency). Considering 2-NFLT and 2-NPYR are mainly produced photochemically and the samples from the bus station were collected in the underground floor with absence of light, they were unevenly found in this site. In this way, 2-NFLT and 2-NPYR levels from the bus station is no further considered. Hence, the nitro-PAHs (and also PAHs) determined inside the bus station are not associated to the atmospheric reactions. Indeed, our results from the bus station show both PAHs and nitro-PAHs studied here are directly emitted by fossil diesel/biodiesel burning. On the other hand, the results determined in the coastal area showed an opposite trend, the nitro-PAH levels, [2-NFLT (7.06 ± 2.08 µg g −1 ) and 2-NPYR (11.7 ± 4.14 µg g −1 )], are higher than the PAH precursors [FLT (9.24 ± 5.77 µg g −1 ) and PYR (7.71 ± 4.27 µg g −1 )]. These results are in good agreement with PAHs reactivity since PYR is less reactive than FLT in nitration reactions 15,16,[49][50][51][52][53] . Also, as can be seen by Fig. S1, air masses arriving the sampling site were subjected to long-range transport (oceanic origin). In fact, during the long-range transport probably PAH-rich air masses were gradually being transformed to their respective nitro-PAH congeners. Indeed, 2-NFLT is known to be formed via reaction between FLT and NO 3 or OH radicals while 2-NPYR is the product of the reaction between PYR and OH radicals only 17,22,37,[49][50][51][52][53] . This route seems to be important for tropical and warm areas, where OH radical formation is likely to be enhanced due to higher sunlight incidence. This implies in the coastal area there are important enrichment of 2-NFLT and 2-NPYR, both photochemically produced, in relation to directly-emitted FLT and PYR by automobiles, as observed when compared with their concentrations found in the bus station.</p><p>On the other hand, 2-NBA formation is mainly done via heterogeneous reaction between benzanthrone (not measured here) and NO 3 or OH radicals on preexisting particles. According to Abbas et al. 54 , there are three different processes which may lead to nitro-PAHs formation from the parent PAHs. They can be formed by (a) electrophilic nitration within the combustion process (e.g. in the exhaust of diesel or gasoline vehicle engines, wood burning and cooking), (b) by gas-phase reactions with atmospheric oxidants (as a secondary and homogeneous process), and (c) by heterogeneous oxidation of particle-bound PAHs (also a secondary process). For instance, 3-NBA is mainly formed by the mechanism described by (a). Only parent PAHs with less than 4 benzene rings and high vapor pressure would be substantially in gas phase in order to react with oxidants in typical atmospheric conditions to form their respective nitro-PAHs, as in (b). Considering benzanthrone vapor pressure is low and boiling point is high (2.21 × 10 −7 mm Hg and 403 °C, respectively) and log K oa is high (10.378) (Table S2), this species would be principally present in PM rather than gas phase under typical atmospheric conditions. In this way, we argue 2-NBA main formation route would be via heterogeneous reactions. However, the 2-NBA formation through heterogeneous reaction still needs to be addressed in future studies.</p><p>In terms of source tracing, it is well accepted 2-NBA, 2-NFLT, and 2-NPYR are predominantly generated via photochemical reactions in the atmosphere while 3-NBA, 3-NFLT, and 1-NPYR is mainly emitted by diesel combustion/vehicles 17,22,37,53,54 . Here again, it implies photochemical reactions are important to explain the nitro-PAHs and PAHs atmospheric levels in the coastal area.</p><!><p>Figure 4 shows ternary correlations, which are useful for improving source identification. For 1-NPYR, 3-NFLT, and 3-NBA we found high correlation (r = 0.8643, p = 0.0322) for the bus station and (r = 0.8303, p = 0.0538) for the coastal area. Since they are mainly directly emitted by diesel combustion, this high correlation implies this source is relevant for them in both sites. In turn, for 2-NPYR, 2-NFLT, and 2-NBA we also see high correlation (r = 0.8652, p = 0.0317) for coastal area, which demonstrates photochemistry actually is an important source for this ambient site. Although in the second case the p-value is little higher than 0.05, indicating a statistical confidence slightly lower than 95%, we should keep in mind the r-values are high enough to be statistically valid when considering ternary correlations.</p><p>Principal component analysis (PCA) were run for both sites (Fig. 5 and SI Table S3). For the bus station PCA explains 66.9% of the whole dataset variance. PC1 accounted for 34.7% of the variance, and it had high positive loadings for particle mass and mass concentration as well as high negative loading for 3-NFLT, and moderate negative loadings for 3-NBA, BaP, and 1-NPYR. In turn, PC2 explained 32.2% of the variance, with high positive loadings for FLT, PYR and BaA. PC1 seems to indicate diesel-biodiesel exhaust direct-emitted as source while PC2, which is represented by less reactive species, then they are able to be constituents of particles direct-emitted by buses and deposited on the floor and be suspended again, which may indicate particle aging and/or particle size growing processes happening in this site. For the coastal site, it was necessary to consider three principal components to explain 82.6% of the total variance. PC1 explains 54.1%, with high negative loadings for particle mass and mass concentration as well as high positive loadings for most of the nitro-PAHs (2-NBA, 2-NFLT, 3-NFLT, 1-NPYR, and 2-NPYR), except for 3-NBA represented in the PC2 (which accounted for 14.9% of the variance), together to FLT and PYR. PC3 accounted for 13.6% of the variance and presents high positive loadings for BaP and BaA. For this site, PC1 represents the photochemical origin (as important secondary source tracers, such as 2-NBA, 2-NPYR and 2-NFLT are better explained in this PC). Although in PC1 3-NFLT and 1-NPYR, which are direct-emitted species, are also presented, it seems through particle aging, some isomers may interconvert into the other (2-NFLT to 3-NFLT and 1-NPYR to 2-NPYR or vice-versa) forming complex interactions. PC2 may indicate direct emission of fuel combustion as probable source since 3-NBA, PYR, and FLT are presented by this PC. Finally, PC3, which has high scores for BaA and BaP, is likely to represent wood combustion as sources.</p><p>Agglomerative hierarchical clustering (AHC) plots (Fig. 6a,b) for the bus station does not show statistically significant dissimilarities for particles collected in the morning, afternoon or night periods nor among species (PAHs and nitro-PAHs). This is due to the fact the bus station does have few different sources contributing to the levels of PAHs and nitro-PAHs in fine particles there, as suggested by the PCA study. On the other hand, for the coastal site (Fig. 6c,d), in the AHC plots there are 3 clusters discriminated. The first cluster is formed by mass concentration, 2-NBA, 2-NPYR, FLT, PYR, BaA, 3-NBA, and 2-NFLT. In turn, the second cluster is only formed by BaP, and the last cluster includes particle mass, 3-NFLT, and 1-NPYR. The first cluster represents the parent PAHs and their nitro derivatives, while the second one is tentatively attributed to wood combustion. Finally, the last cluster is representative of primary emissions.</p><!><p>to four different age groups, namely infants (<1 year), children (1-11 years), adolescents (11-16 years), and adults (>21 years) in the population are summarized in Table 2. BaPeq considering the carcinogenicity contribution are 1.21 and 0.45 ng m −3 as well as BaPeq due to mutagenicity 0.90 and 0.38 ng m −3 for the bus station and coastal site, respectively. Despite the fact there are several studies showing 2-NBA and 3-NBA high levels of carcinogenicity and/or mutagenicity 11,13,18,21,[25][26][27]29,30,36,55,56 are similar to those ones of dinitropyrenes (which, in turn, are considered the most potent carcinogens and mutagens), there are no TEFs and MEFs for the nitrobenzanthrone isomers. At this point, knowing the 2-NBA and 3-NBA carcinogenicity and mutagenicity potencies and not considering them in the ILCR calculations would be a not acceptable underestimation for such relevant target species for human health. This would be interesting to account the nitrobenzanthrones in the ILCR since representing the air toxicity by considering only the 16 priority PAHs is not enough anymore in face to the recent advances in this area and the scientific community recent discussions 56,57 . Even though knowing this is not ideal, we have decided to tentatively use the 1,3-dinitropyrene (1,3-DNPYR) TEF and MEF values as surrogates for 2-NBA as well as 1,8-dinitropyrene (1,8-DNPYR) TEF and MEF as surrogates for 3-NBA (SI Table S4). In our point of view, this is plausible because the 1,3-DNPYR and 1,8-DNPYR carcinogenicity and mutagenicity in assays using mammalian cells, bacteria and rodents are close to the ones from 2-NBA and 3-NBA in the same screening assays 11,19,21,22,[25][26][27][28][29][30] . After that we were able to calculate more realistic BaPeq values (Table 2). In this way, even though we are aware this may be just a proxy to their real values, we propose to adopt 1,8-DNPYR and 1,3-DNPYR TEF and MEF values as 2-NBA and 3-NBA TEF and MEF, respectively. This could be a temporary alternative while studies about evaluation of the real TEF and MEF for the nitrobenzanthrone isomers are not directly measured. Bus station total daily inhalation exposure (E I ) (considering both carcinogenicity and mutagenic contributions), in ng person −1 day −1 according to different age groups, were 14.4, 28.1, 46.3, and 34.7 for infants, children, adolescents and adults, respectively. Total ILCR was 9.48 × 10 −8 (infants), 5.24 × 10 −7 (children), 2.17 × 10 −7 (adolescents), and 9.73 × 10 −7 (adults). These ILCR estimates mean there is a risk of 9.48 infants in a hundred million and ranges up to 9.73 adults in ten million commuting this bus station to develop cancer during their lifetime.</p><p>In the coastal site total E I were 5.66, 11.1, 18.2, and 13.6 ng person −1 day −1 for infants, children, adolescents, and adults respectively. Total ILCR were 3.73 × 10 −8 (infants), 2.06 × 10 −7 (children), 8.64 × 10 −8 (adolescents), and 3.83 × 10 −7 (adults). Coastal site ILCR is about 2.5 times shorter than the bus station ILCR. In the same way, ILCR estimates for this site means there is a chance of 3.73 infants in a hundred million and ranges up to 3.83 adults in a ten million that may get cancer during their 70 years of lifetime.</p><p>If we compare these ILCR to other recent studies 15,16,23,[58][59][60] our estimates could be considered one or two orders of magnitude lower, which primarily may not raise much concern. This is needed further studies in order to obtain more comprehensive ILCR estimates. On the other hand, it should be kept in mind these estimates only address ILCR from about ten polycyclic compounds in fine particles considered in this study. This is well accepted there are thousands of other compounds constituting fine particulate matter and we actually have investigated the large majority of them and much less is known about their toxicity in regard to carcinogenicity and mutagenicity.</p><!><p>Selected fine particulate PAHs and nitro-PAHs were characterized under typical conditions from a bus station and a coastal site. Among nitro-PAHs, 2-NBA and 3-NBA, which are potent carcinogens and mutagens were determined for the first time in the Southern Hemisphere. The main sources for the studied compounds in the bus station were mineral diesel/biodiesel exhaust followed by floor resuspension (which contributed to the particle growing and ageing). In the coastal site, vehicular emission, photochemical formation and wood combustion were the main sources for 2-NBA and 3-NBA as well as the other polycyclic aromatic compounds. Incremental lifetime cancer risk (ILCR) were calculated for both places, which presented low values, showing low cancer risk incidence although the ILCR values for the bus station were around 2.5 times higher than the ILCR from the coastal site.</p><!><p>In this study, we used the chromatographic conditions stated in Santos et al. 45 Briefly describing, we utilized a high-resolution gas chromatograph-high-resolution mass spectrometer detector (HRGC-HRMS) from Shimadzu (GCMS-QP2010Plus, Shimadzu, Japan) with a Rtx-5MS gas capillary column (30 m × 0.250 mm × 0.25 µm, Restek Bellofonte, USA). Oven temperature programing initiated at 70 °C (2 min), then rising from 70-200 °C (30 °C min −1 , 5 min), and 200-330 °C (5 °C min −1 , 0.67 min). Injector temperature was set at 310 °C and transfer line was 280 °C. Analysis was done in GC-MS-SIM, at electron impact mode (EI) (70 eV). Sample preparation was done using a filter piece of 4.15 cm 2 diameter added to a miniaturized micro-extraction device using 500 µL solvent extraction 45,61 . Sample preparation details are found in Supplementary Information.</p><p>Sample collection. PM2.5 samples were collected in two different sites: (i) in the underground floor of a bus station (12°58′S, 38°30′W, 52 m altitude), and (ii) in a coastal area (12°58′S, 38°30′W, 52 m altitude) in Northeastern Brazil [62][63][64] . The PM samples collected in the bus station were mostly subjected to the exhausts from buses, which remained on (in idle point) while waiting for passengers. No substantial additional sources have contributed to the found levels of polycyclics since the underground level of this bus station is a nearly indoor environment. Yet, no air dispersion system was present. This bus station has been previously studied by our research group 45,61,62,64 and our findings show PM sample sources mainly are biodiesel/diesel burning released by buses and dust resuspension. During the sampling period, buses were using the B4 mix as fuel (4% v v −1 biodiesel to fossil diesel, as established by law at that time). In turn, PM2.5 samples were collected around the Todos os Santos Bay, in the Brazilian Navy Base, located in Salvador Metropolitan Area, State of Bahia, Northeastern Brazil. This site is close to industries and the Aratu Harbor and it is also influenced by vehicular fleet from Salvador City and surroundings 62,64 . During sample collection, ambient temperature ranged from 22.5 to 24.9 °C, relative humidity was 72-84%, solar radiation was 145-340 W m −2 , and wind speed ranged 3.9-8.0 m s −1 (Mkoma et al. 62 ). Backward air mass trajectories were calculated starting 48 h before arrival time (00:00 UTC) and 500 m a.g.l. During the sampling time air mass trajectories were of typically oceanic origin passing through urban and industrial areas around the coast (Fig. S1). PM 2.5 samples were collected using a high-volume (Hi-Vol) sampler (Energetica, Brazil) with an inlet for classifying particles smaller than 2.5 μm aerodynamic diameter (Thermo Andersen, USA). Samples were collected on quartz microfiber filters (22.8 cm × 17.7 cm, Whatmann, USA) over 4-12 h periods (7 AM to 2 PM, 2 PM to 7 PM, and 7 PM to 7 AM, completing a 24 h period per day, at the bus station) or during 24 h at the coast, at 1.13 m 3 min −1 . The sampling campaigns lasted 15 consecutive days in each place. After collection, filters were folded in half face-to-face, placed in an aluminum foil envelope then in a zip lock type plastic bag, and finally placed in sealed plastic containers for avoiding any contamination. Following, samples were transported cool to the laboratory and stored in a freezer (−4 °C) until analysis. Field blanks also were considered in this study. Field blank filters were placed in the same containers where sample filters were transported from laboratory to the sampling site and back to laboratory in order to make any minor contamination, if there was any, traceable. Our procedure included analysis of both sample and field blank filters in exactly same way for having any detectable analyte signal in field blank discounted from sample filter results. In this work, we did not detect any analyte in the field blanks.</p><p>Backward air mass trajectories and statistical analysis. Backward air mass trajectory frequencies were calculated during the coastal site sampling time by using the NOAA HYSPLIT database 65,66 . Trajectory frequencies (as number of endpoints per squared grid per number of trajectories) were calculated with frequency grid resolution 1.0° × 1.0°, starting 96 h before arrival time (00:00 UTC) and altitudes ranging from 0 to 99999 m a.g.l. During the sampling period, air mass trajectories were of typical oceanic contribution, passing on Atlantic Ocean through urbanized city centers and industrialized areas around the coast before arriving to our coastal sampling collection site (SI Fig. S1).</p><p>Multivariate statistical analyses, such as Pearson correlation, ternary correlation, Principal Component Analysis (PCA) and Agglomerative Hierarchical Clustering (AHC) were calculated for both dataset by using XLSTAT BASE software package version 19.5 for Microsoft Excel from Addinsoft Ltd (Paris, France). Ternary correlations were done by STATISTICA version 12.0 (Statsoft, USA).</p><p>Incremental lifetime cancer risk assessment. Carcinogenic and mutagenic risk assessments 15,[60][61][62][63][67][68][69] induced by inhalation of PM2. 2-NBA, and 3-NBA) and PAHs (PYR, FLT, BaP, and BaA) were estimated in the bus station and coastal site samples according to calculations done by Wang et al. 60 , Nascimento et al. 61 , and Schneider et al. 67 PAH and PAH derivatives risk assessment is done in terms of BaP toxicity, which is well established [67][68][69][70][71][72][73] . The daily inhalation levels (E I ) were calculated as:</p><p>where E I (ng person −1 day −1 ) is the daily inhalation exposure, IR (m³ d −1 ) is the inhalation rate (m³ d −1 ), BaP eq is the equivalent of benzo[a]pyrene (BaP eq = Σ C i × TEF i ) (in ng m −3 ), C i is the PM2.5 concentration level for a target compound i, and TEF i is the toxic equivalent factor of the compound i. TEF values were considered those from Tomaz et al. 15 , Nisbet and LaGoy 69 , OEHHA 72 , Durant et al. 73 , and references therein. E I in terms of mutagenicity was calculated using equation (1), just replacing the TEF data by the mutagenic potency factors (MEFs) data, published by Durant et al. 73 . Individual TEFs and MEFs values and other data used in this study are described SI, Table S4. The incremental lifetime cancer risk (ILCR) was used to assess the inhalation risk for the population in the Greater Salvador, where the bus station and the coastal site are located. ILCR is calculated as:</p><p>where SF is the cancer slope factor of BaP, which was 3.14 (mg kg −1 d −1 ) −1 for inhalation exposure 60 , EF (day year −1 ) represents the exposure frequency (365 days year −1 ), E D (year) represents exposure duration to air particles (year), cf is a conversion factor (1 × 10 −6 ), AT (days) means the lifespan of carcinogens in 70 years (70 × 365 = 25,550 days) 70,72 , and BW (kg) is the body weight of a subject in a target population 71 .</p><p>The risk assessment was performed considering four different target groups in the population: adults (>21 years), adolescents (11-16 years), children (1-11 years), and infants (<1 year). The IR for adults, adolescents, children, and infants were 16.4, 21.9, 13.3, 6.8 m 3 day −1 , respectively. The BW was considered 80 kg for adults, 56.8 kg for adolescents, 26.5 kg for children and 6.8 kg for infants 70 .</p>

Scientific Reports - Nature

p-SCN-Bn-HOPO: A Superior Bifunctional Chelator for 89Zr ImmunoPET

Zirconium-89 has an ideal half-life for use in antibody-based PET imaging; however, when used with the chelator DFO, there is an accumulation of radioactivity in the bone, suggesting that the 89Zr4+ cation is being released in vivo. Therefore, a more robust chelator for 89Zr could reduce the in vivo release and the dose to nontarget tissues. Evaluation of the ligand 3,4,3-(LI-1,2-HOPO) demonstrated efficient binding of 89Zr4+ and high stability; therefore, we developed a bifunctional derivative, p-SCN-Bn-HOPO, for conjugation to an antibody. A Zr-HOPO crystal structure was obtained showing that the Zr is fully coordinated by the octadentate HOPO ligand, as expected, forming a stable complex. p-SCN-Bn-HOPO was synthesized through a novel pathway. Both p-SCN-Bn-HOPO and p-SCN-Bn-DFO were conjugated to trastuzumab and radiolabeled with 89Zr. Both complexes labeled efficiently and achieved specific activities of approximately 2 mCi/mg. PET imaging studies in nude mice with BT474 tumors (n = 4) showed good tumor uptake for both compounds, but with a marked decrease in bone uptake for the 89Zr-HOPO-trastuzumab images. Biodistribution data confirmed the lower bone activity, measuring 17.0%ID/g in the bone at 336 h for 89Zr-DFO-trastuzumab while 89Zr-HOPO-trastuzumab only had 2.4%ID/g. We successfully synthesized p-SCN-Bn-HOPO, a bifunctional derivative of 3,4,3-(LI-1,2-HOPO) as a potential chelator for 89Zr. In vivo studies demonstrate the successful use of 89Zr-HOPO-trastuzumab to image BT474 breast cancer with low background, good tumor to organ contrast, and, importantly, very low bone uptake. The reduced bone uptake seen with 89Zr-HOPO-trastuzumab suggests superior stability of the 89Zr-HOPO complex.

p-scn-bn-hopo:_a_superior_bifunctional_chelator_for_89zr_immunopet

INTRODUCTION<!>Zr-HOPO Crystal Structure<!>Bifunctional Ligand Synthesis<!>Ligand\xe2\x80\x93Antibody Conjugation<!>Radiolabeling<!>Serum Stability<!>Immunoreactivity<!>Imaging<!>Biodistribution<!>Comparison with Other Ligands<!>Hydroxamate-Based Chelators<!>Nonhydroxamate-Based Chelators<!>CONCLUSIONS<!>Materials and Methods<!>Crystal Structure<!>Synthesis<!>(N1,N4,N9-Tri-tert-butoxycarbonyl)-1,12-diamino-4,9-dia-zadodecane (4)<!>tert-Butyl(4-((tert-butoxycarbonyl)(3-((4-nitrophenethyl)-amino)propyl)amino)butyl) (3-((tert-butoxycarbonyl)-amino)propyl)carbamate (5)<!>N1-(3-Aminopropyl)-N4-(3-((4-nitrophenethyl)amino)-propyl)butane-1,4-diamine (6)<!>1-(Benzyloxy)-N-(3-(1-(benzyloxy)-6-oxo-1,6-dihydropyri-dine-2-arboxamido)propyl)-N-(4-(1-(benzyloxy)-N-(3-(1-(benzyloxy)-N-(4-nitrophenethyl)-2-oxo-1,2-dihydropyridine-3-carboxamido)propyl)-2-oxo-1,2-dihydropyridine-3-carboxamido)butyl)-6-oxo-1,6-dihydropyridine-2-carboxa-mide (7)<!>N-(4-(N-(3-(N-(4-Aminophenethyl)-1-(benzyloxy)-2-oxo-1,2-dihydropyridine-3-carboxamido)propyl)-1-(benzyloxy)-2-oxo-1,2-dihydropyridine-3-carboxamido)butyl)-1-(benzyl-oxy)-N-(3-(1-(benzyloxy)-6-oxo-1,6-dihydropyridine-2-carboxamido)propyl)-6-oxo-1,6-dihydropyridine-2-carboxa-mide (8)<!>4-(11,15-Bis(1-hydroxy-2-oxo-1,2-dihydropyridine-3-car-bonyl)-1-(1-hydroxy-6-oxo-1,6-dihydropyridin-2-yl)-6-(1-hy-droxy-6-oxo-1,6-dihydropyridine-2-carbonyl)-1-oxo-2,6,11,15-tetraazaheptadecan-17-yl)benzenaminium chloride (9)<!>1-Hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydropyridine-2-carboxamido)propyl)-N-(4-(1-hydroxy-N-(3-(1-hydroxy-N-(4-isothiocyanatophenethyl)-2-oxo-1,2-dihydropyridine-3-carboxamido)propyl)-2-oxo-1,2-dihydropyridine-3-carboxamido)butyl)-6-oxo-1,6-dihydropyridine-2-carboxa-mide (p-SCN-Bn-HOPO, 10)<!>Ligand\xe2\x80\x93Antibody Conjugation<!>Radiolabeling Studies<!>Serum Stability Studies<!>Immunoreactivity Assay<!>PET Imaging<!>Biodistribution

<p>Antibodies possess exquisite specificity and affinity for their antigens,1 and as a consequence, positron emission tomography (PET) using targeted antibodies is a molecular imaging technique at the forefront of cancer diagnosis and treatment management.1–6 Zirconium-89 (89Zr), a positron-emitting radionuclide, possesses excellent physical properties for PET imaging when paired with antibodies, namely, an ideal 78.41 h half-life and low energy positron (βavg = 395.5 keV), and is readily attracting attention for this purpose.7–14 In the past several years, a wide variety of preclinical studies have been published15–21 and a number of 89Zr-based antibody imaging agents have been translated into the clinic, including a number of current clinical trials in the US alone.2–4,22–25</p><p>These clinical studies and all preclinical studies use the current standard bifunctional chelator for 89Zr: desferrioxamine B (DFO).16 DFO, a natural bacterial siderophore, is a hexadentate ligand with three hydroxamate groups which provide six oxygen donors for metal binding.26 It possesses an amine tail that can be derivatized for facile conjugation to antibodies and other biomolecular vectors. Although image quality is generally very good, DFO is not the optimal ligand for 89Zr. This is revealed by the observed uptake of radioactivity in the bone.7,16,27,28 This uptake is evidence of in vivo release of 89Zr4+ from the chelator. When unbound, the osteophilic 89Zr4+ cation is readily mineralized into the skeleton.28,29 This accumulation of 89Zr4+ in the bone can dramatically increase radiation dose to the bone marrow, an especially radiosensitive tissue. While the extent of this uptake is less established in the clinic, it is still being investigated and may be of particular concern since 89Zr-immunoPET agents have found specific use in the detection of bone metastasis.30 This concern over in vivo stability sparks the need to develop an improved bifunctional chelator for Zr that will significantly improve 89Zr-antibody PET imaging by providing an improved alternative to DFO, reducing absorbed doses to healthy tissues and therefore safer PET imaging, and enhanced image quality.</p><p>Recently, there has been a surge of interest in the development of an alternative chelator for 89Zr4+ to replace DFO, with several novel ligand systems being reported within the past year or so (Figure 1).31–37 While multiple studies demonstrate the issue of bone uptake seen with 89Zr-DFO complexes and stress the need for improved chelators,16,27,28,34 the first investigation toward designing a better chelator of Zr4+ came from Guérard et al.38 This work examined the coordination chemistry of the Zr4+ cation and confirmed the advantage of an octa-coordinate zirconium complex as Zr4+ was shown to preferentially form complexes with eight oxygen donors contained within four bidentate hydroxamate groups. This study opened the door for the investigation of octadentate ligands to replace the hexadentate DFO chelator with the goal of improving in vivo stability. Thus, far, however, there has been no reporting of a new ligand for 89Zr4+ that has been demonstrated to be viable in vivo for a sufficient length of time for antibody imaging. Several potential ligands require additional development while others simply require further evaluation. Herein, we present the first successful demonstration of an alternative chelator for 89Zr that includes PET imaging and biodistribution data that shows improved stability over DFO across a period of several days in vivo.</p><p>We investigated the potential of a nonhydroxamate-based ligand—3,4,3-(LI-1,2-HOPO) or HOPO—which has four 1,2-hydroxypyridinone groups for metal binding and comes from the actinide sequestration literature.39 As we postulated, the HOPO ligand labeled efficiently and 89Zr-HOPO exhibited equal or superior stability compared to 89Zr-DFO in all chemical and biological assays.34 Not only did the 3,4,3-(LI-1,2-HOPO) ligand show tremendous promise in our preliminary evaluation, but even more recently, stability constants for Zr-HOPO were determined to be on the order of log β = 43, the highest recorded for any Zr complex which attests to the superior stability.40 Therefore, we endeavored to develop a bifunctional variant of the HOPO ligand for further evaluation and application in antibody-based PET imaging. The result of this venture is the bifunctional chelator: p-SCN-Bn-HOPO (Figure 2). This molecule is the HOPO ligand with a para-benzyl-isothiocyanate pendant arm added to one of the secondary amides in order to be directly comparable with the currently most used bifunctional chelator: p-SCN-Bn-DFO (Figure 2). We also report the crystal structure of Zr-HOPO which corroborates the high stability.</p><!><p>Our past work demonstrated the stability of the Zr-HOPO complex in vitro, in vivo, and in silico, which led to the advancement of the HOPO ligand into a bifunctional chelator. However, efforts were also made to confirm the calculated structure. These efforts came to fruition with the successful crystal growth and crystal structure determination of the Zr-HOPO complex (Figure 3). This structure confirmed that the central Zr4+ ion is bonded to eight oxygen donor atoms from the four 1,2-HOPO units to form a neutral complex. The immediate coordination geometry about the Zr is identical to that of the crystal structure reported by Guérard et al. of Zr(Me-AHA)4, where Zr is chelated by four bidentate N-methyl acetohydroxamic acid groups.38 The bond lengths of Zr-HOPO and Zr(Me-AHA)4 are similar as well (Table S.1). Figure S.1 shows the variation of bond lengths in these two crystal structures and in the DFT calculated structure of Zr-HOPO (Zr-HOPOcalc).34</p><p>The Zr-HOPOcalc structure is more contorted and possesses slightly longer Zr–O bond lengths than the single crystal structure (Figure S.2). Optimization of the structure in the gas phase showed several local energy minima, and the lowest energy structure possesses an unfavorable gauche orientation of the –(CH2)4– linker between the middle two chelating groups of the HOPO ligand. In contrast, the Zr-HOPO crystal structure displays an open orientation of the –(CH2)4– linker between the middle two chelating groups. These features point to the flexibility of HOPO backbone and versatility toward metal coordination. The differences in overall structure may likely be a result of crystal packing forces, which the calculations do not take into account. A detailed comparison of the Zr-HOPO crystal structure to the DFT structure as well as to a recently published Eu-HOPO− structure41 is included in the Supporting Information.</p><!><p>Initial attempts were made to attach a linker arm directly to one of the secondary amides of the original 3,4,3-(LI-1,2-HOPO) ligand in order to make it bifunctional; however, our efforts were unsuccessful. Coupling to a single secondary amide proved to be impractical and so we developed a novel synthesis to rebuild the ligand from scratch (Scheme 1). The new method built the pendant arm into the backbone itself before coupling the hydroxypyridinone groups onto it. The synthesis of the bifunctional chelator proved to be challenging, with a particular difficulty in the deprotection and purification steps, but it was ultimately achieved. The final product, p-SCN-Bn-HOPO, was purified by HPLC and characterized by NMR, IR, and HRMS.</p><!><p>p-SCN-Bn-DFO was conjugated to antibodies through the formation of a thiourea bond with the amine side chain of a lysine residue. The p-SCN-Bn-HOPO ligand was designed to be attached in the very same way. Both ligands were conjugated to trastuzumab at a ratio of 5:1 ligand:antibody in the reaction mixture. The average number of chelates per antibody was determined to be 2.0 ± 0.5 for p-SCN-Bn-DFO and 2.8 ± 0.2 for p-SCN-Bn-HOPO through a simplified isotopic dilution assay.</p><!><p>All compounds were radiolabeled under mild conditions using a 89Zr-oxalate solution at pH 7 and room temperature. Reaction progress was monitored using radio-TLC. First, the bifunctional chelators p-SCN-Bn-HOPO and p-SCN-Bn-DFO were radiolabeled on their own without being attached to any targeting vectors to compare their Zr binding ability. Both ligands labeled quantitatively within 1 h without issues. This confirmed that the benzyl isothiocyanate linker arm did not interfere with the metal binding. Next, the chelator-modified trastuzumab complexes were radiolabeled under the same conditions. Both complexes labeled within 1–3 h at room temperature and achieved specific activities of approximately 2 mCi/mg. Radiolabeled antibody conjugates were purified via size exclusion chromatography and spin filtration.</p><!><p>The 89Zr-ligand complexes alone as well as the 89Zr-ligand-antibody complexes were evaluated for stability in human serum at 37 °C. Both 89Zr-ligand complexes showed great stability with 97.7 ± 0.2% of the p-SCN-Bn-DFO complex and 97.5 ± 0.5% of the p-SCN-Bn-HOPO complex intact after 7 d. When the ligands were conjugated to trastuzumab and then labeled, both complexes demonstrated slight decreases in stability, with the 89Zr-DFO-tratuzumab complex showing 94.7 ± 0.7% stability and the 89Zr-HOPO-tratuzumab complex showing 89.2 ± 0.9% stability after 7 d. While 89.2% for the HOPO complex is still reasonably stable, it is notably less than the 94.7% stability of the DFO conjugate. The reason for the change in stability between the 89Zr-ligand complexes and the 89Zr-ligand-antibody complexes is currently unknown, but may be due to the influence of the antibody side chains altering the chelation environment of the metal either during radiolabeling or during the serum incubation.</p><!><p>The viability of the 89Zr-labeled trastuzumab complexes was assayed against BT474 cells to ensure that the conjugation of the chelators did not disrupt the biological activity of the antibody. The 89Zr-DFO-trastuzumab and 89Zr-HOPO-trastuzumab conjugates were found to have immunoreactive fractions of 88.6 ± 2.1% and 92.4 ± 6.8%, respectively.</p><!><p>PET imaging was carried out in order to directly compare the in vivo behavior and pharmacokinetics of the DFO- and HOPO-based 89Zr-trastuzumab radioimmunoconjugates. Female athymic nude mice with subcutaneous BT474 xenografts in their right shoulders were injected with either 89Zr-DFO-trastuzumab or 89Zr-HOPO-trastuzumab (n = 4 for each compound) and imaged over 9 d. The resulting images showed good tumor uptake for both compounds, but with a marked decrease in the appearance of bone uptake for the 89Zr-HOPO-trastuzumab images (Figure 4). While the liver is more visible in the 89Zr-HOPO-trastuzumab images, particularly the maximum intensity projections, this may be due to how the images are scaled individually and not directly comparable in terms of intensity. The reduced bone uptake seen with 89Zr-HOPO-trastuzumab suggests superior stability of the 89Zr-HOPO complex. The difference in in vivo performance in contrast to the in vitro stability study highlights the inadequacy of the serum stability assay alone. This demonstrates the successful use of 89Zr-HOPO-trastuzumab to image BT474 breast cancer with low background, good tumor to organ contrast, and, importantly, very low bone uptake.</p><!><p>Acute biodistribution experiments were performed to further probe the localization and uptake of 89Zr-DFO-trastuzumab and 89Zr-HOPO-trastuzumab. These results corroborate the observations from the PET images with the activity associated with all collected tissues, except the tumors and the bone, decreasing over time (Figure 5). The biodistribution data reveals the liver uptake to be essentially the same for both compounds which suggests that the difference in appearance seen in the images is due to differences in scaling rather than a difference in actual uptake. Both compounds showed good uptake in the tumor with the DFO complex achieving even higher uptake than the HOPO compound (138.2 ± 35.3 vs 61.9 ± 26.4%ID/g at 336 h, Table 1). The difference in tumor uptake between the two compounds is not easily understandable as the immunoreactivity was not significantly different and they are using the same targeting method. Biodistribution data confirmed the lower bone activity of the HOPO conjugate, measuring 17.0 ± 4.1%ID/g in the bone for the 89Zr-DFO-trastuzumab, while the 89Zr-HOPO-trastuzumab only had 2.4 ± 0.3%ID/g at 336 h. The amount of activity seen in the bone with 89Zr-HOPO-trastuzumab is consistently less than the residual blood activity, which means it is possible that there is no specific bone accumulation since the %ID/g values do not increase over time (Figure 6). This is particularly striking when compared with the constantly increasing bone uptake seen with 89Zr-DFO-trastuzumab, which is indicative of accumulation of 89Zr4+ in the skeleton.</p><p>While 89Zr-DFO-trastuzumab has a better tumor:blood ratio than 89Zr-HOPO-trastuzumab (31.4 vs 14.4 at 336 h, respectively), the 89Zr-HOPO-trastuzumab complex has a drastically improved tumor:bone ratio of 25.8 at 336 h compared to that of 89Zr-DFO-trastuzumab (8.1). Both compounds show a high contrast between the tumor and the general background as represented by the blood activity, but 89Zr-HOPO-trastuzumab provides a much better contrast between the tumor and the bone specifically. This benefit of the improved stability of the p-SCN-Bn-HOPO ligand could make a meaningful difference in clinical imaging by enabling easier distinction of bone metastasis.</p><!><p>The search for an alternative chelator for zirconium has led to a number of novel ligand systems based on hydroxamates, picolinates, various hydroxypyridinones, catechols, and terephthalamide groups. Thus, far, however, no single chelator has been fully tested and found to be an improvement over DFO in the context of actual antibody-based long imaging. Several potential ligands have been proposed, but each still requires either further development or evaluation as described below.</p><!><p>These have been a major focus in the development of a new ligand since DFO itself contains such groups. Most recently, Zhai et al.37 have reported a competitive hexadentate ligand based on a derivatized fusarinine C (FSC) molecule (Figure 1). This is a cyclic ligand with a hydroxamate structure very similar to DFO. FSC was conjugated to an RGD peptide and showed improved stability over DFO.37 The prepared 89Zr-FSC-RGD complexes demonstrated improved stability over 89Zr-DFO-RGD in in vitro transchelation studies (93.9% stability vs 42.2%, respectively). The cyclic nature of the ligand likely provides greater kinetic stability as it is more difficult for competing materials to access the metal. However, providing only six donor groups for the binding of Zr4+ allows for the possibility of additional molecules being needed to satisfy the coordination sphere. Currently, the 89Zr-FSC-RGD complex has only been evaluated with short-term in vivo experiments (4 h biodistribution, 24 h PET image) which do not give a full picture of its long-term in vivo stability. Further experiments with longer circulating targeting molecules such as antibodies are necessary to reveal the practicality of this chelator.</p><p>The natural next step, to move toward octadentate hydroxamate-based ligands, has been taken by several groups as well. For example, a series of cyclic (C5–C7) and acyclic (L5–7) tetrahydroxamate ligands (Figure 1) were developed for evaluation with Zr4+. Of the synthesized ligands, the pair with the largest spacer, a seven carbon alkyl chain between hydroxamates, was found to be the best chelator both experimentally in comparison with DFO and computationally. The cyclic ligand C7 in particular showed promise in the stability of the resulting Zr complex, but further development to bifunctionalize the ligand is necessary to prove its utility. In a similar study, an octadentate derivative of DFO called DFO* (Figure 1), containing an additional hydroxamate group was also shown to chelate zirconium well.35 The complex of Zr4+ with this octadentate DFO* was predicted to be more stable than that with hexadentate DFO by DFT calculations. A bifunctional derivative, DFO*–CO2H, was also created and conjugated to bombesin for preliminary evaluation and was shown to be significantly more stable than 89Zr-DFO-bombesin in in vitro challenge studies over 24 h. No additional stability experiments were reported for longer time points or conditions. The potential of this chelator has yet to be determined in vivo or in serum, or in any experiments for longer than 24 h.</p><!><p>These have also been investigated. H6phospa and H4octapa, a pair of octadentate picolinate-based N4O4 ligands (Figure 1), were evaluated for zirconium chelation, but found to be ineffective for radio-labeling with 89Zr.31 The reliance on nitrogen donors for the oxophilic Zr4+ cation was the most likely source of incompatibility.</p><p>YM103 (Figure 1) is a bifunctional isothiocyanate derivative of the hexadentate ligand CP256 which is made up of three 3-hydroxy-4-pyridinone (HPO) groups. YM103 was shown to bind 89Zr4+ and performed well in in vitro studies showing >95% stability in serum; however, the 89Zr-YM103-trastuzumab complex was demonstrated to be unstable in vivo with nearly 30%ID/g of the 89Zr localized in the bone. The release of the 89Zr4+ cation suggested by the high bone uptake is likely due to the lability of a six coordinate Zr complex.</p><p>A pair of nonhydroxamate-based ligands, abbreviated only as BFC 1 and 2 (Figure 1), containing four terephthalamide (TAM) binding groups in large dimacrocyclic structures with built-in amine pendant arms, as well as two short PEG units, were investigated as well.36 While both showed improved stability, BFC 1 was chosen as the preferred ligand due to its superior clearance profile. Follow-up studies will need to be done to evaluate the ability to conjugate this ligand onto a targeting vector and the long-term in vivo stability of the resulting immunoconjugate.</p><p>Additionally, our lab has investigated a catechol-based version of the HOPO ligand: 3,4,3-LICAM (LICAM) (Figure 1, previously unpublished), which was also taken from the actinide literature.39 This variant was ultimately found to be unsuitable for 89Zr4+ due to the incompatibility between the pKa of the catechol group and the solubility of the Zr4+ cation. Despite being octadentate and oxygen-rich, the maximum radiolabeling yield reached for the LICAM ligand was <30%, despite varying temperature, pH, and reaction time. We believe the issue to lie in the fact that the pKa for full deprotonation of the catechol group is 13.0, which requires a higher pH for proper labeling; however, the Zr4+ cation is only soluble at lower pH, and antibody radiolabeling is typically performed at pH ≈ 7. This leads to a precarious balance between keeping the 89Zr4+ in solution and trying to get the coordinating oxygen atoms of the catechol moiety deprotonated. Unfortunately, an appropriate compromise could not be found, so the ligand and catechol groups in general were dropped from further development.</p><p>The current study confirms the stability of the 1,2-hydroxypyridinone-based HOPO chelator and further presents a bifunctional ligand based on the HOPO scaffold. The work reported herein on p-SCN-Bn-HOPO represents the first evidence of a new ligand for 89Zr that demonstrates in vivo stability of an antibody-conjugated complex over an extended period of time.</p><!><p>The 3,4,3-(LI-1,2-HOPO) ligand was found to exhibit excellent stability for 89Zr complexes in a previous study.34 The development of a bifunctional derivative of the HOPO ligand was the next logical step and led to the design and synthesis of p-SCN-Bn-HOPO. This bifunctional ligand was shown to bind 89Zr as expected and was evaluated for conjugation to an antibody, radiolabeling of the ligand–antibody complex, and application of the radioimmunoconjugate in vivo. The p-SCN-Bn-HOPO was comparable to the standard p-SCN-Bn-DFO chelator, achieving specific activity ~2 mCi/mg and remaining ~90% stable through a 7 d incubation in human serum. The greatest distinction between the two compounds was the amount of bone uptake seen in the imaging and biodistribution experiments; the activity measured in the bone for 89Zr-HOPO-trastuzumab was more than 7 times lower than for 89Zr-DFO-trastuzumab. While the absolute uptake in BT474 breast cancer tumors was just over 2 times higher for 89Zr-DFO-trastuzumab, the tumor:bone ratio was more than 3 times higher for 89Zr-HOPO-trastuzumab. This improved contrast between tumor and bone could be advantageous for the detection of bone metastasis and for the general clarity of the images. Furthermore, the lower bone uptake is the ultimate proof that the p-SCN-Bn-HOPO ligand forms a more stable complex with 89Zr4+ than p-SCN-Bn-DFO and reduces the release of free 89Zr4+ in vivo.</p><p>The bifunctional chelator p-SCN-Bn-HOPO was shown to be an effective alternative for the chelation of 89Zr4+ for immunoPET applications. It successfully reduces the levels of radioactivity in the bones of mice compared with the use of p-SCN-Bn-DFO. This study not only led to a superior ligand for 89Zr, but also demonstrates the power of inorganic chemical principles to suggest the best chelation system for a metal which can actually be seen to make a difference in biological applications.</p><!><p>All chemicals, unless otherwise noted, were acquired from Sigma-Aldrich (St. Louis, MO) and used as received without further purification. All instruments were calibrated and maintained in accordance with standard quality-control procedures. High-resolution mass spectrometry was carried out through electrospray ionization using an Agilent 6520 QTOF instrument. 1H and 13C NMR spectra were recorded at varying temperatures on a Bruker Avance III spectrometer equipped with a triple resonance inverse cryoprobe, with 1H and 13C resonance frequencies of 600.13 and 150 mHz or a Bruker DRX spectrometer equipped with a 1H, 13C cryoprobe, with respective resonance frequencies of 500.13 and 125.76 MHz with Topspin software. The NMR spectra are expressed on the δ scale and were referenced to residual solvent peaks and/or internal tetramethylsilane. The HPLC system used for analysis and purification of compounds consisted of a Rainin HPXL system with a Varian ProStar 325 UV–vis Detector monitored at 254 nm. Analytical chromatography was carried out using a Waters Symmetry C18 Column, 100 Å, 5 μm, 4.6 mm × 100 mm at a flow rate of 1.0 mL/min and purification was done with a preparatory Waters Symmetry C18 Prep Column, 100 Å, 5 μm, 19 mm × 100 mm at a flow rate of 17.059 mL/min. IR spectroscopy was performed on a solid sample using an attenuated total reflectance attachment on a PerkinElmer Spectrum 2 FT-IR spectrometer with a UATR Two attachment.</p><p>89Zr was produced at Memorial Sloan Kettering Cancer Center on a TR19/9 cyclotron (Ebco Industries Inc.) via the 89Y(p,n)89Zr reaction and purified to yield 89Zr with a specific activity of 196–496 MBq/mg. Activity measurements were made using a CRC-15R Dose Calibrator (Capintec). For the quantification of activities, experimental samples were counted on an Automatic Wizard (2) γ-Counter (PerkinElmer). The radiolabeling of ligands was monitored using salicylic acid impregnated instant thin-layer chromatography paper (ITLC-SA) (Agilent Technologies) and analyzed on a Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-TLC software (Bioscan Inc., Washington, DC). All in vivo experiments were performed according to protocols approved by the Memorial Sloan Kettering Institutional Animal Care and Use Committee (protocol 08–07–013). Purity of greater than 95% was confirmed using quantitative HPLC analysis for nonradioactive compounds (HOPO and Zr-HOPO) and radio-TLC for radioactive compounds (89Zr-HOPO).</p><!><p>X-ray quality single crystals of Zr-HOPO were obtained from a reaction of ZrCl4 and HOPO in methanol, heating to 55 °C, and slowly cooling the reaction mixture over 2 days. A colorless plate-like crystal with approximate dimensions of 0.04 mm × 0.10 mm × 0.36 mm was selected for geometry and intensity data collection with a Bruker SMART APEXII CCD area detector on a D8 goniometer at 100 K. The temperature during the data collection was controlled with an Oxford Cryosystems Series 700+ instrument. Preliminary lattice parameters and orientation matrices were obtained from three sets of frames. Data were collected using graphite-monochromated and 0.5 mm-Mono-Cap-collimated Mo Kα radiation (γ= 0.71073 Å) with the ω scan method.42 Data were processed with the INTEGRATE program of the APEX2 software42 for reduction and cell refinement. Multiscan absorption corrections were applied by using the SCALE program for the area detector. The structure was solved by the direct method and refined on F2 (SHELX).43 Some solvent molecules, MeOH and H2O, which cocrystallize with the Zr-HOPO, are disordered. The constraints and restraints were applied to keep the geometries and atomic displacements of their groups close to the theoretical values. Non-hydrogen atoms in the whole structure were refined with anisotropic displacement parameters, and hydrogen atoms on carbons were placed in idealized positions (C–H = 0.95–1.00 Å) and included as riding with Uiso(H) = 1.2 or 1.5 Ueq(non-H). The hydrogen atoms on the oxygen and nitrogen atoms were refined isotropically with restrained O–H and N–H distances of 0.84 and 0.86 Å, respectively. The selected crystallographic parameters were listed in Table S.3. The crystallographic information file can be found in the Supporting Information.</p><!><p>Detailed analytical and spectral data are included in the Supporting Information.</p><!><p>The tri-BOC-protected spermine was prepared according to Geall et al.44 To a flask containing spermine (1) (2.02 g, 10 mmol) in 150 mL methanol at −78 °C under argon was added dropwise ethyl trifluoroacetate (1.42 g, 10 mmol) in 100 mL of methanol over 30 min while stirring. Stirring was continued for another 30 min and the reaction mixture was allowed to come to 0 °C. An excess of di-tert-butyl dicarbonate (60 mmol) in 100 mL methanol was added over a period of 1 h. The reaction mixture was stirred at room temperature for 18 h. Concentrated ammonium hydroxide solution was added to the reaction mixture until the pH reached 11 and the reaction was stirred for another 15 h at room temperature. The methanol was evaporated under reduced pressure and the resulting liquid was dissolved in methylene chloride, washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness. The crude compound was purified by silica column chromatography using CH2Cl2:MeOH:conc NH3 70:10:1 to 50:10:1 (v/v/v) yielding the desired product 4 (yield 28%). Products 2 and 3 were used directly and not isolated.</p><p>1H NMR (CDCl3, 400 MHz): δ 3.07–3.21 (m, 10H), 2.68 (t, 2H), 2.08 (bs, 2H), 1.59–1.70 (m, 4H), 1.41–1.46 (m, 31H). 13C NMR (CDCl3, 100 MHz): δ 156.04, 155.55, 79.48, 78.88, 46.80, 43.79, 38.76, 37.35, 32.46, 30.9, 28.42. HRMS calculated for C25H50N4O6 ([M + H]+), 503.38, found 503.3817.</p><!><p>A solution of 4-nitrophenylethyl bromide (0.126 g, 0.55 mmol) in DMF (2 mL) was added to a suspension of 4 (0.201 g, 0.5 mmol) and K2CO3 (0.138 g, 1 mmol) in DMF (5 mL) under N2. The resulting reaction mixture was stirred at 60 °C for 12 h. Solvent was removed under vacuum and the resulting residue was dissolved in methylene chloride, washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness. The crude compound was purified by silica column chromatography using 1% methanol in methylene chloride to give compound 5 as a gummy solid. (Yield = 30%).</p><p>1H NMR (500 MHz, CDCl3): (mixture of rotamers) δ 8.10–8.09 (d, 2H), 7.36 (d, 2H), 3.28–2.58 (m, 20H), 1.80 (bs, 2H), 1.58 (bs, 2H), 1.38–1.36 (m, 27H). 13C NMR (500 MHz, CDCl3): (mixture of rotamers) δ 156.1, 155.9, 129.8, 123.9, 79.3, 78.7, 78.3, 49.85, 49.83, 49.80, 49.76, 49.72, 47.1, 47.0, 46.94, 46.91, 46.89, 46.86, 46.77, 46.72, 46.67, 46.60, 46.52, 46.46, 46.41, 46.38, 46.33, 46.27, 46.26, 46.23, 46.20, 45.69, 45.66, 44.28, 44.20, 43.88, 43.83, 43.78, 43.52, 43.47, 43.40, 43.38, 43.32, 37.76, 37.68, 37.64, 37.60, 37.56, 37.51, 37.48, 37.38, 34.45, 34.40, 34.15, 28.45; HRMS calculated for C33H57N5O8 ([M + H]+), 651.4207, found 652.4387.</p><!><p>A solution of 4 M HCl in dioxane (5 mL) was added to a stirring solution of 5 (0.17g, 0.5 mmol) in CH2Cl2 (10 mL), under nitrogen, at 25 °C. After 2 h, the solution was concentrated in vacuo and co-distilled with toluene (3 × 5 mL) (poly-HCl salt). This compound was not isolated, but rather used directly in the next step.</p><!><p>A solution of 1-(benzyloxy)-6-oxo-1,6-dihydropyr-idine-2-carbonyl chloride (0.789g, 3 mmol) in methylene chloride (15 mL) was added dropwise to a stirred solution of triethylamine (0.835 mL, 6 mmol), 6 (0.351g, 1 mmol), and DMAP (0.006g, 0.05 mmol) in dry methylene chloride (10 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 24 h. The reaction mixture was washed with 10% NaHCO3 solution, followed by water. The organic phase was dried over anhydrous Na2SO4 and was then removed with a rotary evaporator. The crude product was purified through column chromatography over silica gel using 2–4% methanol in dichloromethane eluent producing 5 as brown foam (yield 56%).</p><p>1H NMR (600 MHz, CDCl3): (mixture of rotamers) δ 8.16–7.25 (m, 20H), 7.19–4.86 (m, 16H), 3.82–2.06 (m, 25H), 1.84–0.63 (m, 8H); 13C NMR (600 MHz, CDCl3): (mixture of rotamers) δ 1420.16, 142.14, 139.12, 139.05, 139.0, 138.97, 138.87, 138.82, 138.78, 138.70, 138.66, 138.59, 138.55, 138.50, 133.16, 133.13, 133.06, 130.86, 130.80, 130.72, 130.66, 130.45, 135.41, 135.37, 130.34, 130.32, 130.29, 130.26, 130.23, 129.79, 129.76, 129.73, 129.70, 129.65, 129.62, 129.56, 123.97, 123.92, 123.83, 123.76, 123.74, 123.36, 122.79, 122.65, 122.57, 122.53, 122.52, 122.48, 104.07, 103.62, 103.54, 103.49, 103.40, 103.34, 79.66, 46.97, 46.08, 46.02, 41.99, 40.64, 36.95, 36.93, 36.89, 36.81, 34.61, 33.39, 33.23, 33.11, 26.09, 26.88, 25.52, 25.49, 25.32, 25.18, 25.11, 24.84, 24.80, 24.53, 24.17, 24.13, 23.98; HRMS calculated for C70H69N9O14 ([M + H]+), 1260.5042, found 1260.5038.</p><!><p>To a suspension of Raney nickel in (1:1) MeOH:THF (10 mL), 7 (0.2 g, 0.157 mmol) was added and hydrogenated at balloon H2 pressure at room temperature for 3 h. The catalyst was filtered through a Celite pad under inert atmosphere. The filtrate was evaporated under reduced pressure to obtain the crude amine. This crude product was used directly in the next step without further workup. For characterization purposes, some of this product was HPLC purified as a cream colored solid.</p><p>1H NMR (600 MHz, DMSO-d6): (mixture of rotamers) δ 8.70–8.64 (m, 1H), 7.44–7.33 (m, 27H), 6.93–6.89 (m, 1H), 6.89–6.80 (m, 3H), 6.64–6.62 (m, 4H), 6.31–6.10 (m, 4H), 5.38–5.26 (m, 5H), 5.04–4.99 (m, 3H), 3.60–3.55 (m, 18H), 3.16–3.13 (m, 14H), 1.76–1.21 (m, 10H); 13C NMR (600 MHz, DMSO-d6): (mixture of rotamers) δ 158.77, 158.70, 158.28, 158.25, 158.18, 158.14, 155.63, 155.30, 155.27, 155.09, 155.06, 155.03, 141.89, 141.63, 141.60, 140.70, 140.59, 140.51, 136.82, 136.77, 136.64, 131.65, 161.41, 131.38, 131.35, 127.49, 127.44, 127.38, 127.29, 126.97, 126.87, 126.27, 126.23, 126.20, 120.31, 120.35, 119.75, 117.81, 115.46, 113.1, 101.62, 100.26, 100.1, 76.16, 76.10, 59.81, 52.71, 45.56, 45.49, 43.71, 43.47, 43.31, 41.3, 39.83, 39.65, 34.51, 34.45, 34.18, 34.11, 31.09, 29.71, 25.62, 24.36, 23.29, 22.73, 22.54, 22.43, 21.75, 21.38, 21.29; HRMS calculated for C70H72N9O12 ([M + H]+), 1230.5300, found 1230.5299.</p><!><p>Crude 8 was dissolved in a 1:1 mixture of acetic acid and concentrated HCl (6 mL) at room temperature and heated to 45–50 °C for 18 h. The reaction progress was monitored by LC-MS. The crude product was dried and redissolved in water/acetonitrile and purified by HPLC on a preparative C18 column (Waters Symmetry C18 Prep Column, 100 Å, 5 μm, 19 mm × 100 mm) at 17.059 mL/min using a gradient of 10–23% MeCN in water (both containing 0.1% TFA) with an initial hold at 10% MeCN for 1.33 min and then a ramp to 23% MeCN over 20 min. The product peak was collected from 6.45 to 8.26 min and the eluted solution was lyophilized to recover the product as an off-white solid. The purified ligand was collected in multiple small batches with an approximate combined yield of 70%.</p><p>The purified sample was confirmed by HPLC on an analytical C18 column (Waters Symmetry C18 Analytical Column, 100 Å, 5 μm, 4.6 mm × 100 mm) at 1 mL/min using a gradient of 10–30% MeCN in water (both containing 0.1% TFA) with an initial hold at 10% MeCN for 1.33 min and then a ramp to 30% MeCN over 30 min followed by a ramp to 95% MeCN over 1 min and an isocratic hold at 95% MeCN for 5.66 min.</p><p>1H NMR (500 MHz, CDCl3): (mixture of rotamers) δ 7.44–7.28 (m, 5H), 7.20–7.16 (dd, J1, J2 = 6 Hz, 1H), 7.08–7.01 (m, 2H), 6.54–6.53 (m, 4H), 6.33–6.32 (m, 3H), 6.18–6.21 (m, 0.5H), 5.74–5.67 (m, 0.5H), 3.65–3.51 (m, 4H), 3.51–3.16 (m, 8H), 3.12–2.70 (m, 11H), 1.91–1.38 (m, 10H) ; 13C NMR (500 MHz, CDCl3): (mixture of rotamers) δ 158.8, 158.6, 158.4, 158.1, 157.9, 157.8, 157.77, 157.73, 142.5, 142.49, 142.45, 142.41, 142.32, 142.24, 142.04, 138.10, 138.0, 137.59, 137.51, 130.36, 130.31, 130.18, 130.08, 119.76, 119.45, 117.5, 115.6, 104.3, 102.5, 102.47, 102.41, 50.01, 48.12, 47.9, 46.0, 46.0, 43.7, 42.3, 37.2, 37.0, 36.97, 33.80, 32.65, 32.49, 28.16, 26.90, 26.23, 25.4, 25.4, 25.2, 25.1, 24.4, 24.3, 24.3, 24.2; HRMS calculated for C42H47N9O12 ([M + H]+), 870.3422, found 870.3420.</p><!><p>The benzylamine was transformed into a benzyl isothiocyanate according to Maingot et al.45 NEt3 (0.0012 g, 0.012 mmol) was added to a solution of 8 (0.01 g, 0.011 mmol) in (8:2) acetonitrile and water (1 mL). Next, di-2-pyridyl thionocarbonate (0.011 g, 0.05 mmol) was added at room temperature and stirred vigorously for 1 h. The crude reaction solution was directly purified by HPLC on a preparative C18 column (Waters Symmetry C18 Prep Column, 100 Å, 5 μm, 19 mm × 100 mm) at 17.059 mL/min using a gradient of 5–75% MeCN in water (both containing 0.1% TFA) with an initial hold at 5% MeCN for 1.33 min and then a ramp to 75% MeCN over 30 min. The product peak was collected from 15.01 to 15.5 min and the eluted solution was lyophilized to recover the product as a white solid. The purified ligand was collected in multiple small batches with an approximate combined yield of 32%.</p><p>The purified sample was confirmed by HPLC on an analytical C18 column (Waters Symmetry C18 Analytical Column, 100 Å, 5 μm, 4.6 mm × 100 mm) at 1 mL/min using a gradient of 5–75% MeCN in water (both containing 0.1% TFA) with an initial hold at 5% MeCN for 1.33 min and then a ramp to 75% MeCN over 30 min followed by a ramp to 95% MeCN over 1 min and an isocratic hold at 95% MeCN for 5.66 min.</p><p>1H NMR (500 MHz, CDCl3): (mixture of rotamers) δ 7.37–7.31 (m, 7H), 7.12–7.08 (m, 1H), 6.58–6.52 (m, 4H), 6.34–5.81 (m, 4H), 3.62–3.54 (m, 12H), 3.13–2.83 (m, 11H), 2.01–1.24 (m, 11H) ; 13C NMR (500 MHz, CDCl3): (mixture of rotamers) δ 161.78, 161.71, 161.54, 161.4, 160.65, 160.61, 157.89, 157.83, 157.78, 157.73, 142.59, 142.48, 142.41, 142.33, 142.31, 142.21, 139.58, 138.49, 138.90, 138.81, 138.17, 138.0, 137.57, 137.50, 134.07, 130.76, 130.74, 130.59, 130.52, 128.89, 128.66, 126.41, 126.32, 119.74, 119.53, 119.43,104.33, 102.50, 102.39, 49.62, 48.11, 47.96, 46.49, 46.01, 45.70, 37.24, 37.01, 36.96, 34.04, 32.83, 32.71, 28.15, 27.12, 26.85, 26.22, 25.46, 25.26, 25.17, 24.36, 24.18; HRMS calculated for C43H45N9O12S ([M + H]+), 912.2987, found 912.2987.</p><!><p>Trastuzumab (purchased commercially as Herceptin, Genentech, San Francisco, CA) was purified using prepacked size exclusion chromatography (SEC) columns (Sephadex G-25 M, PD-10 Desalting Columns, 50 kDa, GE Healthcare) and centrifugal filter units with a 50 000 molecular weight cutoff (Amicon Ultra 4 Centrifugal Filtration Units, Millipore Corp., Billerica, MA) and phosphate buffered saline (PBS, pH 7.4) to remove α_α-trehalose dihydrate, L-histidine, and polysorbate 20 additives. After purification, the antibody was taken up in PBS at pH 7.4. Subsequently, ~60 μL of antibody solution (~13 nmol) were diluted to 1 mL with PBS at pH 7.4. The pH of the antibody solution was raised to 8.8–9.0 with 0.1 M Na2CO3 before the slow addition of 5 equiv of p-SCN-Bn-HOPO or p-SCN-Bn-HOPO in ~12 μL of DMSO. The reaction was incubated at 37 °C for 1 h and shaken at 300 rpm, followed by SEC and centrifugal filtration to purify the ligand–antibody conjugate. The final bioconjugates were stored in PBS pH 7.4 at 4 °C.</p><p>Chelate number was determined using an isotopic dilution assay. A stock solution of 1 mM ZrCl4 was made up in 1 M oxalic acid. Approximately ~200 μCi of 89Zr oxalate solution in 1 M oxalic acid was added to 100 μL of the stock solution. This mixture was then neutralized to pH 7 with 1 M Na2CO3 to create a ~200 μL Zr working solution of ~500 μM Zr4+. Triplicate solutions of DFO-Trastuzumab and HOPO-Trastuzumab were prepared containing 300–400 pmol of antibody in 30 μL of PBS. Aliquots of 20, 25, and 30 μL of the Zr working solution were added to the three ligand–antibody samples for each ligand. The solutions were incubated at room temperature with gentle mixing overnight. The next day a volume of 50 mM EDTA equal to 1/9 of the volume of the reaction mixture was added to each sample and left to incubate for 15 min in order to scavenge any nonspecifically bound Zr4+. Samples were then analyzed using radio-TLC to determine the extent of radiolabeling. The average number of chelates per antibody was calculated as the ratio of bound vs unbound radioactive 89Zr × the moles of Zr4+ ÷ the moles of antibody. The experiment was run in duplicate using the same batch of ligand-Trastuzumab conjugate as the in vivo studies.</p><!><p>89Zr was received after target processing as 89Zr-oxalate in 1.0 M oxalic acid. This solution is then neutralized with 1.0 M sodium carbonate to reach pH 6.8–7.2. Both the DFO and HOPO ligands as well as their respective antibody complexes were labeled at various concentrations in water or saline with the neutralized 89Zr solution at room temperature for varying lengths of time, typically 10–180 min. Reactions were monitored via radio-TLC with different stationary phases depending on the nature of the reaction. 89Zr-ligand complexes required Varian ITLC-SA strips (Agilent Technologies) whereas 89Zr-ligand-trastuzumab complexes employed Varian ITLC-SG strips (Agilent Technologies), but both analysis methods used 50 mM EDTA at pH 5 as the mobile phase. 89Zr complexes remained at the origin, while free 89Zr was taken up by EDTA in the mobile phase and migrated along the ITLC strip.</p><!><p>89Zr-ligand and 89Zr-ligand-antibody complexes were prepared according to the radio-labeling protocol as described above. For each 89Zr complex, samples were made consisting of 900 μL human serum and 100 μL of the 89Zr species and were placed in a heat block at 37 °C with agitation. Samples were monitored using radio-TLC before being added to the serum and then after 1 week of incubation. The stability of the complexes was measured as the percentage of 89Zr that was retained at the origin of the ITLC strip and therefore still intact.</p><!><p>The immunoreactivity of the 89Zr-DFO-trastuzumab and 89Zr-HOPO-trastuzumab bioconjugates was determined using specific radioactive cellular-binding assays following procedures derived from Lindmo et al.46,47 To this end, BT474 cells were suspended in microcentrifuge tubes at concentrations of 2.5, 2.0, 1.5, 1.25, 1.0, 0.75, and 0.25 × 106 cells/mL in 500 μL PBS (pH 7.4). Aliquots of either 89Zr-DFO-trastuzumab or 89Zr-HOPO-trastuzumab (50 μL of a stock solution of ~10 μCi in 10 mL of 1% bovine serum albumin in PBS pH 7.4) were added to each tube (n = 3; final volume: 550 μL), and the samples were incubated on a mixer for 60 min at room temperature. The treated cells were then pelleted via centrifugation (600 G for 2 min), aspirated, and washed twice with cold PBS before removing the supernatant and counting the activity associated with the cell pellet. The activity data were background-corrected and compared with the total number of counts in appropriate control samples. Immunoreactive fractions were determined by linear regression analysis of a plot of (total/bound) activity against (1/[normalized cell concentration]). No weighting was applied to the data, and data were obtained as n = 3.</p><!><p>PET imaging experiments were conducted on a microPET Focus 120. Female athymic nude mice with BT474 xenografts on their right shoulders were administered 89Zr-HOPO-trastuzumab (9.25–9.99 MBq [250–270 μCi] in 200 μL 0.9% sterile saline) or 89Zr-DFO-trastuzumab (9.25–9.99 MBq [250–270 μCi] in 200 μL 0.9% sterile saline) via intravenous tail vein injection (t = 0). Approximately 5 min prior to the acquisition of PET images, mice were anesthetized by inhalation of 2% isoflurane (Baxter Healthcare, Deerfield, IL)/oxygen gas mixture and placed on the scanner bed; anesthesia was maintained using 1% isoflurane/gas mixture. PET data for each mouse were recorded via static scans at various time points (n = 4) between 6 h and 9 d. An energy window of 350–700 keV and a coincidence timing window of 6 ns were used. Data were sorted into 2D histograms by Fourier rebinning, and transverse images were reconstructed by filtered back-projection (FBP) into a 128 × 128 × 63 (0.72 × 0.72 × 1.3 mm3) matrix. The image data were normalized to correct for nonuniformity of response of the PET, dead-time count losses, positron branching ratio, and physical decay to the time of injection, but no attenuation, scatter, or partial-volume averaging correction was applied. The counting rates in the reconstructed images were converted to activity concentrations (percentage injected dose per gram of tissue, %ID/g) by use of a system calibration factor derived from the imaging of a mouse-sized water-equivalent phantom containing 89Zr. Images were analyzed using ASIPro VM software (Concorde Microsystems).</p><!><p>Acute in vivo biodistribution studies were performed in order to compare the uptake of 89Zr-HOPO-trastuzumab and 89Zr-DFO-trastuzumab in BT474 tumor-bearing female athymic nude mice. Mice were warmed gently with a heat lamp for 5 min before administration of 89Zr-HOPO-trastuzumab (0.59–0.67 MBq [16–18 μCi] in 200 μL 0.9% sterile saline) or 89Zr-DFO-trastuzumab (0.67–0.74 MBq [18–20 μCi] in 200 μL 0.9% sterile saline) via intravenous tail vein injection (t = 0). Animals (n = 4 per group) were euthanized by CO2(g) asphyxiation at 1, 3, 5, 7, 9, and 14 d. After asphyxiation, 14 organs were removed, rinsed in water, dried in air for 5 min, weighed, and assayed for radioactivity on a gamma counter calibrated for 89Zr. Counts were converted into activity using a calibration curve generated from known standards. Count data were background- and decay-corrected to the time of injection, and the percent injected dose per gram (%ID/g) for each tissue sample was calculated by normalization to the total activity injected. The full data set of organs is also included in the Supporting Information with values represented in %ID without normalization (Table S.5).</p>

PubMed Author Manuscript

Formation of a mixed-valence Cu(<scp>i</scp>)/Cu(<scp>ii</scp>) metal–organic framework with the full light spectrum and high selectivity of CO₂ photoreduction into CH₄

Cu 4 I 4 ) 2.5 [Cu 3 (m 4 -O) (m 3 -I) (pmc) 3 (Dabco) 3 ]$2.5DMF$2MeCN} N (NJU-Bai61, NJU-Bai for Nanjing University Bai group; Dabco ¼ 1,4-diazabicyclo [2.2.2] octane), was synthesized stepwise. NJU-Bai61 exhibits good water/pH stabilities and a relatively large CO 2 adsorption capacity (29.82 cm 3 g À1 at 1 atm, 273 K) and could photocatalyze the reduction of CO 2 into CH 4 without additional photosensitizers and cocatalysts and with a high CH 4 production rate (15.75 mmol g À1 h À1 ) and a CH 4 selectivity of 72.8%. The CH 4 selectivity is the highest among the reported MOFs in aqueous solution. Experimental data and theoretical calculations further revealed that the Cu 4 I 4 cluster may adsorb light to generate photoelectrons and transfer them to its Cu 3 OI(CO 2 ) 3 cluster, and the Cu 3 OI(CO 2 ) 3 cluster could provide active sites to adsorb and reduce CO 2 and deliver sufficient electrons for CO 2 to produce CH 4 . This is the first time that the old Cu(I) x X y L z coordination polymers' application has been extended for the photoreduction of CO 2 to CH 4 and this opens up a new platform for the effective photoreduction of CO 2 to CH 4 .

formation_of_a_mixed-valence_cu(<scp>i</scp>)/cu(<scp>ii</scp>)_metal–organic_framework_with_the_ful

Introduction<!>Results and discussion<!>Conclusions<!>Synthesis of NJU-Bai61p<!>Synthesis of NJU-Bai61<!>Sample activation<!>Photocatalytic reaction<!>Conflicts of interest

<p>Due to climate change, CO 2 capture and conversion has recently, become one of the greatest concerns. 1 In particular, the photoreduction of CO 2 into value-added chemicals (such as CO, HCOOH, CH 4 , and so on) has attracted great attention, because it can be considered as a promising approach for solar-to-chemical energy conversion by mimicking the natural photosynthetic process to achieve a carbon neutral economy. 2 In the past few decades, diverse photocatalysts have been extensively employed for the photocatalytic CO 2 reduction reaction (CO 2 RR). 3 Homogeneous/molecular catalysts exhibit high selectivity and efficiency, but low activity due to catalyst deactivation, 4 whereas heterogeneous/inorganic catalysts show high activity and efficiency, but low selectivity. 5 Very recently, due to their high surface area, inorganic-organic hybrid nature, structural and functional diversity and tunability, metal-organic frameworks (MOFs) may combine the advantages of the traditional homogeneous/ heterogeneous catalysts and are emerging as promising platforms for the photocatalytic CO 2 RR. 6 Since 2011, 7 many MOFs have been designed for the photocatalytic CO 2 RR targeting to improve their efficiency, activity and selectivity by functionalizing organic ligands, optimizing metal ions/clusters, and making MOF-based composites. 8 Although, some achievements have been made, research on MOF-based photocatalysts to date is still in its early stages. In terms of the reductive products, most reported MOFs predominantly produce the 2e À /2H + products of CO/HCOOH. 8a,9 Due to the fact that the photocatalytic reduction of CO 2 into CH 4 is more difficult than with other C1 fuels, because it involves a complex 8e À /8H + reduction process, i.e., multiple steps of hydrogenation and deoxygenation reactions, and requiring the highest kinetic barrier of up to 818.3 kJ mol À1 , 10 the reported MOF catalysts capable of producing even low or moderate yields of CH 4 are still rare. Thus, design of MOFs with high selectivity for the reduction of CO 2 into CH 4 is a great challenge. 11 The Cu(I) x X y L z (where X ¼ Cl, Br or I; L ¼ N, P or S containing organic ligands) are almost the oldest coordination polymers with diversied structures and interesting properties, such as luminescence and semiconductivity, and so on. 12 Very recently, their use has been demonstrated for photocatalytic H 2 evolution. 13 Herein the exploration of these polymers as promising platforms for CO 2 capture and conversion is reported. From a simple hetero-N,O ligand pyrimidine-5-carboxylic acid, a Cu 4 I 4 and Cu 3 OI(CO 2 ) 3 cluster based and semiconductive Cu(I)/Cu(II) mixed-valence MOF (NJU-Bai61) with a full light spectrum, which exhibits good water and pH stabilities and the relatively large CO 2 adsorption capacity (29.82 cm 3 g À1 at 1 atm, 273 K) was successfully constructed. In addition, NJU-Bai61 could photocatalyze the reduction of CO 2 into CH 4 without additional photosensitizers and cocatalysts and with a high CH 4 production (15.75 mmol g À1 h À1 ) and CH 4 selectivity of 72.8%. As far as is known, the CH 4 selectivity is the highest among the reported MOFs in the aqueous solution. Upon light irradiation, its Cu 4 I 4 clusters as photoelectron generators could transfer photoelectrons to the Cu 3 OI(CO 2 ) 3 clusters, whereas the Cu 3 OI(CO 2 ) 3 clusters could provide active sites for adsorbing and reducing CO 2 and act as photoelectron collectors for delivering enough electrons to CO 2 for CH 4 evolution.</p><!><p>From CuI and the Hpmc ligand and using Dabco as the structural directing agent, like many Cu(I) x X y L z , a Cu 4 I 4 cluster-based copper(I) coordination polymer, {(Cu 4 I 4 ) (Hpmc) 2 } N (NJU-Bai61p) was initially obtained. NJU-Bai61p is a 2D layered and 4connected network with sql topology (Fig. S3, ESI †), in which each Hpmc ligand uses its N-donor center to link to a 4-coordinated Cu(I) in a tetrahedral coordination geometry resulting in a [Cu 4 I 4 N 4 ] moiety, leaving its COOH functional group uncoordinated (Fig. S4, ESI †).</p><p>Later on, by changing the acid and extending the time, NJU-Bai61p was further transformed into NJU-Bai61 (Scheme 1). Compared with NJU-Bai61p, the Hpmc ligands in NJU-Bai61 were deprotonated, coordinated with Cu(II) ions in a bridging bidentate mode, facilitating the formation of the Cu 3 OI(CO 2 ) 3 cluster. The Cu 3 OI(CO 2 ) 3 cluster is 7-connected and surrounded by one Cu (Fig. 1d and S7, ESI †). The cages A and B connect alternately with each other to form a 1D channel by sharing quadrilateral windows, whereas the B cages connect with each other to form a 1D cage-stacked chain by sharing the facets including a quadrilateral window and a Cu 4 I 4 cluster (Fig. 1e, f, and S8, ESI †). Therefore, these 1D channels and chains are arranged in an alternating fashion to form a 3D porous framework based on the cages A and B ratio of 1 : 3, in which each cage A shares facets with six cage Bs and each cage B shares facets with two cage As and four cage Bs (Fig. 1g and S9, ESI †). From the viewpoint of structural topology, pmc ligands, Cu 4 I 4 and Cu 3 -OI(CO 2 ) 3 clusters could be regarded as 3-connected triangular nodes, 4-connected tetrahedral nodes, and 7-connected single cap octahedron nodes, respectively. Consequently, NJU-Bai61 is a new (3,4,4,7)-connected network with the point symbol {4 3 $6 12 $8 6 } 2 {4 3 $6 3 } 2 {6 3 } 6 {6 4 $8 2 } 3 (Fig. S10, ESI †).</p><p>The phase purities and thermal stabilities of NJU-Bai61p and NJU-Bai61 were conrmed using PXRD and TG analyses (Fig. S13 and S14, ESI †). As shown in Fig. S15-S17 (ESI †), they are quite stable under water and other organic solvents. Furthermore, they are also stable under the broad variation of the pH values.</p><p>NJU-Bai61p exhibits a visible light adsorption up to 550 nm due to the Cu 4 I 4 cluster to linker charge transfer (CLCT) transition (Fig. 2a and Table S2, ESI †). Very interestingly, NJU-Bai61 shows the widest absorption band among the reported MOFs with the edge up to 1400 nm, which are mainly dominated by intra metal cluster transfer (ICT), CLCT, and metal cluster-tometal cluster charge transfer (CCCT) transitions (Fig. 2a and Table S3, ESI †). The bandgaps of semiconductive NJU-Bai61p and NJU-Bai61 were estimated to be 2.33 eV and 0.92 eV, respectively, (Fig. S18, ESI †), which could be correlated with the calculated HOMO-LUMO gaps of 2.16 eV and 1.25 eV for the corresponding cluster models, respectively, (Tables S4 and S5, ESI †). The solid state of NJU-Bai61 with a periodic boundary condition (PBC) model for the band gap was further calculated, showing a narrow band gap of 0.65 eV (Fig. S19, ESI †). The Mott-Schottky measurements further revealed that they were typical n-type semiconductors and their conduction bands (CB) were À0.55 V and À0.58 V, which were more negative than the reduction potentials for the conversion of CO 2 to CO and CH 4 (Fig. 2b and S20, ESI †). 8a Thus, they are very promising for the CO 2 photoreduction applications.</p><p>The photocatalytic reduction of CO 2 over the activated NJU-Bai61 was further investigated. The amount of CH 4 was 1.26 mmol (i.e., 15.75 mmol g À1 h À1 ) aer 4 h. Except for the small amounts of CO (0.32 mmol, i.e., 4 mmol g À1 h À1 ) and H 2 (0.15 mmol, i.e., 1.87 mmol g À1 h À1 ), no other products, such as HCOOH, CH 3 OH and HCHO, were detected (Fig. 2c, S22 and S23, ESI †). The NJU-Bai61 exhibited a CH 4 selectivity of 72.8% in aqueous solution, which was the highest among the reported MOFs (Table S8, ESI †). No obvious change of the CH 4 activity occurred during the four continuous runs (Fig. S24, ESI †). The XRD patterns obtained before and aer its photocatalytic experiments revealed the structural robustness of the catalyst (Fig. S27, ESI †). The isotopic 13 CO 2 tracing experiment was also performed to conrm that the carbon source of CH 4 did indeed come from the used CO 2 rather than the degradation of organics in the reaction (Fig. 2d). 11b For comparison, the use of NJU-Bai61p as the photocatalyst was also investigated under the same conditions and only CO (1.37 mmol, i.e., 17.13 mmol g À1 h À1 ) and H 2 (1.34 mmol, i.e., 16.75 mmol g À1 h À1 ) were detected aer 4 h (Fig. S25, ESI †). This result may reveal that Cu 3 OI(CO 2 ) 3 clusters in NJU-Bai61 could provide active sites for CH 4 evolution.</p><p>Then in-depth research was carried out to discover the reason underlying the high efficiency of CH 4 evolution. As for NJU-Bai61, the BET surface area was 248.1 m 2 g À1 and the CO 2 uptakes at 273 K and 298 K were 29.82 and 19.69 cm 3 g À1 , respectively, which was helpful for the subsequent CO 2 conversion (Fig. S28-S30, ESI †). The electrostatic potential analysis may further reveal that the Cu(II) centers in Cu 3 -OI(CO 2 ) 3 clusters are the most favorable sites for the nucleophilic attack of CO 2 (Fig. S31, ESI †). The local interactions between Cu(II) sites and CO 2 molecules were investigated using the in situ FTIR technology. The adsorption of CO 2 onto the Cu(II) sites in NJU-Bai61 was a 16 cm À1 red shi of the asymmetric stretching mode of CO 2 (n ¼ 2359 cm À1 ), indicating the stronger binding between the CO 2 and Cu(II) sites (Fig. S33, ESI †). 11b However, for NJU-Bai61p, no shi existed aer CO 2 adsorption (Fig. S32, ESI †). Moreover, this experimental phenomenon was explained by the DFT calculations in which the peaks were also red-shied and the adsorbed CO 2 molecule takes a slightly bent geometry to facilitate the CO 2 activation (Fig. S34 and Table S9, ESI †). 14 Furthermore, its uorescence was quenched in comparison to NJU-Bai61p, indicating that the photo-excited electrons of the Cu 4 I 4 clusters were transferred to the Cu 3 OI(CO 2 ) 3 clusters, making it act as a photoelectron collector to provide electrons for the adsorbed CO 2 (Fig. S35, ESI †).</p><p>An energetically feasible reaction pathway was calculated using DFT with the relative free energy, DG, for each step shown in Fig. 3 and S38 (ESI). † Upon light irradiation, the Cu 4 I 4 clusters in NJU-Bai61 may adsorb light to generate the photoelectrons and transfer them to the Cu 3 OI(CO 2 ) 3 clusters, whereas the Cu 3 OI(CO 2 ) 3 clusters could supply electrons to the adsorbed CO 2 for CH 4 evolution. In the rst step, the adsorbed CO 2 molecule accepted an electron and a proton to generate the COOH*. Then the COOH* combines with the second electronproton pair to generate CO*. The CO* was reduced to the CHO* by accepting two electrons and a proton, and further combined with a total of four electrons and ve protons to generate CH 4 . In the photocatalytic process, the Cu 4 I 4 cluster could serve as a photosensitizer and donated the energy of 2.16 eV to the conversion process of CO* to CHO* at the Cu 3 OI(CO 2 ) 3 cluster which was an endothermic process with the DG of 1.2 eV. Moreover, the stronger CO binding affinity on NJU-Bai61 (E b ¼ À20.13 eV) in comparison with that on only Cu(I)-contained NJU-Bai61p (E b ¼ À8.05 eV) may further stabilize the CO@Cu 3 IO(CO 2 ) 3 complex to complete the CO 2 -to-CH 4 conversion (Fig. S39, ESI †).</p><!><p>In summary, a novel Cu 4 I 4 and Cu 3 OI(CO 2 ) 3 cluster based and semiconductive Cu(I)/Cu(II) mixed-valence MOF with the full light spectrum, NJU-Bai61, was successfully produced, which exhibits good water stability, pH stability and a relatively large CO 2 adsorption capacity. NJU-Bai61 could photocatalyze the reduction of CO 2 into CH 4 , without additional photosensitizers and cocatalysts, but with a high CH 4 production and signicantly high CH 4 selectivity of 72.8% (the highest among the reported MOFs in aqueous solution). It was revealed that the Cu 4 I 4 and Cu 3 OI(CO 2 ) 3 clusters may play the role of photoelectron generators and collectors, respectively. This work rstly expands the old Cu(I) x X y L z coordination polymers' application into the reduction of CO 2 to CH 4 and may open up a new system of MOFs for the reduction of CO 2 to CH 4 with high selectivity.</p><!><p>A mixture of Hpmc (11 mg, 0.09 mmol), CuI (30 mg, 0.16 mmol), Dabco (6 mg, 0.05 mmol), H 2 SO 4 (10 mL), DMF (1.0 mL), and MeCN (3.0 mL) was sealed in a 20 mL Pyrex tube and kept in an oven at 85 C for 1 day. Aer washing with DMF, yellow block crystals were obtained. Yield: 2.5 mg (6%). Selected IR (cm À1 ): 3036, 2666, 2554, 1713, 1586, 1441, 1398, 1330, 1297, 1202, 1170, 1119, 1090, 1054, 996, 908, 837, 749, 695, 667, 568. Elemental analysis (%) calcd. for Cu 2 I 2 C 5 H 4 N 2 O 2 : C 11.89, H 0.80, N 5.54; found: C 11.96, H 1.00, N 5.52.</p><!><p>A single crystal of NJU-Bai61p (10 mg), Dabco (4 mg, 0.036 mmol) and CuI (20 mg, 0.11 mmol) were added to 1.0 mL of DMF and 3.0 mL of MeCN. To this was added 60 mL of HCOOH with stirring. The mixture was sealed in a Pyrex tube and heated to 85 C for 2 d. Dark-red octahedral crystals were obtained and further characterized by PXRD and the results are shown in Fig. S1 (ESI †). Yield: 8.8 mg (25%). Selected IR (cm À1 ): 3392, 3108, 2952, 2883, 2840, 1681, 1652, 1587, 1435, 1377, 1319, 1218, 1170, 1087, 1050, 1000, 924, 840, 805, 764, 700, 612, 583, 468, 420. Elemental analysis (%) calcd. for Cu 13 I 11 C 44.5 H 68.5 -N 16.5 O 9.5 : C 16.66, H 2.15, N 7.20; found: C 16.87, H 2.30, N 6.98.</p><!><p>The as-synthesized sample of NJU-Bai61 was soaked in MeOH for 5 d with refreshing of the MeOH every 8 h. Then, the solventexchanged sample was activated at 70 C and under vacuum for 10 h to obtain the activated NJU-Bai61.</p><!><p>The photocatalytic CO 2 reduction experiments were carried out on an evaluation system (CEL-SPH2N, CEAULIGHT, China), in a 100 mL quartz container. A 300 W xenon arc lamp (300 < l < 2500 nm) was utilized as the irradiation source. The 20 mg MOFs (NJU-Bai61p or the activated NJU-Bai61) were dispersed in 50 mL of a solution of triethylamine and water (TEA/H 2 O ¼ 5 : 45 v/v). The suspension was pre-degassed with CO 2 (99.999%) for 30 min to remove the air before irradiation. The reaction was stirred constantly with a magnetic bar to ensure the photocatalyst particles remained in suspension. The temperature of the reaction was maintained at 25 C by a circulating cooling water system. The gaseous product was measured by gas chromatography (GC-7900, CEAULIGHT, China) with a ame ionization detector (FID) and a thermal conductivity detector (TCD). An ion chromatography (LC-2010 Plus, Shimadzu, Japan) was used for the detection of HCOO À . The concentration of Cu in the solution before and aer catalysis was determined using an ICP-OES system (Optima 5300 DV, PerkinElmer). Before the photocatalytic reaction, the suspension of the activated NJU-Bai61 (220 mg), TEA (5 mL) and H 2 O (45 mL) was pre-degassed with CO 2 (99.999%) for 30 min to remove the air, then 2 mL of the ltrate was removed and a Cu concentration of 0.6 mg L À1 was detected. Thus, the concentration of dissolved Cu ions of the activated NJU-Bai61 was 0.05% before catalysis. Aer the photocatalytic reaction, 2 mL of ltrate was also removed and the concentration of Cu in the ltrate was determined to be 13.8 mg L À1 . Thus, the concentration of dissolved Cu ions of the activated NJU-Bai61 was 1.1%. The cycling experiment was carried out as follows: at the end of each run, the suspension was centrifuged and the supernatant was removed. Then the recovered catalyst was washed with distilled water and dried in air at 60 C before the next cycle.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Activity Enhancement of Defective Carbon Nitride for Photocatalytic Ammonia Generation by Modification with Pyrite

Photocatalytic nitrogen fixation under ambient conditions is currently widely explored in an attempt to develop a sustainable alternative for the Haber-Bosch process. Still, a lack of fundamental understanding of reaction pathways and nitrogen activation mechanisms result in the slow development in this field. In this work we combine defect-rich C3N4, one of the most investigated photocatalysts reported in literature for ammonia generation, with earth abundant and bioinspired FeS2 to improve the activity for photocatalytic ammonia production. By this approach, an activity enhancement of approx. 300 % compared to unmodified C3N4 was achieved. The optimal FeS2 loading was established to be 5 wt.%, with ammonia yields of up to 800 µg L -1 after irradiation for 7 hours. By detailed characterization of the electronic properties of the composites, we deduce that NH3 generation occurs via a novel mechanism involving mainly the reduction of the =N-CN group adjacent to nitrogen vacancies on defect-rich C3N4. FeS2 acts similar to a co-catalyst, improving the activity by π-back-donation from Fe-centers to the imine nitrogen of the defect-rich C3N4, reducing the activation barrier for terminal cyano group reduction upon illumination. ASSOCIATED CONTENTSupporting Information. Additional XRD patterns, XPS data, SEM images, TGA analysis, DRIFT data, more details on salicylate test, hydrogen evolution rate transients, post-catalytic analysis, elemental analysis, TAS transients, PL excitation spectra, PL emission spectra, AUTHOR INFORMATION

activity_enhancement_of_defective_carbon_nitride_for_photocatalytic_ammonia_generation_by_modificati

Introduction<!>Material Synthesis and Characterization<!>𝑓(𝑅) =<!>2⋅𝑅<!>𝑖=1<!>𝐴 𝑆 𝐴 𝐵𝐸 − 𝐴 𝑆𝐸<!>Photocatalysis<!>Material Synthesis and Characterization<!>S K Fe K C K N K<!>Photocatalytic NH3 Generation<!>Post-photocatalytic characterization<!>Charge Carrier Dynamics<!>Table S11).<!>Proposed mechanism for the interaction of FeS2 and defective C3N4 and resulting activity enhancement in photocatalytic ammonia generation<!>Conclusion

<p>The Haber-Bosch process is a large-scale industrial process for the production of ammonia (NH3) from hydrogen (H2) and nitrogen (N2) gas at elevated pressures and temperatures. It is a wellestablished and optimized process, that is crucial for the production of fertilizers. Nevertheless, it still suffers from sustainability issues due to high energy requirements and the utilization of natural gas for the supply of H2 in large, centralized power plants. 1 The photocatalytic nitrogen reduction reaction (NRR), that directly converts N2 into NH3 under (sun)-light irradiation at ambient conditions, presents a feasible alternative. 2,3 However, the NRR is a thermodynamically and kinetically unfavorable process due to the stable and inert nature of the N2 molecule. Furthermore, the similar potentials for N2 reduction and H2 evolution impede a high selectivity in aqueous media so far. This imposes strident demands on an efficient and selective photocatalyst, that are not even close to be met even by the state-of-the art materials, thus highlighting the importance of continued research on the topic. 2,4,5 The low NH3 yields further pose additional difficulties when it comes to accurate quantification, necessitating careful experiment control to avoid impurities and erroneous results. Discrepancies in the experimental conditions and a lack of standardized procedures further limit the comparability and reproducibility of photocatalytic NRR. 6,7,8,9 C3N4, is a low cost, non-toxic polymeric material with good light absorption characteristics, due to a band gap of approx. 2.7 eV, which has received a lot of attention as an n-type photocatalyst during the past decade. 10,11 It is commonly synthesized by thermal polymerization of organic precursor molecules, such as urea, melamine or cyanamide. The polymerization conditions, as well as the choice of the precursors have a strong effect on its structure and properties, by influencing the defect concentration, degree of polymerization, optical band gap, and surface area. 12 Assynthesized C3N4 suffers from high recombination rates. Therefore, alteration of the structure by doping or defect engineering and the formation of heterojunctions have been widely explored to improve the photocatalytic performance of C3N4. 13,14,15,16,17,18,19,20,21 Some of the highest NH3 yields in photocatalytic NRR have been reported using defective C3N4. 22,23,24,25,26 Owing to the polymeric structure of C3N4, the number of possible defects is vast and versatile. 27,28,21,20,18 A frequently exploited strategy for activity enhancement in C3N4 is the introduction of nitrogen vacancies, which are of the same size as the nitrogen atoms in molecular N2, and can thus act as efficient adsorption and activation centres. 27 Another important class of defects are cyano or cyanamide groups, that act as electron-withdrawing groups, assisting in charge separation and suppressing recombination. 14,24,29 The NRR is supposed to proceed via a Mars-van-Krevelen mechanism for both defect types, making a distinction between the influence of cyano groups and nitrogen vacancies difficult. Intercalation of potassium was reported to assist in the replenishment of nitrogen in the structure, more specifically of the cyano groups, resulting in an NH3 production rate of 3.42 mmol g -1 h -1 . 25 Treatment of bulk C3N4 with KOH, or direct incorporation of KOH into the synthesis recently emerged as a promising strategy for both the introduction of vacancies and of cyano groups. Zhou et al. treated bulk C3N4 with KOH in ethanol, followed by solvent evaporation and annealing. They observed an abundance of cyano groups that assisted in charge separation and N2 adsorption. 24 KOH has also directly been involved in the thermal polymerization of urea, likewise promoting the formation of cyano groups. 14 A similar result was obtained by Wang et al., using KOH in the thermal polymerization of dicyandiamide. 25 Etching with KOH was reported to mostly lead to the formation of vacancies.</p><p>Still high NH3 yields of 3.632 mmol g -1 h -1 were reported, together with a quantum efficiency of over 20 %. 22 Another important type of vacancies in C3N4 for NRR are carbon vacancies, graphitized areas and possibly hydroxyl groups. 30,21 Furthermore, although thermal polymerization is an easy and versatile method, residual intermediates were observed the resulting C3N4, causing a high degrees of disorder. 31 Apart from defect engineering, the formation of heterojunctions, preferably of type II, can lead to an improvement of the photocatalytic activity with C3N4. 32 This is due to improved charge transfer characteristics and suppressed recombination, the latter being the dominant cause for the low efficiency in C3N4 materials despite the favorable band gap for visible light absorption. 33 Scientific research on semiconductor materials for photocatalysis is still in its infancy compared to the process optimization in nature. The reduction of molecular N2 has been realized under ambient conditions at the active centers of selected enzymes, termed nitrogenases. Three different classes of nitrogenases are distinguished, based on the composition of the active centers: all contain sulfur and iron atoms, but differ in the nature of additional constituents, one containing vanadium, one molybdenum and one solely iron. 34 This composition led to the exploration of several iron or molybdenum based compounds for the NRR. 35,36,37,38,39,40,41,42,43 One of the simplest iron and sulfur containing compounds is FeS2, which is a non-toxic, stable, and earth-abundant mineral. It shows very high optical absorption and high charge carrier mobility, but its use in photoelectrochemical applications suffers from charge trapping and recombination. 44 Still, FeS2 has been explored for photocatalytic dye degradation. 45,46,47 By itself it is inactive in photocatalytic NRR, due to unsuitable band positions, but it might still offer beneficial contribution in combination with other materials. 48,49 Thus, composites with FeS2 have been employed for dye degradation, H2 evolution, CO2 reduction and NRR. 50,51,52,53,54,55 Apart from improving the light absorption properties in composites, FeS2 can act as an efficient electrocatalyst, which has been shown separately for the H2 evolution, as well as for the NRR. 56,57,58,59,60 There have been some fairly recent reports on the formation of heterojunctions of FeS2 with C3N4 and their application in organic dye and antibiotics removal. 61,62 The reported mechanism and band positions on which the proposed electron transfers are based, are notably different between the reports, requiring further investigation of the interaction between both materials.</p><p>In a bioinspired approach, we report the combination of two earth-abundant semiconducting materials, namely FeS2 and defective C3N4, for photocatalytic N2 reduction to NH3. Exploiting the good light absorption and N2 activation characteristics of the Fe-S system together with the high activity of defective C3N4, NH3 yields of up to 800 µg L -1 can be achieved in the course of the reaction (7 h), which equals to an activity enhancement by approx. 300 % compared to bulk C3N4.</p><p>Even the natural pyrite mineral can be used for this approach. By detailed characterization of the electronic structure of the composites, we propose a novel reaction mechanism, revealing that FeS2 decoration weakens the bonds of C3N4-terminating imine groups in the vicinity of nitrogen defects by back donation, facilitating the reduction of terminal cyano groups under light irradiation towards NH3, with H2 being the only by-product.</p><!><p>C3N4 was prepared via thermal polymerization of melamine. 63 1 g of melamine (Sigma Aldrich, 99%) was calcined at 550 °C for 4 h in a closed crucible in air, using a heating ramp of 5 K/min. The synthesis was repeated several times and the obtained C3N4 powder was ground and thoroughly mixed, before it was used for further modifications and composite formation.</p><p>Vacancy-rich C3N4 (VN-C3N4) was obtained by dispersing 2 g of the as-synthesized C3N4 in 36 mL of 1 M KOH for 3 h under stirring. Subsequently, the material was collected via centrifugation and washed until neutral with ultrapure water. 22 For the composite formation, respective amounts of commercial FeS2 (Sigma Aldrich, 99.8%, 325 mesh) and VN-C3N4 were ground for 10 min in a mortar, under addition of low amounts of ipropanol (p.a.). Subsequently, the mixture was subjected to heat treatment for 2 h at 200 °C in air, to improve interfacial contact.</p><p>The composites were analyzed before and after the photocatalytic experiments. Powder X-ray diffraction (XRD) was measured on a Malvern PANalytical Empyrean device with Cu Kα irradiation (λ1 = 1.5406 Å; λ2 = 1.54443 Å). Acceleration voltage and emission current were set to 40 kV and 40 mA, respectively. Peak assignment was performed with X'Pert Highscore plus.</p><p>Following reference cards were used for the reflection assignment in FeS2 and FeS, respectively: 00-042-1340, 00-023-1123. The diffraction pattern for C3N4 was calculated with Vesta, using crystallographic data from the group of Irvine. 64 Diffuse-reflectance UV/vis spectra were obtained using a Perkin Elmer Lambda 750 spectrometer with a Praying Mantis (Harrick) and spectralon as white standard. The Kubelka-Munk function was used for the calculation of pseudo-absorption. 65</p><!><p>(1−𝑅) 2</p><!><p>For band gap determination, a Tauc plot was used. 66 [𝑓(𝑅) ⋅ (ℎ𝜈)] 1 𝑛 with n = 0.5 for direct band gaps and n = 2 for indirect ones.</p><p>For diffuse reflectance infrared Fourier transformed spectroscopy (DRIFT) a Bruker Alpha II spectrometer and the software OPUS were used. Sample scans were taken from 400 to 4000 cm -1 , with a resolution of 4 cm -1 .</p><p>Fluorescence measurements were conducted on a FluoTime 300 spectrometer from PicoQuant, with the software EasyTau2. Emission spectra were recorded at different excitation wavelengths from a 300 W Xe lamp at room temperature in air. Time correlated single photon counting (TCSPC) spectra were measured using 355 nm laser excitation. The software EasyTau was employed for fitting of the decay curves, using a tailfit with three exponentials, according to:</p><!><p>For steady state measurements, the sample was stuck to carbon tape and placed in a holder for solid powder samples. For quantum yield measurements, dispersions of the respective sample in ethanol were dried as a thin film on the inside of a cuvette, which was placed in an integrating sphere.</p><p>Measurements were conducted for two geometries: "in"signifying placement of the sample film directly in the excitation beam path, and "out"meaning that the film was positioned outside of the direct excitation path. Ideally both would give the same QY, but due to high absorption at ingeometry with relatively thick samples (not all fluorescence will be detected), values for outgeometry are higher and likely represent the more accurate values here. For the calculation of the quantum yield, the intensity of the fluorescence emission was integrated and the area AS divided by the total integral excitation intensity as measured in an empty reference cuvette (ABE) minus the excitation intensity that is not absorbed by the sample, ASE.</p><!><p>X-ray photoelectron spectroscopy (XPS) was performed with a Physical Electronics PHI VersaProbe III Scanning XPS Microprobe device. Monochromatic Al Kα X-ray irradiation with a beam diameter of 100 µm was used, with the beam voltage being set to 15 kV and x-ray power to 25 W. The sample surface was pre-cleaned by Ar-cluster sputtering with a gas cluster ion-beam.</p><p>To avoid surface charging, samples were continuously flooded with slow-moving electrons and Ar + . For survey scans, pass energy and step size were set to 224 eV and 0.4 eV, respectively. Highresolution spectra were measured with a pass energy of 26 eV, a step size of 0.1 eV and a step time of 50 ms. For data analysis a CASA XPS 2.3.17 software was used. The background was corrected using Shirley subtraction. Peak fitting was done with Gaussian-Lorentzian line shapes, with 30 % Lorentz ratio. For charge correction C 1s was set to 284.8 eV.</p><p>Transient absorption spectroscopy data was collected in diffuse reflectance geometry with an LP980 spectrometer (Edinburgh instruments). Pump laser pulse excitation was set at 355 nm (3 rd harmonic of an Nd:YAG laser produced by Ekspla, NT340), while for the probe pulse a 150 W xenon arc lamp was used. Prior to the measurements the powder sample was filled in a cuvette, stored under Argon and sealed directly before the measurement.</p><p>N2 physisorption measurements were conducted on a Quadrasorb Evo device from Anton Paar QuantaTec at 77 K to determine Brunauer-Emmet-Teller (BET) surface areas, using the software ASiQwin for data evaluation. Samples were degassed for 12 h at 120 °C prior to measurements.</p><p>Due to the small surface area, Kr at 77 K was used for FeS2, in an AS-iQ-MP-MP-AG instrument from Anton Paar QuantaTec.</p><p>CHNS elemental analysis was performed with an Unicube instrument from Elementar, using sulfanilamide as standard. Approximately 2 mg of the respective sample were weighed into a tin boat, sealed and combusted at temperatures up to 1143 °C in an oxygen/ argon atmosphere.</p><p>Thermogravimetric Analysis (TGA) with gas evolution detection via mass spectrometry (MS) was conducted with a Netzsch Jupiter STA 449C thermobalance together with a Netzsch Aeolos QMS 403C quadrupole MS, heating the sample at a rate of 5 K/min up to 900 °C in synthetic air.</p><p>Scanning electron microscopy (SEM) images were recorded on a Zeiss Leo 1530 device with an acceleration voltage of 3 kV after sputter-coating with platinum (Cressington Sputter Coater 208 HR). Energy dispersive X-ray diffraction spectroscopy (EDX) measurements were conducted on the same instrument, using and an acceleration voltage of 20 kV. An ultra-dry EDX detector by Thermo Fisher Scientific NS7 was employed.</p><!><p>Photocatalytic NRR was performed in a flow setup using a doped Hg immersion lamp (Z4, 700 W Peschl Ultraviolet) placed in a water-cooled quartz-glass inlay and operated at 350 W. 200 mg of the photocatalyst were dispersed in approx. 30 mL of water by ultrasonic treatment for 10 min. The dispersion was transferred to the glass reactor and diluted to 600 mL by the addition of water and methanol. The total amount of methanol was 20 vol.%. Nitrogen was bubbled through the stirred dispersion at a flow rate of 50 mL/min overnight to flush out residual air. For pre-purification of the inlet gas stream, it was first passed through a 0.1 M KMnO4 solution, followed by a 0.1 M KOH. 8 The dispersion was illuminated for 7 h, during which a constant temperature of 10 °C was ensured by cooling the reactor with the help of a Lauda cryostat (Proline RP845). Evolving gasses were passed through an acid trap containing 10 mL of 1 mM H2SO4, 67 dried and subsequently analysed by a quadrupole mass spectrometer (HPR-20 Q/C, Hiden Analytical). After the reaction, the dispersion was immediately filtered and tested for NH3 using the salicylate test method, a modification of the indophenol blue method. 68,69 For the salicylate test, a stock solution of sodium hypochlorite and a stock solution containing the catalyst, sodium nitroprusside (Carl Roth, >99%), and sodium salicylate (Carl Roth, >99%), were prepared. The solutions were prepared fresh weekly and stored at 4 °C in the dark. For the preparation of the salicylate/ catalyst solution, 2 g of sodium salicylate and 8 mg of sodium nitroprusside were dissolved in 15 mL of ultrapure deionized water, to which 5 mL of a 2 M sodium hydroxide solution was added. For the preparation of the hypochlorite solution, 200 μL of sodium hypochlorite solution (5-10 % Cl, or 12 %, Carl Roth) and 1 mL of 2 M NaOH were given to 18.8 mL of water. In a typical testing procedure, 500 μL of the hypochlorite solution were given to 2 mL of the reaction solution, which was filtered through a 0.2 µm syringe filter beforehand. Then, 500 μL of the sodium salicylate solution were added. The mixture was stored in the dark at room temperature overnight for color development, before being analyzed by UV/vis spectroscopy (Perkin Elmer Lambda 750 spectrometer), using a mixture of the two testing solutions and 20 % aqueous methanol as reference. For the calibration, ammonium chloride (Carl Roth, >99.7%) stock solutions were prepared in a concentration range from 0.1 μg/L to 10 mg/L of NH4 + . Since the NRR was performed in the presence of methanol as a scavenger, 20 % of methanol were present in the calibration as well.</p><p>Ion chromatography (IC) was used to analyse the reaction solution for nitrate by-products. The reaction solution was filtered with a 0.2 µm syringe filter. A 882 Compact IC plus device from Metrohm, equipped with a Metrosep A Supp 4-250/4.0 column and RP 2 Guard/3.5 pre-column were used for the analysis. 4 mM NaHCO3/ 1mM Na2CO3 were used as eluent, lower detection limit was 0.1 mg/L.</p><p>For the quantification of hydrazine, the colorimetric method first reported by Watt and Chrisp was employed. 70 For the testing solution, 0.4 g of p-dimethylaminobenzaldehyde (Sigma Aldrich, 99%) were dissolved in 20 mL of ethanol (p.a.), to which 2 mL of concentrated HCl were added. For the calibration curve, standard solutions of hydrazine sulfate (Sigma Aldrich, >99%) in water/ methanol mixtures were prepared. For the measurement, 1.5 mL of the filtered reaction solutions were mixed with 1.5 mL of the testing solution and stored in the dark for 20 min for color development, before analysis of the absorbance with UV/vis spectroscopy against a reference containing only the testing solution and a water/ methanol mixture.</p><!><p>The X-ray diffraction (XRD) patterns of the KOH-treated C3N4 (VN-C3N4) show the typical two broad reflections at 13° 2θ and at 27.4° 2θ, corresponding to in-plane order and interplanar stacking, respectively, with a d-spacing of approx. 0.326 nm (Figure S2). 64 The structure of C3N4 obtained via thermal polymerization is best described by a model of parallel melon chains connected via hydrogen bonds, as found by Lotsch et al. and confirmed in later studies. The unit cell is orthorhombic with the space group P212121. 71,72,73 This structure model will be assumed in the following. The reduced intensity of the reflection at 13° 2θ (210) upon KOH-etching indicates a reduction of in-plane order. This could be an effect of defect formation and / or consumption of amino groups that lead to partial breakdown and disorder in the heptazine units, which could in turn affect the parallel arrangement of the melon chains. Additionally, the (002) reflection at 27.4° is less intense and shifted to slightly larger diffraction angles, indicating a decrease in interplanar stacking distance and a general loss of order, which has been reported in literature as well and ascribed to either introduced cyano groups or nitrogen vacancies. 13,74,75 A combination of vacancy formation and conversion of amino into cyano groups can well explain the observed changes of both reflections.</p><p>Powder XRD patterns of the formed composites show both the reflections for FeS2 and for VN-C3N4, with the intensity of the reflections for FeS2 increasing with its content (Figure 1). No additional phases could be observed. For further characterization of the morphology, SEM images were recorded. They show the rather large µm size of the FeS2 particles, distributed over a C3N4 matrix (Figure 2 and Figure S3). The FeS2 particles are well distributed and do not aggregate. In some places they are clearly distinguished from the C3N4 matrix, whereas in others C3N4 seems to wrap around the FeS2 particles. EDX confirmed the particles to be phase pure FeS2 with a ratio close to the ideal value of Fe:S 1:2 (Figure 2 and Table S1) and the surrounding matrix to consist of C3N4 with a C/N ratio of approx. 0.52. This is lower than the ideal value of 0.75 and could be an indication of incomplete condensation and thus free amino groups in the sample, although it is also effected by the low sensitivity of EDX for light-weight atoms.</p><p>Physisorption measurements were conducted to evaluate the surface area of the composites with the BET model (Table S2). Pristine C3N4 exhibits the highest surface area of 10.6 m 2 /g, which slightly decreases upon KOH treatment. This is in good agreement with the decreased interlayer stacking distance observed in the XRD patterns and otherwise retention of the morphology. The apparent further decrease of the specific surface area upon composite formation can be explained by a difference in material density and particle size between C3N4 and FeS2, because the specific surface area is measured in m 2 /g.</p><!><p>The optical properties of a photocatalyst are of utmost importance, since they vastly determine the efficiency in light harvesting. The color of the composites gradually gained a grey tinge with increased FeS2 content, compared to the previously pale yellow coloring of both untreated C3N4 and VN-C3N4 (Figure S1). Diffuse reflectance UV/vis measurements were conducted to elucidate the effect of FeS2 addition on the absorption behavior (Figure 3). C3N4 is an indirect n-type semiconductor. 76,77 Therefore, an indirect Tauc plot was used for a more accurate determination of the band gap and compared to the values apparent in the Kubelka-Munk plots. KOH-treatment results in a slight decrease of the band gap from 2.73 to 2.70 eV and marginally improved absorption in the UV region. The band gap reduction -and thus red-shifted absorption -could be caused by the introduction of cyano groups, whose electron-withdrawing properties were reported to lower the conduction band edge and lead to a narrowing of the band gap. 20,78 The increased UV absorption might be caused by improved charge separation due to the decreased layer distance and the introduction of cyano groups, since transitions in the UV region are commonly ascribed to ππ* transitions in sp 2 hybridized centers of the aromatic system. 25,79 The composites exhibit essentially all the same band gap, which is an indication that the major contribution to the light absorption is given by C3N4. This is expected, since its concentration is much higher than that of FeS2. The band gap of the composites is slightly increased but very comparable to VN-C3N4 with a change from 2.70 eV to 2.78 eV (Figure 3). The UV absorption is increased compared to KOH-treated C3N4. Both effects hint at a change in the electronic structure and mobility of electrons in the π-system. The color change and increased absorption of visible light upon addition of FeS2 is reflected by diffuse absorption at higher wavelengths, visible in an offset of the baseline (Figure S5). The effect of band gap widening is especially pronounced for the composites with a FeS2 ratio of 2.5 to 10 wt.% which might indicate optimal charge separation at medium FeS2 loading. All band gaps derived from the Kubelka-Munk and Tauc plots are summarized in Table S3. DRIFT spectra were recorded to further elucidate possible structure changes upon composite formation (Figure 3). The broad signal between 3000 and 3600 cm -1 can be assigned to O-H and N-H stretching vibrations, elucidating the presence of free amino groups that in turn indicate only partial polymerization. The sharp peaks between 1700 and 1200 cm -1 belong to stretching modes of C=N and C-N in the heterocycles, as well as bridging units, and the band at approx. 808 cm -1 can be assigned to the breathing mode of the s-triazine units. 80,24,81 Additionally, a band at 2150 cm -1 could be observed, that is ascribed to the presence of cyano groups, that appear to be present in both the untreated and treated C3N4, as well as in the composites to varying extent. 81,24,20 KOH treatment increases the amount of both cyano groups, as well as -OH and/ or -NHx groups.</p><p>Additionally, the signals arising from stretching vibrations in the heptazine units are of slightly lower intensity for the defective C3N4, with the signal at 1720 cm -1 being more pronounced while the one for deformation vibrations at 808 cm -1 is of decreased relative intensity (Figure S6). This is an indication of structural damage inflicted on the heptazine framework, as would be expected by the formation of defects.</p><p>The spectra for the composites are fairly similar to that of defective C3N4, indicating retention of the structure. An increased absorbance for the vibration of the heptazine units relative to that at 1720 cm -1 was observed for the composites with 10 and 15 % FeS2 in the normalized spectraan opposite effect to that caused by the KOH treatment. The signals for heterocycle-vibrations at higher wavenumbers (closer to 1700 cm -1 ) stem from C=N vibrations, while those at lower wavenumbers (closer to 1100 cm -1 ) arise from C-N vibrations. 82 Therefore, mainly C=N vibrations appear to be affected by the addition of FeS2. We additionally observe a slight shift of the vibrations for the heptazine unit towards lower wavenumbers, that hints at minor changes in the energy of the entire heptazine framework (Figure S6).</p><p>To further investigate the structural evolutions upon KOH etching and composite formation, XPS measurements were conducted on bulk C3N4, VN-C3N4 and a composite containing 5 wt.% of FeS2.</p><p>Survey scans show the expected signals for carbon, nitrogen and low amounts of oxygen in several spots (Figure S6). The latter is mainly due to adventitious carbon at the surface and not OH-groups in the defective C3N4, since oxygen was also observed in several spots on bulk C3N4. C/N ratios for bulk C3N4, and VN-C3N4, are 0.62 and 0.71, respectively, after correction for adventitious carbon, with slight deviations depending on the measured spot (Table S4). This is in good agreement to the C/N ratio derived from XPS analysis in literature and with the expected ratio for melon. 83,81 The increased carbon ratio after KOH treatment might indicate the introduction of nitrogen vacancies. The C/N ratio in the composite is with 0.71 identical to that of VN-C3N4, underlining that the structure remains intact during composite formation.</p><p>Generally, KOH treatment on bulk C3N4 is expected to lead to partial hydrolysis of the structure.</p><p>Yu et al. proposed a deprotonation of an apex amine group, during thermal polymerization in the presence of KOH which led to a breaking of the topmost cycle of the heptazine unit and cyano group formation. The overall amount of amino groups is retained here. 81 The reaction conditions notably differ from those employed in this work, but similar structural changes, specifically the introduction of cyano groups in addition to possible vacancy formation, are expected. Nitrogen vacancies are generally believed to be introduced at C-N=C sites. 25 These kinds of possible defects are mainly considered in the following.</p><p>Two main peaks are observed in the C 1s spectra of untreated C3N4, corresponding to adventitious carbon at 284.8 eV and N-C=N species in the aromatic system at 288.2 eV (Figure 4). The existence of defects gives rise to new peaks. Most important is one at an intermediate binding energy of around 286.1 eV. This is usually assigned to carbon adjacent to amino groups. 80,24 Carbon atoms bound to cyano groups are expected to have a similarthough slightly higherbinding energy, that will overlap with this peak. 72,81 A minor shift from 286.1 eV to 286.2 eV is visible in the spectrum for VN-C3N4, hinting at an increase in the amount of cyano groups, as observed in the DRIFT spectra. Additionally, the intensity ratio of that respective signal to that of C-C changes from 0.21 in pristine C3N4 to 0.27 in the KOH-treated C3N4, which is expected since the total amount of amino groups should be maintained, while additional cyano groups are encompassed by the signal. The ratio of the cyano/ amino groups to that of N-C=N changes from 0.037 to 0.045, which can be explained by carbon atoms of the heptazine unit being converted into -CN upon KOH treatment. Another effect is the observation of a new π-π* satellite at 295.3 eV and a growth in the intensity of the π-π* satellite at ~293 eV, indicating the presence of larger numbers of free electrons in the vacancy rich system. This is in good agreement to the observations from UV spectroscopy and to literature, that claims an increase in the amount of unpaired electrons caused by both vacancy formation and introduction of cyano groups. 84,25 Furthermore, the peak at 298.2 eV is slightly asymmetric for untreated C3N4, which is probably an effect of the extended π-system. Upon KOHtreatment, the asymmetry is reduced, indicating damage inflicted on the aromatic system, which is in good agreement with the assumed breaking of some heptazine units. The formation of a composite of VN-C3N4 and FeS2 results in a marked shift for the signals of both carbon in the -N-C=N and in the cyano group towards higher binding energy (Figure 4 and Table 1). This might indicate electron extraction from the entire heptazine framework, resulting in partial oxidation. Four peaks are identified in the N 1s spectra for bulk C3N4 at approx. 398.6 eV, 399.2 eV, 401.4 eV and ~404 eV, in good agreement to the literature (Figure 4). 80,24,81,25 Correlation of the signals with corresponding nitrogen species is challenging, since peak assignment in the literature is ambiguous. 72 The most prominent peak at 398.6 eV in the spectra for bulk C3N4, and VN-C3N4 both can be assigned to -C-N=C species in the heptazine units (marked as N-(C)2 in the following), and a small peak at 404.9 eV corresponds to a π-π* satellite. The signals between 401 eV and 399 eV can be attributed to amino groups and to the nitrogen atom in the middle of a heptazine unit (N-(C)3), although the exact assignment of the signals to the two nitrogen species is discussed controversially. 25,80,72,83 Additionally, the signal for amino nitrogen should further be fitted by two, since the structure is not ideally graphitic, but closer to that of parallel melon chains, which results</p><p>in the presence of both -NH and -NH2 groups, of which the primary amino group is expected at lower binding energy (Figure 4). 64,72 Cyano groups give rise to a signal at ~400.1 eV, which is indistinguishable from either the signal for the amino groups, or that of N-(C)3. 72 Generally, the nature of the N 1s spectra allows for the possibility of various fits that give good results mathematically but are meaningless in a chemical and physical sense, due to a large number of independent fitting parameters. This is demonstrated in Figure S8. If we assume that the structure of C3N4 obtained via thermal polymerization of melamine lies in between the model of parallel melon chains and of fully condensed sheets, the amount of NH should be smallest, compared to the other nitrogen species. 72 This expectation supports an assignment of the signal at 401.5 eV to NH instead of N-(C)3. Considering, that the signals for primary and secondary amines are likely to be adjacent, we follow assignment of the nitrogen species as: N-(C)2 at 398.7 eV, N-(C)3 at 399.5 eV, NH2 at 400.5 and NH at 401.5 eV, although we stress that a reverse assignment, as suggested in some XPS studies on pristine C3N4 also has its merits. 72,83 To allow for comparison of the spectra for VN-C3N4 and the composites with that for bulk C3N4, we therefore fitted the spectra using several constraints based on structural relationships (Figure S9 and Figure S10).</p><p>Further information about the fitting process is given in the SI.</p><p>The binding energies and atomic ratios for both the C 1s and the N 1s spectra with the most reasonable fitting result are summarized in Table 1. Upon composite formation wit FeS2, a marked shift in all signals towards higher binding energies is apparent, similar to the results observed in the C 1s spectra. This supports the assumption of electron withdrawal from the aromatic system. In order to still derive meaningful insights into structural changes from the N 1s spectra, we decided on normalization (Figure S11). Two things become immediately obvious. One is a shift of the main peak in the N 1s spectra from 398.7 to 399.0 eV upon composite formation, indicating an increase in the binding energy of N-(C)2 in the heptazine units. Additionally, the 'shoulder' at ~401</p><p>eV is less sharply pronounced, indicating larger amounts of nitrogen species at medium binding energy. The same shift towards higher binding energy is observed in the C 1s spectra, proving, that the energy of the entire heptazine-framework is increased upon decoration with FeS2. Additionally, the ππ*-satellite is of increased intensity. The former would indicate partial electron transfer from the C3N4 framework to FeS2, thereby altering the binding energy (oxidizing effect), whereas the latter would indicate an increased number of free π electrons. The lower amount of cyano groups in the untreated C3N4 is also shown by the lower intensity at 286 eV compared to both VN-C3N4 and the composite. These observations confirm the main conclusions drawn from the N 1s spectra above, without relying on arbitrary fitting results.</p><p>Concluding these considerations, XPS analysis supports the introduction of both cyano groups and vacancies upon KOH treatment. Furthermore, it elucidates the changes in the electronic structure upon addition of FeS2 to the system, indicating partial electron extraction from the VN-C3N4 matrix.</p><p>However, especially the number of possible fits for the N 1s spectra necessitates the comparison of the observed changes to the results of other characterization methods. Hydrolysis and introduction of OH-groups similar to what has been observed for hydrothermal treatment with NaOH, involving the breaking of NH-bridging bonds and introduction of OH-groups cannot be totally excluded, but is not likely to have a major influence, since oxygen contents are similar in both C3N4 and VN-C3N4. 73 TGA-MS measurements were conducted on the composite containing 5 wt.% of FeS2 and on both of the constituents, FeS2 and VN-C3N4, to confirm that no structural changes occur during thermal treatment for 2 h at 200 °C, that is part of the composite synthesis (Figure S12). FeS2 was stable up until about 400 °C, above which a gradual extraction of sulfur in the form of SO2 was observed.</p><p>Notably, the lack of an increase in the mass indicates an absence of significant oxidation during the initial heating phase. For VN-C3N4, a major mass loss is observed starting at 570 °C, which is completed at approx. 720 °C. During the heating in synthetic air, NO, H2O and CO2 were found to be the main combustion products up until approx. 650 °C, after which an increase in the evolution of CO and NO2 was observed (Figure S13). The two steps of the combustion process are also apparent in the DSC curves (Figure S12). The TGA curves for the composite show many similarities compared to that of C3N4. However, both the mass loss curves and the ion currents for the evolving gasses are shifted by almost 100 °C to lower temperatures, signifying that the presence of FeS2 boosts the decomposition, likely acting as a catalyst and activating the heptazine units.</p><p>Nevertheless, the observed thermal decomposition only occurs at temperatures far above the 200 °C, thus precluding decomposition during the composite formation.</p><p>The interaction with FeS2 mainly involves the nitrogen in C3N4, as shown in the much decreased ion current for NO2 during the second step of the combustion process, indicating, that more nitrogen is extracted from the structure in the beginning. This is confirmed by the DSC curves,</p><p>where the sharp peak at the end of the combustion process is much less pronounced (at 586 and 712 °C, respectively). While the ion currents for all gaseous combustion products in C3N4 show one signal with a sharp peak current, those of the composites appear as double peaks. Perhaps they correspond to the combustion of areas in close proximity to FeS2 and to areas for which the influence of FeS2 is less. Since the ion currents for both NO and NO2 are significantly lowered compared to that in C3N4 (also in relation to the ion current for CO2, so this observation is not only due to the lower content of C3N4), new nitrogen containing reaction products might be formed, that were not detected.</p><!><p>Photocatalysis was performed in a flow setup using 20 vol.% of methanol as a hole scavenger. The reaction was investigated for VN-C3N4 and composites therewith containing different amounts of FeS2. After the irradiation period of 7 h, the solution was directly filtered and analyzed for NH3 by the salicylate test. Additionally, the concentration of NH3 in an acid trap located behind the reactor was evaluated. The calibration curves can be found in the SI, Figure S14. The quantification of NH3 was performed after stable color development, which was only obtained after several hours in the dark (Figure S15).</p><p>The decoration of VN-C3N4 with FeS2 can significantly enhance the NH3 yield by a factor of 2.1 from 239 µg L -1 to 494 µg L -1 , which equals to 1.9 and 3.9 µmol h -1 for 200 mg photocatalyst. An almost equal improvement was observed for a FeS2 content between 1 and 10 wt.% (Figure 5 and Several control measurements were conducted, to elucidate the source of nitrogen and the selectivity of the reaction (Figure 5 and FigureS18). Firstly, a dispersion of a composite photocatalyst in water/ methanol was tested for NH3 after stirring in the dark. No NH3 was detected in this case. Secondly, a dispersion of VN-C3N4 was filtered, or centrifuged and tested for NH3, to exclude amino groups in the photocatalyst interfering with the test. No NH3 was observed in either control measurement. We also tested a physical mixture of separate FeS2 (5 wt.%) and VN-C3N4</p><p>for photocatalytic NRR ("Mix 5 wt.%"), without performing the grinding and subsequent calcination steps that establish an interfacial contact between the two constituents. The activity was significantly lower than that of VN-C3N4 itself, due to the lower amount of active photocatalyst and lack of interaction between FeS2 and C3N4.</p><p>Additionally, the photocatalytic reaction for the composite containing 5 wt.% of FeS2 was repeated in an argon atmosphere. A similar activity was observed compared to the reaction in N2 atmosphere, strongly suggesting that the nitrogen stems from the C3N4 framework, instead of the feed gas (Figure 5). The slightly higher ammonia yields can be explained by the increased gas flow (100 instead of 50 mL/min). Literature for NRR over vacancy-rich and cyano-rich C3N4 likewise propose a N2 conversion pathway following a Mars-van Krevelen mechanism, therefore this observation is not surprising, 25 however significant NH3 yields for C3N4 under argon atmosphere, like we show, were hardly reported.</p><p>The reaction solutions were additionally tested for nitrogen-containing by-products such as hydrazine and NO3 -. No hydrazine and only trace amounts of NO3around the lower detection limit were found for both VN-C3N4, and the composites (Figure S19, Figure S20). The main side product was H2, with a production rate of around 200 µmol h -1 . The rate was similar for all composites, with a slightly increased H2 formation rate for higher amounts of FeS2 in the sample (Figure S21). FeS2 itself showed a remarkably high H2 production rate of 360 µmol h -1 . Thus, both the activity improvement for NH3 generation and the selectivity are highest for lower FeS2 loadings in the composites.</p><p>A gradual decrease in the H2 evolution rate over time is in good agreement with a possible Marsvan-Krevelen-type of structural changes during the illumination. When the photocatalytic experiment was performed in an argon atmosphere, similar H2 evolution rates were observed as compared to the results in a N2 atmosphere (Figure 5).</p><p>In order to elucidate the formation of methanol oxidation products, UV absorption spectra of the reaction solutions were recorded after the photocatalytic experiments. There is a correlation between the amount of NH3 produced and the amount of UV light absorbance by the filtered reaction solutions at ~205 nm (Figure 6). This absorbance can mainly be attributed to formic acid that is formed alongside NH3 (Figure S22). This is in so far a problem, as it has an effect on the salicylate test, resulting in a significant underestimation of the actual NH3 concentration. 85 The absorbance for a 10 mM solution of formic acid was significantly lower than that observed for the reaction solutions after photocatalysis (Figure S22). Hence, its concentration is definitely not negligible, impeding accurate NH3 quantification. 85 Additionally, the spectra clearly show the presence of other ions that absorb UV-light. The similar wavelengths hamper a clear identification of the responsible ionic species, however. Derivation can help in the distinction between different absorption peaks (Figure S23). Still, the overlap of multiple features renders an accurate identification nearly impossible. Nitrate and nitrite additionally absorb UV light at 203 and at 210 nm, respectively. 86 No clear absorption feature could be identified for those two species, though, which is in good agreement to the ion chromatography results, proving, that only trace amounts are present. The broad absorbance from 230 nm towards higher wavelengths could in part be caused by very fine FeS2 particles, since a filtered FeS2 dispersion gives rise to a noticeable absorbance signal up to 450 nm (Figure S22).</p><p>The observation of significant amounts of formic acid in the reaction solutions, requires a reevaluation of the testing parameters for the salicylate test. Initially we had ensured that we used sufficient amounts of hypochlorite in the test to stay in a concentration range, in which the test is independent of the amount of hypochlorite (Figure S16). Additionally, we always measured a reference sample of known concentration together with the reaction solution to avoid errors based on the testing solutions. However, when we re-measured the reaction solutions for the photocatalytic NRR tests with solutions containing higher amounts of hypochlorite, we found that the determined concentrations were now different, clearly indicating that we previously underestimated the actual ammonia concentrations in the photocatalytic experiments, due to unknown concentrations of formic acid (Figure 7 and Figure S17). Maximum ammonia yields of 819 µg L -1 (6.50 µmol h -1 ) were determined for the composite containing 5 wt.% of FeS2.</p><p>Variations in the relative ammonia yields could be explained by different amounts of formic acid in the reaction solution and thus a varying degree in the influence on the salicylate test. This observation clearly elucidates that the test for NH3 should be repeated several times, ideally with different testing solutions and parameters, and the testing conditions should be verified for each new photocatalyst system. Critical evaluation of the conditions and evolving organic oxidation products is crucial, since the accuracy of the salicylate test can be influenced by a variety of parameters that are too often not considered. For NH3 quantification with Nessler's reagent, the contribution of organic side-products is even more severe, resulting in considerable overestimation of NH3 concentrations and hence also the NRR activity. 85 All concentrations given in the paper are averaged between several quantification measurements with the salicylate test. Since hardly any difference in the NH3 yield in argon and N2 atmosphere was observed after 7 h</p><p>for the most active sample, we devised a longtime measurement, to gain further insights into the reaction mechanism and evaluate if nitrogen vacancies are replenished again under the conditions employed in photocatalysis, confirming a Mars-van-Krevelen mechanism. A dispersion of the composite containing 5 wt.% FeS2 was first illuminated for 14 h in either N2 or argon atmosphere.</p><p>Every two hours, a sample was taken and analyzed for NH3. After 14 h, the lamp was switched off and the solution was stirred in the dark for 6 h under continuous gas flow to allow for nitrogen reincorporation into the structure, before illumination was continued for another 6 h (Figure S24).</p><p>During the first irradiation period, the generated amount of NH3 increased almost linearly with a rate of approx. 82 µg h-1 in N2 and 90 µg h -1 in Ar atmosphere, respectively. Once the lamp was switched off, the measured concentration slightly decreased, likely due to NH3 carried out of the reactor by the gas flow. After continuing the light irradiation, the NH3 generation was increased again with about the same rate, as before the period in the dark. Afterwards, the determined concentrations seem to level out. This effect is likely caused by a combination of slow degradation of the structure and accumulation of oxidation products, that are impeding the NH3 quantification progressively more severely. A similar effect was observed for the H2 evolution rate, indicating that the observed effect is not solely caused by the accumulation of formic acid (Figure S24). The experiment does not prove a re-incorporation of nitrogen into the structure under the photocatalytic conditions.</p><p>In addition to the NH3 concentration reported in the reaction solution, NH3 was detected in the acid trap but not included in the concentrations given here. The amounts of NH3 were maximum around 15 µg and 6 µg total for the experiments in an argon and a nitrogen atmosphere, respectively (Table S8). Still, the use of an acid trap is sensible. The pH of a dispersion of 20 mg of VN-C3N4 in 20 mL of H2O was approx. 9.5. Thus, both NH4 + and NH3 species can occur in significant amounts. For C3N4 the pH is with ~8 closer to neutral, perhaps due to residual OHspecies from the KOH etching, even after washing for at least 10 times with plenty of water. A slightly alkaline pH for C3N4 is expected, due to the large number of amino groups.</p><p>The feasibility of the activity enhancement of VN-C3N4 for photocatalytic NH3 generation by decorating the surface with FeS2 was further underlined by using the natural mineral material of FeS2, that is earth abundant and inexpensive pyrite. Still, composite formation resulted in an increase in the ammonia yield, even though the particles were very large (mm sized grains ground in a mortar) and the surface was likely oxidized due to storage for years in air (Figure S25). Some additional reflections in the XRD patterns even indicated the presence of trace impurities (Figure S28), that do not seem to have a major influence on the photocatalytic activity of the composite.</p><!><p>Both VN-C3N4 and the composites with FeS2 were thoroughly characterized after the photocatalytic experiments, in order to evaluate the stability and to gain further insights into the reduction mechanism. Post-photocatalytic XRD patterns still show the same reflections for phase-pure C3N4 and FeS2 (Figure S26). The intensity for the FeS2 reflections is significantly decreased after the NRR. This effect is a result of a loss of interfacial contact between FeS2 and C3N4 as verified by dispersing a composite in water/ methanol mixtures and subsequently regaining the material via centrifugation (Figure S27). FeS2 is known for its flotation tendency in mineral separation, due to its relative hydrophobicity. 87 Additionally, we tested the stability of FeS2 during both the formation of the composite (with annealing at 200°C) and storage of the sample in air. No changes in the crystal structure of FeS2 were observed (Figure S28).</p><p>UV/vis spectra show a significantly increased absorbance of UV light and a decrease of the band gap (Figure S29). Both VN-C3N4 and the composites showed a pronounced darkening after the NRR that decreased again after storage in air (Figure S30). The effect of increased UV light absorption is less obvious for the composite containing 15 wt.% compared to the other composites, for which the activity was also lowest. All other composites that exhibited a similar activity, show a similar increase in UV absorption, which likely correlates to higher degrees of structural change.</p><p>The increased UV absorption is least pronounced for VN-C3N4 in agreement with the lower activity.</p><p>The band gap decreases very slightly by 0.03 eV for VN-C3N4. For the composite containing 5 wt.%, the decrease is most drastic and the band gap is experiencing a change by 0.07 eV from 2.78 to 2.71 eV. The red shift of the absorption might be caused by defect formation.</p><p>DRIFT spectra for the composites after the NRR experiments show a marked decrease in the vibrations for the heptazine units (Figure S31 and Figure S32), especially in relation to the vibration at 1720 cm -1 , that falls into the range for C=N vibrations. This vibration is further shifted back to slightly higher wavenumbers. A decrease in the relative intensity of the 808 cm -1 vibration agrees with this observation, indicating structural damage to the heptazine framework. Both effects were also observed to a lesser extent upon introduction of vacancies and cyano groups upon KOH treatment and thus confirm further breakdown of the heptazine units during photocatalysis.</p><p>Additionally, a closer look at the vibration at 2147 cm -1 reveals a decrease in the intensity, supporting the assumption that cyano groups are consumed during the photocatalytic experiment (Figure S33). The effect is even more increased for long-term experiments, highlighting the further degradation of cyano groups with prolonged illumination times (Figure 33).</p><p>XP spectra of the composite containing 5 wt.% of FeS2 after the photocatalytic reaction show a slight shift for the carbon species adjacent to cyano-/ amino-nitrogen and the N-C=N peak towards lower binding energies (Figure S34). The intensity for both peaks corresponding to the C3N4 structure decreases markedly in relation to that of C-C, further indicating structural changes, that does not only extend to nitrogen, but is further inflicted on carbon in the structure. The ratio might, however, also partly be influenced by adsorbed organic residues from the sacrificial agent.</p><p>Additionally, the intensity for the satellite peaks is decreased, supporting damage to the aromatic system. The calculated C/N ratio from the survey scan was 0.88, without correction for adventitious carbon, because the amount of C-C or C=C bonds possibly present in the structure after partial extraction of nitrogen is unknown. Thus the C/N ratio is increased compared to 0.80 for the same composite before the photocatalytic experiment. This is a strong evidence for nitrogen extraction from the C3N4 matrix.</p><p>Sample composition was additionally evaluated by elemental analysis before and after photocatalytic experiments (Figure S35). The amount of FeS2 determined in the sample is less than what was expected for all composites. Since the FeS2 particles are rather large and thus limited in number, this effect might simply be due to inhomogeneous distribution over the C3N4 matrix. The amount of sulfur and thus probably also FeS2 was further decreased after the photocatalytic experiment for all composites (Figure S35). This is likely an effect of the imperfect interfacial contact and washing out of the sample, as has already been observed in the XRD patterns.</p><p>For a more detailed comparison, the C/N ratio was calculated for the composite samples before and after the photocatalytic reaction. For all composites, the C/N ratio was about 0.555, which is significantly different from the compositional value of 0.75 and indicates incomplete polymerization and the existence of many free amino groups. The value is in good agreement to the EDX measurements, though. No significant differences of the C/N ratio were observed for different FeS2 loadings. For all composites, the C/N ratio was visibly increased after the photocatalytic reaction, supporting the extraction of nitrogen from the structure. Compared to the C/N ratios obtained from XPS measurements, the nitrogen content in the bulk is significantly lower, due to adsorbed carbon impurities and possibly nitrogen deficiency at the surface.</p><!><p>Due to the large variety of defects that can potentially be present in C3N4, the electronic structure is rather complex and should be studied in detail, in order to gain an understanding of the underlying mechanisms in photocatalysis. To investigate the charge carrier dynamics, transient absorption spectroscopy (TAS) in diffuse reflectance geometry was employed. In Figure 8 the ns-TAS measurement of a composite of VN-C3N4 and 5 wt.% of FeS2 in an argon atmosphere is presented. The positive absorption feature between 650 nm and 900 nm can be assigned to photogenerated electrons in VN-C3N4. 88,89,90,91 The same signal is generally apparent in the measurement of the composite, but an increase of the relative absorption intensity in the range between 650 nm and 750 nm is noticeable, in comparison to the main signal between 750 and 900 nm. This might indicate a change in the electronic structure upon composite formation with FeS2 and an increased relative amount of photo-generated electrons in a second excited state. For a better comparison the main signal of the photogenerated electrons in both materials was analyzed regarding the lifetime of these electrons. The lifetimes monitored at 800 nm of both VN-C3N4 and the composite are comparable and in the order of ns (Figure S36). The signal is best fitted with two different lifetimes, that are 3. We additionally used fluorescence spectroscopy to gain insights into the radiative energy relaxation levels of the excited photocatalyst. Defect states introduce new energy levels into the material and fluorescence can occur from either excited defect states or the conduction band to the valence band, or to defect sites located slightly above it. This would in theory give rise to various emission signals of which many will have highly similar energies. The most dominant emission is that into the ground state. Therefore, low photoluminescence is often an indicator for efficient charge carrier separation and low recombination rates. C3N4 as a polymeric material with a structure in between that of molecules and solid crystals, generally exhibits a prominent blue fluorescence, that has been shown to be significantly influenced by the introduction of defects. 30,93 Upon irradiation at wavelengths below the band gap energy, a broad fluorescence signal between 410 and 640 nm is observed for both VN-C3N4 and the composites, with a maximum emission at 470 nm and a slightly less intense emission at 445 nm, as shown in Figure 9 and Figure S38. The samples could be excited over a wide energy range from 250 to 450 nm (Figure S37) for both emission signals, signifying that radiative recombination proceeds from the same energy levels.</p><p>Notably, the relation between the intensity of fluorescent emission and excitation wavelength is essentially the same for all composites, with VN-C3N4 showing a different behavior. This indicates an alteration of the defect states in VN-C3N4 upon modification with FeS2. To better elucidate these changes, excitation spectra were normalized (Figure S38). The excitation maximum for VN-C3N4 is located at approx. 320 nm which corresponds to band gap transitions according to Gan et al.. 94 The same group ascribes excitation at around 380 nm to transitions from the valence band to lone pairs of -N-(C)3 species. This is in good agreement to the observed increased absorbance for the composites at this wavelength in the UV/vis measurements and also to the maximal fluorescence emission obtained at this excitation wavelength (Figure S39). The ratio of emission intensity at 320 nm excitation to emission intensity at 380 nm excitation in the normalized excitation spectra is significantly decreased for the composites compared to VN-C3N4, supporting the assumption of larger numbers of electrons in lone π and possibly also π* orbitalse.g. of cyano groups -as derived from XPS and UV-light absorption. Addition of >1 wt.% of FeS2 results in a decrease of the fluorescence emission compared to VN-C3N4, especially for the composite containing 2.5 and 5 wt.% of FeS2, which were also the composites that showed the highest activity for photocatalytic ammonia generation, indicating better charge separation compared to defective C3N4 and thereby reduced recombination (Figure 9). The photoluminescence is shifted to lower wavelengths for the composites, which is in good agreement to the slightly increased band gaps and indicates a decrease in interlayer electronic coupling. 88,95 FeS2 itself does not show any fluorescent emission, although a weak emission signal was detected at approx. 460 nm when FeS2 was stored in air for several days, indicating low degrees of surface oxidation. For better evaluation of differences in the signal shape and thus change in the contributions of intrinsic and defect emissions, the emission spectra were normalized (Figure 9 and Figure S38).</p><p>The intensity for the emission at 445 nm in relation to that at 470 nm is clearly increased for the composites. The most prominent fluorescence is often attributed to band gap emission in literature. 93 In contrast, our observations would indicate that the most intense fluorescence signal at 470 nm is instead attributed to radiative emission from defect states and band gap emission likely occurs at 445 nm, which is in good agreement to the band gap energy determined via absorption spectroscopy. An additional tail towards higher wavelengths includes contributions from defect emissions, such as transitions from lone pair states of nitrogen atoms in the s-triazine unit. 94,30 The significantly increased inner band gap emission in relation to defect emission in the composites might indicate the inhibition of radiative recombination at defect sites, likely due to interaction of VN-C3N4 with FeS2 at the defect sites. Charge transfer might assist in exciton separation and retard recombination.</p><p>The emission peak for VN-C3N4 and for each composite is largely independent of the excitation wavelength (Figure 9). A decrease in the relative intensity of the emission peak at 445 nm with increasing excitation wavelength is observed for the composites, along with a slight red-shift of the entire emission signal, that is especially pronounced at excitation with light in the order of the band gap energy. A higher excitation wavelength might increase the relative ratio of charges excited to lower level defect states, thus also increasing the relative ratio of emission from these levels. It has to be noted however, that the absolute emission intensity is decreased, since the light absorption is diminished and less charges are excited (Figure S39).</p><p>The emission signals were fitted with Gauss functions, to allow for detailed investigation of different contributions to the PL spectra. Mostly, emission peaks for C3N4 are decomposed into three signals. 30,74 We additionally used four Gauss functions to account for deviations at higher wavelengths that are apparent in the fit with three species (Figure S40). The determined emission signals were located at ~437 nm, 465 nm and 505 nm, or at 438 nm, 464 nm, 488 nm and 528 nm, respectively. They likely correspond to inner band gap emission, emission from N-(C)3 sites and possibly either transition from electron lone pairs in NH-bridging nitrogen/ N-C bridging nitrogen, or from graphitized areas to the valence band. 94,30,93 The additional defect state emission at 488 nm could perhaps be related to either lone pairs in imine nitrogen in the vicinity of cyano groups/ nitrogen vacancies, or in the cyano groups themselves.</p><p>The fitted emission signals show significant differences for the composites compared to VN-C3N4.</p><p>The relative intensity for the direct band gap emission at 437 nm is considerably increased for the composites, as already observed from the general signal shape (Figure S40). It is also slightly shifted towards lower wavelengths for the composites, in correlation to the increased band gap energy. The relative intensity for the second emission signal at 464 nm is decreased for VN-C3N4</p><p>for the composites compared to VN-C3N4, especially for low to medium FeS2 loading of 5 wt.% or less. This might be attributed to better separation of charges at N-(C)3 sites. The emission signals at 480 nm and 520 nm are slightly increased in relation to the other components for the composites and shifted towards lower wavelengths. The shift could be a result of a variation in the energy of the entire C-N-framework, as observed in XPS and is especially pronounced for the state emission at 480 nm, which supports an emission from defect states at this wavelength.</p><p>The intensity of the fluorescence emission can be an indication for the efficiency of charge separation. However, it is dependent on sample preparation and positioning in the instrument. The lifetime of an emission is regarded as a more reliable parameter for judging charge recombination rates. The lifetime was measured for both detection at 445 and 470 nm (Figure 10 and Figure S41). The decay can be fitted with three exponential functions, as is usually done in literature, as well. 14,30 The shortest fluorescence lifetime (τ3) of about 1.6 ns can be attributed to exciton recombination in the aromatic system (Table S10). 74 A second and third one with about 7 ns (τ2) and 25 ns (τ1) in the reference were attributed to charge carrier migration in the plane or between layers (along the π-stacking direction), respectively. 74 The lifetime for recombination in the aromatic system is slightly shorter for the composites compared to C3N4, which might be due to damaging of the πsystem. Larger changes are observed for the other two lifetimes (Figure S42). An increase in the second lifetime, τ2, for the composites compared to defective C3N4 implies improved intraplanar and intrachain charge separation. This might be due to polarization of the heptazine framework and electron transfer process due to interactions with FeS2. The greatest difference is observed in the time constants for τ1, which increases for the composites (Figure S42). Hence, charge separation in the π system is significantly improved by FeS2. The lifetimes are highly similar for all composites.</p><p>Notably, the highest lifetimes were observed for composites containing 2.5 to 10 wt.% of FeS2, which is in good agreement to the highest photocatalytic activity at these FeS2 loadings.</p><p>Fluorescence decay is slower at 470 nm longer compared to at 445 nm for all samples, supporting the assumption of the main emission at 470 nm mainly being caused by recombination at defect sides.</p><p>The physical mixture of defective C3N4 and FeS2 and the composite with 5 wt.% of FeS2 show almost the same lifetimes, slightly higher for the composite. This implies interaction, likely in the form of charge transfer between both constituents, which is surprising considering that no beneficial effect of FeS2 addition could be observed in the photocatalytic reaction. However, during the fluorescence measurements, solid particles of both C3N4 and FeS2 are in direct contact, while in an aqueous dispersion, no interfacial contact is formed. The quantum yield (QY) was determined at three different excitation energies, to ensure that the increased fluorescent lifetimes truly correlate to improved charge carrier separation (Table 2 and</p><!><p>A decrease in the fluorescence QY with increasing FeS2 content is observed, implying that recombination is indeed reduced in the composites. The QY is highest at 320 nm, in good agreement to direct band gap excitation at this wavelength.</p><!><p>The band positions of FeS2 are located at 0.27 V and 1.23 V vs. NHE, 48 respectively, whereas those of C3N4 are generally around -0.8 V and 1.85 V vs. NHE. 93,14 Hence, a type I heterojunction should be formed, that would result in charge accumulation on FeS2. However, we have shown that FeS2 is inactive for ammonia production. Band bending would additionally impede electron and hole transfer from the conduction band of C3N4 to FeS2 (Figure 11).</p><p>Alternatively, electron transfer could follow a Z-scheme mechanism. Such an arrangement would result in N2 reduction taking place at the valence band of C3N4 and oxidation reactions occurring at the conduction band of FeS2. For a Z-scheme like transfer, the potential difference between the conduction band (CB) of FeS2 and the valence band (VB) of C3N4 is quite large, and the band alignment would be more suitable for hole transfer from C3N4 to FeS2. Additionally, the oxidation However, these considerations do not yet take into account the interaction with additional defect levels that can alter the electronic structure of the composite and promote interactions between C3N4 and FeS2. In order to test this hypothesis, composites containing 5 wt.% of FeS2 and newly synthesized, not KOH treated C3N4 were fabricated and employed in the photocatalytic experiments. No increase in the ammonia yield was observed (Figure S43). It is additionally evident in the DRIFT spectra before and after the photocatalytic experiment, that the signal for the cyano group remains unchanged for bulk C3N4.</p><p>Based on the observations for structural changes in the VN-C3N4 induced by the presence of FeS2, we deduced a mechanism for the activity enhancement.: since KOH treatment is supposed to introduce defect sites that have two nitrogen species with free electron pairs in the vicinity (iminetype nitrogen in =N-CN units and amino groups), 81 an interaction similar to ligand to metal coordination to Fe 2+ appears to be feasible, that results in an activation of the C3N4 structure (Figure 11).</p><p>This model suggestion is based on following observations:</p><p>1) Increased UV absorbance observed for the composites (more π-π*-transitions) (Figure 3).</p><p>2) Band gap widening upon FeS2 addition (Figure 3): possibly due to deformation/ polarization of the structure upon coordination of defects in C3N4 to iron centers due to partial electron transfer (σ-Donor) to FeS2.</p><p>3) The shift of both nitrogen and carbon binding energies towards higher values suggests partial oxidation of the structure, thus supporting the σ-donor effect (Figure 4).</p><p>4) The increased satellite peaks in the XP spectra suggest higher number of electrons in the πsystem, which might be due to π-back-bonding, (Figure 4). Possibly, coordination of the cyano groups and/ or amino groups to Fe 2+ centers induces a marked electron withdrawing effect and partial charge transfer to iron, reducing the electron delocalization.</p><p>5) Fluorescence quenching and decreased QY indicate improved charge transfer (Figure 9). 6) Decreased ratio of defect emission (from lone electron pair states) in fluorescence measurements (Figure 9) indicates interaction with and possibly partial charge transfer from defects in VN-C3N4 to FeS2.</p><p>7) Increased lifetimes for the longest lifetime, τ1, for the composite (Table S10): electrons in the π-system might be influenced by the interaction with iron. Thus, electrons excited into π* states of the aromatic system are possibly stabilized due to increased occupation of antibonding orbitals. 11) VN-C3N4 has significantly more free electron pairs than C3N4 that can coordinate to FeS2.</p><p>12) π-back-donation from Fe-centers to imine bonds are known, as is back-donation and charge transfer with cyano group. 96,97,98 13) ML charge transfer could be induced upon radiation, increasing the electron density in =N-CN groups.</p><p>14) The oxidation products in the photocatalytic reaction are the same for both, composites and VN-C3N4, (Figure 6), suggesting oxidation on VN-C3N4</p><p>15) The HER activity is decreased for the composites (Figure S21), even though FeS2 is present, indicating that electrons in FeS2 are used for other redox reactions, such as π-back-bonding and re-oxidation of Fe 3+ to Fe 2+ . Fe 3+ might form during light-induced ML charge transfer.</p><p>16) The activity enhancement was not observed for bulk C3N4 decorated with FeS2, indicating an interaction of FeS2 with the vacancies introduced by KOH-treatment. 17) Interaction of the composite with N2 is indicated in the TGA-MS measurements, possibly due to coordination to Fe 2+ . Such an effect could be advantageous for re-incorporation of nitrogen into the structure, similar to what has been observed for K + coordination. 25 The conditions for interaction with N2 (gas atmosphere vs. poorly dissolved nitrogen in aqueous solutions) is significantly different in TGA-MS compared to photocatalytic reaction, though.</p><p>The coordination as proposed in Figure 11 would activate both, the imine nitrogen and nitrogen in the cyano group. In VN-C3N4, only the nitrogen in the cyano group would be slightly activated.</p><p>Conversion of the entire =N-CN group would also result in a loss of C as observed in XPS (Figure 4) and the formation of carbon-containing oxidation products (Figure S24). Vacancies would be necessary for the interaction, therefore the effect was absent for composites with bulk C3N4, even though it also contains some amounts of cyano groups. Thus, photocatalytic ammonia generation with defective C3N4 is most likely always a product of self-degradation, which can be enhanced by back-donation with suitable coordinating species.</p><!><p>We have shown that a combination of defect engineering of C3N4 and subsequent composite formation with FeS2 can significantly improve the activity of C3N4 for photocatalytic ammonia generation, resulting in an activity enhancement of approx. 300 % compared to unmodified C3N4.</p><p>The optimal FeS2 loading was established to be 5 wt.%. The system only employs inexpensive, earth abundant and non-toxic materials. Knowledge about the exact structure of C3N4 and the presence and character of defects proved to be crucial, as they significantly influence the interactions between the two constituents. Charge transfer between FeS2 and C3N4 was established to proceed only at the defect sites, resulting in an electronic activation of the structure. NH3 generation was found to occur via a novel type of mechanism, involving reduction of the =N-CN group adjacent to nitrogen vacancies. A replenishment of nitrogen in the structure could, however, not yet be verified.</p><p>FeS2 acts akin to a co-catalyst, boosting the activity, although the mechanism is different to that of (metal) electrocatalysts typically employed for photocatalytic HER or OER. Here, π-back-donation from Fe-centers to imine nitrogen and amino groups of the defect-rich C3N4 reduces the activation barrier for the reduction of terminal cyano groups upon illumination.</p><p>Although there are numerous reports on C3N4 for the NRR, the complexity of the system, with a broad variety of defects, that can theoretically be present, necessitates a strict control of the synthesis parameters and thorough characterization experiments. We deduce that photocatalytic ammonia generation with defective C3N4 is most likely always a product of self-degradation, which can be enhanced by back-donation with suitable coordinating species to reduce the activation barrier.</p>

ChemRxiv

Growth of Giant Peptide Vesicles Driven by Compartmentalized Transcription–Translation Activity

AbstractCompartmentalization and spatial organization of biochemical reactions are essential for the establishment of complex metabolic pathways inside synthetic cells. Phospholipid and fatty acid membranes are the most natural candidates for this purpose, but also polymers have shown great potential as enclosures of artificial cell mimics. Herein, we report on the formation of giant vesicles in a size range of 1 μm–100 μm using amphiphilic elastin‐like polypeptides. The peptide vesicles can accommodate cell‐free gene expression reactions, which is demonstrated by the transcription of a fluorescent RNA aptamer and the production of a fluorescent protein. Importantly, gene expression inside the vesicles leads to a strong growth of their size—up to an order of magnitude in volume in several cases—which is driven by changes in osmotic pressure, resulting in fusion events and uptake of membrane peptides from the environment.

growth_of_giant_peptide_vesicles_driven_by_compartmentalized_transcription–translation_activity

<!>Conflict of interest<!>

<p>T. Frank, K. Vogele, A. Dupin, F. C. Simmel, T. Pirzer, Chem. Eur. J. 2020, 26, 17356.</p><p>One of the most prominent goals of bottom‐up synthetic biology is the creation of synthetic cells. [1] Such systems are envisioned to display a set of properties and capabilities that are associated with extant living cells, namely (i) compartmentalization, (ii) growth, self‐maintenance and self‐replication, (iii) signaling, communication and sensing, and (iv) the potential for evolution through replication and transfer of genetic information.</p><p>Compartmentalization is an essential prerequisite for the remaining properties, and has been achieved using a wide variety of different approaches. [2] Cell‐scale reaction containers have been created from phospholipid membranes, [3] using fatty acids [4] or polymers, [5] emulsion droplets, [6] coacervates, [7] or even using microfluidics‐based DNA chips. [8]</p><p>Several synthetic cell models already displayed at least some of the desired properties listed above. For instance, Szostak and co‐workers reported on fatty acid vesicles—serving as primordial cell models—, which were able to grow and divide upon external feeding with fatty acids. [9] In further work, enzyme‐free copying of nucleic acid templates was shown inside fatty acid vesicles. [10] Kurihara et al. succeeded to show DNA amplification inside lipid‐based giant vesicles, which were able to grow when membrane precursors were added to the outside solution. [11] Growth was also observed for phospholipid vesicles externally fed with fatty acids, which contained a cell‐free protein synthesis reaction. [12] Other research groups focused on in situ phospholipid biosynthesis inside of liposomes. For instance, Hardy et al. catalytically synthesized phospholipids from simpler precursors, which also resulted in membrane growth. [13]</p><p>More closely mimicking lipid synthesis in natural cells, Scott et al. established parts of the complex phospholipid synthesis pathway inside of liposomes. To this end, all the required enzymes were encoded on DNA templates and produced inside of the liposomes via cell‐free gene expression. [3a] Due to their similarity to biological cell membranes, phospholipid membranes appear to be the most natural candidates for compartmentalization of synthetic cell‐mimicking systems. From a technical point of view, however, phospholipids have several drawbacks. For instance, phospholipids form membranes with a relatively high resistance to a change in membrane area, and even minor stretching causes membrane rupture. [1b] Membranes composed of lipid mixtures can have more favorable mechanical properties, but in vesiculo production of mixed membranes would be even more challenging than for homogeneous membranes.</p><p>Other membrane‐forming molecules such as amphiphilic block co‐polymers or polypeptides represent an interesting alternative to phospholipids.[ 1b , 14 ] Such membranes are mechanically quite robust and even capable of storing elastic energy. [15] Furthermore, membrane forming peptides can be easily produced inside of vesicles [16] using cell‐free transcription‐translation systems. [17] A particularly interesting class of polypeptides that can be used for membrane formation are elastin‐like polypeptides (ELPs).[ 1b , 18 ] The commonly used sequence motif (VPGXG)n (shorthand notation: Xn) is derived from tropoelastin, where X is any natural amino acid except proline and n is the number of pentapeptide repeats. Depending on the amino acid used for X the peptide displays different hydrophobicity. [19]</p><p>We have recently shown that ELPs can form ≈200 nm sized vesicular structures for the compartmentalization of biochemical reactions such as transcription of RNA aptamers and the expression of fluorescent proteins. [16a] In the present work we demonstrate the fabrication of much larger, cell‐sized polymersomes using a solvent evaporation method. [20] In contrast to the glass beads method previously used by Vogele et al. [16a] the necessary peptide film was formed on the inner glass surface of a round‐bottom flask, which resulted in vesicle sizes in the μm scale (Figure 1). Importantly, ELP polymersomes encapsulating transcription (TX) or transcription‐translation (TX‐TL) reactions displayed a strong increase in size when they were externally supplied with additional membrane peptides. In several cases, fusion events between adjacent vesicles were observed as well.</p><p>a) Scheme of the sequence design of the amphiphilic ELP. b) Graphical depiction of the workflow for the solvent evaporation method to produce giant ELP vesicles.</p><p>Transcription of RNA aptamers inside the vesicles resulted in mixed growth behaviors, in which some vesicles started to shrink at one point, while others continued to grow. By contrast, when expressing the fluorescent protein YPet, 97 % of the vesicles continually increased in size; for the remaining two vesicles no clear shrinkage or growth could be observed. Our experiments hence demonstrate biochemically driven growth of peptide‐based synthetic cellular structures, which could set the stage for competition and selection dynamics emerging among such compartments.</p><p>We used an ELP with the sequence (R5Q5)2‐F20 (in shorthand notation) as the amphiphilic membrane component (Figure 1, Supporting Information section 3). This sequence design ensures that the peptides contain a well‐structured hydrophobic tail and a random coil polar head group under our standard experimental conditions. Peptide expression and purification were performed as described previously. [21]</p><p>Controlled formation of giant peptide vesicles was carried out through solvent evaporation based on a protocol originally introduced by Marsden et al.[ 16b , 20 ] Initially, the amphiphilic ELPs were lyophilized in a round‐bottom flask to completely remove water from the peptides (Figure 1 b). The peptides were then re‐dissolved by the addition of tetrahydrofuran (THF) and sonication. For encapsulation, the internal solution (IS), which contained a defined amount of sucrose in purified water, was added to the THF/ELP mixture, followed by agitation and incubation at room temperature. Because of the amphiphilic nature of the ELPs used, droplets of the inner solution are formed, which are covered and stabilized by an ELP layer. Subsequent formation of a peptide double‐layer at the droplet interface, and hence the formation of vesicles, was accomplished by the removal of the THF solvent through evaporation. Nonetheless, residual solvent within the ELP double layer cannot be ruled out entirely.</p><p>After formation the vesicles were mixed with an outer solution (OS) to dilute residual IS. The OS contained an isotonic amount of glucose as well as a low percentage of Triton X‐100 (0.01 %). Due to the higher density of the IS, the vesicles sedimented at the bottom of a microscopy sample chamber and could thus be easily observed for several hours. If an osmotic shock is applied the vesicles vanish (Figures S5, S6).</p><p>The solvent evaporation method resulted in two populations of vesicles of different sizes, namely small vesicles (SV) with radii far below 1 μm and larger vesicles with sizes spanning two to three orders of magnitude, which we will collectively refer to as giant vesicles (GV). In order to characterize the size of the vesicles across these scales, we utilized transmission electron microscopy (TEM), dynamic light scattering (DLS) as well as light microscopy (LM). For the SVs, a mean radius of 0.03 μm ±0.01 μm was determined using TEM (Figure 2), where the estimated uncertainty is the standard deviation of the distribution. The GV fraction displayed a wide range of radii between 400 nm and several micrometers. Due to the size resolution limits of the DLS and LM characterization methods, we were not able to acquire a quantitative size distribution across the whole range, but they allowed us to determine the lower and upper size limits of the GV population (Figure 2 b). In LM, we occasionally also observed vesicles with radii much larger than 10 μm. A rationale for the occurrence of the SV and GV populations is the presence of two alternative processes of vesicle formation. SVs are presumably created through spontaneous formation in aqueous solution from ELP monomers, whereas the GVs are generated only through the application of the solvent evaporation method.</p><p>a) Size distribution of ELP vesicles analyzed with TEM. Sample size: N=100. Inset: TEM image of ELP vesicles. Scale bar: 100 nm. b) Size distributions of vesicles using DLS (orange) and LM (cyan). Sample sizes: N=100. Inset: LM image of ELP vesicles. Scale bar: 50 μm.</p><p>We next studied the capability of the GVs for fusion and growth. We speculated that—similarly as previously observed with fatty acid vesicles [4] —an osmotic imbalance could lead to an influx of water and thus promote vesicle growth. In our experiments the imbalance was created through biopolymerization of polyelectrolytes. We therefore transcribed the fluorogenic RNA aptamer dBroccoli inside the vesicles by encapsulating a transcription mix containing T7 RNA polymerase, rNTPs, template DNA, the ligand of the aptamer, DFHBI (3,5‐difluoro‐4‐hydroxybenzylidine imidazoline), and sucrose (Supporting Information section 1.2). In order to be able to control the start of the transcription reaction, we separately produced two types of vesicles, which were either missing the template DNA or the T7 RNA polymerase (Figure 3 a). In the experiments, these vesicles were mixed, resulting in the transcription of fluorescent aptamers only inside of vesicles, which were generated through fusion of the two vesicle types. We added DNase I to the OS to prevent transcription by accidentally released transcription mix. In addition, the surrounding OS was supplemented with a low percentage of Triton X‐100 (0.01 %) and an isotonic amount of glucose to balance the initial osmotic pressure in the vesicles. Control experiments showed that no TX activity was observed in the absence of Triton X‐100, which apparently promoted vesicle fusion. Surprisingly, Triton X‐100 alone cannot induce peptide vesicle fusion (Figure S4), and we suppose that a slight osmotic imbalance is necessary for successful fusion events. It was essential to add additional ELP monomers (200 μm) to the OS to facilitate vesicle growth through their incorporation into the membrane. We found that in the absence of external ELPs, the observed vesicles were generally smaller and less abundant. Furthermore, the vesicles were not stable during the experiments and tended to shrink.</p><p>a) Schematic illustration of vesicle fusion between vesicles containing complementary components of a transcription mix, which is used to start the synthesis of dBroccoli RNA aptamers in situ. b) Time series of LM images (overlay of bright field and fluorescence) of growing ELP vesicles. Scale bars: 40 μm. c) Typical time traces for vesicle radius (solid lines) and fluorescence intensity (dashed lines) of continuously growing vesicles (green) and growth followed by shrinkage (purple). d) Exemplary time traces for vesicle radius (solid) and fluorescence (dashed) of fusing vesicles (I–III) indicated in b). After the fusion of II and III, the combined (II+III) fuse with I.</p><p>The initial vesicle population was highly polydisperse in size, which likely resulted in large variations in the contents of the compartments generated via fusion of the two types of vesicles. This in turn was expected to result in a broad distribution of transcriptional activities among the vesicles. [22] As shown in Figure 3 b and Video S1, vesicles containing the transcription mix strongly varied in number and size over a time period of several hours. Next to a strong increase in size of the GVs, the appearance of small "satellite" vesicles around the GVs was observed. These are potentially generated by spontaneous budding events, [23] membrane instabilities followed by budding due to osmotic imbalances and Triton X‐100 or interactions with the microscopy glass slide. However, similar vesicles were observed to emerge throughout the whole micrograph, which suggests that they could also simply originate from growing SVs, whose size initially was below the observation limit.</p><p>The observed growth of the vesicles is consistent with our expectation that compartmentalized RNA polymerization is accompanied by an increasing osmotic pressure in the vesicles. [4] In the absence of bio‐polymerization reactions no vesicle growth was observed, even with externally provided ELPs (Figure S4, Video S2). Figure 3 c shows example time traces for growing vesicles and for growth followed by shrinkage when RNA aptamers are transcribed inside a vesicle. When in close proximity, two or more GVs can also fuse and thereby rapidly increase in size. We find that in all fusion events, the final intensities, radii and volumes are slightly less than expected from the sum of the fusing vesicles (Figures 3 d, S8, Video S3), implying that some of the vesicle content leaks out during fusion events. The excess membrane resulting from fusion may either be lost to solution, or incorporated into a multilamellar membrane structure.</p><p>In order to understand the dynamics of vesicle growth and shrinkage, we have to consider the interplay of compartmentalized RNA polymerization, the incorporation of externally provided ELPs into the membrane and water influx. As shown in Figure 4 a, fluorescence intensities and volumes are almost linearly correlated, which indicates that the concentration of the transcribed RNA molecules in the vesicles stays approximately constant. This in turn suggests that water influx into the growing vesicles is fast enough to compensate for the excess osmotic pressure generated by the newly formed polyelectrolytes.[ 4 , 24 ] As can be seen in the inset of Figure 4 a, the global linear trend is occasionally interrupted by phases of alternating growth and shrinkage, where the correlation between intensity and volume becomes non‐linear.</p><p>a) Representative plots of total vesicle fluorescence intensity vs. vesicle volume over a time course of 500 min. The different colors indicate different vesicles (N=20). The dashed line shows a linear fit to all 20 vesicles (see Supporting Information). Inset: intensity vs. volume of two exemplary vesicles showing growth, shrinkage and growth one after another. b) Typical time traces for vesicle radius (solid lines) and fluorescence intensity (dashed lines) of a growing vesicle (purple) and a vesicle showing alternating growth and shrinking phases (green). For all curves a 4‐point moving average filter was used. Numbers 1–5 indicate the corresponding LM images. c) Time series of LM images (overlay of bright field and fluorescence). The green and purple frame indicate the corresponding trace in b) Please note that data analysis started after t=75 min due to ongoing sedimentation. The corresponding video S4.2 starts at t=0 min. Scale bars: 40 μm.</p><p>Our experimental observations further suggest that the availability of a supply of ELPs (as monomers, micelles or small vesicles) in their immediate vicinity determines whether vesicles will grow or shrink. Some vesicles are found to repeatedly grow and shrink, and finally even disappear (Figure S8, Videos S4.1 and S4.2). Figures 4 b,c show two representative examples for vesicles surrounded by either many or by a few smaller vesicles. It can be clearly seen that in both cases the smaller vesicles slowly disappear whereas the largest vesicle continually grows. We suppose that the large sizes of the central vesicles are reached by the consumption of the surrounding "prey" vesicles. As soon as the supply of prey vesicles is depleted, the central vesicle starts to shrink (Figure 4 b, green).</p><p>In another set of experiments, we encapsulated a bacterial cell extract‐based protein expression system (TX‐TL) mixed with sucrose solution and a plasmid encoding the yellow fluorescent protein YPet into the GVs (Supporting Information section 1.2). The OS contained glucose, ELPs, and kanamycin to inhibit translation outside of the GVs by potentially present non‐encapsulated TX‐TL components. The vesicle fluorescence (Figures 5 a,b; S11) and the corresponding Video S5 show a similar behavior as for vesicles containing only a transcription reaction (Figures 3 c,b; S8, S11). In contrast to the TX mixture, TX‐TL had to be prepared already before encapsulation, resulting in YPet synthesis prior to the measurement. Bulk measurements in a fluorescent plate reader using the same TX‐TL protocol show an increase in fluorescence after roughly 20 min (Figure S13), whereas the time delay between preparation and microscopic observation of the vesicles lasted up to 45 min.</p><p>a) LM images (overlay of bright field and fluorescence) of vesicles containing TX‐TL after 15 min (left) and 24 h (right). The fluorescence is produced by YPet. Scale bars: 50 μm. b) Typical radius vs. time traces of vesicles expressing YPet. c) Fluorescence intensity vs. volume of 77 vesicles expressing YPet over 3 h. The dashed line shows a linear fit to all 77 vesicles.</p><p>As in the TX case the correlation between YPet fluorescence and volume is approximately linear (Figure 5 c), which again suggests a balance between in vesiculo production of biopolymers and osmotically driven growth. In contrast to the TX mix, the much more complex cell extract contains nearly the complete proteome of BL21 rosetta E. coli cell, in which case the osmotic pressure of the vesicle will be influenced by a more complex network of biochemical reactions. Only very few cases of shrinking vesicles were observed, but most vesicles enter a plateau phase of constant volume and constant YPet fluorescence after completion of the TX‐TL reaction, which indicates that an osmotic equilibrium has been attained between the inner and the outer solution.</p><p>In conclusion we have demonstrated the generation of cell‐sized peptide vesicles, which upon encapsulation of cell‐free transcription and protein expression reactions exhibit volume changes over at least an order of magnitude in several cases. The size changes appear to be caused by a combination of fusion events and osmotically driven growth, when fed with membrane components from the outside. Vesicle growth promoted by internal bio‐polymerization reactions is thus much more pronounced than previously observed for other membrane systems, which may be related to the high permeability of the ELP membranes for water combined with their considerable mechanical stability.</p><p>It is conceivable that usage of more complex membrane compositions will facilitate the implementation of other cell‐like behaviors such as compartmental division and reproduction. In fact, we already observed occasional budding events in our experiments (Supporting Information, Figure S10, Video S6), which may be taken as precursors for such processes. As the growth of our peptide vesicles is coupled to internal gene expression activity, our results also may lay the ground for a competition between different peptide compartments based on the efficiency of the compartmentalized reactions.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Study of Ruthenium-Contamination Effect on Oxygen Reduction Activity of Platinum-based PEMFC and DMFC Cathode Catalyst

We outline a systematic experimental and theoretical study on the influence of ruthenium contamination on the oxygen reduction activity (ORR) of a Pt/C catalyst at potentials relevant to a polymer electrolyte fuel cell cathode. A commercial Pt/C catalyst was contaminated by different amounts of ruthenium, equivalent to 0.15-4 monolayers. The resulting ruthenium-contaminated Pt/C powders were characterized by Energy-Dispersive X-ray Spectroscopy (EDS), X-ray Photoelectron Spectroscopy (XPS) and Scanning Transmission Electron Microscopy (STEM) to verify ruthenium contamination. A rotating disk electrode (RDE) technique was used to study the influence of ruthenium on oxygen reduction kinetics. Density functional theory (DFT) calculations were performed to estimate the oxygen reduction activity of the platinum surface with increasing ruthenium coverage, simulating ruthenium-contaminated Pt/C. The binding energies of O and OH on the surfaces were used for activity estimations.It was found that the specific activity of the ORR at 0.85V vs RHE exhibited a pseudoexponential decay with increased ruthenium contamination, decreasing by ~45% already at 0.15 monolayer-equivalent contamination. The results of the DFT calculations were qualitatively in line with experimental findings, verifying the effect of O and OH binding energies and the oxophilic nature of ruthenium on ORR and the 2 ability of the chosen approach to predict the effect of ruthenium contamination on ORR on platinum.

study_of_ruthenium-contamination_effect_on_oxygen_reduction_activity_of_platinum-based_pemfc_and_dmf

Introduction<!>Catalyst synthesis<!>Electrochemical characterization<!>Physicochemical characterization<!>Theoretical methods and computational details<!>Modelling of Ru on Pt(111)<!>Modelling of O and OH on RunPt(111)<!>Binding energies, reaction energies and activity<!>Structure and composition analysis<!>Experimental study of ORR catalytic activity<!>Ruthenium coverage of platinum surface<!>O binding on Run/Pt(111) surface<!>OH binding on Run/Pt(111) surfaces<!>Binding energies for O and OH<!>Analysis of ruthenium effect on ORR<!>PEMFCs and DMFCs<!>Conclusions

<p>Hydrogen-fed Polymer Electrolyte Membrane Fuel Cells (PEMFCs) and their closely related Direct Methanol Fuel Cells (DMFCs) are considered to be promising energy generators for electric vehicles (EVs), backup or off-grid power and mobile electronic devices [1,2]. Despite intensive research over the last three decades, performance, durability and cost issues are still major obstacles to successful widespread commercialization of PEM-type FCs.</p><p>One of the reasons for cost challenges of PEMFCs and DMFCs is the fact that carbonsupported nano-size platinum (Pt/C) is used as the catalyst on the anode and cathode.</p><p>In reformate-based PEMFCs, the hydrogen stream to the anode contains CO produced by reforming or partial oxidation of hydrocarbons or alcohols and by a reverse-shift reaction of CO2 [3,4]. In DMFCs, direct oxidation of methanol on the anode to CO2 progresses with production of mainly CO as an intermediate. Even very low concentrations of CO (10ppm) in a reformate-based H2 poison Pt/C catalysts [5] by strongly adsorbing on the platinum surface, hence reducing the available electrochemically active surface area and seriously inhibiting catalysis of H2 oxidation.</p><p>Likewise, in DMFCs, CO (an intermediate of methanol oxidation) is adsorbed on platinum, preventing catalysis of methanol oxidation. To solve this CO-poisoning problem, platinum-ruthenium (PtRu) alloys are used as anode catalysts. The ruthenium component provides the oxygen-containing species needed to oxidize CO to CO2 and release the platinum surface for further fuel oxidation [6,7].</p><p>However, PtRu catalysts were found to be prone to preferential dissolution of ruthenium [8], especially in the presence of methanol [9]. Ruthenium dissolution from the anode catalyst results in a loss of ruthenium and changes to its Pt:Ru ratio. This change leads to reduced CO tolerance and methanol oxidation activity [10] of the catalyst (for reformate-based PEMFCs and DMFCs, respectively) that will translate to higher overpotentials of the anode [11].</p><p>Ruthenium ions leaving the anode enter the Nafion membrane and cross it to deposit on the platinum cathode catalyst. Ruthenium crossover and its deposition on the cathode in DMFCs were first reported by Piela et al. of the Los Alamos Group [12]. Their pioneering work showed, with the use of XRF and CO-stripping, the presence of ruthenium in the membrane and the cathode already after MEA humidification (the socalled current-less contamination) and after operating a DMFC under various operating conditions (the so-called current-assisted contamination). Cathode contamination resulted in a voltage drop of 25mV in an H2/air-operated fuel cell across the entire current density range. This voltage drop was ascribed to lowered catalytic activity of oxygen reduction on the Ru-contaminated cathode. Schoekel et al. reported ruthenium dissolution and deposition on the cathode already during fabrication of the MEA by decal transfer process [13]. Rapid ruthenium contamination of the cathode was recorded during early operation time (two hours) of a DMFC, and was attributed to dissolution of highly soluble ruthenium species in the anode catalyst (Johnson Matthey HiSPEC 12100). Following that, a slower contamination process was recorded, attributed to dissolution of less soluble ruthenium oxides or ruthenium from the platinum-ruthenium alloy phase.</p><p>The negative effect of ruthenium contamination on the catalytic activity of the ORR demonstrated by Piela et al. [12], is consistent with studies of ORR kinetics on ruthenium and PtRu surfaces. Anastasijević et al. [14] studied the ORR mechanism and kinetics on a ruthenium rod. Their results clearly showed that ruthenium has poor ORR activity at potentials relevant to the PEMFC cathode. A similar conclusion can be drawn from the studies of ORR on electrodeposited ruthenium on a gold disk by Metikoš-Huković et al. [15] and ruthenium nanoparticles by Cao et al. [16]. Stamenkovic et al. [17] demonstrated the poor ORR activity of the polycrystalline PtRu (1:1) alloy electrode in comparison to the polycrystalline platinum electrode. The negative impact of ruthenium presence on ORR catalytic activity can also be seen in the case of Pt/Ru nanoparticles with exposed ruthenium on the surface [18,19]. Gancs et al. have studied the effect of platinum contamination by ruthenium on ORR catalysis [20]. In their study, different concentrations of ruthenium ions were used to contaminate commercial Pt/C by spontaneous deposition of ruthenium. With the use of different concentrations of ruthenium ions, several degrees of Ru-contamination were produced, thus enabling the study of the effect of contamination degree on CV polarization curves and ORR kinetics. Continuous suppression of Hupd stripping peaks with increased Ru-contamination was recorded, as well as severe decrease in ORR kinetics as evidenced by RDE polarization curves and Tafel plots. Quite close Tafel slopes were recorded for clean and Ru-contaminated Pt/C (-122 mV/dec vs 113 mV/dec respectively), indicating an identical ORR mechanism (at least at low overpotentials) for which ruthenium contamination is not a factor. Interestingly, at ruthenium coverage of 0.18 monolayer (ML), Ru ORR kinetics reached a minimum value and a maximum overpotential of ~160 mV was recorded.</p><p>In this work we studied the influence of ruthenium contamination on ORR kinetics with the use of a commercial Pt/C catalyst (Johnson Matthey HiSPEC8000) that was Rucontaminated. Various and precisely known amounts of ruthenium were deposited on platinum by electroless deposition at 90°C with methanol as the reducing agent. This resulted in Pt/C catalysts contaminated with different coverage levels of ruthenium.</p><p>This deposition method was chosen to mimic the existing conditions in a DMFC cathode (approximated working temperature and presence of methanol which had crossed from the anode). The effect of ruthenium on ORR performance was measured by cyclic voltammetry with an RDE. We believe that this simple approach has allowed us to correlate between precisely known ruthenium contamination of Pt/C and its ORR kinetics behavior. To explain the effect of Ru contamination on the ORR activity, DFT simulations were performed for the adsorption of Ru atoms on the Pt(111) surface and their effect on the O and OH binding energies. We then followed the analysis performed by Nørksov et al. [21] to correlate the calculated binding energies with a model estimate for the ORR activity. We showed that the theoretically obtained ORR activity trends have good qualitative agreement with the experimental trend. The combined experimental and theoretical work leads to a deeper understanding of the effect of platinum contamination by a sub-monolayer to a few monolayers of ruthenium on ORR kinetics and potential losses in PEMFCs and DMFCs.</p><!><p>The Ru-contaminated catalysts were prepared by electroless deposition of ruthenium on commercial 50%Pt/C (HiSPEC8000, Johnson Matthey) with methanol as the reducing agent. For each catalyst, the total amount of deposited ruthenium was equivalent to 0.15-4 (0.15, 0.22, 0.6, 1, 2, 4) monolayers of ruthenium. The calculation of the required amount of ruthenium for each catalyst was made on the basis of the atomic radius of ruthenium and an approximation of spherical platinum nanoparticles with a surface area of 60 m 2 gPt -1 (manufacturer's data). The catalysts were named according to the amount of deposited ruthenium: 0.15ML Ru/Pt, 0.22ML Ru/Pt, 0.6ML Ru/Pt, 1ML Ru/Pt, 2ML Ru/Pt, 4ML Ru/Pt.</p><p>For the synthesis, 0.2 g of HiSPEC8000 was dispersed by vigorous magnetic stirring in an aqueous solution of 1M methanol at room temperature. The suspension was heated to ~90 °C while being refluxed. A desired amount of RuCl3•3H2O was dissolved in 10 mL of 0.4 M HCl solution and added to the suspension at a rate of 1 mL every 15 minutes while the suspension temperature was maintained at ~90 °C. On completing the addition of ruthenium solution, the mixture was refluxed for an additional 30 minutes and then cooled to room temperature. The catalytic powder was recovered by centrifugation, washed with DI water until no chloride ions could be detected and dried.</p><!><p>All electrochemical experiments were performed at a controlled temperature (25±1 °C) with the use of a custom-made three-compartment glass cell with an Ag/AgCl/3 M KCl reference electrode in a Luggin-capillary compartment and a platinum wire as a counter electrode. A 0.5 M H2SO4 solution was used as the electrolyte. All potentials are reported on the reversible-hydrogen-electrode (RHE) scale.</p><p>Measurements of the electrochemically active surface area (ECSA) of Ru/Pt/C were carried out by the Cuupd stripping method [22], described in detail in our previous publication [23]. The working electrode was a 1 cm×5 cm glassy-carbon rectangle. The catalytic ink consisted of 10 mg of catalyst powder, 5 %(w/w) Nafion solution, 7.5 mL DI H2O, 2.5 mL EtOH and XC72 that was added to obtain a concentration of 0.2-0.3 %(w/w) solids in the ink. This concentration range of solids allowed obtaining a stable ink. The Nafion volume was adjusted to be ~30 %(v/v) of the solids in the inks. The ink was dispersed for 60 minutes in an ultrasonic ice-water bath, with additional fiveminute dispersion by pulse sonication (also in an ice-water bath) with the use of a horn sonicator (Heilscher UP200st). Immediately after sonication, 10 μL of the catalytic ink was applied to the lower part of the working electrode.</p><p>Nitrogen (99.999% purity) was bubbled through a 0.5 M H2SO4 solution for 30 minutes before electrochemical experiments and then passed over the solution during the entire procedure. Prior to ECSA measurements, the working electrodes were conditioned in order to clean their surface. A common conditioning procedure of Pt and Pt/C catalysts consists of repetitive cycling in deaerated electrolyte over a potential range of 0-1.2/1.4 volts until a stable voltammogram is obtained [24]. Cycling of Ru/Pt/C catalysts above 750mV might lead to substantial ruthenium dissolution that would result in unintended surface modification of these catalysts [9,25]. Hence, to reduce the possibility of ruthenium dissolution, the conditioning procedure of Ru/Pt/C catalysts was limited to 0-750 mV potential range [26].</p><p>For ORR measurements, a 5mm-diameter glassy-carbon RDE (Pine Instruments, USA) with Ageo=0.196 cm 2 was used. The RDE was polished to a mirror finish with a 0.05 µm Al2O3 particle suspension on a moistened polishing Micro-Cloth (both from Buehler). The electrode was mounted on an interchangeable RDE holder connected to an electrode rotator (MSRX electrode rotator, Pine Instruments, USA).</p><p>The catalytic ink for ORR measurements consisted of catalyst powder, XC72 powder, 5 %(w/w) Nafion solution, DI H2O and IPA. The Nafion volume was adjusted to 30 %(v/v) of the solids in the inks. A catalytic loading of ~20 µgPGM cmgeo -2 on the RDE was used and the weight of catalyst powder, DI H2O and IPA volumes (~30 %(v/v) IPA [27]) were adjusted accordingly. As in the case of ECSA measurements, addition of XC72 to obtain a concentration of 0.2-0.3 %(w/w) solids in the ink enabled the preparation of a stable ink and also a uniform catalyst coating on the RDE. The ink was ultrasonically dispersed by the same procedure as the ink used for ECSA measurements, afterwards 10 µL of the ink was applied on the RDE.</p><p>Several studies have shown that a uniform catalytic film has a beneficial effect on the currents obtained during RDE ORR polarization [28,29]. We have examined different drying procedures of the ink droplet in order to obtain the most uniform film. In our laboratory environment a stationary drying procedure at room temperature with IPA environment consistently produced the most uniform catalytic films. Subsequently, this drying method was used during this research. Nitrogen (99.999% purity) was bubbled through a 0.5 M H2SO4 solution for 30 minutes before electrochemical experiments and then passed over the solution during conditioning and background measurement. iR drop between the working and reference electrodes was measured and consistently found to be ~4 Ω. Working electrodes with Ru/Pt/C were conditioned as mentioned above. Working electrodes with Pt/C were conditioned by cycling over a potential range of 0-1.2 V. For background measurements, the working electrodes were cycled for five cycles over 0-1V (for Ru/Pt/C) or 0.025-1.2 V potential range (for Pt/C) at 20 mV s -1 . Background measurements were also used to measure ECSA of Pt/C by the Hupd stripping method [30].</p><p>Before ORR measurements, the O2 (99.999% purity) was bubbled through the electrolyte for 30 minutes and then passed over the electrolyte during ORR polarization.</p><p>The RDE potential was cycled between 1 and 0 V at 20 mV s -1 while the RDE was rotated at 2500 rpm in the O2-saturated electrolyte. The current at 0.85 V during anodic sweep polarization, after mass transport, background and iR corrections, was taken as a measure of the ORR activity, 𝑗 𝑘 0.85 𝑉 [31].</p><!><p>Detailed procedures of EDS and XPS measurements were described previously [32,33].</p><p>The measurements were made at ten (EDS) and four (XPS) points, respectively, in each sample of homemade catalysts and the results showed no significant inhomogeneity.</p><p>The reported results are an average of the measurements.</p><p>Transmission Electron Microscope (TEM) imaging was performed with an FEI F20 Philips-Tecnai STEM operated at 200 kV. Samples were prepared by manually pressing the grid (200-mesh grid, EMS) against the sample powder. TEM images were used to construct particle-size distributions of the catalysts by measuring diameters of at least 80 individual particles with ImageJ software [34].</p><p>Elemental mapping was performed with the use of an FEI Titan 80-200 STEM equipped with a CS-probe corrector (CEOS GmbH). "Z-contrast" conditions were achieved with the use of a probe semiangle of 25 mrad and an inner collection angle of the detector of 68 mrad. During STEM-EDS elemental mapping, HAADF detector and Pt L and Ru L peaks were used. Samples were prepared by placing a drop of diluted sample on a 400mesh carbon-coated copper grid.</p><!><p>Our total energy calculations were carried out with the use of DFT simulations within the Perdew-Burke-Ernzerhof generalized gradient approximation (PBE-GGA) as exchange-correlation energy functional, and the all-electron projected augmented wave (PAW) [35,36] method as implemented in the Vienna Ab initio Simulation Package (VASP) code [37,38]. For the calculations, a plane wave cut-off energy of 500 eV and k-point grids of 10×10×10 and 6×6×1 for the bulk and slab surface cells, respectively, were used. Geometric relaxation was considered to be complete once the atomic forces on each atom were smaller than 0.02 eV Å -1 , and a total energy convergence of 10 −6 eV for the structural energy minimization was achieved.</p><p>For the bulk fcc Pt, the calculated equilibrium lattice constant is 3.968 Å, which is consistent with theoretical findings from the Aflow database [39] and other theoretical [40,41] and experimental [42,43] results. The calculated bulk Ru hcp parameters were a=2.721 Å and c=4.293 Å, also in agreement with the theoretical [38,44] and experimental [45] results.</p><!><p>The adsorption of Ru atoms was modelled with different coverage levels (of 0.11, 0.22, 0.44, 1ML, which correspond to 1, 2, 4 and 9 atoms in the surface unit cell) on Pt(111)</p><p>applying the repeated slab geometry model with a 3×3 surface unit cell and five layers in the slab separated by a vacuum region of about 25 Å.</p><p>A single Ru ad-atom was placed at the hollow and bridge sites over Pt(111) surface as they are considered to be the most favorable positions. Then, the adsorption of additional Ru atoms, such as 2, 4 and 9 Ru atoms, was modelled on the Pt(111) surface.</p><p>In order to take into account all possible Ru geometries, as a starting point for geometrical relaxation, the Ru atoms were placed as planar and three-dimensional clusters (that is, pyramid-like configurations for Ru4 and Ru9) and separately diffused atoms over the Pt(111) surface. Then, geometrical optimization of the initial structures was performed, allowing all atoms to move but freezing the two bottom atomic Pt layers. Finally, the lowest energy structures were selected according to the total energies of the optimized structures.</p><!><p>The initial structures for mO (m = 1, 2, 3, and 4) atoms on the Run/Pt(111) surfaces were built on the following basis: O atoms were placed at hollow, bridge and top sites over Ru and Pt atoms. Then the systems were allowed to relax, again freezing the two bottommost Pt layers. The initial structures for mOH (m = 1, 2, 3, and 4) species on the Run/Pt(111) surfaces were constructed on the basis of the optimized mO/Run/Pt(111) structures.</p><!><p>The following steps were applied for the calculations. First, the binding energies (Eb)</p><p>for O atoms on Run/Pt(111) surfaces were calculated as Eb=(E(mO/RunPt(111))-</p><p>, where the first, second and third terms are the total energies of the mO/Run/Pt(111), Run/Pt(111) and the O atoms in gas-phase, respectively. m is the number of O atoms in the system, the same definition for binding energy was applied also to OH (OH replacing O in all equations). For the estimation of ORR activity, we follow the approach by Nørksov et al. [21], for completeness we repeat the main principles of this approach here. The oxygen reduction reaction can be written as:</p><p>As described in [21], in the simplest way, it is possible to consider the following processes at the surface:</p><p>Here "*" implies the pure surface, and O* and OH* imply the surface with the adsorbed species. Following [21], we analyze the reaction energies for the reactions:</p><p>We assume that the hydrogen evolution reaction:</p><p>is in equilibrium for a potential 𝑈 0 = 0 relative to the standard hydrogen electrode. It is hence evident that the free energy change of reaction 6 is the minus of the change in reaction 4, and that the difference of the free energies of reactions 6 and 5 yields the free energy change of reaction 3. The reaction energies (∆EO and ∆EOH) for reactions 5 and 6 for 1 and 4 O and OH on all the surfaces were computed on the basis of the total energies of the species as:</p><p>In the case of m oxygen atoms (or OH species), we normalized Δ𝐸 𝑂 and Δ𝐸 𝑂𝐻 by m. In order to account for the effect of the surrounding water molecules in the environment, the VASPSol [46,47] solvation model for water was utilized.</p><p>The free energy difference was calculated as ∆G = ∆EO/∆EOH + ∆ZPE ̶ T∆S, where ∆EO/∆EOH is the reaction energy, ∆ZPE and ∆S are the changes in zero-point energies and in entropy, due to the reaction, respectively. The second and third terms in the expression are calculated by DFT and were taken by us from [21], where ∆ZPE -T∆S are 0.35eV for (OH* + ½H2) and 0.05eV for (O* + H2).</p><p>For a general potential 𝑈 0 , ∆Gx(U0) can be calculated as</p><p>A potential of 𝑈 0 = 1.23𝑉 is assumed for the reaction of Eq. 1 to be in equilibrium.</p><p>From the discussion above it is clear that Δ𝐺 2 describes the free-energy change in reaction 4, and Δ𝐺 1 describes the change in reaction 3, reaction 2 is partially described by Δ𝐺 0 . In addition, the activation barrier for O2 dissociation at the surface, Ea, was taken according to the universal relation found in [21,48]. This relation connects the reaction activation energy and the stability of the reaction intermediates, according to Ea = 1.8*∆EO ̶ ̶ 2.89 eV. While this relationship was established for pure surfaces, we have also extended it to our case.</p><p>The values of ∆G0(U0), ∆G2(U0), ∆G1(U0), and Ea can be used to calculate the different reaction-rate constants according to: 𝑘 𝑖 ∼ 𝑘 0 𝑒 − Δ𝐺 𝑖 𝐾 𝐵 𝑇 . We can assume that the slowest step determines the overall rate of the reaction. Hence, we [21] define the activity, A, [21,49] by the logarithm of the rate constants, as</p><p>A is proportional to the logarithm of the lowest reaction-rate constant.</p><!><p>Weight and atomic compositions based on SEM-EDS and XPS analyses (before and after sputtering) of Ru-contaminated catalysts are shown in Table I. Both SEM-EDS and XPS show an increasing at% of ruthenium as its experimentally planned monolayer number increases, from Ru4Pt96 (SEM-EDS) and Ru7Pt93 (XPS) for 0.15ML Ru/Pt to Ru56Pt44 (EDS) and Ru65Pt35 (XPS) for 4ML Ru/Pt. The only exception to this trend being 0.22ML Ru/Pt for which a lower at% of ruthenium than for 0.15ML Ru/Pt was detected by XPS analysis. We believe this anomaly is related to insufficient sensitivity of XPS at such low at% of ruthenium in both samples. All catalysts show higher at% of ruthenium on the surface compared to the situation after five minutes of sputtering (determined by XPS) and in the bulk (determined by SEM-EDS). The combined SEM-EDS and XPS results are an indication that ruthenium was deposited on platinum nanoparticles during the synthesis and not as separate nanoparticles.</p><p>Representative TEM images and size-distribution histograms (insets) of Rucontaminated catalysts, and HiSPEC8000 are presented in Figures S1(a-i) in Supplementary Material. The average particle sizes of Ru-contaminated catalysts are similar to HiSPEC8000 up to 1ML Ru/Pt (3.7-3.9 nm) and increasing substantially to 4.5-4.6 for 2ML Ru/Pt and 4ML Ru/Pt (Table I). Several Ru/Pt catalysts show increased particle agglomeration compared to HiSPEC8000. Suspecting that the agglomeration was caused by exposure to hot reflux during the synthesis, a suspension containing HiSPEC8000 and methanol but not ruthenium salt was refluxed for the same time as during ruthenium deposition. Indeed, the resulting powder (named HiSPEC8000_M)</p><p>shows significant agglomeration in TEM images (Figure S1(i) in Supplementary Material) that also manifests itself in ECSA measurements presented later. This expansion originates from ruthenium oxidation/reduction [50] and high pseudocapacitance of hydrous ruthenium oxides [51]. particle size and their agglomeration. It appears that 1ML Ru/Pt had a lower particle agglomeration that led to a higher ECSA value compared to other Ru/Pt catalysts with similar particle sizes. On the other hand, 0.6ML Ru/Pt had higher particle agglomeration that led to lower ECSA. ECSA values of 2ML and 4ML Ru/Pt are on the lower side compared to other Ru/Pt catalysts. This was to be expected in light of the higher amount of ruthenium contamination in these catalysts that led to higher particle sizes.</p><!><p>ORR polarization curves of the examined catalysts are shown in Figure 3 Assuming that the decrease in ORR activity is originating from the oxophilic nature of ruthenium that covers the platinum, we used DFT calculation of the binding energies of O and OH to estimate the ORR activity on model Run/Pt(111) surfaces that represent Ru-contaminated Pt/C catalysts. The results of the DFT studies and their correlation to experimental results will be presented in the next sections.</p><!><p>Initially, the surface energies of the clean Ru(0001) and Pt(111) surfaces were calculated. Our surface energy value for the Ru(0001) was found to be higher than that of the Pt(111) surface, that is 169 meV/A 2 and 95 meV/A 2 , respectively, which is close to experimental data [53] and in agreement with other theoretical calculations from the literature [54,55].</p><p>Next, the adsorption energy of Ru atoms at the clean Pt(111) was calculated. It was found that in the lowest-energy structure, ruthenium ad-atom binds to the hollow site on the Pt(111) surface, which corresponds to the ABCA Pt stacking. In addition, it was S2 and S3. It is known from the literature that there is formation of Ru monatomic layers at low coverages [56] and bilayer islands and three-dimensional clusters at higher levels of coverage [56][57]. Our surface cell was slightly too small to show the effect of multilayer formation and hence in our simulations, the ruthenium atoms typically tended to form a monolayer. A Bader charge analysis reveals that the Ru atoms tend to give some of their electrons to the Pt surface.</p><p>For Ru1/Pt(111) we found that the Ru atom had a Bader charge of +0.34e. For the Ru2/Pt(111), the charge was +0.29e per Ru atom (total of +0.57e), for Ru4/Pt(111), the charge was +0.21e per Ru atom (total of +0.85), and for full coverage of Ru9/Pt(111)</p><p>we found a charge of +0.11e per Ru atom (total of +1.01e for the Ru monolayer).</p><!><p>As found previously [58][59][60][61], and also supported by us, the O atom has a greater tendency to bind on hollow sites on both the pristine Pt(111) and Ru(0001) surfaces.</p><p>On the Run/Pt(111) surfaces it was found that O atoms generally tend to bind on the Ru atoms, which can be explained by the stronger binding of O atom on the Ru(0001) (with calculated binding energy, Eb, of -5.97 eV) compared with that of binding on the Pt(111) (calculated Eb of -4.25 eV). The lowest energy structures for mO on Run/Pt(111) surfaces are presented in Figure 4.</p><p>Since we examine the thermodynamic limit of lowest energy structures, the binding energy of a single oxygen atom will not be much affected by the Ru-atom coverage level, even if the coverage is one Ru atom per million Pt surface atoms, the oxygen would still tend to bind to the Ru atom. To fully account for an actual scenario, one needs to build a kinetic simulation which takes into account the adsorption on alternative surface sites. Here we use an alternative approach of saturating the surface with oxygen atoms. At some point, the next oxygen atom cannot bind to the Ru atom(s)</p><p>and binds instead to a Pt surface atom, hence showing Ru-coverage-dependent behavior.</p><p>On A Bader charge [62,63] analysis of mO on the Ru1/Pt(111) surface was performed. At</p><!><p>The binding of OH species to the Run/Pt(111) surfaces follows trends similar to that of the binding of O atoms, namely, OH species first bind to Ru atoms and occupy all possible sites on these atoms. However, in addition, OH can also form hydrogen bonds with either other OH or surface atoms. This can further stabilize some surface-adsorbed structures. The structures for the OH binding on the Run/Pt(111) are presented in Figure S4 in Supplementary Material.</p><!><p>The binding energies (Eb) for mO and mOH (m = 1-4) species on the clean Pt(111) and Ru(0001) surfaces, and on the Run/Pt(111) (n = 1,2,4,9) surfaces, are presented in Figure 5. For the pristine surfaces it was found that the Eb of a single O atom on Pt(111) is weaker than on Ru(0001): -4.25 eV and -5.97 eV, respectively, which is consistent with literature results [56,59,64].</p><p>For the Run/Pt(111) surfaces it is found that the oxygen binding energy, Eb, decreases with the number of adsorbed oxygen atoms. This trend is especially strong for the Ru1/Pt(111) (Eb decreases from -5.86 eV for a single oxygen to -4.87 eV with four oxygens) and Ru2/Pt(111) (from -6.21 to -5.10 eV) surfaces. This can be explained by the saturation of Ru sites which forces some of the oxygen atoms to be adsorbed on Pt atoms and not on the Ru atoms. This decrease in Eb still exists but is less prominent for the case of Ru9/Pt(111) (Eb changes from -6.35 to -5.92 eV), where all the O atoms are uniformly distributed above the full-coverage Ru monolayer. Here, and also on the clean Pt(111) and Ru(0001) surfaces, another mechanism, of electrostatic repulsion between the adsorbed oxygen atoms, can explain the smaller decrease in the binding energy.</p><p>The Eb for mO atoms on the Run/Pt(111) is stronger than on Pt(111) and closer to the Eb of Ru(0001). This finding can be related to the strain and ligand effects on the Run/Pt(111) systems, that play an important role in controlling the surface reactivity [65], and is in agreement with the experimentally known oxophilic nature of ruthenium (compared to platinum). For Ru4/Pt(111), the binding energy, Eb, for 4O atoms approaches that of Ru(0001) because all the oxygen atoms tend to adsorb on Ru sites. However, it is statistically possible that one or more of the O atoms can also bind to Pt atoms at a higher energy state. We therefore considered an additional higher energy structure, defined as Ru4/Pt(111)*, where one of the O atoms is adsorbed on the Pt surface and not on the Ru4 cluster.</p><p>The Eb for OH species shows a trend similar to that of the Eb of O atoms, except for the case of 4OH/Run/Pt(111), in which we found a stronger binding of 4OH relative to 3OH on most surfaces. This can be explained by the contribution of the hydrogen bonds between the hydrogen and Pt/Ru atoms on these surfaces. and 4O/OH (Figure 6 and Figure S6 in Supplementary Material) the addition of Ru atoms is followed by a decrease in Δ𝐸 𝑂 and Δ𝐸 𝑂𝐻 (i.e., increased binding of O and OH) that leads to a decrease in ORR activity. It is also evident from Figures S5 and S6, that the effect of Ru coverage on Δ𝐸 𝑂 , Δ𝐸 𝑂𝐻 and the estimated ORR activity is more pronounced for the case of 4O/OH. The reason for this is that a single O/OH will always have an available Ru site to adsorb on, while in the case of 4O/OH, the Ru sites become saturated at low Ru coverages.</p><!><p>The DFT calculations presented above clearly show that indeed the oxophilic nature of ruthenium (compared to platinum), that manifests itself in increased O and OH binding, is the root cause of the inferior ruthenium ORR activity at potentials relevant to PEMFC. According to the Sabatier principle, too-strong binding of O and OH on ruthenium (compared to platinum) reduces the possibility of O and OH hydrogenation that is needed in order to complete O2 reduction to H2O. Moreover, because of its oxophilic nature, ruthenium is oxidized at much lower potentials compared to platinum.</p><p>Hence, at PEM-cathode operating potentials ruthenium is oxidized, cannot adsorb O2 molecules and facilitate their reduction. Besides being effectively ORR-inactive, ruthenium deposited on platinum (as in the case of Ru-contaminated platinum studied here) masks three platinum atoms, preventing them from adsorbing O2 molecules and effectively reducing the available platinum sites (i.e., available surface area) for ORR. In Figure 7b it can be seen that the deposition of a 0. 7b). In order to answer this question, it is necessary to examine the mechanism of O2 reduction on a platinum surface and the preferred deposition of Ru sub-monolayers on Pt surfaces.</p><p>Although ORR is a multistep reaction with a quite complex mechanism that is still somewhat in debate [21,66,67], the breaking of an O−O bond (i.e., O2 dissociation) and the formation of O−H bonds [66] involving a four-electron process must occur in order to achieve a complete reduction of O2. Among multiple possible configurations for O2 adsorption on a platinum surface, adsorption on two adjusted platinum sites (the socalled bridge side-on) is generally favored for the promotion of O2 dissociation [68,69].</p><p>As mentioned above, ruthenium adsorbed on platinum masks three platinum sites, preventing those sites from adsorbing O2 in any configuration. We shall name this type of platinum deactivation a direct deactivation. Nine platinum sites surrounding the masked three platinum sites lose some of their potential neighbors for bridge side-on adsorption. This may lead to increased probability for O2 adsorption in a less favorable configuration for O2 dissociation and thus negatively affecting ORR kinetics. We shall name this type of deactivation an indirect deactivation. Hence, one ruthenium atom has the potential to deactivate to some degree, twelve atoms of platinum and not only three atoms. We believe this to be the reason for the massive decrease in the ORR activity for 0.15ML Ru/Pt.</p><p>We shall now turn our attention to the deposition of ruthenium sub-monolayers on platinum surfaces. As was mentioned previously, ruthenium deposition on platinum has a tendency to create monolayer clusters at low coverages and bilayer islands and threedimensional clusters at higher coverages (Volmer-Weber growth). It is likely that for 0.22 and 0.6 equivalent monolayers such islands will be formed, hence effectively reducing the number of platinum sites deactivated by each ruthenium atom and reducing the decrease rate in ORR activity with increased ruthenium deposition.</p><p>Additional reduction of deactivated platinum sites for each ruthenium atom (and subsequent reduction in ORR activity decrease rate) is expected as a result of the overlap between the deactivated platinum sites. It is reasonable to assume that such overlap will occur at sufficiently high ruthenium coverage and will grow as ruthenium coverage is increased until the ORR activity will reach a plateau value that is similar to the ORR activity of ruthenium. We believe that the proposal described above provides</p><!><p>Even though RDE ORR experiments cannot precisely predict an FC cathode polarization, they can be used to obtain a first-order approximation of it. Hence, the RDE ORR polarization data obtained during this research can be used to assess the overpotential penalty of an FC cathode originating from ruthenium contamination.</p><p>To assess the added overpotential, we shall look at Figure 8, which presents masstransport, background and iR-corrected Tafel plots for HiSPEC8000 and 0.15ML Ru/Pt. The latter was chosen because of the similarity of its ruthenium content to ruthenium contamination found by Piela et al. [12]. In order to approximate the overpotential, we compare the potentials of both RDEs at the same current densities that polarize the RDEs to typical potential ranges of DMFC and PEMFC cathodes during FC operation. The catalyst loadings on RDEs for both catalysts were similar, ~20 µgPGM cmgeo -2 , hence any overpotential for 0.15ML Ru/Pt can be associated with ruthenium contamination.</p><p>Looking at the potential range typical to operating DMFC cathode, ~0.8V [70,71,72,73], it can be seen that the approximated overpotential penalty (marked as ηRu at Figure 8) for 0.15ML Ru/Pt is roughly 45mV. Assuming a cell voltage of 0.45V during DMFC operation, this penalty will translate to roughly 10% decrease in power density. The overpotential penalty grows to roughly 75 mV over a potential range typical to an operating PEMFC cathode, ~0.65V. Taking into account the small overpotential of a PEMFC anode and assuming a cell voltage of 0.6V during operation, we can approximate the penalty in power density at slightly more than 10%.</p><!><p>In this work we studied the effect of ruthenium contamination on ORR on platinum, focusing on the implications on PEMFC and DMFC cathode-relevant potentials. To obtain our objective, a commercial 50%Pt/C catalyst was contaminated by precisely known amounts of ruthenium. The contamination range varied from relatively low contamination, equivalent to 0.15ML of ruthenium, to severe contamination, equivalent to 4ML of ruthenium.</p><p>The contaminated catalysts were examined with the use of physico-chemical methods to verify and quantify ruthenium contamination. It was found that ruthenium was deposited on the surface of platinum nanoparticles in a core-shell-like structure without creating separate ruthenium nanoparticles on the carbon support.</p><p>Hupd-stripping region analysis showed a gradual suppression of the platinumcharacteristic hydrogen region that was correlated with increased ruthenium coverage of the platinum. While Hupd-stripping peaks from (110) and (100) platinum planes could be seen for 0.15ML Ru/Pt, only small remnants of platinum-characteristic features could be seen for 1ML Ru/Pt, while higher ruthenium contamination showed ruthenium-characteristic features. RDE ORR polarization showed the negative effect of ruthenium on ORR at potentials relevant to the PEMFC/DMFC cathode, exhibiting a progressive shift of onset potentials and mixed-kinetics/mass-transport regions toward more negative potentials and decrease of ORR specific activity with increasing contamination of ruthenium.</p><p>However, in contrast to gradual changes in Hupd features, ORR specific activity showed a drastic ~45% decrease already for 0.15ML Ru/Pt and, in general, a pseudoexponential decay with increased ruthenium coverage.</p><p>With the use of our DFT studies and previously published experimental results [20], we showed that the negative effect of ruthenium on ORR could be attributed to the masking of platinum sites by adsorbed ruthenium atoms, as well as to the oxophilic nature of ruthenium, that was found to bind O and OH much more strongly than platinum, reducing its effectiveness in ORR catalysis. Potentially unfavorable configuration of O2 adsorption on platinum sites that immediately surround the ruthenium-masked platinum sites, the formation of bilayer islands and three-dimensional clusters and overlap between the deactivated platinum sites were proposed as a possible explanation for the exponential-like decay of ORR on ruthenium-contaminated catalysts.</p><p>The results of this research stress the negative impact of ruthenium dissolution from the anode and its crossover to the cathode in reformate-based PEMFCs and DMFCs. The dramatic reduction in ORR activity -almost 50% -and the subsequent reduction in power density that accompanied the smallest ruthenium contamination, emphasizes the need for development of PtRu catalysts with higher stability, Ru-pre-leaching procedures during catalyst/GDE/MEA preparation and control of anode potential during FC operation in order to avoid or at least to reduce the performance penalty caused by ruthenium crossover.</p>

ChemRxiv

Structure elucidation of uniformly 13C labeled small molecule natural products

Utilization of 2H, 13C, and 15N isotopically labeled proteins and peptides is now routine in biomolecular NMR investigations. The wide-spread availability of inexpensive, uniformly 13C enriched glucose now makes it possible to isolate uniformly 13C labeled natural products from microbial fermentation. We now wish to describe an approach for the rapid structural characterization of uniformly 13C labeled natural products that avoids the pitfalls of relying on parameters typically employed in biomolecular NMR studies.

structure_elucidation_of_uniformly_13c_labeled_small_molecule_natural_products

Introduction<!>1H\xe2\x80\x9313C HSQC: a cornerstone of structure elucidation<!>CT-HSQC and its limitations<!>Analysis of the limits of CT approach<!>Optimization of CT for a general case<!>1H decoupled 13C\xe2\x80\x9313C COSY<!>Universally optimized 1H\xe2\x80\x9313C CT-HSQC<!>Structure elucidation of enterocin<!>Enterocin fermentation and isolation<!>Conclusions

<p>Natural products from microbial and fungal fermentations are a rich source of bioactive molecules that can provide an effective entry point into the development of therapeutic agents for a wide variety of human and animal health issues.[1] Although the field of natural products isolation and structure elucidation is, in many respects, mature, it is still a generally time-consuming process that sometimes may lead to structural ambiguity. In the past decade, natural product-based drug discovery has fallen into disfavor with most major pharmaceutical research companies.[2] In this report, we present a streamlined approach to rapid structure characterization of natural products utilizing uniform (U) 13C labeling. This strategy has become feasible because of the increased availability and affordability of labeled substrates such as U-13C labeled glucose. Using labeled substrates in fermentation media produces metabolites highly enriched with 13C. With the 13C label in place, the backbone of virtually any natural product can be quickly established by standard and long-range optimized 1H-decoupled 13C–13C COSY experiments.[3–5] The 1H NMR chemical shifts can be subsequently assigned through the use of an F1-decoupled HSQC experiment. The constant time CT-HSQC described by Bax and coworkers[6] provides a method to achieve F1 decoupling in the HSQC experiments of uniformly 13C-labeled peptides and proteins. Herein, we expand the theoretical framework of the CT-HSQC approach to make it universally applicable to practically any U-13C labeled organic small molecule. The resulting structure elucidation strategy is demonstrated both theoretically and experimentally using the natural product enterocin,[7] 1, as an example. Practical limitations of the experimental approach will also be described in this report.</p><p> </p><!><p>Natural abundance 1H–13C HSQC experiments[8,9] have represented a cornerstone of small molecule structure elucidation protocols for more than 20 years. The experiment offers high sensitivity, straight-forward and robust setup, while providing a 1H/13C fingerprint of the molecule. Multiplicity-editing adds additional information content relating to the number of protons covalently attached to each carbon atom. Recently developed pure shift analogs of the HSQC experiment have served to further increase resolution and sensitivity.[10–12] In addition to 1H and 13C chemical shifts, HSQC spectra provide a wealth of structurally relevant information. In fact, there are successful examples of using just 1H and multiplicityedited 1H–13C HSQC data for automated structure verification purposes.[13]</p><p>Uniform 13C labeling of small molecules offers opportunities to access quaternary 13C chemical shifts, as well as 1H–13C and 13C–13C coupling constant information, the latter of which are not easily accessible at 13C natural abundance.[14] At the same time, U-13C labeling introduces additional JCC and JCH pathways during t1 evolution in the HSQC experiment, which are negligible at natural abundance. Evolution of JCC and JCH couplings affects sensitivity, lineshape, and the phase of HSQC cross-peaks and complicates data interpretation, and therefore is undesirable and to be avoided. The constant time (CT) approach was first introduced in 1992 to avoid JCH evolution and to minimize JCC evolution in 1H–13C HSQC of uniformly 13C-labeled proteins and peptides.[6] The success of CT-HSQC experiment is heavily reliant on the structural uniformity of amino acid residues, which equates to uniformity of the corresponding JCC coupling constants.</p><p>Applying the same approach to a much broader range of 13C–13C coupling constants and chemical shifts is non-trivial. Recently, a modified version of 1H–13C HSQC for U-13C labeled small molecules was published.[15] However, the modified experiment still focuses on the same narrow range of JCC couplings as the original CT publication and hence has limited general applicability. We believe that the issue can benefit from a thorough theoretical analysis of potential modulations introduced by JCC couplings, similar to the analysis performed for the ADEQUATE experiments.[16]</p><!><p>The constant time (CT) pulse sequence element and an example of an HSQC pulse sequence are shown in Fig. 1. Constant time pulse sequence elements simplify, but do not remove, JCC evolution. Evolution caused by JCC introduces a modulation to the magnetization that is a cosine function of the JCC coupling constant and CT value: (1)M(J)=cos(π×AJCC×CT)×cos(π×BJCC×CT)×cos(π×CJCC×CT).</p><p>Here, the terms AJCC, BJCC, and CJCC denote one-bond 13C–13C homonuclear coupling constants between the carbon of interest and up to three neighboring carbons, A, B, and C. Long-range nJCC couplings can also contribute to the modulation significantly in some cases because some nJCC couplings can be rather large, ranging up to >16 Hz.[17]</p><p>In the case of amino acids and, consequently, peptides and proteins, the mathematical description of the CT-HSQC experiment can be significantly simplified because of structural uniformity, which translates into uniformity of the corresponding JCC coupling constants. All aliphatic 1JCC values will be within a 32–40 Hz range, and long-range nJCC couplings are small enough in amino acids to consider them negligible. One-bond and long-range couplings to the carbonyls of the peptide backbone are selectively suppressed, which is possible because of narrow and predictable range of their 13C chemical shifts.[6] The simplified formula uses average aliphatic 1JCC value and a number of covalently bound carbon neighbors, N: (2)M(J)≈cosN(π×Jav×CT).</p><p>Choosing CT to match 1/Jav (26.6 ms) allows multiplicity-editing based on the number of covalent carbon neighbors. CT matched to 2/Jav (53.2 ms) leads to correlation responses with the same phase.[6] Long-range nJCC are again considered negligible with a corresponding cosine term close to 1.</p><p>One can easily see that while this approach is powerful, provided that the assumption regarding JCC is valid, the method quickly fails, becoming unusable in the more general case of an organic small molecule. Small molecule 1JCC values typically are in 25–70 Hz range,[18,19] with known outliers as low as 6–15 Hz for cyclopropyl and 15–20 Hz for cyclobutyl carbons and as high as 85–95 Hz for protonated acetylenic carbons.[19,20] Similarly, long-range nJCC constants are also more highly variable and can range to as large as 16 Hz,[17] thus contributing significantly to the modulation of magnetization. As an example, at an optimization of 53.2 ms for the constant time period as in the original publication,[6] a long-range coupling of 9 Hz will essentially null the modulation function, thereby eliminating an HSQC correlation.</p><!><p>In the general of case of an organic molecule there are four possible situations: 0, 1, 2, and 3 carbon neighbors. We will provide a mathematical treatise for every case. The effect of nJCC couplings will be considered later.</p><p>N= 0: No JCC couplings.</p><p> (3) M 0 ( J ) ≡ 1 . </p><p>N= 1: One JCC coupling.</p><p> (4) M 1 ( J ) = cos ( π × J CC × CT ) . </p><p>N= 2: Two JCC couplings.</p><p> (5a) M 2 ( J ) = cos ( π × A J CC × CT ) × cos ( π × B J CC × CT ) . </p><p>Applying a trigonometric transformation cos(x) × cos(y) = 0.5 × [cos(x + y) + cos(x − y)] transforms (5a) from the multiplicative to the additive function: (5b)M2(J)=0.5×[cos(π×CT×(AJCC+BJCC))+cos(π×CT×(AJCC−BJCC))].</p><p>The resulting arguments are now (AJCC + BJCC) and (AJCC − BJCC) instead of AJCC and BJCC.</p><p>It is now easy to see that within the range of one-bond carbon– carbon couplings typically encountered with small organic molecules the function M2(J) can adopt any value between −1 and +1, including null. Moreover, even small changes in the 1JCC value may dramatically impact the result and thereby response intensity. For example, consider a CT value of 53.2 ms used in the original publication[6] (matched to the average aliphatic 1JCC coupling for proteins and peptides): (6)IfAJCC=50HzandBJCC=38Hz→M2(J)=−0.5, (7)IfAJCC=47HzandBJCC=38Hz→M2(J)=−0.0, (8)IfAJCC=44HzandBJCC=38Hz→M2(J)=+0.5.</p><p>Considering that 38, 44, 47, and 50 Hz are all within the typical range of 1JCC coupling constants for organic molecules, one can easily see how an otherwise minor 3 Hz difference can cause dramatic variations in both sign and relative intensity of cross-peaks in a CT-HSQC spectrum. This hypothetical example clearly illustrates challenges of using CT-HSQC for a uniformly 13C-labeled organic molecule such as a natural product.</p><p>N= 4: Three JCC couplings.</p><p> (9a) M 3 ( J ) = cos ( π × A J CC × CT ) × cos ( π × B J CC × CT ) × cos ( π × C J CC × CT ) . </p><p>Equation (9a) can be simplified considering that the only case when a protonated carbon can have three other covalently bound carbons if it is an sp3 carbon. In this case, direct carbon–carbon 1JCC couplings will be confined to a relatively narrow range, and original CT assumption can still be used here: (9b)M3(J)=cos3(π×JCC×CT).</p><p>Graphical representation of M1(J) and M3(J) are shown in Fig. 2, and M2(J) behavior is shown in Fig. 3. For all cases CT is set to 53.2 ms as in the original publication.[6] All functions have intensity and sign variations that are dependent on 1JCC coupling constants.</p><p>Zero crossings are especially undesirable because it means that certain coupling constants will result in null magnetization, effectively erasing the corresponding HSQC cross-peak.</p><!><p>It is clear from the analysis of Eqns (3–9) that there is no 'ideal' setting of CT that will adequately cover the typical range of one-bond carbon–carbon couplings in organic molecules without modulation of the function crossing zero (often more than once) within this range. Figure 4 provides a visual aid showing the M2(J) function at varied CT values.</p><p>As a first approach to identifying a 'universal' CT setting, one could try to find a compromise CT value that would be sufficient for most but not all possible one-bond couplings, similar to the approach utilized for the inverted 1JCC 1, n-ADEQUATE experiment.[21] Using similar methodology, we calculated that the CT value of 20 ms would represent such compromise. With this optimization, M2(J) will cross zero at 25 Hz and 75 Hz (see Fig. 5), which may be considered acceptable because 25 and 75 Hz are on the edges of the typical range of one-bond couplings encountered for most natural products.[18–20] However, further analysis of M3(J) behavior shows that choosing a 20-ms optimization for CT is a rather poor choice in terms of a universal optimization. With 20-ms CT optimization, 1JCC coupling constants in the range from 19 to 31 Hz (as well as from 69 to 81 Hz) will result in response intensity below 5% of maximum possible value, which for practical purposes can be considered null (see Fig. 5). Having 1JCC coupling constants ranges of 19–31 Hz and 69–81 Hz as null regions is very undesirable because they overlap with the typical range of one-bond 13C–13C couplings in organic small molecules and natural products. Large long-range 13C–13C couplings would also attenuate down the signal intensity, although less drastically. With the optimization of CT for 20 ms, the intensity loss because of long-range carbon– carbon couplings, nJCC, of 16, 10, and 7 Hz is calculated to be 40%, 20%, and 10%, respectively. Of course an investigator could still use a 20-ms optimization for the CT parameter if no 1JCC couplings are smaller than 32 Hz or larger than 68 Hz are expected in the molecule, but it is definitely not universal for organic small molecules and natural products.</p><p>A closer look at the formula (5b) makes it clear that in order to decouple JCC evolution in a truly uniform fashion, one has to satisfy all possible ranges of (AJCC + BJCC) and (AJCC − BJCC). The only way to accomplish this goal is by creating a very slow cosine wave, which is achieved with a 5 ms optimization of the CT interval (see Fig. 6). This setting represents truly universal optimization: in a coupling range of 0–96 Hz it yields both constant phase and reasonable signal intensity (zero crossing is at 100Hz). Additionally, this optimization is very insensitive to smaller nJCC couplings: even the largest nJCC of 16 Hz would cause an intensity loss of less than 3%.</p><p>Optimization of the CT interval for a universal value of 5 ms has one major disadvantage: a short constant time interval limits t1, which in turn limits the resolution of the indirectly determined frequency domain F1 to 200 Hz. Linear prediction during processing can somewhat improve F1 digital resolution. However, this disadvantage does not affect the structure elucidation protocol for 13C-labeled molecules, because high resolution 13C data are available via 1D 13C and 2D 13C–13C COSY spectra. These points will be further considered below in the results and discussion section.</p><!><p>The first step in the carbon backbone determination of a uniformly 13C labeled natural product is to acquire a 1H decoupled 13C–13C COSY spectrum.[4] These data quickly provide information on the carbon–carbon connectivity of all neighboring carbon atoms. Because of the high incorporation of 13C label, these experiments can be acquired in a short experiment time as shown below using a 7.8-mg sample of enterocin (1). One-bond carbon–carbon correlations afford partial structures, which can be successively linked together through heteroatoms utilizing long-range (nJCC, n = 2,3) 13C–13C correlations via a 13C–13C COSYLR[5] experiment. In the case of 13C–13C COSY, the choice of the t1 evolution time governs the size of the 13C–13C coupling constant leading to the observation of a cross-peak. As the t1 interval becomes longer, progressively smaller 13C–13C coupling constants will afford correlations. For a given spectral width, the t1 evolution time can be increased by either increasing the number of F1 increments or by using COSYLR with an explicitly specified t1 evolution delay. Both of these approaches provide comparable data, but COSYLR proved to be more practical because it allows one to set any t1 evolution interval while keeping the number of F1 increments small and governed only by needed resolution in F1 indirectly acquired dimension.</p><p>Figure 7 shows the 1H decoupled 13C–13C COSY spectra optimized for one-bond and for long-range 13C–13C couplings. The spectra were acquired in 35 min and in 4 h and 45 min, respectively using a Bruker 600-MHz AVANCE III triple resonance spectrometer equipped with 1.7-mm MicroCryoProbe.™</p><!><p>Structure elucidation paradigms for U-13C labeled natural products generally utilize a 1H-decoupled 13C–13C COSY as the primary structural tool, while CT-HSQC data are employed in a supplementary role to establish 1H–13C direct correlations. Figure 8 shows CT-HSQC spectra acquired with CT intervals of 5 and 26.6 ms acquired on a 7.8 mg sample of U-13C labeled enterocin. The acquisition times were 16 and 85 min, respectively, using a Bruker 600-MHz spectrometer equipped with a 1.7-mm MicroCryoProbe.™</p><p>One can easily appreciate the robustness of the universally optimized CT-HSQC spectrum acquired with CT optimized to 5 ms: no correlations are missing, phase characteristics are good, and there are only small variations in relative peak intensities. Other CT settings are noticeably less reliable. For example, a CT-HSQC spectrum acquired with 7.5 ms optimization has some peaks missing and other peaks exhibit severe phase distortions (see Figure S1 in the Supplementary Information). The CT optimization of 26.6 ms (default value for proteins and peptides[6]) results in a spectrum with multiple missing correlations and with inverted phase for other responses. Experimental results in these three spectra confirm the theoretical considerations developed above. All missing or inverted responses are in quantitative agreement with nulls and inversions of the response intensity governed by the Eqn (1) (see Figures S1–S4 in the Supplementary Information).</p><!><p>A 'rough' carbon skeleton of the enterocin molecule was assembled using 1H decoupled 13C–13C COSY optimized for large couplings (see Figs. 7 and 9). This skeleton consisted of several substructures that are formed whenever a contiguous carbon– carbon chain is interrupted by a heteroatom (Fig. 9). The resultant substructures were connected together using long-range 1H decoupled 13C–13C COSYLR data and 1H–13C connectivities from the optimized CT-HSQC spectrum (see Figs. 8 and 9). The total acquisition time for all necessary experiments (short-range 1{H} 13C–13C COSY, long-range 1{H} 13C–13C COSYLR, CT-HSQC, 1D 1H, and 1D 13C) was 5 h 35 m for a 7.8 mg sample of U-13C labeled enterocin, with 13C–13C COSYLR requiring 4 h 45 m, which represents 85% of total acquisition time.</p><!><p>The ascidian, Didemnum psammathode, was collected in October, 2010, in the Florida Keys (24° 37.487′, 81° 27.443′). For cultivation, a sample of ascidian (1 cm3) was rinsed with sterile seawater, macerated using a sterile pestle in a micro-centrifuge tube, and dilutions were made in sterile seawater, with vortexing between steps to separate bacteria from heavier tissues. Dilutions were separately plated on three media: ISP2, R2A, and M4. Each medium was supplemented with 50 μg/ml cycloheximide and 25 μg/ml nalidixic acid. Plates were incubated at 28 °C for at least 28 days. Strain WMMB285 was identified as a Streptomyces sp. based on the 16S rDNA sequence (GenBank JX960758.1).</p><p>A 10 mL seed culture (25 × 150 mm tubes) in medium ASW-A (20-g soluble starch, 10 g glucose, 5 g peptone, 5 g yeast extract, 5 g CaCO3 per liter of artificial seawater) was inoculated with strain WMMB285 and shaken (200 RPM, 28 °C) for seven days. The seed culture was used to inoculate 100 mL of 13C-enriched ASW-A media (10 g U-13C-glucose/L) containing Diaion HP20 (4% by weight). After 7 days, filtered HP20 and cells were washed with water and extracted with acetone. The acetone extract was subjected to liquid–liquid partitioning using 30% aqueous methanol and chloroform (1:1). The chloroform partition was fractionated by size exclusion chromatography applying Sephadex® LH-20 as stationary phase and a solvent mixture of chloroform:methanol 50:50 as mobile phase. Five fractions were obtained, and the fraction containing enterocin was purified by HPLC.</p><p>A Shimadzu LC system was used, equipped with a LC-20AT pump, SPD-M20A diode array detector, a SIL-20AC HT auto sampler, and a FRC-10A fraction collector. The LC time program consisted on a gradient of methanol 15%/water 85% to methanol 60%/water 40% in 8 min, then increased from methanol 60%/water 40% to methanol 100% in 1 min, and held for 3 min. The column used was a Phenomenex Onyx monolithic column, 100 × 4.6 mm. The flow rate used was 3 mL/min. Deoxyenterocin eluted at 2.45 min and was collected in fractions 3 to 7, while enterocin, eluted at 3.70 min and was collected in fractions 9 to 11.</p><!><p>We have demonstrated a rapid and efficient structure elucidation protocol for U-13C labeled natural products. The protocol we propose consists of 1H decoupled 13C–13C COSY optimized for shortand long-range couplings and CT-HSQC optimized for use with natural products and organic small molecules. We developed a theoretical framework and have demonstrated the experimental verification of the techniques for expanding the scope of CT-HSQC experiment from narrow amino acid applications[6] to a protocol that is universally applicable for labeled natural products and organic small molecules. The only trade-off of the universally optimized CT-HSQC is lower F1 resolution, which, should not be a major impediment to the use of the protocol for structure elucidation because of the availability of high resolution 13C information from 1D 13C and 2D 13C–13C COSY data.</p>

PubMed Author Manuscript

Discovery of C-3 Tethered 2-oxo-benzo[1,4]oxazines as Potent Antioxidants: Bio-Inspired Based Design, Synthesis, Biological Evaluation, Cytotoxic, and in Silico Molecular Docking Studies

The discovery of C-3 tethered 2-oxo-benzo[1,4]oxazines as potent antioxidants is disclosed. All the analogs 20a-20ab have been synthesized via “on water” ultrasound-assisted irradiation conditions in excellent yields (upto 98%). All the compounds have been evaluated for their in vitro antioxidant activities using DPPH free radical scavenging assay as well as FRAP assay. The result showed promising antioxidant activities having IC50 values in the range of 4.74 ± 0.08 to 92.20 ± 1.54 μg/mL taking ascorbic acid (IC50 = 4.57 μg/mL) as standard reference. In this study, compounds 20b and 20t, the most active compound of the series, showed IC50 values of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, respectively in comparison with ascorbic acid. In addition, the detailed SAR study shows that electron-withdrawing group increases antioxidant activity and vice versa. Furthermore, in the FRAP assay, eight compounds (20c, 20j, 20m, 20n, 20r, 20u, 20z, and 20aa) were found more potent than standard reference BHT (C0.5FRAP = 546.0 ± 13.6 μM). The preliminary cytotoxic study reveals the non-toxic nature of active compounds 20b and 20t in non-cancerous 3T3 fibroblast cell lines in MTT assay up to 250 μg/mL concentration. The results were validated via carrying out in silico molecular docking studies of promising compounds 20a, 20b, and 20t in comparison with standard reference. To the best of our knowledge, this is the first detailed study of C-3 tethered 2-oxo-benzo[1,4]oxazines as potential antioxidant agents.

discovery_of_c-3_tethered_2-oxo-benzo[1,4]oxazines_as_potent_antioxidants:_bio-inspired_based_design

Introduction<!><!>Introduction<!><!>Introduction<!>General experimental<!>General procedure for the synthesis of functionalized diketo-acid 18a-h<!>General procedure for the synthesis of functionalized (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20a-ab)<!>(Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20a)<!>(Z)-3-[2-(4-methoxy-phenyl)-2-oxo-ethylidene]-3,4-dihydro-benzo [1, 4] oxazin-2-one (20b)<!>(Z)-6-chloro-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20c)<!>(Z)-6-chloro-3-(2-(4-fluorophenyl)-2-oxoethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20d)<!>(Z)-6-chloro-3-(2-(4-chlorophenyl)-2-oxoethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20e)<!>(Z)-6-chloro-3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)- 3,4-dihydro-2H-benzo[b][1,4]oxazine-2-One (20f)<!>(Z)-3-(2-(4-bromophenyl)-2-oxoethylidene)-6-chloro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20g)<!>(Z)-6-chloro-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20h)<!>(Z)-6-chloro-3-(2-(4-methoxyphenyl)-2-oxoethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20i)<!>(Z)-3-(2-(4-fluorophenyl)-2-oxoethylidene)-6-methyl-3,4-dihydro-2H-benzo[1,4]oxazin-2-one (20j)<!>(Z)-3-[2-(2,4-dichloro-phenyl)-2-oxo-ethylidene]-6-methyl-3,4-dihydro-benzo[1,4]oxazin-2-one (20k)<!>(Z)-3-[2-(4-methoxy-phenyl)-2-oxo-ethylidene]-6-methyl-3,4-dihydro-benzo[1,4]oxazin-2-one (20l)<!>(Z)-8-bromo-6-methyl-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20m)<!>(Z)-8-bromo-3-(2-(4-fluorophenyl)-2-oxoethylidene)-6-methyl-3,4-dihydro-2H-benzo[b][1,4] oxazin-2-one (20n)<!>(Z)-8-bromo-3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)-6-methyl-3,4-dihydro-2H-benzo [b][1,4]oxazin-2-one (20o)<!>(Z)-8-bromo-3-(2-(4-bromophenyl)-2-oxoethylidene)-6-methyl-3,4-dihydro-2H-benzo[b] [1,4]oxazin-2-one (20p)<!>(Z)-8-bromo-6-methyl-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20q)<!>(Z)-6-methyl-3-(2-(4-nitrophenyl)-2-oxoethylidene)-3,4-dihydro-2H-benzo[1,4]oxazin-2-one (20r)<!>(Z)-3-(2-(4-nitrophenyl)-2-oxoethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20s)<!>(Z)-6-nitro-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20t)<!>(Z)-3-(2-(4-fluorophenyl)-2-oxoethylidene)-6-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20u)<!>(Z)-3-(2-(4-methoxyphenyl)-2-oxoethylidene)-6-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20v)<!>(Z)-7-nitro-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20w)<!>(Z)-3-(2-(4-fluorophenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20x)<!>(Z)-3-(2-(4-chlorophenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20y)<!>(Z)-3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20z)<!>(Z)-3-(2-(4-bromophenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one(20aa)<!>(Z)-3-(2-(4-methoxyphenyl)-2-oxoethylidene)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (20ab)<!>DPPH radical scavenging antioxidant assay<!>Ferric reducing antioxidant power (FRAP) assay<!>Cell toxicity assay<!>In silico molecular docking studies<!>Chemistry<!><!>Chemistry<!><!>DPPH radical scavenging antioxidant activity and SAR studies<!><!>DPPH radical scavenging antioxidant activity and SAR studies<!><!>Ferric reducing antioxidant power (FRAP) activity and SAR studies<!>Cell toxicity study<!><!>In silico molecular docking simulation studies<!><!>In silico molecular docking simulation studies<!>Conclusion<!>Author contributions<!>Conflict of interest statement

<p>"Antioxidant" are primarily reducing agents/compounds which refer to the activity of numerous vitamins, minerals and phytochemicals (such as vitamin E, vitamin C and glutathione etc.) by providing protection against the damage caused by reactive oxygen species (ROS) (Park and Pezzutto, 2002; Trombino et al., 2004; Govindarajan et al., 2005; Zhang et al., 2006). Antioxidants (either natural or synthetic) are molecules, which are capable of neutralizing free radicals as well as ROS by acting at several levels such as: prevention, interception, and repair (Lehtinen and Bonni, 2006; Khan et al., 2011; Bayoumi and Elsayed, 2012). Thus, the search for antioxidants has been stimulated due to their significant importance in human health (Balakin et al., 2004; Mitra et al., 2009). Moreover, it is known that ROS like superoxides (O22-), peroxyls (ROO−), hydroxyls (HO−), alkoxyls (RO−), nitric oxides (NO−), play a important role in disturbing metabolic pathways associated with several pathological conditions, such as cardiovascular diseases, metabolic disorders, and even carcinogenesis (Lai et al., 2001; Cheng et al., 2011). Therefore, the human body is capable to neutralize ROS by antioxidant defense mechanisms by eradicating an excess of ROS from the cell (Apel and Hirt, 2004; Zhang et al., 2010; Mittal et al., 2014). An imbalances between the detoxification of ROS with respect to their production leads to a phenomena known as "oxidative stress (OS)" which is correlated to several diseases such as stroke (Simao et al., 2015), myocardial infarction (Hassan et al., 2015), cancer (Aldawsari et al., 2016), Parkinson's disease (Wood-Kaczmar et al., 2006) and Alzheimer's disease (Nunomura et al., 2006). Therefore, the development of natural as well as synthetic antioxidants, which are able to scavenge ROS and keep cell integrity via prevention or reduction of the impact of OS on cells, is now currently an recognized area of research interest.</p><p>During the last decade, benzoxazines, benzodioxine, and its derivatives have emerged as a possible antioxidants (Largeron et al., 1999, 2001; Czompa et al., 2000; Sadiq et al., 2015). Several naturally occurring antioxidants (Abdel-lateif et al., 2016; Aziz and Karboune, 2016) such as dimboa 1 (Niemeyer, 2009; Adhikari et al., 2013; Glenska et al., 2015) and sylbin 2 (Kosina et al., 2002; Varga et al., 2006; Surai, 2015; Vavríkova et al., 2017) have been identified as promising antioxidant agents. Likewise, several synthetic molecules bearing benzoxazines as whole or as part in their structure, have also been identified as potential antioxidant agents such as exifone 3 (Largeron et al., 1995; Largeron and Fleury, 1998), isatoic anhydrides i.e., benzoxazine-2,4-diones 4 (Sáncheza et al., 2014), 2-hydroxy-1,4-benzoxazin-3(4H)-one 5 (Harput et al., 2011), and some analogs 6a-b (Largeron et al., 1999) etc. as shown in Figure 1. This encourages us to synthesize non-naturally occurring benzo [1,4] oxazines analogs.</p><!><p>Structures of some natural as well as synthetic compounds (1-6) having antioxidant activity.</p><!><p>Moreover, several natural products like Curcumine 7 (Barclay and Vinqvist, 2000), Quinolines 8 (Detsi et al., 2007; Savegnago et al., 2013; Oliveri et al., 2015), Chalcones 9 (Qian et al., 2011; Shakil et al., 2013; El Sayed Aly et al., 2014), Resveratrol 10 (Scartezzini and Speroni, 2000), Rosmarinic acid 11 (Fadel et al., 2011; Zhu et al., 2014), Trolox 12 (Hall et al., 2010a), Coumarins-chalcone hybrid 13 (PérezCruz et al., 2013; Mazzone et al., 2016), and Quercetin 14 (Kumar et al., 2007) etc. were also reported as antioxidants. However, due to several drawbacks such as poor solubility, less abundance and severe toxicity; their antioxidant properties were found to be relatively lower. Thus, there is still an urgent need to develop a potent antioxidant by designing a new scaffold via structural modification and incorporation of functional group present in these antioxidants. Hence, based on above fact, we have designed prototype 15 i.e., C-3 tethered 2-oxo-benzo[1,4]oxazine, incorporating similar sub-structural units assuming that the resulting structure will be a new class of potent antioxidant agent (Figure 2).</p><!><p>Design strategy for the target compound 2-oxo-benzo[1,4]oxazine 15 as an antioxidants.</p><!><p>In the continuation toward the search of new class of antioxidants; we were interested to explore the designed prototype 15. Therefore herein, we report the synthesis via our methodology (Jaiswal et al., 2017), antioxidant activity, and SAR of a series of C-3 tethered 2-oxo-benzo[1,4]oxazine analogs 20a-20ab. Although compounds 20a-i, 20l, 20t-w, 20y, and 20aa-ab have been earlier reported in the literature but were prepared by other routes (Iwanami et al., 1971; Mashevskaya et al., 2002; Gein et al., 2008; Xia, 2008; Xia et al., 2008; Stepanova et al., 2011, 2013; Maslivets and Maslivets, 2012). Moreover, their antioxidant activities are also not reported so far in the literature. To the best of our knowledge, the antioxidant activities of all the synthesized compounds 20a-20ab, were evaluated for the first time using DPPH radical scavenging assay taking ascorbic acid as standard reference and FRAP assay using BHT as standard reference. In addition, the cytotoxic studies of active compounds were also performed. Moreover, we also report the validation of our results via in silico molecular docking studies of compounds 20a, 20b and 20t in comparison with standard reference ascorbic acid.</p><!><p>All glass apparatus were oven dried prior to use. Melting points were taken in open capillaries on complab melting point apparatus and are presented uncorrected. Ultrasonic irradiation was performed in a Elmasonic S 30 (H) ultrasonic water bath cleaner and the reaction vessel was positioned in the maximum energy area in the cleaner and the removal or addition of water was used to control the temperature of the water bath. Infrared spectra were recorded on a Perkin-Elmer FT-IR Spectrum 2 spectrophotometer 1H NMR and 13C NMR spectra were recorded on ECS 400 MHz (JEOL) NMR spectrometer using CDCl3, CD3ODandCD3SOCD3 as solvent and tetramethylsilane as internal reference. Electrospray ionization mass spectrometry (ESI-MS) and HRMS were recorded on Xevo G2-S Q Tof (Waters, USA) Spectrometer. Column chromatography was performed over Merck silica gel (particle size: 60-120 Mesh) procured from Qualigens? (India), flash silica gel (particle size: 230-400 Mesh). All chemicals and reagents were obtained from Sigma Aldrich (USA), Merck (India) or Spectrochem (India) and were used without further purification.</p><!><p>Substituted acetophenone 16a-h (2.00 mmol, 1eq.) were taken in toluene (50 ml) and NaH (2.20 mmol, 1.1 eq.) was added carefully. After stirring this reaction mixture at 0°C for 30 min dimethyl oxalate (2.20 mmol, 1.1 eq.) were added and reflux for 6 h. The progresses of the reaction were monitored by TLC using 9:1 Hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was quenched with distilled water and extracted with ethyl acetate (3 × 50 ml); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The organic layer was combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified by recrystalization using EtOAc/Hexane (v/v = 20:80), which afforded the pure desired diketo-ester 17a-h in 78-92% yields. Compounds 17a-h was used for next step without any further purification.</p><p>To a solution of 17a-h (1.00 mmol, 1eq.) in MeOH:THF: H2O (10 ml, 7:2:1), added LiOH.H2O (1.20 mmol, 1.2eq) into the reaction mixture and stirred it for 4 h at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, it was quenched with 3N HCl solution and extracted with ethyl acetate (3 × 30 mL); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The combined organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the corresponding crude product. These crude products were further purified by recrystalization with EtOAc/Hexane, which afforded diketo-acids 18a-h in excellent yields (up to 97%). Compounds 18a-h were used for next step without any further purification.</p><!><p>To a solution of the compound 18a-h (0.20 mmol; 1eq.) in water (2.0 mL) was added compound 19a-f (0.20 mmol; 1eq.) and the reaction mixture was irradiated under ultrasonic sonicator at 80°C temperature for about 75-90 min (depending upon the substrate employed). The progress of the reaction was checked by TLC using 9:1 Hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was extracted with ethyl acetate (3 × 50 ml); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The organic layers were combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified either by recrystalization using Hexane/EtOAc (v/v = 90:10) or by flash column chromatography method over silica gel using 7.5:2.5 to 9:1 Hexane/ethyl acetate as an eluent which afforded the pure desired (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]-oxazin-2-one 20a-ab having good yields (80–98%).</p><!><p>Yellow solid; yield: 51.97 mg (98%), Rf (EtOAc/Hexane; 20:80) = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 185–186°C; FT-IR (KBr, νmax/cm−1) 3434, 1754, 1614, 1594, 1270, 1113; 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 7.4 Hz, 2H, Ar-H), 7.55–7.46 (m, 3H, Ar-H), 7.21–7.05 (m, 5H, C=CH, Ar-H); 13C NMR (100 MHz, CDCl3) δ 191.6 (C=O), 156.3 (O=C-O), 141.3 (C=CH), 139.1 (Ar-C), 138.3 (Ar-C), 132.8 (NH-C), 128.8 (Ar-C-NH), 127.7 (Ar-C), 126.0 (Ar-C), 124.0 (Ar-C), 123.8 (Ar-C), 117.2 (Ar-C), 116.0 (Ar-C), 94.7 (C=CH); HRMS (ESI) calcd. for C16H11NO3 [M+H]+: 266.0739; found 266.0734.</p><!><p>Yellowish solid; yield: 51.2 mg (88%), Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. 200–203°C; FT-IR (KBr, νmax/cm-1) 3435, 1756, 1602, 1112; 1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 8.8 Hz, 2H, Ar-H), 7.17 (t, J = 7.1 Hz, 2H, Ar-H), 7.09–7.05 (m, 2H, C=CH, Ar-H), 7.02–6.95 (m, 3H, Ar-H), 3.87 (s, 3H, O-CH3); 13C NMR (100 MHz, CDCl3) δ 190.5 (C=O), 163.5 (Ar-C-OCH3), 156.6 (O=C-O), 141.2 (Ar-C-N), 138.6 (Ar-C-O), 131.2 (C=CH), 130.0 (Ar-C), 125.9 (Ar-C), 124.0 (Ar-C), 123.7 (Ar-C), 117.2 (Ar-C), 115.8 (Ar-C), 114.0 (Ar-C), 94.7 (C=CH), 55.6 (O-CH3); HRMS (ESI) calcd. for C17H13NO4 [M+H]+: 296.0845; found 296.0849.</p><!><p>Yellowish solid; yield: 56.93 mg (95%), Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 185–187°C; FT-IR (KBr, νmax/cm−1) 3434, 1761, 1555, 1622, 1174; 1H NMR (400 MHz, CDCl3) δ 8.00–7.98 (m, 2H, Ar-H), 7.58–7.55 (m, 1H, Ar-H), 7.50-7.47 (m, 2H, Ar-H), 7.13–7.03 (m, 4H, Ar-H, C=CH); 13C NMR (100 MHz, CDCl3) δ 191.8 (C=O), 155.8 (O=C-O), 139.8 (Ar-C-N), 138.4 (Ar-C-O), 138.1 (C=CH), 133.0 (Ar-C), 131.1 (Ar-C-Cl), 128.9 (Ar-C), 127.8 (Ar-C), 124.8 (Ar-C), 123.8 (Ar-C), 118.3 (Ar-C), 115.8 (Ar-C), 95.7 (C=CH); HRMS (ESI) calcd. for C16H10ClNO3 [M+2]+: 301.7085; found 301.7089.</p><!><p>Yellowish solid; yield: 59.08 mg (93%); Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 155–157°C; FT-IR (KBr, νmax/cm−1) 3434,1754,1634,1601,1495, 1226,1160; 1H NMR (400 MHz, CDCl3) δ 8.06-8.02 (m, 2H, Ar-H), 7.20-7.13 (m, 4H, Ar-H), 7.08-7.04 (m, 2H, C=CH, Ar-H);13C NMR (100 MHz, CDCl3) δ 190.4 (C=O), 167.2 (Ar-C-F), 155.8 (O=C-O), 139.8 (Ar-C-N), 138.6 (Ar-C-O), 131.3 (Ar-C-Cl), 130.5 (C=CH), 130.4 (Ar-C), 124.8 (Ar-C), 123.9 (Ar-C), 118.4 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 115.8 (Ar-C), 95.4 (C=CH); HRMS (ESI) calcd. for C16H9ClFNO3 [M+H]+: 318.0255; found 318.0259.</p><!><p>Yellowish solid; yield: 63.9 mg (96%); Rf (EtOAc/Hexane; 20:80) = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 182–185°C; FT-IR (KBr, νmax/cm−1) 3434, 1761, 1631, 1586, 1088; 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 8.2 Hz, 2H, Ar-H), 7.45 (d, J = 8.1 Hz, 2H, Ar-H), 7.13–7.00 (m, 4H, C=CH, Ar-H); 13C NMR (100 MHz, CDCl3) δ 190.4 (C=O), 155.7 (O=C-O), 139.8 (Ar-C-Cl), 139.4 (Ar-C-N), 138.7 (Ar-C-O), 136.3 (C=CH), 131.2 (Ar-C), 129.2 (Ar-C), 129.1 (Ar-C), 124.6 (Ar-C), 124.0 (Ar-C), 118.3 (Ar-C), 115.9 (Ar-C), 95.3 (C=CH); HRMS (ESI) calcd. for C16H9Cl2NO3 [M+2]+: 334.9959; found 334.9956.</p><!><p>Yellowish solid; yield: 65.9 mg (89%); Rf (EtOAc/Hexane; 20:80) = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 135–137°C; FT-IR (KBr, νmax/cm-1) 3432, 3075, 2923, 1626, 1583, 1105; 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 8.4 Hz, 1H, Ar-H), 7.47 (d, J = 1.6 Hz, 1-H, Ar-H), 7.34 (dd, J = 1.6 Hz, 8.4 Hz, 1H, Ar-H), 7.17-7.09 (m, 3H, Ar-H), 6.82 (s, 1H, C=CH); 13C NMR (100 MHz, CDCl3) δ 192.1 (C=O), 155.4 (O=C-O), 139.9 (Ar-C-Cl), 138.4 (Ar-C-Cl), 137.7 (Ar-C-N), 137.2 (Ar-C-O), 132.7 (C=CH), 131.3 (Ar-C), 130.8 (Ar-C), 130.7 (Ar-C), 127.6 (Ar-C), 124.4 (Ar-C), 118.4 (Ar-C), 116.1 (Ar-C), 99.3 (C=CH); HRMS (ESI) calcd. for C16H8Cl3NO3 [M+2]+: 368.9570; found 368.9577.</p><!><p>Yellowish solid; yield: 70.7 mg (93%); Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 175–177°C; FT-IR (KBr, νmax/cm−1) 3436, 2924, 1755, 1632, 1583, 1007; 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.9 Hz, 2H, Ar-H), 7.62 (d, J = 7.9 Hz, 2H, Ar-H), 7.13 – 7.00 (m, 4H, Ar-H, C=CH); 13C NMR (100 MHz, CDCl3) δ 190.5 (C=O), 155.6 (O=C-O), 139.8 (Ar-C-N), 138.8 (Ar-C-O), 136.8 (C=CH), 132.1 (Ar-C-Cl), 131.2 (Ar-C), 129.3 (Ar-C-Br), 128.1 (Ar-C), 124.6 (Ar-C), 124.0 (Ar-C), 118.3 (Ar-C), 115.9 (Ar-C), 95.2 (C=CH); HRMS (ESI) calcd. for C16H9BrClNO3 [M+2]+: 377.9454; found 377.9458.</p><!><p>Yellowish solid; yield: 56.5 mg (90%); Rf (EtOAc/Hexane; 20:80) = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 160–162°C; FT-IR (KBr, νmax/cm-1) 3434, 2925, 1624, 1766, 1494, 1178; 1H NMR (400 MHz, CDCl3) δ 7.94–7.92 (m, 2H, Ar-H), 7.30 (d, J = 8.0 Hz, 2H, Ar-H), 7.14-7.04 (m, 4H, C=CH, Ar-H), 2.44 (s, 3H, Ar-CH3);13C NMR (100 MHz, CDCl3) δ 191.6 (C=O), 156.0 (O=C-O), 144.0 (Ar-C-CH3), 139.8 (Ar-C-N), 138.2 (Ar-C-O), 135.6 (C=CH), 131.2 (Ar-C), 129.7 (Ar-C), 128.0 (Ar-C), 125.0 (Ar-C), 123.7 (Ar-C), 118.3 (Ar-C), 115.8 (Ar-C), 95.9 (C=CH), 21.8 (-CH3); HRMS (ESI) calcd. for C17H12ClNO3 [M+H]+: 314.0506; found 314.0509.</p><!><p>Yellowish solid; yield: 56.6 mg (86%); Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 178-180°C; FT-IR (KBr, νmax/cm−1) 3434, 1764, 1628, 1594, 1018; 1H NMR (400 MHz, CDCl3) δ 7.99-7.97 (m, 2H, Ar-H), 7.10-7.06 (m, 2H, Ar-H), 7.02–6.99 (m, 2H, Ar-H), 6.97-6.94 (m, 2H, Ar-H, C=CH), 3.87 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3) δ 190.5 (C=O), 163.7 (Ar-C-OCH3), 156.1 (O=C-O), 139.6 (Ar-C-N), 137.8 (Ar-C-O), 131.1 (C=CH), 130.9 (Ar-C), 130.1 (Ar-C), 125.0 (Ar-C), 123.4 (Ar-C), 118.2 (Ar-C), 115.6 (Ar-C), 114.1 (Ar-C), 95.7 (C=CH), 55.6 (-OCH3); HRMS (ESI) calcd. for C17H12ClNO4 [M+2]+: 331.7345; found 331.7349.</p><!><p>Yellowish solid; yield: 53.7 mg (90%); Rf (EtOAc/Hexane; 20:80) = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 145–147°C; FT-IR (KBr, νmax/cm−1) 3433, 2930, 1770, 1624, 1596, 1128; 1H NMR (400 MHz, CDCl3) δ 8.02 (dd, J = 5.6, 8.8 Hz, 2H, Ar-H), 7.17–7.07 (m, 3H, Ar-H), 6.98–6.90 (m, 3H, C=CH, Ar-H), 2.36 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 190.0 (C=O), 166.9 (Ar-C-F), 156.4 (O=C-O), 139.3 (Ar-C-CH3), 136.1 (Ar-C-N), 134.7 (Ar-C-O), 130.3 (C=CH), 130.2 (Ar-C), 124.9 (Ar-C), 123.3 (Ar-C), 116.9 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 115.8 (Ar-C), 94.1 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H12FNO3 [M+H]+: 298.0801; found 298.0807.</p><!><p>Yellowish solid; yield: 65.4 mg (94%), Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 142–145°C; FT-IR (KBr, νmax/cm−1) 3436, 2913, 1755, 1618, 1570, 1083; 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 8.3 Hz, 1H, Ar-H), 7.45 (d, J = 2.0 Hz, 1H, Ar-H), 7.33–7.30 (m, 1H, Ar-H), 7.10–7.08 (m, 1H, Ar-H), 6.94–6.92 (m, 2H, Ar-H), 6.73 (s, 1H, C=CH), 2.36 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 191.6 (C=O), 155.9 (O=C-O), 139.5 (Ar-C-CH3), 139.2 (Ar-C-Cl), 137.5 (Ar-C-Cl), 137.2 (Ar-C-N), 136.2 (Ar-C-O), 132.5 (C=CH), 130.7 (Ar-C), 130.6 (Ar-C), 127.4 (Ar-C), 125.4 (Ar-C), 123.0 (Ar-C), 117.0 (Ar-C), 116.4 (Ar-C), 98.0 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H11Cl2NO3 [M+2]+: 349.0116; found 349.0112.</p><!><p>Yellowish solid; yield: 57.7 mg (93%), Rf (EtOAc/Hexane; 20:80) = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. 180–182°C; FT-IR (KBr, νmax/cm−1) 3452, 1755, 1625, 1581; 1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 8.8 Hz, 2H, Ar-H), 7.06 (d, J = 8.0 Hz, 1H, Ar-H), 7.00 (s, 1H, Ar-H), 6.96 (d, J = 9.4 Hz, 2H, Ar-H), 6.88 – 6.86 (m, 2H, Ar-H, C=CH), 3.88 (s, 3H, OCH3), 2.34 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 190.4 (C=O), 163.4 (Ar-C-OCH3), 156.8 (O=C-O), 139.3 (Ar-C-CH3), 138.7 (Ar-C-N), 136.0 (Ar-C-O), 131.3 (C=CH), 129.9 (Ar-C), 124.4 (Ar-C), 123.6 (Ar-C), 116.8 (Ar-C), 116.0 (Ar-C), 114.0 (Ar-C), 94.5 (C=CH), 55.6 (OCH3), 21.1 (CH3); HRMS (ESI) calcd. for C18H15NO4 [M+H]+: 310.1001; found 310.1009.</p><!><p>Yellowish solid; Yield: 65.7 mg (92%); Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 190–192°C; FT-IR (KBr, νmax/cm−1) 3417, 1767, 1626, 1583, 1282, 1177; 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 7.2 Hz, 2H, Ar-H), 7.58-7.47 (m, 3H, Ar-H), 7.13 (s, 1H, Ar-H), 7.07 (s, 1H, C=CH), 6.85 (s, 1H, Ar-H), 2.34 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 191.7 (C=O), 155.7 (O=C-O), 138.6 (Ar-C-N), 138.1 (Ar-C-O), 136.9 (C=CH), 136.6 (Ar-C-CH3), 132.9 (Ar-C), 128.9 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 124.7 (Ar-C), 115.5 (Ar-C), 110.2 (Ar-C-Br), 95.2 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H12BrNO3 [M+2]+: 359.0001; found 359.0008.</p><!><p>Yellowish solid; Yield: 67.6 mg (90 %); Rf (EtOAc/Hexane; 20:80) = 0.80; Purification of crude product was done by recrystalization using EtOAc/Hexane; m.p. 230–232°C; FT-IR (KBr, νmax/cm−1) 3417, 1771, 1631, 1285, 1159; 1H NMR (400 MHz, CDCl3) δ 8.03-7.99 (m, 2H, Ar-H), 7.17-7.13 (m, 3H, Ar-H), 6.99 (s, 1H, Ar-H), 6.84 (brs, 1H, C=CH), 2.34 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 190.1 (C=O), 167.0 (Ar-C-F), 164.5 (Ar-C-F), 155.6 (O=C-O), 138.7 (Ar-C-N), 136.9 (Ar-C-O), 136.6 (C=CH), 134.5 (Ar-C-CH3), 134.4 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 128.3 (Ar-C), 124.6 (Ar-C-Br), 116.1 (Ar-C), 115.9 (Ar-C), 115.5 (Ar-C), 110.2 (Ar-C), 94.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H11BrFNO3 [M+2]+: 376.9906; found 376.9909.</p><!><p>Yellowish solid; Yield: 79.7 mg (94%); Rf (EtOAc/Hexane; 20:80) = 0.85; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 224–226°C; FT-IR (KBr, νmax/cm−1) 3417, 1763, 1626, 1561, 1294, 1127; 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 8.4 Hz, 1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.33 (d, J = 8.0 Hz, 1H, Ar-H), 7.18 (s, 1H, Ar-H), 6.88 (s, 1H, C=CH), 6.79 (s, 1H, Ar-H), 2.35 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 191.9 (C=O), 155.2 (O=C-O), 138.7 (Ar-C-Cl), 137.5 (Ar-C-N), 137.3(Ar-C-O), 137.0 (C=CH), 136.8 (Ar-C-CH3), 132.7 (Ar-C-Cl), 130.8 (Ar-C), 130.7 (Ar-C), 128.8 (Ar-C), 127.5 (Ar-C), 124.3 (Ar-C), 115.7 (Ar-C), 110.3 (Ar-C), 98.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H10BrCl2NO3 [M+2]+: 426.9221; found 426.9227.</p><!><p>Yellowish solid; Yield: 77.6 mg (89%); Rf (EtOAc/Hexane; 20:80) = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 259–260°C; FT-IR (KBr, νmax/cm−1) 3417, 1768, 1629, 1562, 1283, 1138; 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.8 Hz, 2H, Ar-H), 7.62 (d, J = 8.4 Hz, 2H, Ar-H), 7.16 (s, 1H, Ar-H), 7.00 (s, 1H, Ar-H), 6.86 (s, 1H, C=CH), 2.35 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 190.4 (C=O), 155.6 (O=C-O), 139.0 (Ar-C-N), 137.0 (Ar-C-O), 136.9 (C=CH), 136.7 (Ar-C-CH3), 132.2 (Ar-C), 129.3 (Ar-C), 128.5 (Ar-C), 128.1 (Ar-C), 124.5 (Ar-C), 115.6 (Ar-C), 110.3 (Ar-C), 94.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H11Br2NO3 [M+2]+: 436.9106; found 436.9100.</p><!><p>Yellowish solid; Yield: 70.7 mg (95%); Rf (EtOAc/Hexane; 20:80) = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 219–220°C; FT-IR (KBr, νmax/cm−1) 3417, 1764, 1630, 1602, 1281, 1182; 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.4 Hz, 2H, Ar-H), 7.26–7.24 (m, 2H, Ar-H), 7.08 (s, 1H, Ar-H), 7.00 (s, 1H, Ar-H), 6.80 (s, 1H, C=CH), 2.39 (s, 3H, CH3), 2.30 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 191.4 (C=O), 155.8 (O=C-O), 143.9 (Ar-C-CH3), 138.3 (Ar-C-N), 136.8 (Ar-C-O), 136.5 (C=CH), 135.6 (Ar-C-CH3), 129.6 (Ar-C), 128.0 (Ar-C), 127.9 (Ar-C), 124.8 (Ar-C), 115.4 (Ar-C), 110.1 (Ar-C), 95.3 (C=CH), 21.8 (CH3), 20.9 (CH3); HRMS (ESI) calcd. for C18H14BrNO3 [M+2]+: 373.0157; found 373.0152.</p><!><p>Yellowish solid; yield: 57.2 mg (82%), Rf (EtOAc/Hexane; 20:80) = 0.75; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 211–213°C; FT-IR (KBr, νmax/cm−1) 3446, 3072, 1758, 1621, 1515, 1183; 1H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 8.4 Hz, 2H, Ar-H), 8.13 (d, J = 8.4 Hz, 2H, Ar-H), 7.12 (d, J = 7.6 Hz, 1H, Ar-H), 7.01–6.96 (m, 3H, Ar-H, C=CH), 2.37 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 188.8 (C=O), 155.9 (O=C-O), 149.9 (Ar-C-NO2), 143.3 (C=CH), 140.5 (Ar-C-N), 139.7 (Ar-C-O), 136.4 (Ar-C), 128.6 (Ar-C), 125.8 (Ar-C), 124.0 (Ar-C), 123.9 (Ar-C), 117.2 (Ar-C), 116.7 (Ar-C), 94.1 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H12N2O5 [M+H]+: 325.0746; found 325.0748.</p><!><p>Yellowish solid; yield: 52.2 mg (84%); Rf (EtOAc/Hexane; 20:80) = 0.75; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 207–209°C; FT-IR (KBr, νmax/cm−1) 3448, 3069, 1758, 1621, 1515; 1453; 1H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 8.8 Hz, 2H, Ar-H), 8.15 (d, J = 7.2 Hz, 2H, Ar-H), 7.26 – 7.17 (m, 4H, Ar-H), 7.04 (s, 1H, C=CH); 13C NMR (100 MHz, CDCl3) δ 188.9 (C=O), 155.8 (O=C-O), 150.1 (Ar-C-NO2), 143.3 (Ar-C-N), 141.7 (C=CH), 140.5 (Ar-C-O), 128.7 (Ar-C), 126.2 (Ar-C), 125.1 (Ar-C), 124.1 (Ar-C), 123.4 (Ar-C), 117.5 (Ar-C), 116.6 (Ar-C), 94.3 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0596.</p><!><p>Yellowish solid; yield: 55.21 mg (89%); Rf (EtOAc/Hexane; 20:80) = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 198–200°C; FT-IR (KBr, νmax/cm−1) 3436, 2928, 1762, 1625, 1581, 1142; 1H NMR (400 MHz, CDCl3) δ 8.03-7.96 (m, 4H, Ar-H), 7.61-7.49 (m, 3H, Ar-H), 7.31 (d, J = 8.8 Hz, 1H, Ar-H), 7.15 (s, 1H, C=CH); 13C NMR (100 MHz, CDCl3) δ 192.1 (C=O), 155.1 (O=C-O), 145.2 (Ar-C-NO2), 144.9 (C=CH), 137.7 (Ar-C-N), 137.6 (Ar-C-O), 133.4 (Ar-C), 128.9 (Ar-C), 127.9 (Ar-C), 124.7 (Ar-C), 118.9 (Ar-C), 118.0 (Ar-C), 111.5 (Ar-C), 96.9 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0593.</p><!><p>Yellowish solid; yield: 61.71 mg (94%); Rf (EtOAc/Hexane; 20:80) = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. > 250°C; FT-IR (KBr, νmax/cm−1) 3435, 3107, 1759,1622, 1594, 1156; 1H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H, Ar-H), 8.11 (d, J = 5.2 Hz, 2H, Ar-H), 7.92 (d, J = 6.4 Hz, 1H, Ar-H), 7.44 – 7.36 (m, 3H, Ar-H), 6.92 (s, 1H, C=CH); 13C NMR (100 MHz) δ 188.7 (C=O), 166.6, (Ar-C-F), 156.0 (O=C-O), 145.9 (Ar-C-NO2), 144.6 (C=CH), 139.5 (Ar-C-N), 135.1 (Ar-C-O), 130.9 (Ar-C), 125.8 (Ar-C), 118.9 (Ar-C), 117.7 (Ar-C), 116.6 (Ar-C), 116.4 (Ar-C), 113.0 (Ar-C), 94.5 (C=CH); HRMS (ESI) calcd. for C16H9FN2O5 [M+H]+: 329.0495; found 329.0490.</p><!><p>Yellowish solid; yield: 58.51 mg (86%); Rf (EtOAc/Hexane; 20:80) = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7:3) as an eluent; m.p. 195–197°C; FT-IR (KBr, νmax/cm−1) 3435, 2926, 1599, 1758, 1633, 1594; 1H NMR (400 MHz, CDCl3) δ 8.02–7.93 (m, 4H, Ar-H), 7.29 (d, J = 8.9 Hz, 1H, Ar-H), 7.10 (s, 1H, C=CH), 6.98 (d, J = 8.7 Hz, 2H, Ar-H), 3.89 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3) δ 190.7 (C=O), 164.0 (Ar-C-OCH3), 155.3 (O=C-O), 145.2 (Ar-C-NO2), 144.9 (Ar-C-N), 137.0 (Ar-C-O), 130.7 (C=CH), 130.3 (Ar-C), 124.9 (Ar-C), 118.6 (Ar-C), 117.8 (Ar-C), 114.2 (Ar-C), 111.2 (Ar-C), 97.0 (C=CH), 55.7 (OCH3); HRMS (ESI) calcd. for C17H12N2O6 [M+H]+: 341.0695; found 341.0692.</p><!><p>Yellowish solid; yield: 55.22 mg (89%); Rf (EtOAc/Hexane; 20:80) = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 240–242°C; FT-IR (KBr, νmax/cm-1) 3436, 1763, 1622, 1596, 1268; 1H NMR (400 MHz, CDCl3) δ 8.06 – 8.00 (m, 4H, Ar-H), 7.83–7.81 (m, 1H, Ar-H), 7.62 (t, J = 7.3 Hz, 1H, Ar-H), 7.54 (t, J = 7.5 Hz, 2H, Ar-H), 6.99 (s, 1H, C=CH); 13C NMR (100 MHz, CDCl3) δ 190.7 (C=O), 156.1 (O=C-O), 142.2 (Ar-C-N), 141.1 (Ar-C-O), 139.2 (Ar-C-NO2), 138.3 (C=CH), 133.6 (Ar-C), 131.3 (Ar-C), 129.6 (Ar-C), 128.0 (Ar-C), 121.4 (Ar-C), 117.4 (Ar-C), 112.6 (Ar-C), 96.0 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0595.</p><!><p>Yellowish solid; yield: 52.7 mg (80%); Rf (EtOAc/Hexane; 20:80) = 0.70; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 235–237°C; FT-IR (KBr, νmax/cm-1) 3411, 3090, 1773, 1626, 1516, 1473; 1H NMR (400 MHz, DMSO-d6) δ 8.18 - 8.08 (m, 4H, Ar-H), 7.88 – 7.86 (m, 1H, Ar-H), 7.42–7.38 (m, 2H, Ar-H), 7.02 (s, 1H, C=CH); 13C NMR (100 MHz, DMSO-d6) 188.7 (C=O), 163.7 (Ar-C-F), 155.5 (O=C-O), 141.8 (Ar-C-N), 140.6 (Ar-C-O), 138.7 (Ar-C-NO2), 134.4 (C=CH), 130.7 (Ar-C), 130.6 (Ar-C), 130.5 (Ar-C), 120.9 (Ar-C), 116.9 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 112.0 (Ar-C), 95.3 (C=CH); HRMS (ESI) calcd. for C16H9FN2O5 [M+H]+: 329.0495; found 329.0499.</p><!><p>Yellowish solid; yield: 59.7 mg (87%); Rf (EtOAc/Hexane; 20:80) = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 205–207°C; FT-IR (KBr, νmax/cm-1) 3432, 2925, 2860, 1633, 1525, 1776, 1075; 1H NMR (400 MHz, DMSO-d6) δ 8.08 (d, J = 8.4 Hz, 4H, Ar-H), 7.89-7.87 (m, 1H, Ar-H), 7.63 (d, J = 8.4 Hz, 2H, Ar-H), 7.01 (s, 1H, C=CH); 13C NMR (100 MHz, DMSO-d6) δ 188.9 (C=O), 155.4 (O=C-O), 141.9 (Ar-C-N), 140.7 (Ar-C-Cl), 138.9 (Ar-C-O), 136.5 (Ar-C-NO2), 130.7 (C=CH), 129.8 (Ar-C), 129.5 (Ar-C), 129.2 (Ar-C), 120.9 (Ar-C), 117.0 (Ar-C), 112.1 (Ar-C), 95.2 (C=CH); HRMS (ESI) calcd. for C16H9ClN2O5 [M+H]+: 345.0200; found 345.0207.</p><!><p>Yellowish solid; yield: 64.6 mg (85%); Rf (EtOAc/Hexane; 20:80) = 0.70; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 208–210°C; FT-IR (KBr, νmax/cm−1) 3433, 3088, 1770, 1620, 1522, 1470, 1072; 1H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H, NH), 8.09–8.08 (m, 2H, Ar-H), 7.95–7.93 (s, 1H, Ar-H), 7.78–7.58 (m, 3H, Ar-H), 6.62 (s, 1H, C=CH); 13C NMR (100 MHz, DMSO-d6) δ 190.3 (C=O), 155.3 (O=C-O), 142.2 (Ar-C-N), 140.8 (Ar-C-Cl), 138.5 (Ar-C-O), 137.7 (Ar-C-NO2), 136.2 (C=CH), 131.2 (Ar-C-Cl), 131.0 (Ar-C), 130.5 (Ar-C), 130.1 (Ar-C), 127.9 (Ar-C), 120.8 (Ar-C), 117.3 (Ar-C), 112.1 (Ar-C), 98.8 (C=CH); HRMS (ESI) calcd. for C16H8Cl2N2O5 [M+H]+: 378.9810; found 378.9818.</p><!><p>Yellowish solid; yield: 67.7 mg (87%); Rf (EtOAc/Hexane; 20:80) = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 230–232°C; FT-IR (KBr, νmax/cm−1) 3435, 3093, 1769, 1619, 1521; 1H NMR (400 MHz, DMSO-d6) δ 8.09-7.96 (m, 4H, Ar-H), 7.83-7.44 (m, 3H, Ar-H), 7.01(d, J = 4.8 Hz, 1H, C=CH); 13C NMR (100 MHz, DMSO-d6) δ 188.9 (C=O), 154.7 (O=C-O), 141.8 (Ar-C-N), 140.1 (Ar-C-O), 137.9 (C=CH), 136.6 (Ar-C-NO2), 131.5 (Ar-C), 130.0 (Ar-C), 129.1 (Ar-C-Br), 126.6 (Ar-C), 120.7 (Ar-C), 116.5 (Ar-C), 111.4 (Ar-C), 95.7 (C=CH); HRMS (ESI) calcd. for C16H9BrN2O5 [M+2]+: 389.9695; found 389.9691.</p><!><p>Yellowish solid; yield: 55.4 mg (81%); Rf (EtOAc/Hexane; 20:80) = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7:3) as an eluent; m.p. 218–220°C; FT-IR (KBr, νmax/cm−1) 3437, 2927, 2854, 1632, 1517; 1H NMR (400 MHz, DMSO-d6) δ 8.07-8.02 (m, 4H, Ar-H), 7.73 (d, J = 10.8 Hz, 1H, Ar-H), 7.09 (d, J = 8.8 Hz, 2H, Ar-H), 7.02 (s, 1H, C=CH), 3.89 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 189.1 (C=O), 163.4 (Ar-C-OCH3), 155.8 (O=C-O), 141.7 (Ar-C-N), 140.6 (Ar-C-O), 138.1 (C=CH), 131.1 (Ar-C-NO2), 130.7 (Ar-C), 130.1 (Ar-C), 121.2 (Ar-C), 116.8 (Ar-C), 114.5 (Ar-C), 112.2 (Ar-C), 95.9 (C=CH), 55.8 (OCH3); HRMS (ESI) calcd. for C17H12N2O6 [M+H]+: 341.0695; found 341.0699.</p><!><p>In DPPH radical scavenging method the plant extract (0.75 mL) at different concentrations ranging from 10 to 100 μg mL−1 was mixed with 1.5 mL of a DPPH methanolic solution (20 mg L−1). Pure methanol was taken as control and ascorbic acid (vitamin C), vitamins A and E were used as a reference compounds. The absorbance was measured at 517 nm after 20 min of reaction. The % of DPPH decolouration of the sample was calculated according to the formula (Sharma and Bhat, 2009).</p><p>The decolouration was plotted against the sample extract concentration and a logarithmic regression curve was established in order to calculate the IC50. The results are expressed as antiradical efficiency (AE), which is 1000-fold inverse of the IC50 value AE=1000/ IC50.</p><!><p>The FRAP reagent was prepared by the addition of freshly prepared 20.0 mM FeCl3.6H2O solution, 10.0 mM of ferric-tripyridyltriazine (TPTZ) solution and 300 mM sodium acetate buffer (pH 3.6) in a ratio of 1:1:10 (v/v/v). After that, Sample (our synthesized 2-oxo-2-phenylethylidenes-linked 2-oxo-benzo[1,4]oxazines 20a-ab) was added to 3 ml of freshly prepared FRAP reagent and this reaction mixture was incubated at 37°C temperature for 30 min. and the absorbance was measured at 593 nm. It is also noted that, a freshly prepared solution of FeSO4 was used for calibration of standard curve. The FRAP antioxidant capability were evaluated in terms of C0.5FRAP (the antioxidant capability of samples related to their concentration, which is equivalent to that of FeSO4 at 0.5 mmol/L) (Benzie and Strain, 1996).</p><!><p>Cell toxicity of active C-3 tethered 2-oxo-benzo[1,4]oxazine analogs were accessed using 3T3 fibroblast cell lines in MTT assay via the reported protocol of Danihelová et al. (2013) [For details, see supporting information].</p><!><p>Molecular modeling studies of C-3 tethered 2-oxo-benzo [1, 4] oxazine derivatives 20a-ab were carried out using molecular modeling software Sybyl-X 2.0, (Tripos International, St. Louis, Missouri, 63144, USA). Drawing of structures and simple geometry optimization were performed with Chem Bio-Office suite Ultra v12.0 (2012) (Cambridge Soft Corp., UK). Docking of all compounds was carried out on the human antioxidant enzyme in complex (PDB ID: 3MNG) (Hall et al., 2010b; Bayoumi et al., 2012; Yapati et al., 2016). The Surflexdoc module in Sybyl was used to construct a 3D model of the structures.</p><p>To find the possible bioactive conformations of C-3 tethered 2-oxo-benzo[1,4]oxazine derivatives, molecular modeling studies were performed using the Sybyl X 2.0 interfaced for the synthesized compounds, which exhibited promising and lower antioxidant activity in vitro to find the preferred binding conformations in the receptor. The starting coordinates of the human antioxidant enzyme in complex with the competitive inhibitor DTT (PDB: 3MNG) were taken from the Protein Data Bank (http://www.rcsb.org/pdb). Program automatically docks ligand into binding pocket of a target protein by using protomol-based algorithm and empirically produced scoring function. The protomol is very important and necessary factor for docking algorithm and works as a computational representation of proposed ligand that interacts into binding site. Surflex-Dock's scoring function have several factors that play an important role in the ligand-receptor interaction, in terms of hydrophobic, polar, repulsive, entropic and solvation, and it is a worldwide well-established and recognized method. The most standard docking protocols have ligand flexibility into the docking process, while counts the protein as a rigid structure. Present molecular docking study involves the several steps viz., import of protein structure into Surflex and addition of hydrogen atoms; generation of protomol using a ligand-based strategy. During second step, two parameters first called protomol_bloat, which determines how far the site should extend from a potential ligand; and another called protomol_threshold, which determines deepness of the atomic probes, used to define the protomol penetration into the protein) were specified to form the appropriate binding pocket. Therefore, protomol_bloat and protomol_threshold was set to 0 and 0.50, respectively. In reasonable binding pocket, all the compounds were docked into the binding pocket and 20 possible active docking conformations with different scores were obtained for each compound. During the docking process, all of the other parameters were assigned their default values.</p><!><p>The synthetic scheme for the synthesis of desired C-3 tethered 2-oxo-benzo[1,4]oxazine analogs 20a-20ab using our reported procedure (Jaiswal et al., 2017) is depicted in Schemes 1, 2.</p><!><p>Synthesis of starting substrate functionalized diketo-acid (18a-h).</p><p>Ultrasound-assisted green synthesis of C-3 tethered 2-oxo-benzo [1,4]oxazine analogs (20a-20ab).</p><!><p>The base-mediated reaction of acetophenone 16a-h with dimethyl oxalate in toluene for 6h furnished the diketo-ester 17a-h in 70-80% yields. Conversion of these diketoesters 17a-h to 2, 4-dioxo-4-phenylbutanoic acid 18a-h were achieved by hydrolysis with LiOH.H2O in MeOH:THF:H2O (4:3:1) solvent. The reaction of nitro/alkyl/halide-substituted 2, 4-dioxo-4-phenylbutanoic acid 18a-h with nitro/alkyl/halide-substituted 2-aminophenol 19a-f in water furnished C-3 tethered 2-oxo-benzo[1,4]oxazines 20a-20ab in 74-98% yields after purification either by flash column chromatography or by recrystallization method (Scheme 2, Figure 3; see Supplementary Figures 1–28 for details). All the synthesized compounds were well characterized by 1H-NMR and 13C-NMR spectroscopy, FTIR and HRMS analysis.</p><!><p>Structures of all synthesized C-3 tethered 2-oxo-benzo [1, 4]oxazines (20a-20ab).</p><!><p>All the synthesized C-3 tethered 2-oxo-benzo[1,4]oxazine analogs 20a-20ab were evaluated for in vitro antioxidant activities using DPPH radical scavenging assay compared with standard reference ascorbic acid (Table 1). The choice of the reference compounds is based on hydrophilic nature of ascorbic acid and the maximum inhibition of the DPPH radical in IC50 value (μg/mL) by all the compounds 20a-20ab. The DPPH radical scavenging assay is generally utilized as a quick and reliable parameter to investigate the antioxidant activities of diverse heterocycles (Baydar et al., 2007). DPPH is a stable free radical, that can easily accept a hydrogen radical or an electron to become a stable molecule (Blois, 1958). In the methanolic medium, DPPH has odd electron configuration having a strong absorption band at 515 nm, whereas this absorption decreases slightly in the presence of free radical scavengers, and it results color change to yellow from deep purple (Eklund et al., 2005; Sharma and Bhat, 2009). The radical trapping ability strongly depends on the structural availability of the radical trapping site. The steric hindrance as well as electron density plays a dynamic role in the antioxidant activity since they may prevent the test molecule from reaching the radical site of DPPH and thus results in low activity (Faria et al., 2006).</p><!><p>Antioxidant activity of synthesized compounds 20a-ab by DPPH radical scavenging assay and FRAP assay.</p><p>Results are expressed as a mean ± standard deviation (n = 3). bDPPH radical scavenging activities are expressed as IC50 concentrations of the compounds (μg/mL) required to inhibit 50% of the radicals and the maximum inhibition values and Positive control for DPPH assay=Ascorbic acid; cPositive control for FRAP assay=BHT. The bold value indicates promising antioxidant compounds.</p><!><p>Kareem et al. proposed two mechanisms involved in DPPH assay; first one is the hydrogen atom transfer (HAT) mechanism and the second one is the single electron transfer (SET) mechanism (Kareem et al., 2015). Similar to their interpretation, it can be speculated that, for DPPH assay, a dominant HAT mechanism is assumed and the favored hydrogen abstraction sites are enamine –NH group, preferably with conjugation to the side chain of phenacyl group (-COPh), as the latter could stabilize by the resulting radical from additional resonance structures.</p><p>The generic scaffold of the newly synthesized C-3 tethered 2-oxo-benzo[1,4]oxazines, as illustrated in Figure 4, consists of a two fused cyclic ring A and B linked with ring C via α, β-unsaturated ketone having electron-withdrawing group (EWG) and/or electron-donating group (EDG) either at ring A or at C. The active group -CO-C=C-NH- enables resonance between the ring B and C, leading to multiple resonance structure, which may be further initiated by the attached substituents of ring A and C and in situ enhances the radical scavenging activity through the removal of hydrogen atom from NH of ring B via HAT mechanism. It has been found that electron withdrawing substituent NO2 at ring A or C increase the antioxidant activity which may be due to resonance based stabilizing effects. Therefore, based on the structures and their antioxidant activities, it was found that the compounds have either no substitution at ring A and C or have EWG/EDG at ring A and C plays a very important role in deciding their DPPH radical scavenging activities. Hence, based on the substituent's (either EWG or EDG) at ring A and C of 2-oxo-benzo[1,4]oxazines 20a-20ab, and their antioxidant activities, their structure-activity relationship can be explained by grouping all compounds into two groups:</p><!><p>No substitution or EDG at ring A or ring C: In the first group of compounds having no substitution at ring A and C i.e., the model compound 20a, exhibited promising antioxidant activity (IC50 = 10.20 ± 0.08 μg/mL) in comparison with standard reference Ascorbic acid (IC50 = 4.57 μg/mL) [entry 1]. Then, by putting EDG (OMe group) at ring C, as in compound 20b, further increases activity (IC50 = 6.89 ± 0.07 μg/mL) [entry 2]. Reversing the order i.e., halogen substitution at ring A and no substitution at ring C do not cause any further increase in antioxidant activity as shown by compound 20c (entry 3). Furthermore, when we incorporated halogen substituents (Cl, F, Br, 2,4-dichloro) or CH3 substituent either at A or C as in the case of compounds 20c-l; a decrease in the antioxidant activity was observed due to high electron density in compounds 20d-g, 20j and 20k (entry 4-12). Furthermore, It was observed that two EDG at ring A (20m-q; entry 13-17) exhibited moderate antioxidant activity having IC50 value in the range of 18.86 ± 0.72 to 44.32 ± 0.45 μg/mL. In this series (20m-q; entry 13-17), ring C having Fluorine substituent i.e. compound 20n exhibited good antioxidant activity (IC50 = 16.86 ± 0.72 μg/mL) in comparison with other compounds (20m and 20o-q).</p><p>To our surprise; when we incorporated EWG group i.e., NO2 group at ring C and EDG group i.e., CH3 group at ring A (compound 20r); the antioxidant activity was regained and shows IC50 value of 12.23 ± 0.05 μg/mL nearly equivalent to 20a (entry 18).</p><p>It is to be noted that EDG at ring A decreases antioxidant activity as shown in entries 3-17; so, we synthesized compound 20s (having no any substitution at ring A and EWG i.e., NO2 group at ring C), which, contrary to our expectations, displayed further decrease in antioxidant activity (IC50 = 21.27 ± 0.28 μg/mL) (entry 19).</p><p>EWG (-NO2 Group) at ring A: Since 20r having EWG (NO2) at ring C showed promising antioxidant activity; inspired by this observation, we prepared 20t-v having NO2 group at C-4 position of ring A. 2-oxo-benzo[1,4]oxazine 20t having no substitution at ring C, showed excellent antioxidant activity having IC50 value of 4.74 ± 0.08 μg/mL (entry 20). Since 20b having OMe substituent at ring C, was also found to show excellent antioxidant activity; thus, we synthesized 20u and 20v having Fluoro as well as OMe substituent, respectively at ring C. Unfortunately, antioxidant activity diminishes (entry 21 and 22). In addition, we also prepared 2-oxo-benzo[1,4]oxazines 20w-20ab having EWG group (NO2) at C-5 position of ring A further to investigate SAR study. Compound 20w having no substitution at ring C showed promising antioxidant activity having IC50 value of 12.53 ± 0.09 μg/mL (entry 23). On incorporating Fluoro group at ring C; activity of 20x increases (IC50 = 10.18 ± 0.10 μg/mL, entry 24). Furthermore, when we incorporated halogen substituents (Cl, Br, 2,4-dichloro and OMe) at ring C as in the case of compounds 20y-20ab; a decrease in the antioxidant activity was observed (entry 19-23).</p><p>Overall, we can interpret that no substitution or EWG at ring A or ring C enhances antioxidant activity of all the synthesized 2-oxo-benzo[1,4]oxazines 20a-20ab. Whereas EDG either at ring A or C diminishes antioxidant activity. Our SAR results depict that 20b and 20t, the best compounds of the series, showed antioxidant activity comparable to standard reference Ascorbic acid.</p><p>SAR analysis of synthesized 2-oxo-benzo[1,4]oxazines.</p><!><p>The FRAP assay was deliberated using the method as illustrated by Benzie and Strain (Benzie and Strain, 1996). It reveals that the reducing potential of an antioxidant molecule, which reacts with a complex of ferric tripyridyltriazine [Fe3+−TPTZ] and develops a colored ferrous tripyridyltriazine [Fe2+-TPTZ]. The reducing nature of an antioxidant depends on their property to donate a hydrogen atom for the breaking of the free radical chain, which is responsible for oxidative stress etc.</p><p>All the synthesized C-3 tethered 2-oxo-benzo[1,4]oxazine analogs 20a-20ab were assessed for FRAP assay taking BHT as standard reference; as depicted in Table 1. In this study, the trend with respect to ferric ion reducing activities of all the screened compounds i.e., 20a-20ab showed that eight compounds (20c, 20j, 20m, 20n, 20r, 20u, 20z, and 20aa) were found more potent than BHT (C0.5FRAP = 546.0 ± 13.6 μM).</p><p>In summary, all the compounds (20a-20ab) displayed good to moderate activity in comparison with BHT in the range of C0.5FRAP = 328.6 ± 25.8 μM to 916.8 ± 21.4 μM in in-vitro antioxidant FRAP assay except compounds 20e, 20f, 20l, 20q, 20v, and 20ab which showed C0.5FRAP greater than 1000 μM. Compounds having EWG i.e. NO2 substituent at ring C (20r and 20s) showed different potency than standard reference BHT. While 20r having CH3 at C-4 position of ring A displayed potent activity than BHT; 20s was found to be less active. Anomaly was observed in the case of compounds having no substitution at ring C. While 20c and 20m showed greater potency; compounds 20a, 20t and 20w were found less active than BHT.</p><!><p>Out of 28 compounds, three most active compounds i.e., 20b, 20t, and 20x were then selected for their cytotoxic studies. As depicted in Figure 5, compounds 20b, 20t, and 20x were accessed for their cytotoxic study using 3T3 fibroblast cell lines in MTT assay (Danihelová et al., 2013). The result showed that these compounds were non-toxic in nature (>65% cell viability) even at 250 μg/mL concentration and therefore, displays permissible values of cell viability.</p><!><p>Percentage cell viability test.</p><!><p>Finally, the biological results were validated via in silico molecular docking studies of two most active compounds (20b and 20t). Since 20b has OMe group at ring C and 20t has NO2 group at ring A; it is worthwhile to compare the docking studies of active compounds with molecules having no substitution either at ring A or C. Therefore, we have also selected compound 20a for our in silico molecular docking simulation studies. For that purpose, Peroxiredoxins (Prdxs), a family of small human antioxidant enzyme, was selected as our target protein. Peroxiredoxins contain essential cysteine residues as catalyst and thioredoxin as an electron donor, which help in scavenging peroxide and are involved in the metabolic cellular response to ROS (Neumann et al., 2003; Monteiro et al., 2007).</p><p>The binding affinities and interactions of C-3 tethered 2-oxo-benzo [1, 4] oxazine derivatives with the human antioxidant enzyme were investigated through molecular docking simulations. Binding affinities were predicted by the Sybyl docking total score upon docking with the Surflex-Dock program (Sybyl X 2.0).Compounds were docked into the active site of the known the human antioxidant enzyme target peroxiredoxins (Prxs) DTT complex (PDB ID: 3MNG) were taken from the Protein Data Bank (http://www.rcsb.org/pdb) (Hall et al., 2010b; Bayoumi et al., 2012; Yadav et al., 2014a,b; Yapati et al., 2016).</p><p>Docking studies were carried out to evaluate the binding affinity and interactions with their target proteins. Hydrogen bonds (H-bonds, with a donor-receptor distance of 3Å) between the ligand and amino acids in the binding site of the protein were used for the ranking of compounds. The mode of interaction of the co-crystallized ligand dithiothreitol (DTT) within the crystal structure of enzyme in complex was used as a reference binding model. The root mean-square deviation (RMSD) of each docking pose was compared to the co-crystallized ligand and used for ranking and for RMSD calculation. The co-crystallized DTT molecule was re-docked onto the same binding site and the most probable binding mode was selected as that with the highest docking total score of 4.8921. An RMSD value 0.6772Å between the predicted and crystal binding mode indicates the high reliability of Surflex-Dock for this protein target.</p><p>On the other hand, docking results for 20a, 20b, and 20t against the antioxidant target protein Prxs showed a high binding affinity docking score indicated by a total score of 3.8470 (Figure 6A), 3.6567 (Figure 6B) and 4.2709 (Figure 6C) forms a H-bond (NH2…O) of length 1.8Å to the backbone of hydrophobic aliphatic residue that is, Glycine-46. In the docking pose of the 20a, 20b, and 120t and Prxs complex, the chemical nature of binding site residues within a radius of 3Å with diverse properties was aromatic (hydrophobic), for example, Phe-120, (Phenylalanine); hydrophobic, for example, Leu-116, Ile-119, Leu-149, Leu-112(Leucine), Gly-46, Gly-148(Glycine); (polar, hydrophobic, positive charged) residues, for example, Arg-127 (Arginine); nucleophilic (polar, hydrophobic), for example, Thr-147 and Thr-44 (Threonine), nucleophilic (polar uncharged), for example Cys-47 (Cysteine); and hydrophobic (polar, uncharged) residues, for example, Pro-40 and Pro-45 (Proline) as a result, the bound compound showed a strong hydrophobic interaction with Prxs, thus leading to more stability and activity in this compound.</p><!><p>Binding interactions of compound 20a, 20b, 20t and reference drug Ascorbic acid upon docking onto human antioxidant enzyme target peroxiredoxins (Prxs) (PDB ID: 3MNG). The formation of a H-bond of length 1.8Å to residue Gly-46 in the binding site was predicted in the case of 20a, 20b, and 20t along with the formation of four H-bond of length 2.2, 1.8 and 2.1 Å to residue Thr-147, Gly-46 and Thr-44 in the binding site was predicted in the case of reference drug Ascorbic acid. (A) A top docking energy (total score) of 3.8470 was predicted for 20a; (B) A top docking energy (total score) of 3.6567 was predicted for 20b. (C) A top docking energy (total score) of 4.2709 was predicted for 20t. (D) A top docking energy (total score) of 3.4829 was predicted for Ascorbic acid.</p><!><p>The docking results for the ascorbic acid (standard compound) with the antioxidant target protein Prxs showed a low binding affinity docking score, indicated by a low total score of 3.4829 with three H-bond (hydrogen bond) formation of length 2.2, 1.8 and 2.1Å to the Thr-147, Gly-46 and Thr44 (Figure 6D). The ascorbic acid-Prxc-docked complex also showed a similar type of binding site residues within a radius of 3Å of bound ligand such as Thr-147, Leu-116, Pro-40, Phe-120, Leu-112, Thr-44, Gly-46, Pro-45, Cys-47, Arg-127, Leu-149 shown in Figure 6D. Thus, the docking procedure of Surflex-dock software (Sybyl-X 1.3) in reproducing the experimental binding affinity seems reliable, and therefore predicted as true positive.</p><p>Thus, it can be inferred based on docking simulation studies that the most active compounds i.e., 20b and 20t having IC50 value of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, showed the binding affinity docking score of 3.6567 and 4.2709, respectively (Figures 6B,C), which were found to be comparable to the binding affinity docking score of standard reference ascorbic acid (Figure 6D). While comparing the binding energy docking score of 20a (unsubstituted both at ring A and C) i.e., 3.8470 (Figure 6A) with ascorbic acid and the two most active compounds; the results were also found to be comparable. Thus, the in silico docking results of 20b and 20t successfully validated the in vitro experimental studies.</p><!><p>In summary, we disclose C-3 tethered 2-oxo-benzo [1, 4]oxazine analogs 20a-20ab as a of potent antioxidant agents. Compound 20b and 20t, the most active compounds of the series, showed promising antioxidant activity having IC50 value of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, respectively, in DPPH radical scavenging assay in comparison with ascorbic acid (IC50 = 4.57 μg/mL). Whereas in FRAP assay, eight compounds (20c, 20j, 20m, 20n, 20r, 20u, 20z, and 20aa) were found more potent than BHT (C0.5FRAP = 546.0 ± 13.6 μM). The active compounds were also found non-toxic in 3T3 fibroblast cell lines in MTT assay. Our in silico molecular docking results reveal that 20b and 20t showed excellent docking total scores against human antioxidant enzyme target as compared to ascorbic acid. Thus, the in silico docking simulation studies effectively validated the in vitro experimental results.</p><!><p>SC was responsible for the study of concept and design of the project. VS and PKJ were responsible for the performing synthetic reactions, acquisition and analysis of data. MS, MM, and AKS were responsible for the pharmacological in vitro activity evaluation of synthesized compounds. DKY and Saloni performed docking studies. SM and MHK provided molecular modeling facility. SC and PKJ drafted the manuscript. All authors read and approved the final manuscript.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

Photocatalytical removal of inorganic and organic arsenic species from aqueous solution using zinc oxide semiconductor\xe2\x80\xa0

The photocatalytic removal of arsenite [As(III)] and monomethylarsonic acid [MMA(V)] was investigated in the presence of UV light (350 nm) and aqueous suspensions of ZnO synthesized by the sol\xe2\x80\x93gel technique. Photocatalytic removal of these potent arsenic compounds results in the effective and rapid mineralization to less toxic inorganic arsenate [As(V)]. The effect of ZnO loading and solution pH on the treatment efficiency of the UV/ZnO photocatalytic process was evaluated. The optimal conditions for the removal of 5 mg L\xe2\x88\x921 [As(III)] and [MMA(V)] aqueous solutions were observed at catalyst loadings of 0.25 and 0.50 g L\xe2\x88\x921 with solution pH values of 7 and 8, respectively. Under these conditions, the activity of photocatalyst sol\xe2\x80\x93gel ZnO was compared with TiO2 Degussa P25 and commercial ZnO catalyst. The results demonstrate that the high adsorption capacity of ZnO synthesized by sol\xe2\x80\x93gel gives enhanced removal of arsenic species from water samples, indicating that this catalyst is a promising material for treatment of arsenic contaminated groundwater.

photocatalytical_removal_of_inorganic_and_organic_arsenic_species_from_aqueous_solution_using_zinc_o

1. Introduction<!>2.1 Materials<!>2.2 Synthesis of ZnO<!>2.3 Adsorption experiments<!>2.4 Photocatalytic experiments<!>3.1 Effect of the pH on the adsorption of arsenic species<!>3.2 FTIR study of adsorbed As species<!>3.3 Effect of the pH on the photocatalytic removal of arsenic species<!>3.4 Effect of the ZnO loading<!>3.5 Control experiments<!>3.6 Comparison of the photocatalytic activity of sol\xe2\x80\x93gel ZnO, commercial ZnO and TiO2 Degussa P25<!>4. Conclusions

<p>Arsenic is a carcinogenic and highly toxic element for humans. Chronic arsenic exposure via drinking water has been reported in several countries around the world.1 Both inorganic and organic forms of arsenic have been found in natural waters.2 Arsenic exists mainly as [As(III)] and [As(V)] oxyanions, arsenite (H3AsO3) and arsenate (H3AsO4) in the aquatic environment. Under groundwater conditions, [As(III)] is the predominant form of arsenic, which is much more toxic and mobile than [As(V)]. Although the inorganic species are predominant in natural waters, organoarsenic compounds, such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)], are problematic pollutants in groundwater at sites with a history of pesticide manufacturing and improper disposal.</p><p>A relatively high concentration of arsenic in the aquatic system has many implications for the health of humans, animals and plants. The World Health Organization (WHO) has established a guideline of 0.01 mg L−1 as the maximum allowable level of arsenic in drinking water.3 A simple, low-cost and effective technology for arsenic removal is thus highly desirable to ensure safe drinking water to the people in contaminated areas. Commonly used removal technologies include coagulation and precipitation employing iron and aluminum salts, adsorption onto activated alumina and activated carbon, ion exchange and reverse osmosis.1,4 These techniques have however been found to be more efficient for [As(V)] as a result of the stronger adsorption affinity of negatively charged [As(V)] oxyanions to solid surfaces compared to the neutral [As(III)] molecule.1 Current treatments require oxidation of [As(III)] to [As(V)] prior to adsorption of the arsenic species to achieve efficient total arsenic removal. TiO2 photocatalytic oxidation has been used for the efficient oxidation of [As(III)]/organic arsenic species to [As(V)]5–12 followed by adsorption of [As(V)] by TiO 9,10 The photocatalytic removal of [MMA(V)] and [DMA(V)] using Degussa P25 and nanocrystalline TiO2 has also been investigated.11,12 In this context, TiO2 materials have been recognized as an excellent material for photocatalysis due to their photoactivity, modest cost, nontoxic nature, and large band gap.13 Although TiO2 is generally considered as the most important photocatalyst, ZnO is also an attractive alternative to TiO2 due to the similar band gap energy (3.2 eV) and its lower cost for large-scale water treatment.14 Moreover, larger quantum efficiency and higher photocatalytic activity have been reported for ZnO compared to TiO2 for the photocatalytic destruction of specific pollutants.15–18 However enhancing the photocatalytic activity of ZnO for water treatment is desirable to obtain better removal efficiency. Different synthesis techniques have been adopted to prepare catalysts with suitable characteristics that can reduce the recombination rate of photogenerated electrons and holes.19 The sol–gel method allows flexibility in parameter control with its relatively slow reaction process. This technique allows tailoring the photocatalysts to obtain desired structural characteristics such as compositional homogeneity, grain size, particle morphology and porosity.20</p><p>The primary aim of this work was to accomplish the photocatalytic removal of [As(III)] and [MMA(V)] from aqueous solution using ZnO synthesized by sol–gel as the catalyst under UV radiation at different solution pH and catalyst loading. The activity of sol–gel ZnO on the removal efficiency of arsenic species was compared with TiO2 Degussa P25 and commercial ZnO. To the best of our knowledge, the removal of [As(III)] and [MMA(V)] from aqueous solution by the UV/ZnO system has not been previously reported.</p><!><p>Sodium arsenate (Na2HAsO4·7H2O, [As(V)]) and sodium arsenite (NaAsO2, [As(III)]) were purchased from Sigma-Aldrich. Monosodium methanearsonate (CH3AsO3Na2·6H2O, [MMA(V)]) was purchased from Chem Service Inc. (West Chester, PA, USA). Stock solutions of different arsenic species (100 mg L−1)were prepared in volumetric glassware. The required working standards were prepared daily from the stock solutions. All other reagents were reagent grade and used as received. HCl was trace metal grade from Fisher. All solutions were prepared with ultra-pure water (18 MΩ cm−1) from a Millipore Milli-Q system. Titanium dioxide was P25 (80% anatase and 20% rutile, Degussa, Germany) and commercial ZnO (minimum purity 99%) was purchased from Acros Organics.</p><!><p>It was prepared by the sol–gel technique using zinc acetate, Zn-(CH3COO)2·2H2O, as a precursor according to a procedure reported in previous work.21 Briefly, 13.5 g of zinc acetate was dissolved in 216 mL of water. An aqueous solution (50% v/v) of NH4OH was added under continuous stirring to reach a pH of 9.0. The reaction mixture was kept at room temperature until gel was formed. Once the colloidal ZnO was observed, it was aged for 24 h and filtered; then the material was washed with 0.1 M NH4NO3 and dried by heating the sample slowly to 90 °C until the solvent was completely evaporated. The dried powder (fresh sample) was annealed at 350 °C for 5 h in air. BET surface areas were calculated from N2 adsorption-desorption isotherms obtained in an Autosorb-1 instrument (Quantachrome Co., Boynton Beach, FL, USA). The band gap energy (Eg) values of the studied catalysts were calculated from the UV-Vis diffuse reflectance spectra using a Thermo Scientific Evolution 300 UV-Vis spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) equipped with an integrating sphere TFS-Praying Mantis. The powder X-ray diffraction patterns of the ZnO catalysts (commercial and prepared by the sol–gel method) were observed in accordance with the zincite phase of ZnO (International Center for Diffraction Data, JCPDS).22</p><!><p>FTIR spectra of sol–gel ZnO and arsenic species adsorbed on the catalyst samples were obtained in transmission mode using a Perkin Elmer model Paragon 1000 PC Spectrometer. The spectra were all recorded in the form of KBr pellets.</p><!><p>The catalyst was added to a Pyrex cylindrical reaction vessel (12 × 1 in, 160 mL capacity) containing an aqueous solution of 5mgL−1 [As(III)] or [MMA(V)]. The pH of the solution was adjusted to the desired values (5 to 9) using diluted solutions of HNO3 (0.1 M) and NaOH (0.1 M). Under stirring, the mixture was kept in the dark for 60 min to ensure equilibrium adsorption prior to illumination. Thereafter, the photocatalytic reaction was carried out in a Rayonet Model RPR-100 reactor equipped with 16 phosphor-coated low-pressure mercury lamps that had a spectral energy distribution with a maximum intensity at λ = 350 nm, and yielding an incident light intensity of 5.2 ± 0.1 × 106 photon s−1 cm−3. The temperature was kept close to room temperature by a cooling fan and the reaction rate was followed by taking aliquots at desired time intervals. The catalyst was separated by filtration through a 0.45 μm syringe filter and subsequently analyzed to determine the individual arsenic species concentrations present in the solution. Arsenic speciation analysis was performed within 6 h of sampling, using a PS Analytical Millenium Excalibur Atomic Fluorescence System (AFS, PSA 10.055) coupled to a high-performance liquid chromatography (HPLC) instrument. An anion exchange column, PRP X-100 (250 mm × 4.6 mm × 10 μm), was used for the separation of arsenic species. The mobile phase was a 15 mM monosodium phosphate solution (pH 5.7). The flow rate was 1.0 mL min−1, and the injection volume of the sample was 100 μL. The separated As species were subjected to hydride generation with HCl (12.5% v/v, 2 mL min−1) and NaBH4 (1.4% w/v in 0.1 M NaOH, 2 mL min−1). This was followed by the introduction of Ar into a gas/liquid separation chamber to efficiently carry the gases to the atomic fluorescence spectrometer.23 The effect of the pH of the solution (5–9) and the catalyst loading (0.25 and 0.50 g L−1) on the photocatalytic removal of [As(III)] and [MMA(V)] was studied. All the experiments were performed in triplicate.</p><!><p>The solution pH can have a pronounced effect on the speciation of arsenic species and the surface charge of sol–gel ZnO, and therefore plays a key role in the ZnO adsorption and removal of arsenic species. In order to better understand the process, the adsorption of [As(III)] and [MMA(V)] was studied as a function of the solution pH (structures shown in Table 1).</p><p>In the initial adsorption experiment an aqueous solution of 5 mg L−1 arsenic at a ZnO loading of 0.25 g L−1 was placed in the dark to equilibrate for 60 min followed by the filtration and analysis. The results are summarized in Table 2.</p><p>The adsorption process is favored for both species in the range of pH from 7 to 9. At pH 7 the adsorption of [As(III)] was 53 ± 4%, while the adsorption of [MMA(V)] was 33 ± 3%. The pKa values of H3AsO3 [As(III)] are 9.2, 12.1, and 12.7 and those of [MMA(V)] (CH5AsO3) are 4.1 and 8.71, respectively. At the pH range of 7–9, [As(III)] exists predominantly in the neutral H3AsO3 form while [MMA(V)] exists predominantly as a monoanion. Under the neutral or slightly basic conditions (pH between 7 and 9) of the experiments, the catalyst surface charge is mainly positive since the reported zero point charge (zpc) of ZnO is 9.0 ± 0.3.24 While electrostatic interactions are important in the adsorption process especially for the anion [MMA(V)] species, the [As(III)] is neutral at pH ≤ zpc and thus the adsorption is related to surface complexation rather than to electrostatic interactions.25 The surface hydroxyl groups of ZnO can act as chelating groups for [As(III)] species.11</p><p>On the other hand, the adsorption of [MMA(V)] is mainly the result of the electrostatic attraction between the positively charged catalyst surface and the negatively charged [MMA(V)] species. The presence of the methyl groups in [MMA(V)] reduces capacity and efficiency of the adsorption and thus overall removal compared to [As(III)] adsorption onto ZnO. The adsorption behavior of [As(V)] at pH 7 was also evaluated since this is the final product from the photocatalytic oxidation of [As(III)] and [MMA(V)] using the TiO2 catalyst.1,4 The adsorption percentage of [As(V)] was 95 ± 3% using 0.25 g L−1 of ZnO after 60 min in the dark. The pKa values of H3AsO4 [As(V)] are 2.3, 6.9, and 11.5,1 thus, at pH 7, [As(V)] predominantly exists in a di-anion H2AsO42− form while the surface of the catalyst at the same pH has an overall positive charge. Thus strong electrostatic attraction and surface complexation lead to strong adsorption of [As(V)] onto the catalyst. The presence of four oxygen atoms on [As(V)] available for complexation to the catalyst surface increases the potential adsorption capacity onto ZnO compared to [As(III)] with three oxygen atoms. The adsorption capacity onto sol–gel ZnO followed the order [As(V)] > [As(III)] > [MMA(V)]. We proposed that the adsorption processes of arsenic species onto sol–gel ZnO are mediated by operative mechanisms similar to those reported for TiO2.26 The adsorption mechanisms for TiO2 surfaces involve the formation of bidentate inner sphere complexes for [As(V)], [As(III)], and [MMA(V)].27–29</p><!><p>FTIR spectra of sol–gel ZnO before and after adsorption with 5 mg L−1 arsenic species for 6 h were studied in order to determine the behavior of arsenic species on the catalyst. The solution pH was 7 for [As(III)] and [As(V)] species, and 8 [MMA(V)], respectively. The FTIR spectrum of sol–gel zinc oxide is shown in Fig. 1a. The adsorption peaks of low intensity at 720–600 cm−1 and the broad absorption band centered at 450 cm−1 had been attributed to the ZnO stretching frequency of the Zn–O bond.30 A new band at 830 cm−1 in the FTIR spectrum of sol–gel ZnO was found after adsorption and reaction, which matched well with the stretching frequencies of the As–O band in the arsenite group (see Fig. 1d). The spectrum of ZnO–As(III) displayed a peak that weakly adsorbed at 780 cm−1 due to the symmetry stretching vibration of As–OH. The results agree well with previous FTIR studies of As(III) adsorption on iron oxides published by Goldberg and Johnston.31</p><p>The spectrum after sorption of [MMA(V)] (Fig. 1b) showed a broad peak at around 839 cm−1 (stretching frequency of the As–O band), and the peak at 730 cm−1 due to the symmetric stretching vibration of As–OH disappeared. This could be due to an apparent increase in molecular symmetry upon the sorption or to the peak shifting to a lower wave number. Peaks from a methyl group were not observed in the ZnO–MMA(V) spectrum. This is likely due to the weak peak signal from a single methyl group being overshadowed by the intense As–O peak.31,32 During the adsorption process of [MMA(V)], the As–O(H) group from [MMA(V)] may go through ligand exchange reactions with OH groups from the ZnO surface, forming inner-sphere complexes. Similar results were observed for [As(V)] (Fig. 1c). During As(V) sorption on sol–gel ZnO, the As–O peak (945 cm−1) downshifts to 862 cm−1 as a result of inner-sphere complex formation at the catalyst surface.31 Previous results indicated that arsenic species are adsorbed onto the TiO2 surface by an inner-sphere mechanism.27–29 From the FTIR spectrum analysis results, it was concluded that specific adsorption mechanism should occur at the aqueous solution of arsenic species and the solid sol–gel ZnO interface.</p><!><p>Since maximum adsorption of [As(III)] and [MMA(V)] species was achieved between pH 6 and 8, this pH range was selected for further studies. According to a previous study, the stability of ZnO can be maintained at this pH range.24 Fig. 2 shows the effect of pH on the removal of [As(III)] and [MMA(V)].</p><p>The ZnO mediated photocatalytic treatment of [MMA(V)] was lower compared to As(III) oxidation. In the presence of UV light, the complete ZnO photocatalytic oxidation of As(III) was achieved within 180 min and it was not significantly influenced for the pH. Hence, further experiments for As(III) were performed at pH 7, near the pH of natural waters. At pH 8 the ZnO photocatalysis of [MMA(V)] resulted in 94% removal after 360 min, and thus pH 8 was selected for further experiments with [MMA(V)].</p><!><p>Experiments were conducted to assess the effect of catalyst loading on the overall [As(III)] and [MMA(V)] removal rate. The evaluated catalyst loadings were selected on the basis of preliminary experiments using a catalyst mass ranging from 0.10 to 1.00 g L−1. The lowest amount of catalyst was found to be ineffective for arsenic species removal. On the other hand, the removal of arsenic species was completed at the highest loading level (1 g L−1); however, the process was controlled by the adsorption. Thus, the catalyst dosages of 0.25 and 0.50 g L−1 were studied for a 5 mg L−1 arsenic species concentration in aqueous solutions. The removal efficiencies of As(III) and [MMA(V)] are depicted in Fig. 3. The removal of As species increased with increasing catalyst loading. The increase in the catalyst loading dose provides more active sites on the photocatalyst surface, which in turn increases the number of hydroxyl radicals.8 On a reported study using isopropanol and acetonitrile as radical scavengers and iodine anions as hole scavengers was demonstrated that the reaction mechanism for the photocatalytic oxidation on the ZnO surface mainly proceeded by HO˙ radicals and to a lesser extent by the contribution of holes.33 This result was in agreement with the mechanism proposed by Daneshvar et al. for the photocatalytic degradation of a reactive dye using ZnO.16 For [As(III)], 120 min of treatment results in complete removal employing 0.25 and 0.50 g L−1 sol–gel ZnO loading. While the oxidation of [As(III)] is significantly enhanced in the presence of 0.50 g L−1 catalyst, a lower loading of sol–gel ZnO was selected for the removal of [As(III)] in order to reduce the amount of used catalyst. The photocatalytic removal of [MMA(V)] was 92% in 120 min when the loading of sol–gel ZnO was 0.50 g L−1, illustrated in Fig. 3. However, when the amount of catalyst was lowered to 0.25 g L−1, the efficiency of photocatalytic oxidation of [MMA(V)] declined to 70% removal. The lower removal observed in [MMA(V)] compared with [As(III)] is a consequence of the lower adsorption capacity of the [MMA(V)] species on the soly–gel ZnO surface. The trend suggests that the optimal catalyst loading for removal of As species in a sol–gel ZnO suspension under our experimental conditions was 0.25 g L−1 for [As(III)] and 0.50 g L−1 for [MMA(V)].</p><!><p>The UV photolysis on the removal of arsenic species was evaluated for an initial concentration of arsenic species of 5 mg L−1. The oxidation of [As(III)] and [MMA(V)] to [As(V)] occurred by UV irradiation in the absence of sol–gel ZnO, but to a much less extent with only 17–18% of [As(III)] and [MMA(V)] oxidized in 6 h. Otherwise, the adsorption capacity of [As(III)] and [MMA(V)] without irradiation under the same reaction conditions was 53 and 33%, respectively. The adsorption of arsenic species occurred within the first hour and remained almost unchanged for up to 6 h. In contrast, the photocatalytic oxidation of [As(III)] and [MMA(V)] was highly efficient in the suspensions as illustrated in Fig. 2 for optimum catalyst loading. These control experiments clearly illustrated that the removal of [As(III)] and [MMA(V)] predominantly involves ZnO surface mediated photocatalysis.</p><p>The photocatalysis of arsenic species using TiO2 has been adequately modeled by the first-order reaction rate expression.7,12,34,35 Then the kinetic parameters for removal of [As(III)] and [MMA(V)] can be calculated according to eqn (1): (1)ln(C0∕C)=kt where C0 is the equilibrium concentration after adsorption; C the concentration at time t; and k the observed first-order rate constant.</p><p>The plots of ln(C0/C) as a function of time gave linear relationships, with regression coefficients, R2, >0.9725 (see Table 3). The rate constants k were obtained as 1.66 × 10−2 and 3.84 × 10−2 h−1 for [MMA(V)] and [As(III)], respectively. The results were consistent with the observation that the initial photocatalytic oxidation of [As(III)] was faster than the [MMA(V)] removal process.</p><!><p>For the evaluation of the photocatalytic activity of sol–gel ZnO on the removal of [As(III)] and [MMA(V)], Degussa P25 and commercial ZnO were used as the reference under identical experimental conditions. The photocatalytic removal of [As(III)] is shown in Fig. 4a.</p><p>The results clearly indicated that Degussa P25 was found to be the most active in the oxidation of [As(III)]. By using sol–gel ZnO, the complete removal of [As(III)] was achieved in 150 min. The order of photocatalytic activity was Degussa P25 > sol–gel ZnO > commercial ZnO. While these results clearly demonstrated that [As(III)] was readily removed by these catalysts, it was important to identify the reaction by-products of the treated solutions. The results are shown in Fig. 4b. During the oxidation of [As(III)], [As(V)] species was formed in the solution. This behavior was in agreement with previous reports on a TiO2 surface where the photocatalytic oxidation of [As(III)] was extremely fast, involving oxidation of the arsenic atom to the pentavalent oxidation state.10,36–40 By using TiO2, the As(V) concentration that was formed as a by-product increased up to 4.9 mg As L−1 in the first 2 h and remained constant thereafter. The observed difference between TiO2 and sol–gel ZnO semiconductors was due to the excellent adsorption capability of sol–gel ZnO semiconductor shown for [As(V)], thus the [As(V)] concentration in the treated solution was largely decreased in spite of the continuous generation of [As(V)] by the oxidation of [As(III)]. Overall, the results demonstrated complete removal of [As(V)] even below the WHO drinking water limit of 10 μg L−1 using the sol–gel ZnO catalyst.</p><p>Similar behavior was observed during the removal of [MMA(V)] (see Fig. 5a).</p><p>The results clearly demonstrated that photocatalytic oxidation of [MMA(V)] was faster in TiO2 than in sol–gel ZnO. By using commercial ZnO, the removal efficiency of [MMA(V)] was approximately 55% after 300 min. Although TiO2 exhibited better photocatalytic performance on the removal of [MMA(V)] than sol–gel ZnO, the [As(V)] generated during the photocatalytic removal process remained in solution since it was not adsorbed on the TiO2 surface (Fig. 5b). By using commercial ZnO, 0.7 mg L−1 [As(V)] remained in solution, conversely, complete adsorption of [As(V)] on the catalyst surface was observed with sol–gel ZnO.</p><p>The BET surface area and band gap energy (Eg) of the studied catalyst are shown in Table 4. With respect to Degussa P25, the ZnO catalysts showed lower BET surface area values. As can be observed in Fig. 4 and 5, the sol–gel ZnO showed better performance than commercial ZnO and TiO2 on the removal of these two arsenic species. This fact can be a consequence of the structural defects formed on the semiconductor during the sol–gel preparation method, which in turn produced a shift on the Eg values. As can be seen in Table 4, the Eg value of sol–gel ZnO slightly decreased compared to the band gap value of commercial ZnO due to the elimination of the OH groups that are bound to the synthesized material. The sol–gel method using inorganic salts as precursors causes formation of M–OH2 bonds which produces through the condensation several types of bridges and an infinite network.20,21 Additionally, the hydroxyl groups that are lost during the thermal treatment cause a certain degree of defects and yield oxygen vacancies that act as active sites improving the catalytic activity of sol–gel ZnO with respect to TiO2 Degussa P25.21 According to Zheng et al.,41 defects in an oxide semiconductor have an important role in photocatalysis since the oxygen defects could benefit the efficient separation of electron–hole pairs in the photocatalytic process.</p><p>Thus, the high adsorption capacity observed in the ZnO synthesized by the sol–gel method provided the advantage that this material could be used as a combined catalyst and adsorbent to remove toxic arsenic species from groundwater samples.</p><!><p>The heterogeneous photocatalysis using ZnO prepared by sol–gel is effective for the oxidation of inorganic and organic arsenic species leading to the complete removal of [As(III)] and [MMA(V)] from aqueous solutions under UV irradiation. The [As(V)] generated as a by-product during the removal of these arsenic species was completely and strongly adsorbed onto the ZnO surface under a variety of conditions. This material has promising potential to consistently produce arsenic free water (<10 μg L−1) from highly arsenic contaminated water. The photocatalytic method using ZnO prepared by the sol–gel technique may be applied as a relatively inexpensive method to treat arsenic contaminated groundwater of emerging regions of the world.</p>

PubMed Author Manuscript

Boehmite Nanofibers as a Dispersant for Nanotubes in an Aqueous Sol

By exploiting the dispersibility and rigidity of boehmite nanofibers (BNFs) with a high aspect ratio of 4 nm in diameter and several micrometers in length, multiwall-carbon nanotubes (MWCNTs) were successfully dispersed in aqueous solutions. In these sols, the MWCNTs were dispersed at a ratio of about 5-8% relative to BNFs. Self-standing BNF-nanotube films were also obtained by filtering these dispersions and showing their functionality. These films can be expected to be applied to sensing materials. TextOne-dimensional materials such as nanofibers, nanotubes, and nanowires have received a great deal of attention in the field of nanotechnology.[1] Depending on their composition, these materials can exhibit interesting properties that are not found in bulk materials owing to their specific shape, which consists of a high aspect ratio and a narrow diameter composed of a limited number of molecules. Because of their unique

boehmite_nanofibers_as_a_dispersant_for_nanotubes_in_an_aqueous_sol

<p>characteristics, these materials have been the subject of extensive research motivated by not only fundamental scientific interest but also the potential practical applications such as electronic materials, sensors, and fillers for composite materials. [2][3][4][5][6][7] Nanofibers and nanotubes can be produced by various methods, such as electrospinning, decomposition of biological materials, and hydrothermal treatment. [8][9][10][11] A common problem encountered while handling these materials is aggregation. Similar to the case of quantum dots and nanoparticles, which readily form secondary particles and lose their interesting physical properties, nanofibers and nanotubes are susceptible to bundle formation. One solution for maintaining the unique functions of nanofibers and nanotubes in macroscale is to disperse them in liquids or polymers, which necessitates the use of dispersants and surface modification, although the optimal method must be determined for each material. For example, various techniques have been proposed for the dispersion of carbon nanotubes in a liquid, such as surface oxidation, polymer coating, and the use of surfactants or an appropriate dispersion medium. [12][13][14][15][16][17][18] Each of these methods for dispersing one-dimensional materials has both advantages and disadvantages, and the most appropriate choice can depend on the specific application.</p><p>Our group has studied porous materials by using (pseudo)boehmite nanofibers (BNFs) composed of aluminum oxide hydroxide (AlOOH), which has a high aspect ratio of 4 nm in diameter and several micrometers in length. [19][20][21][22] Nanofibers prepared by the solvothermal method can be stably dispersed in concentrated aqueous acetic acid for several months. [23,24] However, when a base or phosphoric acid is added under appropriate conditions, the nanofibers form a three-dimensional network without bundling, and the dispersed sol becomes a gel. By supercritical drying of the wet gel obtained from this reaction, ultralow-bulk-density transparent porous monoliths (aerogels of 5 mg cm −3 or less) can be obtained, which have potential applications as optical materials. [21] By dispersing functional materials such as fluorescent molecules or nanoparticles in the gel before freeze-drying, a transparent monolith (cryogel) containing the functional material dispersed in the nanofiber network can also be obtained. [19,22] These materials are expected to apply to the fabrication of sensing materials. As the nanofiber dispersion sol is fluid, it is also possible to disperse the functional material using ultrasonication. However, because the BNFs themselves are rigid materials, they are expected to hinder the aggregation of the functional material. In particular, when the functional material is also a one-dimensional material, the movement in the dispersion is mostly restricted. Indeed, the addition of multi-walled carbon nanotubes (MWCNT) to a BNF dispersion was found to afford a relatively stable dispersion. The results of this study demonstrate the applicability of already dispersed one-dimensional materials as dispersants.</p><p>To investigate the dispersibility several BNF dispersions with different concentrations were prepared, and MWCNTs were then added and dispersed by sonication. The obtained BNF-MWCNT sols remained black even after ultracentrifugation at 10,000 ×g for 5 minutes, which indicated that the MWCNTs were still dispersed (Figure 1). In contrast, when the MWCNTs were added to water or aqueous acetic acid, ultracentrifugation resulted in pelletization of the MWCNTs and the formation of a colorless and transparent supernatant. These results confirmed that the presence of BNF affected the dispersion state of the MWCNTs under aqueous conditions. Table 1 shows the MWCNT concentrations in the supernatants obtained after ultracentrifugation of BNF-MWCNT dispersions containing various BNF concentrations. MWCNTs were dispersed at a weight ratio of 5-8% relative to BNF. A lower BNF concentration in the dispersion resulted in a reduction in the amount of dispersible MWCNT and an increase in the error. Figure 1c shows transmission electron microscopy (TEM) images obtained after casting the obtained BNF-MWCNT dispersion on a grid. The MWCNTs were kept in the dispersed state by multiple BNFs, and no noticeable aggregation was observed. Interestingly, the addition of the MWCNTs was found to lead to a change in the viscosity relative to the BNF dispersion.</p><p>In the system with the highest viscosity (sample X2.5 in Table 1), the viscosity decreased from 76 Pa s to 49 Pa s. This reduction was ascribed to the homogeneous dispersion of the MWCNTs between the BNFs, which weakened the hydrogen-bonding interactions. From these results, it is considered that the dispersed MWCNTs became physically entangled between the rigid BNFs in the dispersed state. Even when this BNF-MWCNT dispersion was allowed to stand for one month, almost no change in the dispersion state was observed. In a similar manner to paper, the structure of which consists of intertwined cellulose fibers, one-dimensional materials such as nanofibers, nanotubes, and nanowires are known to be capable of forming films upon removal of the solvent by filtration and evaporation. With respect to BNFs, Kodaira et al. fabricated free-standing films with visible-light reflectivity and thermally insulating properties by preparing nanofiber bundles under appropriate conditions and subsequently depositing them. [25] In the case of the BNF-MWCNT dispersion, it was possible to prepare a black self-supporting film by vacuum filtration through a polycarbonate membrane filter (Figure 2). Scanning electron microscopy (SEM) images confirmed that this film had a structure in which the BNFs and MWCNTs were arranged in layers. As BNFs are composed of aluminum oxide hydroxide and act as an insulator, the obtained film exhibited only the low electrical conductivity of the MWCNTs. Instead of MWCNTs, polydiacetylene nanotubes (PDANTs), which have been reported as a vapochromic material, [26][27][28] could also be dispersed with the BNTs to prepare a similar film.</p><p>Although PDANTs themselves can be dispersed in water or alcohols, the resulting composite films were stable to immersion in these solvents. This observation indicated that the PDANTs in the film were successfully confined in a dispersed state with the BNFs. As with the BNF-MWCNT film, the PDANT-MWCNT film was also found to have a structure in which the nanofibers formed overlapping layers. Exposure of this composite film to water or 2-propanol vapor revealed that it had vapochromic properties (Figure 3, Movies S1 and S2). Although the color change was small and different from that which occurs for pure PDANTs, the basic functionality remained even after film formation. Therefore, the use of BNF as a dispersant/carrier is expected to be a promising method for fabricating functional films when additional one-dimensional materials with unique functionalities are synthesized in the future. By changing the pH of the BNF dispersion, wet gels can be obtained instantaneously. We have reported studies to prepare ultralow-density transparent porous monoliths by drying those BNF gels. In the case of the BNF-MWCNT and BNF-PDANT binary dispersions, wet gels were also obtained without conspicuous aggregations in the internal structure. However, when these wet gels were subjected to supercritical drying, considerable shrinkage occurred, and the reproducibility of the obtained aerogels was low. This was because the addition of the nanotubes reduced the bonding between the BNFs and the networks became non-uniform. At present, the formation of monoliths based on BNF composites remains difficult.</p><p>In summary, MWCNTs were successfully dispersed in aqueous solution by exploiting the dispersibility and rigidity of BNFs. In these dispersions, the MWCNTs were dispersed at a ratio of about 5-8% relative to BNF. Self-standing BNF-nanotube films were also obtained by filtering these dispersions, allowing the introduction of functionalities such as chromic properties depending on the type of dispersed nanotubes.</p><p>These dispersions can potentially be applied in the fabrication of functional films and used as inks and fillers. Unlike existing methods such as chemical modification, the application of this method to one-dimensional materials such as single-walled carbon nanotubes, which are susceptible to bundle formation, remains challenging, and further research is required. In future work, the described method for physically creating a dispersed state by utilizing the geometric properties of a one-dimensional material is expected to lead to the development of new dispersants.</p>

ChemRxiv

Slt, MltD and MltG of Pseudomonas aeruginosa as Targets of Bulgecin A in Potentiation of \xce\xb2-Lactam Antibiotic

The interplay between the activities of lytic transglycosylases (LTs) and penicillin-binding proteins (PBPs) is critical for the health of bacterial cell wall. Bulgecin A (a natural-product inhibitor of LTs) potentiates the activity of \xce\xb2-lactam antibiotics (inhibitors of PBPs), underscoring this intimate mechanistic interdependence. Bulgecin A in the presence of an appropriate \xce\xb2-lactam causes bulge deformation due to the formation of aberrant peptidoglycan at the division site of the bacterium. As Pseudomonas aeruginosa, a nefarious human pathogen, has 11 LT paralogs, the answer as to which LT activity correlates with \xce\xb2-lactam potentiation is important and is currently unknown. Growth of P. aeruginosa PAO1 strains harboring individual transposon-insertion mutants at each of the 11 genes for LTs, in the presence of the \xce\xb2-lactam antibiotic ceftazidime or meropenem, implicated the gene products of slt, mltD, and mltG (of the eleven), in bulge formation and potentiation. Hence, the respective enzymes would be the targets of inhibition by bulgecin A, which was indeed documented. We further demonstrated by imaging in real time and by SEM that cell lysis occurs by the structural failure of this bulge. Upon removal of the \xce\xb2-lactam antibiotic prior to lysis, P. aeruginosa experiences delayed recovery from the elongation and bulge phenotype in the presence of bulgecin A. These observations argue for a collaborative role for the target LTs in the repair of the aberrant cell wall, the absence of activities of which in the presence of bulgecin A results in potentiation of the \xce\xb2-lactam antibiotic.

slt,_mltd_and_mltg_of_pseudomonas_aeruginosa_as_targets_of_bulgecin_a_in_potentiation_of_\xce\xb2-la

<!>Potentiation of \xce\xb2-lactams by Swarm-motility Assays<!>Bulgecin A Binding Affinity for LT Targets<!>Potentiation of \xce\xb2-lactams by Growth-curve Assays<!>Inhibition of Cell Wall Repair<!>Bulge Deformation is the Site of Cell Lysis<!>MIC Determinations<!>METHODS

<p>The bacterial cell wall is a polymer formed by the crosslinking of the peptide stems of glycan strands.1 The glycan strands consist of the repeating N-acetyl glucosamine (GlcNAc) β-1,4-tethered to N-acetyl muramic acid (MurNAc) disaccharide (Figure 1A). The peptide stem is found on the MurNAc saccharide. The enzymes inactivated by the β-lactam antibiotics are the penicillin-binding proteins (PBPs), the catalysts of the transpeptidation reaction that crosslinks these stems. Ceftazidime (CAZ) is a cephalosporin β-lactam that in Gram-negative bacteria inactivates primarily a specific PBP required for the synthesis of the septum.2 In Pseudomonas aeruginosa a particular PBP inhibited by CAZ is PBP3, an essential enzyme of this pathogen.3 PBP3 is an enzyme of the divisome, the multi-protein assembly that spatially and temporally controls the synthesis of the septal peptidoglycan cell wall. Meropenem (MEM) is a carbapenem β-lactam that inactivates both the PBP2 and PBP3 of P. aeruginosa. PBP2 is an enzyme of the elongasome (the multi-protein assembly involved in the creation of the side-wall peptidoglycan) and of the nascent divisome. Although the sequence of structural transformations catalyzed by the elongasome and the divisome is unknown, an early observation was that net peptidoglycan synthesis required concomitant peptidoglycan disassembly and reassembly.4 A recent observation is that the bactericidal event induced by the loss of PBP function results from the failed synchronization of these two seemingly opposing processes.5 These observations have drawn sharp attention to the role(s) played by a second key enzyme family, that of the lytic transglycosylases (LTs) (Figure 1A), which catalyze peptidoglycan disassembly.6,7</p><p>A confounding aspect to the understanding of the role of the LTs is the plurality of LTs. There are eight LTs in Escherichia coli, up to eight LTs in Neisseria gonorrhoeae, and 11 LTs in P. aeruginosa.7,8 The entire LT family of 11 enzymes is conserved in virtually all sequenced P. aeruginosa genomes.9 Single-gene knockouts of each LT of P. aeruginosa give viable phenotypes.10 Evolution of paralogous LT genes can result in the formation of families of proteins within a genome that share highly conserved—both at the protein sequence and three-dimensional structure level—catalytic domains. Notwithstanding the acquisition of additional domains in various LTs—presumably for the purposes of regulation, sequestration, and/or substrate breadth—the members of the LT family upon in vitro assay appear to have overlapping catalytic activities.11,12 If viability is lost as a result of a single-gene knockout, the gene is deemed as "essential." A single gene is deemed "non-essential" if viability is not lost. However, this reasoning fails when the gene function is so critical that evolution builds redundancy within the genome, so as to preserve the function. The LT superfamily exemplifies this phenomenon.7,13 As overall LT function is critical, LTs are worthy targets for interference in manifestation of antibacterial effect.</p><p>The bulgecins are natural-product inhibitors of LTs (Figure 1B).14,15 The bulgecins cause septal bulges in Gram-negative bacteria in the presence of β-lactam antibiotics, resulting in enhanced bactericidal activity.16 Bulgecin A was shown first to inhibit a soluble LT (Slt70) of E. coli.17 At the time of this observation Slt70 was the only known LT. With the current recognition that each Gram-negative bacterium has a superfamily of LTs, we undertook the assessment of the LT superfamily of the nefarious human pathogen P. aeruginosa for inhibition by bulgecin A. The key question was identification of the subset of the LTs inhibited by bulgecin A to achieve bactericidal potentiation of the β-lactams. We addressed this question using the two β-lactam antibiotics, CAZ and MEM, having different PBP selectivities.</p><!><p>We previously reported that bulgecin A potentiates CAZ against P. aeruginosa as visualized by confocal microscopy of swarm-motility assays.16,19 In this assay the β-lactam antibiotic (alone, or with bulgecin A) is spotted distant from a P. aeruginosa colony. As the bacteria swarm outward, the swarm-colony edge that faces the antibiotic spot encounters an increasing antibiotic gradient. These bacteria undergo structural deformation (in response to the antibiotics) and eventual cell lysis. Cell lysis is visualized by the red fluorescence arising from encounter of the bacterial DNA, spilled into the agar upon cell lysis, with propidium iodide embedded in the agar. In this assay CAZ alone induces an elongated cell phenotype as a result of the failure of septum formation due to the loss of PBP3 function by CAZ inactivation. In the presence of both CAZ and bulgecin A, a different shape phenotype was observed, that of a periodic structural bulge within the elongated cell phenotype. This bulged phenotype correlated with a more rapid progression to cell lysis.</p><p>We performed this same experiment with MEM (Figure 2A). A bulgecin A–MEM mixture was plated 20 mm away from a P. aeruginosa colony. The swarm edge (white arrow, top left panel) of the colony was imaged at 8 h and at 12 h. We saw again an elongated cell phenotype with MEM alone (MEM also inhibits PBP3). Bulge formation in the elongated cell phenotype was not observed in the cells exposed to MEM alone, but was observed with the bulgecin A–MEM combination at 8 h (Figure 2A). At 12 h both conditions showed bulge structures (Figure 2B). Notwithstanding that the MEM-induced bulge structures formed in both conditions, we again saw earlier and more extensive cell lysis of bacteria encountering the MEM–bulgecin A combination compared to MEM alone. The previously reported results for the CAZ–bulgecin A combination at 14 h (Figure 2C) and 22 h (Figure 2D) are shown for comparison.</p><p>These observations showed that loss of LT function by the presence of bulgecin A enhanced the bactericidal action of both β-lactams. We acquired from the Manoil Library the transposon (Tn)-insertion mutant for each of the 11 LTs of P. aeruginosa.20,21 The background wild-type P. aeruginosa PAO1 strain for this library, and the 11 Tn-insertion strains in this same background, each were transformed for visualization by the mini-Tn7 chromosomal, constitutive, GFP-expressing insertion (Table S1).22</p><p>The first experiment was the individual challenge of each strain by CAZ and by MEM separately in the swarming motility plate assay. The data for CAZ are given in Figure S1. Of the 11 mutants, early (6 h) septal bulge formation was detected foremost in the slt::Tn strain (Figure S1A). Bulge formation was also evident in the membrane-bound lytic transglycosylase D (MltD) Tn-insertion strain mltD::Tn. Slt is a soluble LT of the periplasm, while MltD is a lipoprotein of the periplasm. The nine remaining mutants (for the lipoprotein LTs MltA, MltB, MltF, MltF2, MltG, RlpA; and the soluble LTs SltB1, SltB2, and SltB3) showed no discernable bulge formation. At 9 h minor bulge formation was detected for the mltA::Tn, mltF::Tn, mltG::Tn, sltB1::Tn, and sltB3::Tn strains. However, the degree of bulge formation in these five strains was significantly less than that seen for slt::Tn and mltD::Tn (Figure S2). The results of the Tn-insertion mutants for the genes sltB1 and rlpA (for LTs SltB1 and RlpA, respectively) are shown in Figure S1A as representative data. After 9 h, cell lysis was seen in both the slt::Tn and mltD::Tn strains (Figure S1B) as assessed by the formation of red fluorescence. Cell lysis was not detected in the wild-type control at this time point, nor in the nine other LT Tn mutants (Figure S2; Figure S1 shows the mltG::Tn, sltB1::Tn, and rlpA::Tn strains as representative data). These observations implicate functional redundancy between Slt and MltD, with the appearance of septal bulges in both of their Tn-insertion strains as a foreshadow of progression to bactericidal lysis (vide infra) in the presence of the challenge by β-lactam antibiotic CAZ.</p><p>This identical experiment was repeated with MEM as the β-lactam. Two notable experimental changes were made with MEM compared to CAZ experiments. The first is a spatial change as MEM was spotted 20 mm (compared to 15 mm for CAZ) placement of β-lactam from the site of inoculation of the bacterium. A longer distance between the colony and MEM was required as a result of the significantly faster MEM-induced cell lysis compared to CAZ. Accordingly, the second change was temporal—the MEM experiments required different observation times. The results from the wild-type strains in the presence of MEM (Figure 2A) show that MEM induces large bulge-like structures in cells. In the slt::Tn and mltG::Tn strains (Figure S3; but not the remaining nine Tn-insertion mutants, Figure S4) bulge formation was seen at 8 h. At 10 h significant bulge formation was detected in the slt::Tn, mltG::Tn, and mltD::Tn strains. Cell lysis was most prevalent in the slt::Tn strain. Although these bulges have not formed after 10 h in the other Tn-insertion mutant strains, they form if the experiment is given time to continue. Progression to MEM-induced bulge formations is most rapid in the slt::Tn, mltD::Tn, and mltG::Tn Tn-mutant strains. This result identified Slt, MltD, and MltG as the targets of bulgecin A.</p><!><p>We purified these enzymes to homogeneity (P. aeruginosa MltD and MltG were prepared as the soluble enzymes MltDsol and MltGsol by genetic deletion of their respective lipoprotein signal sequences). Their binding ability for bulgecin A was assessed by mass spectrometry analyses (Figure 3). RlpAsol and SltB1, two LTs whose respective Tn-insertion strains lacked bulge formation for either CAZ or MEM, were used for comparison. As the Pseudomonas MltGsol gave a weak MS signal, we used E. coli MltGsol (Ec MltGsol, ECK1083: 35,864 Da) in place of the MltGsol of P. aeruginosa (38% sequence identity, 97% query coverage). Bulgecin A binds to Slt and MltDsol at single-digit micromolar concentrations (Slt and MltDsol, Kd values of 8.5 ± 1.1 μM and 1.4 ± 0.3 μM, respectively). Bulgecin A also binds well to Ec MltGsol (Kd of 24 ± 5.1 μM). The Kd values for SltB1 (Kd of 160 ± 21 μM) and RlpA (1200 ± 280 μM) were higher. The lack of bulge formation upon exposure to CAZ in P. aeruginosa lacking the activities of MltG, SltB1, and RlpA (the respective Tn-insertion mutants) indicates that the loss of function of these three enzymes individually is not linked directly to the loss of function of PBP3 (the primary PBP target of CAZ). In contrast, the loss of function of Slt, MltD, and MltG is linked to the loss of function of PBP2, the primary PBP target of MEM.</p><!><p>We explored the effect of CAZ and MEM challenges to the individual Tn-insertion strains by growth-curve assays in liquid medium. We showed previously that bulgecin A potentiates CAZ (MIC = 0.5 μg mL−1) at a sub-MIC concentration (0.28 μg mL−1) of CAZ. Wild-type P. aeruginosa in the presence of bulgecin A and CAZ, and also in the presence of bulgecin A and MEM, showed normal growth (not delayed growth) until mid-log phase, at which time a lysis event occurred (Figure 4, red trace). None of the 11 single-Tn-insertion strains showed potentiation in the presence of sub-MIC CAZ (data for the slt::Tn, mltD::Tn, mltG::Tn, sltB1::Tn, and rlpA::Tn strains are shown in Figure 4). The only difference (compared to wild type) among the 11 strains in the presence of sub-MIC of CAZ was a reproducible delay of growth (but without bulge formation) for the mltF::Tn strain (Figure S10). The reason for this delay is unknown. The important conclusion is that the loss of activity of any one LT in P. aeruginosa—be it by Tn insertion into the gene (or by logical extension, inhibition of a single given LT) does not potentiate CAZ at the selected sub-MIC concentration of 0.28 μg mL−1. The MIC for MEM of the parent strain is 0.5 μg mL−1. In the presence of a sub-MIC concentration of MEM (0.14 μg mL−1) potentiation is observed for either the slt::Tn or mltG::Tn strain. As was seen with CAZ, the delay-of-growth (but without bulge formation) phenomenon is observed with MEM for the mltF::Tn strain (Figure S10).</p><p>The observation for sub-MIC CAZ seemingly contrasts with the observation of both bulge formation and cell lysis in the Tn-insertion strains for mltD (similar to slt::Tn) as shown in Figure S1. We attribute the lysis shown in Figure S1 to exposure to an increasing concentration gradient of drug as the colony swarms toward the antibiotic spot. In contrast, when the strains are grown in liquid medium at a fixed sub-MIC concentration of CAZ, cell growth is unhindered (Figure 4). The antibiotic efficacy of CAZ against P. aeruginosa is diminished by β-lactam induction of expression of the native AmpC β-lactamase.23, 24 Therefore, the CAZ concentration will diminish over time. In contrast, with sub-MIC MEM (a poorer substrate of AmpC than CAZ) we see potentiation with two single Tn mutants (slt::Tn and mltG::Tn). This result is consistent with a more stable titer of MEM in the MEM-exposed growth-curve assays. The results of both the swarm assays and the growth-curve assays, in the presence of either CAZ or MEM challenge, implicate bulge formation as a pre-requisite for early lysis. We conclude that the potentiation by bulgecin at the sub-MIC of CAZ used (0.28 μg mL−1) results from concurrent loss of a subset (that of Slt and MltD) LT activities. Potentiation at the sub-MIC of MEM (0.14 μg mL−1) is seen upon Tn-insertion inactivation of either the slt or mltG genes.</p><!><p>Lytic transglycosylases of Gram-negative bacteria have a central role in sensing the cell-wall damage resulting from encounter with β-lactam antibiotics. In addition, LTs may have a key role in the repair of this damage, such as by removal of the accumulation of nascent non-crosslinked glycan strands.5 Failure of this repair of the peptidoglycan contributes to the eventual cell lysis. Indeed, the products (referred to as anhydromuropeptides) of LT catalysis are enriched in non-crosslinked pentapeptide stems.12 We obtained support for a function of the LTs in the repair of damaged peptidoglycan. A bacterial swarm assay used the parent P. aeruginosa PAO1 strain inoculated at a 20 mm distance from a co-spot of CAZ (5 μg) and bulgecin A (350 μg). Confocal-microscopic imaging at 14 h verified that the bacteria at the swarm edge facing the antibiotic spot were elongated and showed septal bulging. These damaged cells were collected by aspiration using 5 μL of FAB-glucose broth. These cells were diluted 3-fold so as to decrease the concentration of CAZ. The diluted cells were imaged on a glass slide to verify a homogenous culture of septal-bulge-containing cells (data not shown). A portion (1 μL) of these cells was placed onto a swarm plate without either CAZ or bulgecin A. A second 1 μL portion was mixed with a solution of 350 μg of bulgecin A, and was added to a second plate. The two plates were imaged at their swarm edges at 2 h and 5 h time after inoculation (Figure S11). Bacteria premixed with bulgecin A exhibit delayed recovery compared to bacteria on the control plate (Figure S11A). Moreover, the bacteria plated on the bulgecin A plate displayed significantly more bulges and less recovery from the damage caused by CAZ leading to more cell death (Figure S11B). Bulgecin A delays recovery of the bacteria during the period following antibiotic exposure. These observations affirm, at the whole-cell level, an important role of LTs in cell-wall repair.</p><!><p>We propose two mechanisms that may contribute to the ability of bulgecin A to potentiate CAZ. LTs initiate the signaling pathway that results in induction of the expression of the AmpC β-lactamase resistance enzyme.5,25,26 Accordingly, bulgecin A-treated cells may arrest (or delay) this potent resistance mechanism. Second, we have experimental evidence that the bulge structure seen in cells exposed to both CAZ and bulgecin A is a point of structural weakness. A movie of the slt Tn-insertion mutant exposed to CAZ on a swarm plate captures this vulnerability (Movie1). Figure 5 shows four snapshots from this movie. The white arrow in Figure 5A (at 5 o'clock) points to a bulge that is in the process of expansion. This bulge grows (Figure 5B) and bursts subsequently (Figure 5C). The red fluorescence that appears (Figure 5C, 33 s into the movie) indicates spillage of the DNA from the bacterium upon the burst. The culminating image implicates loss of the structural integrity of the bacterium, seen now only in the form of the red fluorescence plume (at 74 s, Figure 5D). Examination of other bacteria in the field of these images suggests the possibility of compromised viability prior to the burst of their bulges. In Figure 5B the green fluorescence of the cells immediately flanking (above) the bulge at 5 o'clock appears to fade, presumably as a result of cessation of GFP biosynthesis (Figure 5B). These images attest that the coordinated actions of a PBP inhibitor (the β-lactam) and of an LT inhibitor (bulgecin A) compromise, in the form of osmotically fragile bulges, the integrity of the peptidoglycan of the bacterium.</p><p>The structural character of the bulge defect was examined by scanning electron microscopy (SEM). In the absence of either β-lactams or bulgecin A, the images of the PAO1 strain of P. aeruginosa strain show rods with the typical (2 µm length, 1 μm width) dimensions and with the customary crenellated exterior surface (Figure 6A). The presence of bulgecin A alone gives an image that is indistinguishable (Figure 6B). The presence of CAZ alone gives lengthened rods as a result of blocked septation (Figure 6C). The presence of both CAZ and bulgecin A gives an initial mid-cell bulge (Figure 6D) that progresses to substantial size (Figure 6E) prior to structural failure (Figure 6F). The presence of both MEM and bulgecin A effects an initial mid-cell bulge (Figure 6G) that is (other than the very different cell lengths) qualitatively similar to the image of Figure 6D. The MEM/bulgecin A combination progresses to a cell whose appearance (Figure 6H) suggests greater structural defect than is seen with CAZ/bulgecin A (Figure 6E) prior to cell lysis (Figure 6I). Prior to lysis both images of the β-lactam/bulgecin A-treated cells (Figure 6E and 6H) show an exterior surface similar to the control images (Figure 6A and 6B). This similarity suggests that bulge growth coincides with the underlying presence of the entirety of the outer-membrane-creating biosynthetic apparatus. As the visualization of cell lysis indicates that the interior of the bulge contains DNA (Figure 5), bulge formation is interpreted as a fundamentally derailed pattern of bacterial growth, as a consequence of the synergistic perturbation of peptidoglycan biosynthesis resulting from the simultaneous loss of critical PBP and LT functions.</p><!><p>P. aeruginosa is an opportunistic pathogen.27-30 As all of the above experiments used the non-pathogenic P. aeruginosa PAO1 strain, the relevance of bulgecin potentiation of β-lactam antibiotic efficacy was assessed by MIC determinations for ten clinical P. aeruginosa strains (Table S2). The preponderance of the MIC data of this table shows a 2-fold to 4-fold reduction in the β-lactam MIC value in the presence of 100 μg mL−1 bulgecin A. The potentiation of CAZ, and of MEM, by bulgecin A is not unique to P. aeruginosa PAO1. These experiments address three fundamental questions. The first question is whether this relationship has clinical implication. While the determination of MIC values against pathogenic strains is not a complete answer, the data of Table S2 indicate that the fundamental, molecular-level basis for this potentiation exists among a range of P. aeruginosa strains. Complementary study on LT inhibition in other Gram-negative bacteria indicates that an intimate molecular-level relationship between the PBPs and the LTs is retained in other Gram-negative bacteria.31-33 The second question is the nature of the bulge. The SEM images (Figure 6) support an interpretation of the bulge as a misshape arising from fundamentally flawed cell growth. Given the pronounced curvature of the bulge, and the emerging recognition of the outer membrane as a load-bearing component of the bacterial cell envelope,34 it is hardly surprising that cell lysis coincides with structural failure of the bulge (Figure 5). Given the extensive covalent tethering of the peptidoglycan to the proteins and lipids of the Gram-negative outer membrane,35 structural derailing of ordered peptidoglycan biosynthesis could concurrently derail the many events of outer-membrane biosynthesis.36</p><p>The third question is the molecular origin of the LT inhibitor-PBP inhibitor potentiation. This study identifies in particular three LTs (Slt, MltD, and MltG) of the 11 found in P. aeruginosa as complicit in β-lactam-induced bulge formation. Both the MS analyses (Figure 3) and the selectivity seen with respect to the structural consequence of β-lactams with the LT-Tn mutants (Figure S1 and S3), pair the Slt and MltD LTs as complementary targets and quite possibly LTs with redundancy of physiological function. Moreover, the restoration of LT activity (by withholding bulgecin A) coincident with the progressive regain of the normal cell shape of P. aeruginosa (Figure S11) further affirms a contribution of LT activity to cell-shape creation.37 The absence of a discernible morphological phenotype with respect to most of the other LTs could follow from the absence of a direct PBP-LT paired function, and (with respect to bulgecin A as an LT inhibitor) the fact that bulgecin A is a selective and not a universal inhibitor of the LT family.38</p><p>The points of similarity and difference between the two β-lactams used in this study, ceftazidime and meropenem, attest to the complexity of the paired LT-PBP functions. A key similarity for both pair is bulge formation at the apparent mid-cell location (where the biosynthesis of the septum initiates). This location for bulge formation arguably is not surprising with respect to ceftazidime as the PBP inhibitor. Ceftazidime inhibits preferentially PBP1a and PBP3.39 PBP3 is a component of the divisome machinery. Sub-inhibitory concentrations of selective PBP3-inactivating β-lactams give dramatically elongated/filamentous cells (Figure 2) as a result of unimpeded elongation and failed septation. Sub-MIC concentrations of meropenem preferentially inhibit both PBP2 and PBP3,40 resulting in an initial mid-cell bulge in the rod bacterium (Figure 6) that ultimately transitions to a spherical cell, likely as a result of the loss of function of at least the two PBPs. Although PBP2 is most often described as a key PBP of the elongasome of the Gram-negative bacteria,41,42 PBP2 also co-localizes with PBP3 at the nascent septum.41,43 This concurrence at the apparent site of bulge synthesis may indicate that the loss of LT function (by bulgecin A inhibition) compromises the transition between elongasome and divisome peptidoglycan biosynthesis. The seam of peptidoglycan corresponding to this transition is significantly more structurally fragile than either the peptidoglycan of the sidewall, or the peptidoglycan of the poles.44 Meropenem additionally inactivates l,d-transpeptidase-dependent peptidoglycan synthesis.45,46 Its multiple targets may account for the ability of meropenem–bulgecin A to more rapidly progress to lysis compared to the case of ceftazidime–bulgecin A.</p><p>An unexpected observation with meropenem (but not ceftazidime) was the appearance of a bulge phenotype with the mltG::Tn strain (Figure S3). Although this observation is consistent with our evaluation of bulgecin A as an inhibitor of (E. coli) MltG (Figure 3), the observation does not reconcile with the inchoate understanding of the function of the MltG LT. MltG is the most recently identified member of the Gram-negative LT family, and is suggested to cut to size the glycan strands of nascent septal peptidoglycan.31 The assignment of a molecular basis for bulge formation upon MIC exposure of the mltG::Tn strain to meropenem is not possible at this time.</p><p>The key accomplishment of this study is its sharp identification of three of the 11 LTs of P. aeruginosa—Slt, MltD, and MltG—for intensive experimental study using β-lactam–bulgecin A pairs. The β-lactam family is structurally diverse with a corresponding diversity of PBP inactivation patterns.47,48 These studies must also reconcile the fact that loss of function of particular LTs can coincide also with increased β-lactam resistance.10,32,49,50 The experimental approach of this study provides a pathway to identify the β-lactam structures having a PBP profile that aligns optimally with concurrent loss of LT function while minimizing AmpC β-lactamase induction;25,51,52 that minimizes AmpR- and LT-dependent cross-talk with other resistance networks;53 that favors the LT loss-of-function appearance of peptidoglycan defects facilitating antibiotic permeation;10 that directs the structural manipulation of the LT inhibitor toward optimal potency and selectivity;32,54 and that will allow a molecular-level understanding as to basis for elongasome and/or divisome misfunction upon simultaneous PBP and LT incapacitation.</p><!><p>Complete descriptions of the strains used in the study, the confocal imaging, non-denaturing mass spectrometry chromatograms, growth curve assay negative data for the remaining Tn-insertion mutant strains, cell-wall recovery study, and MIC determinations is provided in the SI Materials and Methods.</p>

PubMed Author Manuscript

Triplet Acceptors with a D‐A Structure and Twisted Conformation for Efficient Organic Solar Cells

AbstractTriplet acceptors have been developed to construct high‐performance organic solar cells (OSCs) as the long lifetime and diffusion range of triplet excitons may dissociate into free charges instead of net recombination when the energy levels of the lowest triplet state (T1) are close to those of charge‐transfer states (3CT). The current triplet acceptors were designed by introducing heavy atoms to enhance the intersystem crossing, limiting their applications. Herein, two twisted acceptors without heavy atoms, analogues of Y6, constructed with large π‐conjugated core and D‐A structure, were confirmed to be triplet materials, leading to high‐performance OSCs. The mechanism of triplet excitons were investigated to show that the twisted and D‐A structures result in large spin–orbit coupling (SOC) and small energy gap between the singlet and triplet states, and thus efficient intersystem crossing. Moreover, the energy level of T1 is close to 3CT, facilitating the split of triplet exciton to free charges.

triplet_acceptors_with_a_d‐a_structure_and_twisted_conformation_for_efficient_organic_solar_cells

<!>Introduction<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!>Conclusion<!>Conflict of interest<!>

<p>L. Qin, X. Liu, X. Zhang, J. Yu, L. Yang, F. Zhao, M. Huang, K. Wang, X. Wu, Y. Li, H. Chen, K. Wang, J. Xia, X. Lu, F. Gao, Y. Yi, H. Huang, Angew. Chem. Int. Ed. 2020, 59, 15043.</p><!><p>OSCs have rapidly developed over the past decades owing to their advantages of low cost, flexibility, and light weight.1 With the efforts on the materials development and device engineering, the power conversion efficiency (PCE) of OSCs have now reached over 17 %.2 The working mechanism of OSCs includes photon absorption and exciton generation, exciton diffusion, exciton split and charge generation, charge transport, and charge collections. Thus, the PCE of OSCs equals to the time of the efficiency of each step. Obviously, one of the important strategies of improving the efficiency of OSCs is to increase the exciton diffusion distance. Although triplet excitons can travel longer than singlet excitons, the role and mechanisms of triplet excitons in OSCs are still elusive.3 Thus, it is of importance to develop triplet materials to investigate the mechanisms.</p><p>The generation of T1 state depends on the enhancement of the intersystem crossing (ISC) from the lowest singlet state (S1) to T1.4 According to the perturbation theory, the rate constant (k ISC) of ISC is given by Equation (1):(1)kISC∝⟨1Ψ|H^SO|3Ψ⟩/exp(ΔE2ST)</p><p>where ⟨1 Ψ|Ĥ SO|3 Ψ⟩ is the spin–orbit coupling matrix element, Ĥ SO is the spin–orbit coupling Hamiltonian, and ΔE ST is the energy gap between the singlet and triplet states. This equation suggests that large spin–orbit coupling value and small ΔE ST can afford high k ISC. Incorporation of heavy atoms into the π‐conjugated systems can enhance the spin–orbit coupling, facilitating the ISC to generate triplet excitons. Various triplet materials containing heavy atoms have been developed for high‐performance OSCs. For example, Yang et al. reported a triplet platinum porphyrine‐based donor materials for OSCs, affording an efficiency of 2.1 %,5 while Huang and co‐workers introduced iridium into the backbone of PTB7 to significantly improve the efficiency of OSCs with over 40 %, affording an efficiency of 8.71 %.6 In 2017, Huang et al. reported the first tellurophene‐based triplet acceptors for OSCs.7 The diffusion distances of triplet excitons were estimated to be 30 nm, which is comparable to other triplet fullerene derivatives. As a result, an efficiency of 7.52 % was achieved, which is much higher than that of the thiophene analogue based OSCs. According to Equation (1), reducing ΔE ST is another important method to increase k ISC to achieve triplet materials. One of important strategies to minimize ΔE ST is combining nonplanar donor (D) and acceptor (A) units in conjugated systems, which has been employed to construct various organic room‐temperature phosphorescence8 and thermally activated delayed fluorescence materials.9 However, these type of triplet materials have never been used for OSCs since their twist structures usually led to weak light absorption intensities and low charge transport mobilities, which is detrimental for efficiency of OSCs.</p><p>Herein, two twisted‐conformation molecular semiconductors with A‐D‐A′‐D‐A structure (H1 and H2), analogues of Y6,2c, 10 were constructed based on a large π‐conjugated fused core (Figure 1 b,c), which were shown to be triplet acceptors with strong light absorption, supported by steady and transient photoluminescence, and absorption spectroscopy, electron paramagnetic resonance (EPR), magneto‐photocurrent (MPC), and time‐dependent density function theory (TD‐DFT). The results revealed that the D‐A structure with nonplanar conformation reduced the ΔE ST and facilitated the ISC, yielding triplet excitons efficiently. Moreover, the energy level of the T1 state is rather close to the 3CT state, which is beneficial for the split of triplet excitons to free charges. Finally, the large π‐conjugated fused core of the acceptors afforded strong light absorption, which is beneficial for the photocurrents. As a result, high‐performance OSCs based on these acceptors were fabricated to afford efficiencies of over 15 %, demonstrating that the triplet excitons were generated and split in the blend films to contribute to the PCE, supported by magneto‐photocurrent and transient spectroscopy.</p><!><p>The molecular structures of a) donor polymer PBDB‐T and PM6, b) Y6, and c) H1, H2. d) Normalized UV‐vis absorption spectra of acceptors as solutions. e) Normalized UV‐vis absorption spectra of donors and acceptors as thin films. f) Energy levels of PBDB‐T, PM6, H1, H2 and Y6 obtained from CV.</p><!><p>The compounds H1 and H2, synthesized through Knoevenagel condensation (Supporting Information, Scheme S1), were fully characterized with 1H and 13C NMR spectroscopy and elementary analysis. Figure 1 d shows that the absorption spectra of Y6, H1, and H2 in solution were rather close to each other, in the range of 400–800 nm with an absorption peak at 735 nm, while the absorption peaks of the thin films demonstrated around 90 nm red‐shift (Figure 1 e), indicating that Y6, H1, and H2 possess strong intermolecular interactions and electronic coupling in the solid state.10a The absorption coefficient were estimated to be 9.95×104 cm−1, 1.01×105 cm−1, and 1.03×105 cm−1 for H1, H2, and Y6 films, respectively (Supporting Information, Figure S6), which can be attributed to the large π‐conjugation.</p><p>The electrochemical properties of these acceptors were investigated by cyclic voltammetry (CV; Supporting Information, Figure S8). According to the equation E HOMO/LUMO=−e(E onset,ox/red+4.71 eV), the energy levels of HOMO/LUMO for H1, H2, and Y6 are calculated to be −5.54/−3.84 eV, −5.62/−3.94 eV, and −5.76/−4.03 eV, respectively (Figure 1 f). Obviously, Y6 possesses the lowest energy levels owing to the strong electron‐withdrawing properties of four fluorine atoms in the end groups.</p><p>Time‐resolved transient photoluminescence was employed to estimate the excited state lifetime of these acceptors. Figure 2 a showed that Y6, H1, and H2 in 2‐methylfuran solution possess a short excited‐state lifetime of 11.67 ns, 13.36 ns, and 10.41 ns at 298 K, respectively. However, the lifetime increased sharply to 6.07 μs, 8.15 μs, and 7.66 μs, respectively, when the solution samples were cooled down to 77 K (Figure 2 b). This observation suggested that ISC is efficient in these acceptors, generating a large amount of triplet excitons.11</p><!><p>a) Time‐resolved transient photoluminescence decay traces of H1, H2, and Y6 at 298 K. b) Time‐resolved transient photoluminescence decay traces of H1, H2, Y6 at 77 K. c) Transient absorption spectrum of H1 in degassed chloroform. d) Decay traces of H1 probed at 550 nm. e) Magneto‐photocurrent of H1, H2, and Y6 pristine films; the device structure is ITO/ZnO/prinstine film/MoO3/Ag. f) Electron paramagnetic resonance spectra of H1 in dark and under light conditions.</p><!><p>Transient absorption spectroscopy is another effective method to investigate the dynamics of triplet excitons. As shown in Figure 2 c, transient spectra in degassed chloroform solution share two photoinduced absorption (PIA) bands at around 530 nm and 850 nm, standing for the kinetic process of excited state absorption (ESA),12 and a strong ground state bleaching (GSB) peak at around 660 nm, consistent with the solution absorption spectra. The decay traces of H1, H2, and Y6 are shown in Figure 2 d and the Supporting Information, Figure S9, and their lifetimes were evaluated to be 42 μs, 55 μs, and 41 μs, respectively, which is consistent with the lifetime measured by time‐resolved transient photoluminescence. Thus, the ESA peaks were reasonably assigned to the upper transitions from T1 to Tn of these acceptors.13</p><p>TD‐DFT calculation was applied to probe the generation of the triplet excitons. Two halves of the molecules share a dihedral angel of 16.87°, 16.90°, and 16.88° for H1, H2, and Y6, respectively (Supporting Information, Figures S10–S12), suggesting their twisted structures, which may be beneficial for reducing the ΔE ST.14 As discussed above, the ISC process is decided by ΔE ST and spin–orbit coupling constants, where a small ΔE ST and a large spin–orbit coupling constant may lead to an efficient ISC. Detailed calculated data of excited states energy levels and spin–orbit coupling constants are summarized in the Supporting Information, Tables S1–S6. The ΔE ST of H1 between S2 and T3 is only 0.0672 eV, and the spin–orbit coupling constant of around 0.1 between S2 and T3 is exhibited. These data combined are comparable to afford a high k ISC,15 thus providing an efficient ISC channel.</p><p>Magneto‐photocurrent experiments were performed to investigate the triplet properties of these acceptors.16 Magneto‐photocurrent can be defined as MPC=(I(B)−I(0))/I(0),17 in which I(B) and I(0) are the photocurrent in the presence and absence of magnetic field, respectively. The results of the pristine acceptor films are presented in Figure 2 e. The magnetic field can manipulate singlet‐to‐triplet ratio through Larmor precession, which will influence photocurrent effectively.18 All three acceptor films exhibited a negative signal as the magnetic field strength increases, which indicate triplet excitons are more likely to be produced at excited states, and the effects originate from the triplet‐charge reaction,19 which decreased photocurrent.</p><p>Electron paramagnetic resonance measurements can be applied to detect signals and analyze information of the states and excitons because it is a spin‐sensitive technique.20 The dark and under light electron paramagnetic resonance spectra were shown in Figure 2 f and the Supporting Information, Figure S13. An electron paramagnetic resonance signal was observed under light around 351 mT for these acceptors, suggesting their paramagnetic properties,21 and the corresponding g‐factors for H1, H2, and Y6 are 2.00412, 2.00499, and 2.00462 respectively. Considering the fingerprints and the magnetic field width of approximately 1.5 mT, the electron paramagnetic resonance signal can be attributed to triplet CT state (3CT) polaron pairs,22 which shall be transformed from 1CT polaron pairs through ISC. These results further illustrated the triplet nature of these acceptors.</p><p>PBDB‐T and PM6 were chosen as the donors to couple with the three acceptors since they have complimentary light absorption and matched energy levels. OSCs based on H1, H2, and Y6 were fabricated with a conventional structure of ITO/PEDOT:PSS/active layer/PDINO/Al to study their performance on OSCs. The typical current density‐voltage (J–V) curves and the external quantum efficiency (EQE) curves are shown in Figure 3 a,b, and the device performance parameters are summarized in Table 1. After preliminary optimization, a best PCE of 14.16 % for PBDB‐T:H1 devices was obtained with a V OC of 0.76 V, a J SC of 25.74 mA cm−2 and a FF of 71.40 %, which are close to reported results of Y14.23 When H1 is replaced by H2, the PCE climbed over 15 % with a V OC of 0.79 V, a J SC of 25.82 mA cm−2, and a FF of 73.86 %. The enhancement of J SC can be ascribed to the stronger absorptivity, while the enhancement of FF may be attributed by the better crystallinity of the chlorine atom,24 which lead to a higher electron mobility. The improvement of V OC is however abnormal, since H2 have a lower LUMO energy level compared to H1, which will be discussed later in the following parts. These results suggested that both H1 and H2 can be applied as high‐performance acceptors for efficient OSCs. However, the PBDB‐T:Y6 based solar cells only afforded a relatively low efficiency of 9.88 % (Supporting Information, Table S7). Thus, PM6 was used as the donor to couple with Y6, affording a high performance OSCs with a best PCE of 15.35 % (a V OC of 0.83 V, a J SC of 25.24 mA cm−2 and a FF of 74.07 %), which is comparable to the reported results.10a</p><!><p>a) J–V curves and b) EQE curves of the H1, H2 and Y6 based OSCs. c) Magneto‐photocurrent of H1, H2 and Y6 based OSCs. d) Transient absorption spectrum of PBDB‐T:H1 blend film. e) Decay traces of PBDB‐T:H1 blend film probed at 770 nm. f) EL and EQE spectra of PBDB‐T:H1 based devices.</p><p>Detailed photovoltaic parameters of the OPV cells based on ten devices.</p><p>Devices</p><p>V OC [V]</p><p>J SC [mA cm−2]</p><p>FF</p><p>PCE [%]</p><p>PBDB‐T:H1</p><p>0.76±0.01</p><p>25.74±0.21</p><p>0.71±0.02</p><p>14.06(13.70±0.13)</p><p>PBDB‐T:H2</p><p>0.79±0.01</p><p>25.82±0.19</p><p>0.73±0.01</p><p>15.12(14.89±0.19)</p><p>PM6:Y6</p><p>0.83±0.01</p><p>25.24±0.25</p><p>0.74±0.02</p><p>15.35(15.10±0.21)</p><!><p>The EQE curves (Figure 3 b) illustrated that the devices have a broad photoresponse range from 300 nm to 950 nm, which is consistent with the UV/Vis absorption of the blend films. All devices exhibited a high EQE of over 70 % from 450 nm to 850 nm, and the maximum EQE value are close to 85 %, suggesting an efficient process of photoelectron conversion for all devices. The integrated J SC results calculated for these devices are 24.92, 25.13, and 24.56 mA cm−2 for H1, H2, and Y6 based devices, respectively, which are close to the J SC from J–V measurements.</p><p>The magneto‐photocurrent on PBDB‐T:H1, PBDB‐T:H2, and PM6:Y6 OSC devices were then measured (Figure 3 c). All of the measurements exhibit a positive signal, and the magneto‐photocurrent are gradually increased with the rising field strength. In fact, since the electron and hole dissociation and recombination are spin‐dependent in the photovoltaic process for OSCs,25 such a line‐shape denotes that the dominant mechanism behind the increase of photocurrent is the exciton dissociation at charge transfer states owing to the increase of the magnetic field strength.26 Judging from Figure 3 c, the full width at half maximum (FWHM) of magneto‐photocurrent for PBDB‐T:H2 based devices seems to be the narrowest in comparison to the rest. It also exhibits the largest magneto‐photocurrent effect among these three, suggesting that the exciton dissociation at charge transfer states is more efficient in PBDB‐T:H2 based solar cells. The results are consistent with the device performance parameters given in Table 1, where the PBDB‐T:H2 based solar cells produce the highest J SC.</p><p>Transient absorption spectra experiments were employed to further analyze the dynamics of excitons in the blend films. All blend films were sealed with PMMA film to avoid oxygen quenching in air. The films were excited by a 600 nm laser beam (45 μW). Figure 3 d showed two strong GSB peaks at around 640 nm and 850 nm, which are consistent with absorption peaks of the pristine films of donors and acceptors, respectively. Moreover, a wide range of PIA band is observed between these two peaks, which stands for the charge‐transfer process.12 The decay lifetime was investigated for the strongest signal at 770 nm, and the decay traces (Figure 3 e; Supporting Information, Figure S15) demonstrate that the lifetime has dropped to 65 ns, 30 ns, and 45 ns for H1, H2, and Y6 in blend films, respectively. These excitons can usually be classified as triplet excitons, since singlet excitons will not be able to possess such a long lifetime.27 Considering the long lifetime of the excitons, geminate recombination were suppressed in the blend film,28 while non‐geminate recombination was observed to be rather weak, based on the light intensity dependence measurements (Supporting Information, Figure S18). Since geminate and non‐geminate recombination is both negligible in the blend film, these combined observations indicate that triplet excitons can be generated and split into free charges at the D/A interfaces.7</p><p>EQE and electroluminescence (EL) quantum efficiency were used to measure the energy losses and state energy levels to further understand the mechanism of the photovoltaic devices. The energy loss (ΔE) was divided into three different parts according to Equation (2):28 (2)ΔE=qΔV=Eg-qVOC=(Eg-qVSQOC)+(qVSQOC-qVradOC)+(qVradOC-qVOC)=(Eg-qVSQOC)+qΔVrad,belowgapOC+qΔVnon-radOC=ΔE1+ΔE2+ΔE3</p><p>in which VSQOC stands for the maximum voltage under the SQ limit, VradOC stands for the open‐circuit voltage when there is only radiative recombination existing, ΔVrad,belowgapOC stands for the energy loss for radiative recombination due to below gap absorption, and ΔVnon-radOC stands for the voltage loss of non‐radiative recombination.29 ΔE 1 is the radiative recombination energy loss above the band gap, which is unavoidable in all kinds of solar cells. ΔE 2 is the radiative energy loss below the band gap, owing to the nonstop function absorption. ΔE 3 is the non‐radiative energy loss, which comes from the non‐radiative recombination.30 It could be calculated by Equation (3):(3)ΔE3=qΔVrad,belowgapOC=-kTln(EQEEL)</p><p>The detailed data of the energy loss in devices of these two acceptors are enlisted in Table 2. The optical band gap (E g) can be extracted from EQE spectra.31 Both devices exhibited very close ΔE 1 value of 0.26 eV according to Shockley–Queisser (SQ) theory.32 As a result, VSQOC of H1 and H2 devices can be evaluated to be 1.15 V and 1.16 V, respectively. ΔE 2 can be derived from EQE spectra, and both devices have a steep curve of high sensitive EQE (Figure 3 f; Supporting Information, Figure S20), which shows that H1 and H2 possess a similar low ΔE 2 value of 0.07 eV and 0.06 eV, respectively.33 According to Equation (2), ΔE 3 was estimated based on EQEEL. H1 and H2 devices process EQEEL of 2.8×10−6 and 7.4×10−6, respectively (Supporting Information, Figure S21), which are relatively high in OSCs.29 Thus, the corresponding ΔE 3 are 0.33 eV and 0.31 eV for H1 and H2, respectively. This observation supported that the H1 based solar cells unusually possess a smaller V OC than H2 based ones, although H1 has a higher LUMO energy level than H2.10c</p><!><p>Detailed data of V OC loss and excited states energy levels of the PBDB‐T:H1 and PBDB‐T:H2 based devices.</p><p>Material</p><p>E g [eV]</p><p>ΔE [eV]</p><p>qVSQOC [eV]</p><p>ΔE 1 [eV]</p><p>ΔE 2 [eV]</p><p>ΔE 3 [eV]</p><p>S1 [eV]</p><p>T1 [eV]</p><p>CT [eV]</p><p>H1</p><p>1.41</p><p>0.66</p><p>1.15</p><p>0.26</p><p>0.07</p><p>0.33</p><p>1.41</p><p>1.06</p><p>1.35</p><p>H2</p><p>1.42</p><p>0.63</p><p>1.16</p><p>0.26</p><p>0.06</p><p>0.31</p><p>1.42</p><p>1.08</p><p>1.38</p><!><p>The energy levels of the CT state and excited state were evaluated to understand the roles of these states in the photovoltaic performances. We take S1 to be equivalent to E g here, and the energy levels of S1 of H1 and H2 are thus determined to be 1.41 eV and 1.42 eV, respectively. The T1 energies of H1 and H2 are 1.06 eV and 1.08 eV, respectively, based on the emission band of films occurred at low temperature (Supporting Information, Figure S22).34 The small ΔE ST between S1 and T1 may strongly promote ISC process,35 resulting in large amounts of triplet excitons in the system. Moreover, through fitting the low‐energy shoulder of the EL and EQE spectra,36 CT state energy levels of H1 and H2 are determined to be 1.35 eV and 1.38 eV,31 which is close to the T1 state. Consequently, triplet excitons may be allowed to form 3CT, which provides sufficient time to subsequently dissociate excitons into free charges and thus contributes to the photovoltaic performance.</p><p>To further understand the effect of the end‐groups on the fill factors, charge transport mobilities of blend films were investigated by space charge limit current (SCLC) method (Supporting Information, Figure S23). The hole mobilities for H1, H2, and Y6 based blend films are 5.17×10−4, 5.24×10−4, and 5.76×10−4 cm2 V−1 s, respectively, while the electron mobilities are 3.47×10−4, 4.21×10−4, and 3.89×10−4 cm2 V−1 s, respectively. The electron mobility of H2 based blend films is higher than that of H1 based ones, which may be because the H2 possesses stronger accumulation with its chlorine end‐groups, resulting more balanced hole/electron mobility and a higher FF.</p><p>Grazing‐incidence wide‐angle X‐ray scattering (GIWAXS) was used to probe the molecular packing of neat PBDB‐T, H1, and H2 pristine films and PBDB‐T:H1 and PBDB‐T:H2 blend film, and the results of the 2D GIWAXS patterns are shown in Figure 4 and the Supporting Information, Figure S24. Neat PBDB‐T film exhibited strong crystallinity with a (100) lamellar peak both in the out‐of‐plane (OOP) direction at q=0.28 Å−1 (d≈22.5 Å) and in the in‐plane (IP) direction at q=0.40 Å−1 (d≈15.8 Å). The polymer donor film also presents a π–π peak in the OOP direction at q=1.65 Å−1 (d≈3.8 Å). Neat H1 and H2 film both have a π–π stacking peak in the OOP direction at around q=1.75 Å−1 (d≈3.60 Å), and signal of H2 is even much stronger, which could be because the chlorine end‐group processes better accumulation than fluorine end‐group.10c Also, two peaks in the IP direction were observed in these two neat films at around q=0.25 Å−1 (d≈25.2 Å) and q=0.42 Å−1 (d≈15.0 Å). The peak at q=0.25 Å−1 (d≈25.2 Å) can be identified as lamellar peak, while the peak at q=0.42 Å−1 (d≈15.0 Å) may be ascribed to the backbone ordering owing to π–π stacking of the end‐group. The annealed blend film of PBDB‐T:H1 and PBDB‐T:H2 showed a strong peak at q=1.75 Å−1 (d≈3.60 Å) in the OOP direction, which is consistent with the π–π peak of neat acceptor films, and the π–π peak of neat donor film disappeared in the blend films, suggesting crystallinity of PBDB‐T has been weakened. Meanwhile, in these blend films, we can observe an enhancement of the lamellar peak of the polymer in the IP direction at q=0.28 Å−1 (d≈22.5 Å), and the backbone peak of acceptors in the IP direction at q=0.42 Å−1 (d≈15.0 Å) also vanished in blend films, meaning that the acceptor tends to arrange and stack alongside the polymer donor in the blend film.</p><!><p>a),b) 2D GIWAXS patterns of a) PBDB‐T:H1 and b) PBDB‐T:H2 blend films. c),d) Intensity profiles along the c) in‐plane and d) out‐of‐plane directions.</p><!><p>The surface morphology of films were studied by atomic force microscopy (AFM) and transmission electron microscopy (TEM). As shown in the Supporting Information, Figure S25, blend films of PBDB‐T:H1 and PBDB‐T:H2 both exhibited smooth surface with similar low root‐mean‐square (Rq) values of 1.34 nm and 1.55 nm, indicating the good miscibility between donor and acceptor. TEM images of blend films (Supporting Information, Figure S26 show the nanofiber structure with small phase domain, which is beneficial for the charge transport and separation, thus leading to a high J SC and FF.</p><!><p>Two novel acceptors H1 and H2 were synthesized, which along with Y6 were shown to be triplet materials, and the OSCs afforded a highest PCE of 15 %. Steady and transient photoluminescence and absorption spectroscopy showed that these materials possess strong light absorption and long lifetime excitons, while TD‐DFT calculations revealed that the twisted conformation and D‐A structure led to a considerable k ISC. Magneto‐photocurrent and electron paramagnetic resonance experiments further revealed the triplet nature of these acceptors. The triplet exciton pairs were generated and split in the blend films to contribute to the PCE, supported by magneto‐photocurrent, transient spectroscopy, EQE, and EL spectroscopy. This work sheds light on understanding the working mechanism of triplet excitons in OSCs.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

A Bioinspired Diversification Approach Toward the Total Synthesis of Lycodine-type Alkaloids

Nitrogen heterocycles (azacycles) are common structural motifs in numerous pharmaceuticals, agrochemicals, and natural products. Many powerful methods have been developed and continue to be advanced for the selective installation and modification of nitrogen heterocycles through C\xe2\x80\x93H functionalization and C\xe2\x80\x93C cleavage approaches, revealing new strategies for the synthesis of targets containing these structural entities. Here, we report the first total syntheses of the lycodine-type Lycopodium alkaloids casuarinine H, lycoplatyrine B, lycoplatyrine A, and lycopladine F as well as the total synthesis of 8,15-dihydrohuperzine A through bioinspired late-stage diversification of a readily accessible common precursor, N-desmethyl-\xce\xb2-obscurine. Key steps in the syntheses include oxidative C\xe2\x80\x93C bond cleavage of a piperidine ring in the core structure of the obscurine intermediate and site-selective C\xe2\x80\x93H borylation of a pyridine nucleus to enable cross-coupling reactions.

a_bioinspired_diversification_approach_toward_the_total_synthesis_of_lycodine-type_alkaloids

INTRODUCTION<!>Preparation of the Key Diversifiable Precursor.<!>Synthesis of (\xe2\x88\x92)-Casuarinine H, (\xe2\x88\x92)-8,15Dihydrohuperzine A, and (+)-Lycoplatyrine B.<!>Synthesis of Lycoplatyrine A and Lycopladine F.<!>CONCLUSION

<p>The Lycopodium alkaloids are a diverse group of natural products found in plants of the widely distributed Lycopodium genus, commonly known as clubmosses.1,2 Since the isolation of the first of these alkaloids, lycopodine, in 1881,3 a wealth of biosynthetically related alkaloids have also been isolated and characterized. These natural products are organized into four main classes (lycodine, lycopodine, fawcettimine, and a miscellaneous class) on the basis of their distinct carbon backbones, which arise as a consequence of C–C bond formation and rearrangement events during their putative biosyntheses.2 Many Lycopodium alkaloids possess intriguing and complex molecular architectures, and also display promising bioactivity profiles. The archetypical lycodine alkaloid huperzine A (1, Figure 1a), for example, is a potent and selective acetylcholinesterase (AChE) inhibitor and also demonstrates non-cholinergic neuroprotective effects.4–6 This bioactivity is of interest for the symptomatic treatment of Alzheimer's disease and other neurodegenerative disorders.2,4–6 The combination of interesting structural features and note-worthy bioactivity continue to drive synthetic studies toward Lycopodium alkaloids and their analogues.7–12</p><p>Synthetic strategies that enable late-stage structural modification and diversification of a common advanced intermediate can provide versatility that facilitates efficient access to a range of products that might otherwise each require significant synthetic investment. A rapidly growing catalog of C–H bond functionalization technologies has powerfully expanded the processes available for such structural alterations, typically elaborating around the periphery of a molecule.13 Alternatively, C–C bond cleavage and functionalization strategies represent a key complementary approach which can be applied to remodel not only the periphery but also the core carbon skeleton of organic compounds.14 Although C–C cleavage tactics typically result in a decrease in molecular complexity -- in contrast to Corey's retrosynthetic paradigm15 -- they can lead to the identification of new retrosynthetic disconnections. In turn, such methods could enable rapid access to a diverse range of natural products or bioactive agents from a single compound, which, albeit more structurally complex, is easily obtained through chemical synthesis, biosynthesis, or synthetic biology.</p><p>The ubiquity of nitrogen heterocycles in pharmaceuticals,16 agrochemicals, and alkaloids17 render them attractive structural motifs for diversification to efficiently access underexplored chemical space.18 Therefore, a variety of methods for both the introduction and selective functionalization of azacycles continue to be reported.19–21 Inspired by these contributions, we envisioned nitrogen heterocycles as versatile synthetic handles that would enable the expedient preparation of a collection of lycodine-type alkaloids (2–4, 8, 9, Figure 1a) from a common, readily prepared, precursor through a series of programmed oxidation and C–C bond cleavage events in analogy to their biosynthesis.2,22</p><p>Although the complete biosynthetic pathways to the Lycopodium alkaloids remain to be fully elucidated,23 biochemical studies have suggested that these compounds derive from phlegmarine (13), which arises from the coupling of pelletierine (11) and 4-(2-piperidyl) acetoacetate (12), both of which originate from L-lysine (10, Figure 1b).2</p><p>Subsequent closure of ring B through bond formation between C13 and C4 furnishes the characteristic [3.3.1]-bicyclic scaffold of the lycodine class. A series of oxidative modifications, which include oxidation of the A-ring to the corresponding pyridone (e.g., in N-desmethyl-β-obscurine, 6) or pyridine (e.g., in lycodine, 7), C-ring cleavage, and excision of C9 further diversifies the parent scaffold, yielding a range of alkaloids including 1–6 (Figure 1b, blue arrows).</p><p>On the basis of these presumed biosynthetic events, we envisioned a retrosynthesis (Figure 1c) in which 8,15-dihydrohuperzine A (3)24 could arise from casuarinine H (2)25 through olefin isomerization, whereas lycoplatyrine B (4)26 could be accessed from 2 through semi-reduction of the pyridone. Casuarinine H (2) was traced back to functionalized tricyclic intermediate A through decarboxyolefination. In turn, A could be formed from the readily accessible key precursor N-desmethyl-β-obscurine (6) through oxidative functionalization and cleavage of the C9–N bond.</p><p>Another small set of structurally unique lycodine alkaloids bearing substitution at the C2 position of the pyridine A-ring (e.g., lycoplatyrine A,26 8, and lycopladine F,27 9) is proposed to arise biosynthetically through electrophilic substitution on lycodine (7) or the corresponding dihydropyridine by a Δ1-piperidinium or Δ1-pyrrolinium cation (or the corresponding imines; Figure 1b, green arrows).26,27 Subsequent oxidative cleavage of the pyrrolidine ring in 14 is suggested to provide lycopladine F (9), analogous to the oxidative ring cleavage pathway that leads to metabolic products of nicotine.28 Overall, we envisioned lycoplatyrine A (8) and lycopladine F (9) could be accessed through cross-coupling of appropriate C(sp3) nucleophiles with a functionalized lycodine analog (B), which again would be prepared from the key obscurine scaffold 6. The required deoxygenation of precursor 6 and site-selective functionalization at C2 would rely upon precedent demonstrated by our laboratories in the total synthesis of the dimeric lycodine alkaloids complanadine A and B.29,30</p><!><p>Our investigations commenced with the development of a robust synthesis of N-desmethyl-β-obscurine (6), the late-stage common intermediate for the synthesis of all of the alkaloids described here. A convergent route featuring a diastereoselective formal (3+3)-cycloaddition to form the three contiguous stereocenters and two C–C bonds in ring B of 631 was adapted from literature protocols by Schuster,32 Caine,33 Dake34 and Jung35 as well as our own previous studies.29</p><p>The coupling partner that would lead to ring A, dihydropyridone 17, was prepared from β-ketoester 15 through a Michael addition into acrylonitrile followed by decarboxylation to give nitrile 16. Subsequent nitrile hydration and cyclization in vacuo delivered 17 in 18% overall yield (Scheme 1a).29,31</p><p>The C/D ring cycloaddition partner 22 was prepared from (+)-pulegone (18) in six steps and 28% overall yield (Scheme 1b).32,33 The sequence was initiated by Weitz–Scheffer-type epoxidation of the exocyclic olefin group of 18, which provided a 1:2 mixture of epoxide isomers (19).38,39 Subsequent nucleophilic opening of the epoxide with sodium thiophenolate and concomitant retro-aldol reaction delivered the phenylthioether,33 which was selectively oxidized to sulfoxide 20 with sodium perborate.34 α-Alkylation of 20 with acrylonitrile, followed by thermal syn-elimination of phenylsulfenic acid gave enone 21,35,36 which was protected as the ethylene glycol ketal and reduced with LiAlH4 to deliver primary amine 22.32 The two building blocks (17 and 22) were ultimately coupled upon heating with perchloric acid (Scheme 1c). Under these conditions, oxygen-sensitive α,β-unsaturated iminium ion 22a and the open-chain enolamide 17a are presumably formed in situ and undergo the desired formal cycloaddition to furnish N-desmethyl-α-obscurine (5).29,31,37 Boc-protection of the piperidine nitrogen in 5 and dehydrogenation of the dihydropyridone ring using lead(IV) acetate provided N-Boc-β-obscurine (23) in 49% yield over three steps.</p><p>As an alternative to the oxidation of Boc-protected 5 using stoichiometric lead(IV) acetate, we investigated a photocatalytic dehydrogenation protocol.40,41 Our preliminary results demonstrated that N-Boc-5 was readily oxidized to 23 (57% yield) in the presence of an iridium(III) photoredox catalyst (Ir[dF(CF3)ppy]2(dtbbpy)PF6) with potassium persulfate as the terminal oxidant upon irradiation with blue light (λ = 450 nm) under anoxic conditions. In the absence of light or the photoredox catalyst, only traces of product (6%) were formed in the best case, whereas under aerobic conditions complete decomposition of the substrate was noted (see Section 3.1 in the Supporting Information). Despite attempts to optimize this reaction, we were unable to obtain yields comparable with those achieved with lead(IV) acetate (90%). Therefore, the latter conditions were employed for the preparation of large quantities of material. Finally, pyridone O-triflation of 23 delivered fully protected β-obscurine scaffold 24 in 78% yield.29</p><!><p>Our envisioned route toward the lycodine alkaloids casuarinine H (2), 8,15-dihydrohuperzine A (3), and lycoplatyrine B (4) required the identification of suitable conditions to effect the bioinspired oxidative cleavage of the C9–N bond in protected tetracycle 24 or a related obscurine congener. To this end, we pursued several conditions for C–N cleavage and functionalization that included biocatalytic and transition metal-mediated approaches.</p><p>Biocatalytic methods were explored as a means to achieve a protecting group-free oxidation of the C9–N bond, reminiscent of the proposed biosynthetic tailoring process. Although the requisite biosynthetic enzymes have not been identified, we posited that other established biocatalysts capable of oxidizing C–heteroatom bonds could accept the bicyclo[3.3.1]nonane scaffold of 5 as a substrate while retaining site-selectivity. A screening set comprised of 14 commercial and in-house heterologously expressed copper-42,43 and flavin-dependent oxidases,44–46 a pyrroloquinoline (PQQ) dependent dehydrogenase,47 a horseradish peroxidase (HRP),48 and a laccase/TEMPO redox mediator system49 was assembled. However, overview screenings under representative conditions did not identify any oxidation activity with unprotected substrate 5 (see Section 3.2 in the Supporting Information for details).</p><p>We therefore sought to examine other established chemical conditions for the oxidation of carbamate-protected saturated nitrogen heterocycles. While methods employing iron50 and copper51 redox mediators in combination with peroxides failed to generate the anticipated enamine or enamide products, we observed that sub-stoichiometric quantities of RuO2 with sodium periodate as stoichiometric oxidant in a mixture of tBuOH and water resulted in piperidine oxidation to yield 25 (Scheme 2a).48,53 Although oxidation under these conditions by the presumed in situ generated RuO4 catalyst was expected to give the corresponding amino acid (i.e., following hydrolysis of an intermediate C-ring iminium ion and oxidation of the resulting aldehyde), cyclic imide 25 was obtained in 86% yield. Additional experiments demonstrated that the electronically deactivating triflyl group on the pyridone oxygen was critical to the success of the piperidine oxidation-- oxidation of derivatives of 25 bearing methyl-, benzyl-, or SEM- groups instead of the triflyl moiety proved unsuccessful under identical conditions.</p><p>We envisioned that hydrolysis of imide 25 followed by decarboxyolefination of the resulting carboxylic acid could offer an attractive strategy to excise C9 and install the required unsaturation at C10–C11. Treatment of 25 with aqueous LiOH at the elevated temperatures required for imide hydrolysis resulted in undesired concomitant triflate cleavage. Therefore, a methyl ether was introduced in place of the triflate prior to imide hydrolysis to yield carboxylic acid 26. Unfortunately, subjecting 26 to classic Kochi oxidative decarboxylation conditions54 failed to deliver alkene 27. Additionally, an attempted Hunsdiecker-type decarboxyhalogenation55 resulted in the C-ring contracted pyrrolidine 28 (Scheme 2b), presumably the result of an SN2 displacement of the intermediate alkyl halide. While more recently developed decarboxyolefination conditions using metallo-organo-56 or organo-photocatalysts57 in combination with cobalt-based dehydrogenation catalysts furnished olefin 27 in 50% yield, a competing protodemetalation pathway leading to ethyl derivative 29 hindered further optimization of this process. Alternatively, desired terminal olefin 27 was obtained in higher yield (65%) through a Pd(0)-catalyzed decarbonylative elimination of an in situ-generated mixed anhydride of 26.58 Deprotection of 27 using TMSI12 completed the first total synthesis of the neuroprotective compound (−)-casuarinine H (2, Scheme 2c).25 Semireduction of the pyridone moiety in 2 with samarium metal in aqueous HCl59 cleanly yielded (+)-lycoplatyrine B (4)26 in 84% yield, also constituting the first total synthesis of this Lycopodium alkaloid. Furthermore, treatment of terminal olefin 27 with an in situ-generated palladium hydride catalyst effected isomerization to the internal (E)-alkene in 81% yield.60 A subsequent TMSI-mediated deprotection delivered (−)-8,15-dihydrohuperzine A (3).24,61</p><p>The spectroscopic data for synthetic (−)-casuarinine H (2), (+)-lycoplatyrine B (4), and (−)-8,15dihydrohuperzine A (3) were in full agreement with those reported upon isolation of these natural products from the producing organisms.24–26 Taking advantage of this late-stage diversification approach, the target alkaloids 2–4 were prepared in 16 to 17 steps (longest linear sequence, LLS) and 1.7–4.5% overall yield from (+)-pulegone.</p><!><p>For the synthesis of Lycopodium alkaloids bearing substituents at C2 (i.e., 8–9), we envisioned a cross-coupling approach in which the key β-obscurine intermediate 24 would be elaborated to a selectively C2-functionalized lycodine derivative to serve as a common coupling partner. Accordingly, protected β-obscurine 24 was deoxygenated in the presence of a palladium catalyst and ammonium formate as reductant to deliver N-Boc lycodine (30). Subsequent iridium-catalyzed meta-selective C–H borylation29,62 of the pyridine A-ring and bromodeborylation63 furnished 2-bromolycodine (31) (Scheme 3a).</p><p>Lycoplatyrine A (8) features a C2 piperidine substituent as an epimeric mixture of undetermined absolute configuration,26 which we anticipated could be installed through the coupling of 31 with an α-functionalized piperidine derivative (Scheme 3b). We specifically envisioned the application of a method recently disclosed by our laboratory in which α-hydroxy-β-lactams such as 32 serve as surrogates for α-metallated N-heterocycles in a palladium-catalyzed coupling with aryl halides.20 This method was particularly attractive due to the mild and stereospecific nature of the cross-coupling, although the use of pyridyl bromides had not been previously demonstrated. As proposed, the coupling of 31 with racemic lactam 32, prepared from the corresponding piperidine-derived 2-oxophenylacetamide through a Norrish-Yang reaction,20 delivered 33 as a mixture of epimers at C2'. Cleavage of the 2-oxophenylacetamide and Boc-protecting groups under sequential basic and acidic conditions yielded lycoplatyrine A (8) as a 1:1.5 mixture of the anticipated C2' epimers.</p><p>According to the previously proposed mechanism for this coupling, the hydroxy group of the lactam coordinates to the palladium center before irreversible C–C bond cleavage (β-carbon elimination) driven by the release of ring strain in 32 delivers a C2'-palladated species in a stereoretentive manner (Scheme 3b, grey box).20,64 We therefore anticipated that the use of enantiomerically pure lactams (2'S)- and (2'R)-32 would enable the stereospecific piperidinylation of the lycodine scaffold at C2, and thus allow the assignment of absolute configurations at C2' in naturally occurring alkaloid 8.</p><p>To obtain α-hydroxy-β-lactam 32 in enantioenriched form, we first investigated enzymatic resolution methods. Despite extensive reaction engineering, selectivity for an enzymatic hydrolytic kinetic resolution65,66 of acetylated tertiary alcohol 32 with pig liver esterase (PLE) and lipase A from C. antarctica (CalA) was poor and therefore not synthetically useful (E ≤ 7) (see Section 3.5 in the Supporting Information). Alternatively, enantiomerically resolved lactams (2'S)- and (2'R)-32 were obtained from preparative chiral supercritical fluid chromatography (SFC).20 Coupling of lactams (2'S)- and (2'R)-32 with lycodine bromide 31 gave single epimers of 33 in 65% yield, which were deprotected to provide single epimers of lycoplatyrine A (8) in 4.7% overall yield over 16 steps from (+)-pulegone (LLS). Comparison of the spectral data of single epimers of synthetic 8 with data for naturally-derived 8 revealed a slight excess of the (2'S)-8 epimer in material isolated from natural sources (d.r. 1.3:1).26 The cross-coupling product obtained using racemic 32 was also enriched in the same epimer (d.r. 1.5:1, vide supra), suggesting that the chiral lycodine scaffold exerts a low level of enantiodiscrimination and enantiotopic face discrimination in both the synthetic and natural coupling processes.</p><p>Indeed, our success in preparing single epimers of lycoplatyrine A (8) rested on the stereospecific coupling of α-hydroxy-β-lactams as surrogates for α-metallated piperidines, which otherwise typically suffer from low yields and poor stereoselectivities in the metalation step.67,68 Although an analogous β-lactam-based cross-coupling for five-membered nitrogen heterocycles is precluded due to the inaccessibility of the five-membered analogues of 32 with established photochemical methods,69 α-metallated pyrrolidines are excellent stereoselective coupling partners. These reagents set the stage for the preparation of the pyrrolidine analog of lycoplatyrine A ("pyrrolo-lycoplatyrine A", 14), which is hypothesized to be an intermediate in the biosynthesis of other lycodine-derived congeners including lycopladine F (9).27 For the synthesis of N-Boc pyrrolo-lycoplatyrine A (36), we turned to a method by Campos and coworkers70 for the stereoselective α-arylation of N-Boc-pyrrolidine (34) (Scheme 3c). Enantioselective ortho-lithiation of 34 in the presence of either (+)- or (−)-sparteine,71 transmetallation to form the corresponding organozinc species (35), and subsequent palladium-catalyzed coupling to lycodine bromide (31) delivered single C2'-epimers of the desired product (36) in high yield (88%). Subsequent deprotection provided each of the two C2'-epimers of pyrrolo-lycoplatyrine A (14) in 15 steps from (+)-pulegone (7% overall, LLS).</p><p>We sought to similarly access lycopladine F (9) via a direct coupling approach where the necessary amino acid moiety is appended at C2 of lycodine bromide (31, Scheme 3d). To this end, iridium-catalyzed photoredox conditions effected activation of bis-protected glutamic acid 37 through single-electron oxidation of the cesium carboxylate, followed by decarboxylative C–C bond scission and nickel-catalyzed C(sp3)–C(sp2) coupling with aryl bromide 31 to deliver protected lycopladine F (38) in 84% yield.72 A low nickel loading (1 mol%) was necessary to attenuate consumption of bromide 31 in a non-productive protodehalogenation pathway and achieve good yields of 38. Removal of both Boc protecting groups followed by hydrogenolytic cleavage of the benzyl ester in the presence of trifluoroacetic acid yielded lycopladine F (9) in 71% yield as a 1:1 mixture of epimers (4.8% yield over 16 steps LLS). The analytical data obtained for the synthetic material matched those reported for the natural material, which was isolated from Lycopodium complanatum as a 3.5:1 mixture of (2'S):(2'R)-epimers.27 We expect access to pyrrolo-lycoplatyrine A (14) and lycopladine F (9) to set the stage for studies into the biosynthesis of the latter natural product.27</p><!><p>In summary, we have developed the first total syntheses of lycodine alkaloids casuarinine H (2), lycoplatyrine B (4), lycoplatyrine A (8), and lycopladine F (9) and a total synthesis of 8,15-dihydrohuperzine A (3) employing the readily accessible tetracycle N-desmethyl-β-obscurine (6) as a common intermediate. A series of bioinspired modifications of the piperidine C-ring in 6, including oxidative ring cleavage, C–C bond scission with carbon atom excision, and olefin isomerization delivered tricyclic congeners 2–4. Conversion of the pyridone A-ring in 6 to the corresponding pyridine (7) and site-selective C–H functionalization to ultimately afford bromopyridine 31 enabled direct cross-couplings with saturated azacycles or an amino acid to complete the syntheses of C2-derivatized lycodine alkaloids lycoplatyrine A (8) and lycopladine F (9). The general late-stage peripheral derivatization and C–C functionalization strategies outlined herein may provide a basis for synthetic access to an even wider range of Lycopodium alkaloids. Our synthetic studies toward these compounds should also set the stage for a broader, more systematic assessment of their biosynthesis and bioactivity.25,26,61 Biological activities exerted by these natural products include a range of neuroprotective effects such as those observed for huperzine A,4,5 for example the attenuation of both glutamate-induced neurotoxicity and free radical-mediated oxidative stress.</p>

PubMed Author Manuscript

Focal Adhesion-Chromatin Linkage Controls Tumor Cell Resistance to Radio- and Chemotherapy

Cancer resistance to therapy presents an ongoing and unsolved obstacle, which has clear impact on patient's survival. In order to address this problem, novel in vitro models have been established and are currently developed that enable data generation in a more physiological context. For example, extracellular-matrix- (ECM-) based scaffolds lead to the identification of integrins and integrin-associated signaling molecules as key promoters of cancer cell resistance to radio- and chemotherapy as well as modern molecular agents. In this paper, we discuss the dynamic nature of the interplay between ECM, integrins, cytoskeleton, nuclear matrix, and chromatin organization and how this affects the response of tumor cells to various kinds of cytotoxic anticancer agents.

focal_adhesion-chromatin_linkage_controls_tumor_cell_resistance_to_radio-_and_chemotherapy

1. Introduction<!>2. Microenvironmental Signals Including Integrin Signaling Regulate Cellular Radio- and Chemosensitivity<!>3. The Impact of Focal Adhesion-Chromatin Linkage on Tumor Resistance against Irradiation and Cytotoxic Drugs

<p>Resistance to radiotherapy, chemotherapy, and novel molecular drugs still represents one of the major obstacles in cancer therapy [1–3]. Limited effectiveness of therapy inevitably results in progressive disease or recurrence, thereby reducing the chance of cure for the patients. Phenotypically, two types can be distinguished: pretherapeutically existing and acquired resistances [4, 5]. Acquired resistance to irradiation is not known, but anticancer drugs, both conventional and molecular, frequently induce defense mechanisms [6–8]. To optimize the efficacy of cytotoxic agents, it is necessary to ameliorate drug delivery to the tumor and to better understand the underlying molecular mechanisms causing the resistance or evolving the defense process [5, 9].</p><p>In order to address the latter, we and others focused on a particular cellular substructure called focal adhesion (FA) [10–17]. FAs are membrane areas, which cells employ to interact with the surrounding extracellular matrix (ECM) via integrin adhesion receptors [10, 17–22]. Due to their multiprotein composition including growth factor receptors, signaling, and adapter proteins, FAs are huge hubs for signaling downstream to control critical cell functions such as cell survival, proliferation, differentiation, and invasion [11, 13, 14, 18, 20, 22–30]. The highly complex interplay between all of these signaling molecules secures homeostasis of single cells as well as of tissues in the context of responses to external signals from the microenvironment.</p><p>In tumor cells, according to the hallmarks of cancer [31], the proper physiological communication with the extracellular space is massively disturbed as a consequence of gene mutations and epigenetic modifications. Despite tumor growth-driving gene mutations, malignant cells often retain a high degree of susceptibility to certain extracellular factors [13, 31–34]. Prime examples are microenvironmental signals induced by cell adhesion to the ECM, adhesion to neighbouring cells, and growth factor receptor-ligand interactions, which all contribute to tumor progression and resistance to cytotoxic injury resulting from chemotherapeutic drugs and irradiation [12, 32, 35].</p><p>Keeping these facts in mind, a lot of effort was put in the improvement of in vitro models that best reflect in vivo growth conditions [11, 33, 36–40]. Since the advent of ECM-based 3D cell culture assays, a large body of evidence has suggested that conventional monolayer models do not reflect the complexity of tissues, phenotypes of cells, and modifications in transcriptome, proteome, phosphoproteome, protein-protein interactions, and signal transduction as the 3D models [11, 40–53].</p><p>From the therapeutic point of view, flat monolayer cell cultures contain an ECM-integrin-cytoskeleton connection very different from 3D grown cells [22, 40, 45, 46, 50, 51, 54, 55]. Moreover, cell growth in 3D ECM shows additional features like 3D multicellular spheroid growth [38, 56, 57]. Common to all 3D conditions is that the responsiveness to extracellular signals, drug, and radiation sensitivity as well as the physical forces between ECM and cytoskeleton for controlling chromatin organization and gene expression is very different from cells cultured in 2D. In this paper, we discuss cell-adhesion-mediated radio- and chemoresistance in the context of signaling and interplay between ECM, integrins, cytoskeleton, nuclear matrix, and chromatin organization.</p><!><p>Next to genetic alterations, the microenvironment plays an important role for carcinogenesis, tumor progression, and development of therapy-resistant phenotypes [31]. A closer look at the initiators and promoters of this multistep process suggests that a combination of both extra- and intracellular events commonly occurs to activate proto-oncogenes and deactivate tumor suppressor genes [31]. With regard to carcinogenesis, the particular reasons for cancer development can only be assumed in the minority of cases. Exploring a "mature" tumor provides a picture of the accumulated alterations in the various molecular determinants, which maintain unlimited growth and cause both existing and de novo therapy resistance mechanisms. In addition to the aforementioned genetic modifications, various soluble and structural microenvironmental factors like cytokines, chemokines, growth factors, and ECM essentially contribute to anticancer therapy defense mechanisms [13, 58–63].</p><p>Importantly, the ECM has structural, signaling, and storage functions. Thus, cells communicate with the surrounding ECM by mechanotransduction, by integrin-mediated adhesion, and by growth factor release and subsequent binding to their cognate receptors [21, 64–68]. For the role of mechanotransduction, only one issue has been evidently shown: changes in ECM stiffness induce perturbations of normal cell physiology preparing the ground for malignant transformation [42, 50, 69–71]. Open questions are, for example, how changes in ECM expression pattern of tumors impact on tumor cell behavior or how therapy-related alterations in tumor structure influence integrin-ECM interactions and intracellular signaling. For cell-adhesion-mediated radioresistance (CAM-RR) and cell-adhesion-mediated drug resistance (CAM-DR), integrins play critical roles [59, 72, 73].</p><p>Integrins are transmembrane receptors consisting of an α and a β chain. The 18 α and 8 β subunits form 24 known αβ-heterodimers dependent on cell type and function [20]. Integrin signals are transferred via the cell membrane in both directions. The binding activity of integrins is regulated from the inside and is called inside-out signaling; the interaction of integrins with ECM proteins for signal transduction into the cell is called outside-in signaling [10, 17–22]. These interactions essentially contribute to the regulation of various cellular functions like proliferation, survival, adhesion, differentiation, migration of cells, and tissue integrity [29, 41, 74–77].</p><p>For many years, it remained unknown how integrin signaling mediates tumor cell resistance. Well known were increased survival and reduced apoptosis in irradiated or drug-treated tumor cells of varying origin like head and neck, lung, pancreas, glioma, colon, breast, cervix, prostate, myeloma, and leukemia [58, 78–83]. But which signaling cascades do transmit these biochemical prosurvival signals? Physiologically, a large set of signal transduction and adapter molecules assembles at the cytoplasmic integrin domain upon integrin binding to ECM [17, 20]. Formation of mature FAs is critical for robust cell adhesion to ECM as well as accessibility to the intracellular signaling network for optimized regulation of key cellular processes [10, 16, 17]. For this signaling, integrins and growth factor receptors need to cooperatively and mutually interact [84]. Both adapter and nonreceptor bound signaling proteins are recruited to integrin or growth factor receptor tails upon activation. Through proteins such as focal adhesion kinase (FAK), small GTPases of the Rho family, PI3K/Akt, JNK, and ERK as well as the ternary protein complex consisting of integrin-linked kinase (ILK), PINCH1 and alpha-parvin (IPP), talin, alpha-actinin, and vinculin, biochemical signals are transferred as a result of integrin/RTK commitment [14, 15, 17, 85]. Despite prosurvival signaling, it remains to be solved what exact impact morphology has on cell survival. ECM-integrin-actin cytoskeletal and cell-cell-intermediate filament connections determine cell morphology, which consequently define function and integrity of single cells and tissues [33, 34, 37, 42, 62, 86]. A variety of molecules involved in these interactions have been shown to be altered in cancer. For example, integrins are overexpressed in many human cancers originating from the head and neck region [87], lung [88], prostate [89], ovary [90], and breast [91], while E-cadherin, as one of the key cell-cell contact proteins, is frequently reduced in its expression or absent [87, 92, 93].</p><p>These expression changes are highly likely to impact on tumor cell behavior. We know that this physical linkage between ECM and cytoskeleton via integrins is crucially involved in translating mechanical into chemical signals and in controlling cell morphology [37, 64, 68, 71, 94]. In vivo, the ECM determines the shape and stiffness of tissues [34, 62, 95]. Under conventional cell culture conditions, ex vivo cultured cells grow attached to artificial surfaces like cell culture plastic. Missing physiological cell-matrix contacts, as optimally met in a 3D environment, has dramatic impact on cell shape and cellular behavior in vitro [12, 13, 37, 41, 69, 86, 96]. A large number of studies demonstrated that 2D cultured cells lose important features of their original phenotype due to severe genetic, epigenetic, and signal transduction changes [42, 48, 71, 97]. As this is similarly true for normal and cancer cells, one might realize that tumor cells have a preserved susceptibility for external signals originating from the ECM or soluble extracellular factors. Obviously, these facts are contrary to observations demonstrating an independency from external input signals by autonomous activation of intracellular pathways or activating mutations in proto-oncogenes leading to anchorage-independent growth [13, 31, 32]. Doubtless, mutation-driven, constitutively activated oncogenes overrun antiproliferative signals from the outside, but the myriad of additional stimuli affecting the cells is very well perceived and processed.</p><p>Taking these features into account, one can easily imagine that ECM and integrins contribute to the regulation of the cellular reaction to genotoxic injury. Onoda et al. found that nonlethal irradiation of melanoma cells induces alphaIIb/beta 3 integrin upregulation and increased adhesion to fibronectin [98]. Further studies corroborated these findings in a variety of normal and transformed human cell lines [59, 72, 78, 99–101]. However, clinically important is the fact that integrin-mediated cell adhesion to the surrounding ECM confers resistance to ionizing radiation, cytotoxic drugs, and molecular agents [59, 72, 102–104]. In addition to CAM-DR [59] and CAM-RR [72], a new paradigm was entitled "Environment-Mediated Drug Resistance" by Meads and colleagues (EMDR) [60]. Intriguingly, these three phenomena have been confirmed in irradiated or drug-treated cells from various tumor entities like glioma, leukemia, and melanoma as well as carcinomas of the pancreas, lung, and head and neck [18, 25, 28, 59, 72, 105–107].</p><p>Besides increased cell survival, ECM attachment prolonged radiogenic G2/M cell cycle arrest [103, 108] and reduced the number of residual DNA double-strand breaks (DSBs) and lethal chromosomal aberrations [40]. Also apoptotic cell death of small cell lung cancer cells was diminished under adhesion to laminin, fibronectin, or collagen type IV upon treatment with cytotoxic drugs [83].</p><p>Based on these findings, efforts are undertaken to uncover the underlying mechanisms and identify the cellular mediators and determinants involved in CAM-RR and CAM-DR. To elucidate therapeutic possibilities, small interfering RNA (siRNA) knockdown and antibody-mediated integrin inhibition are evaluated in different tumor cell lines with promising effects. In breast carcinoma, head and neck carcinoma, glioma, and leukemia cells, beta1 integrin targeting resulted in enhanced radiosensitivity and apoptosis [26, 27, 47, 80, 102, 109]. The pseudokinase ILK was clearly identified as antisurvival molecule in an attempt to classify the pro- and antisurvival function(s) of molecules acting downstream of integrins in cancer cells exposed to radiotherapy (reviewed in [110, 111]). Amongst others, this prosurvival group of molecules consists of FAK, JNK1, Akt1, PINCH1, and Caveolin-1 [55, 104, 112–117]. For example, overexpression of FAK protects 3D grown head and neck squamous cell carcinoma (HNSCC) cells from radiation-induced cell death [118], while siRNA-mediated silencing or pharmacological inhibition of FAK increases the radiosensitivity of different tumor cell lines from pancreatic cancer [112], breast cancer, colorectal cancer [119], and HNSCC [45, 55]. Furthermore, human melanoma cells become more sensitive to the chemotherapeutic agent 5-fuorouracil when FAK expression is downregulated [120]. In pancreatic cells, a reduction of FAK expression using microRNA for RNA interference leads to decreased FAK phosphorylation and repressed chemoresistance to gemcitabine [121]. Another interesting key player in this field is the LIM domain-containing particularly interesting new cysteine-histidine-rich 1 protein (PINCH1). Recent work from our group showed that knockdown of PINCH1 diminished the chemo- and radioresistance of diverse human carcinoma cells in vitro and in vivo [43, 122]. Mechanistically, PINCH1 was identified as novel Akt1 regulator by serving as platform for a regulatory interaction between protein phosphatase 1α (PP1α) and Akt1 [43]. According to the radiosensitization upon PINCH1 depletion, increased numbers of radiogenic DSBs were found in PINCH1 knockdown cell cultures indicating a role of PINCH1 in DNA repair processes [122]. On the basis of these findings, identification and targeting of molecules such as FAK or PINCH1 that critically regulates the cytotoxic drug and radiation response of tumor cells is a promising concept to overcome radio- and chemoresistance of tumor cells to improve cancer patient survival.</p><p>Additionally and of high importance for the current concepts of multimodal therapies, integrin-mediated cell-ECM interactions confer reduced efficacy of novel molecular agents/small molecules. In HNSCC, Eke and colleagues showed that adhesion to fibronectin attenuates the antiproliferative effect of a potent pharmacological epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor [104]. Recent findings provide evidence for an essential role of ILK for EGFR targeting in HNSCC [113] and that EGFR overexpression mediates hypersusceptibility to the anti-EGFR antibody cetuximab in 3D grown HNSCC cell lines in a FAK-dependent manner [55].</p><p>In summary, integrin-mediated adhesion to ECM protects cancer cells from varying types of cell death intended by radiotherapy, chemotherapy, and molecular drugs. Monotherapeutic targeting of integrins and intracellular signaling molecules to overcome adhesion-mediated resistance is already part of first clinical trials [13]. Cilengitide, a peptide potently blocking ανβ3 and ανβ5 integrin, is currently under evaluation in clinical phase II trials in glioblastoma and other malignancies [123, 124]. Identification of novel potential cancer targets and evaluation of targeted approaches against these targets require extensive examination and, most importantly, consideration and usage of preclinical tumor models, which best reflect clinical circumstances.</p><!><p>Regardless of normal or malignant cells, extracellular factors control critical cellular functions like survival, proliferation, and differentiation in a tissue-specific context [23, 37, 71, 97]. Studies using ex vivo cell cultures show the loss of morphological and functional properties in an artificial environment such as cell culture plastic as compared to ECM scaffolds [38, 69, 71]. Interesting studies in diverse tumor cell lines and normal cells showed that 3D growth in a matrix modifies gene and protein expression, cell survival, proliferation, differentiation, and metabolism in comparison to conventional 2D monolayer cell cultures [40, 42, 43, 46, 48, 116]. In line with these findings, osteosarcoma cells are protected against doxorubicin treatment [125] and head and neck and non-small-cell lung cancer cells display a reduced radiation sensitivity when grown in a 3D matrix in contrast to 2D [11, 40]. Beside these effects on cell survival upon cytotoxic injury, 3D growth conditions result in differential gene expression [126]. Global reorganization of chromatin through varying ECM compositions has been shown to be accompanied by changes in gene expression [94, 95, 127, 128]. Early work from Barcellos-Hoff and colleagues indicated that although polarized monolayers are formed, mammary epithelial cells fail to express milk proteins in 2D [23]. In 3D laminin-rich ECM, however, they formed alveolar-like structures with a central lumen and secreted milk proteins like casein [48, 49, 71, 95]. In this context, genes with ECM-responsive elements (EREs) were identified and helped to explain how the ECM participates in the regulation of gene expression [94, 128–130]. Furthermore, the pattern of gene expression is controlled by chromatin organization, which in turn is regulated by posttranslational modifications, that is, acetylation, phosphorylation, and methylation of nucleosomal histones [131, 132].</p><p>By integrating the above into an ECM-integrin-actin-nuclear membrane-nuclear matrix scenario, the ECM serves as one of the most powerful determinants of chromatin organization and gene expression [94, 130]. Concurrently, histones are upregulated as shown in 3D grown neuroblastoma cells [133] and tumor spheroids of melanoma cells [134], and histone acetylation is decreased to cooperatively control gene expression [46, 86, 135]. These highly dynamic actions are facilitated by histone acetyltransferases (HAT) and histone deacetylases (HDAC) [136, 137]. Gene expression in less condensed, euchromatic DNA regions is associated with histone hyperacetylation, while transcriptional repression occurs in more dense, heterochromatic DNA regions, by deacetylation [132, 138]. Recent own data demonstrate that growth in 3D ECM scaffolds decreases the levels of histone H3 acetylation in line with enhanced expression of the heterochromatin protein HP1α indicating a higher amount of heterochromatin [40]. Additionally, Le Beyec et al. highlighted the impact of cell-shape-induced changes in histone acetylation [46]. Cells cultured on polyHEMA showed a round cell morphology that led to histone deacetylation as consequence of changes in cell morphology but not adhesion [46]. These observations indicate that modifications in cell morphology impact on gene expression and thereby fundamentally determine tissue homeostasis and cellular responsiveness to external stress signals in a microenvironment-specific context [69].</p><p>Hence, it is most likely that cells cultivated in 3D show also differences in pathways of DNA repair after treatment with DNA-damaging agents in comparison to 2D. Little is known about the distribution of radiogenic DSBs within areas with different chromatin condensation status. With regard to the increased radiation survival caused by reduced numbers of residual DSBs and a lower number of chromosomal aberrations, 3D cell growth induces larger amounts of heterochromatin in comparison to 2D (Figure 1) [40]. Furthermore, these data show DSB localization in eu- and heterochromatic DNA regions to be similar in 3D and tumor xenografts. Conversely to this 1 : 1 distribution, 2D cells show a 2 : 1 eu- to heterochromatin DSB distribution [40]. These results underline the findings that 3D cell culture models better mimic the in vivo situation than conventional 2D monolayers.</p><p>How are ECM and nuclear matrix linked? Cytoskeletal filaments physically bridge between integrins or other cell adhesion molecules and the nuclear matrix including chromatin structures (Figure 2) [17, 18, 64, 127, 139]. Thus, both cell-matrix-activated signal transduction and mechanical forces sensed at the surface promote structural rearrangements in the cytoplasm and in the nucleus [68, 127, 140]. The linkage between cytoskeletal filaments and the nuclear matrix was identified as a complex termed linker of nucleoskeleton and cytoskeleton (LINC) and contains nesprins, sun, and lamin proteins [68, 141–143]. Nesprins 1 and 2 are nuclear membrane proteins that bind actin filaments and interact with sun proteins at the inner nuclear membrane. To control nuclear organization and gene function according to external stimuli, lamin proteins, which are connected with the inner nuclear membrane, form a nuclear scaffold that can bind chromatin directly or indirectly via other nuclear proteins [142, 144].</p><p>Through this complex interplay between ECM, integrins, cytoskeleton and nuclear matrix, many changes such as genome reorganization and differential gene expression, alterations in cell morphology, and integrin-mediated signal transduction occur in response to microenvironmental factors [13, 23, 33, 129]. Importantly, this focal adhesion-chromatin linkage contributes to existing and acquired therapy resistance in cancer. An increased understanding of the underlying molecular mechanisms and the implementation of better translational cancer models will assist our efforts to optimize and personalize cancer therapy.</p>

PubMed Open Access

Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development

Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that over-express active Stat3 leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers.

alkylation_of_cysteine_468_in_stat3_defines_a_novel_site_for_therapeutic_development

<!>Screening of potential inhibitors of Stat3 DNA-binding activity<!>Order-of-addition is critical for inhibition of Stat3-DNA complex formation by compound C48<!>Compound C48 alkylates cysteine 468 of Stat3<!>Stattic alkylates Stat3<!>Compound C48 affects Stat3 nuclear localization and Stat3-mediated gene expression<!>C48 inhibits Stat3 signaling and induces apoptosis in human cancer cells<!>C48 suppresses the in vivo growth of MDA-MB-468 human breast cancer cells in a xenograft mouse model<!>C48 suppresses the growth of C3L5 murine breast cancer cells in a syngeneic mouse model<!>DISCUSSION<!>Virtual ligand screening<!>Molecular biology<!>Analytical size exclusion chromatography<!>Mass Spectrometry LC/MS analysis<!>Cell lines and cell culture<!>Western blot analysis<!>Nuclear translocation assay and confocal microscopy<!>Luciferase assay system for the measurement of Stat3- and Stat1-mediated transcriptional activity<!>Nuclear extract preparation and EMSA<!>Tumor models<!>Flow cytometric analysis of Annexin V/PI<!>Note Added in Proof<!>Structure of Stat3 inhibitor compounds and effect of C48 on STAT DNA-binding activity<!>Surface representation of Stat3 bound to DNA<!>Effect of C48 on wildtype and mutant STAT homologs<!>Modification of Stat3 by C48<!>Effect of C48 on Stat3 nuclear translocation<!>Effect of C48 on Stat3- and Stat1-mediated transcriptional activity<!>Effect of C48 on cell viability and Stat3 signaling pathway proteins<!>Effect of C48 on in vivo growth of a human breast tumor xenograft and a syngeneic mouse model of murine breast adenocarcinoma

<p>Signal Transducers and Activators of Transcription (STATs) comprise a family of transcription factors that transmit signal responses triggered by cytokines, growth factors and hormones (1-3). Upon ligand engagement, STATs are recruited to cell surface receptors and become phosphorylated at a single carboxy-terminal tyrosine by receptor-intrinsic, receptor-associated or non-receptor tyrosine kinases. Tyrosine phosphorylation of STATs then initiates their nuclear translocation, resulting in binding of activated STAT dimers to specific promoter regions in DNA and regulation of target gene expression (4-7). Although STATs exist as non-phosphorylated and phosphorylated dimers, it has been speculated that the conformational change that occurs after tyrosine phosphorylation stabilizes the dimer and improves its ability to bind to specific DNA regions (8-13).</p><p>The STAT family includes seven members, namely Stat1–Stat4, Stat5a, Stat5b, and Stat6 (1). In normal cells, STAT signaling is usually transient and tightly regulated by cytokines and growth factors. However, constitutive activation of some STATs, including Stat3 and Stat5, has been detected in a large number of diverse human cancer cells and primary tumor tissues, including blood malignancies and solid tumors (14, 15). Overwhelming evidence indicates that hyperactivation of Stat3 signaling leads to enhanced tumor cell proliferation and survival (16), and that this signaling pathway is a valid target for the development of cancer therapeutics. Moreover, Stat3 itself has also been identified as an oncoprotein (17) and a valid target for cancer drugs. In contrast, aberrant Stat1 signaling has been associated with suppression of cell growth rather than malignant transformation, and thus can act as a potential tumor suppressor (18). Because of the opposing effects of Stat1 and Stat3 on oncogenesis, and their close structural similarity, it is important to develop drugs that can distinguish between Stat1 and Stat3.</p><p>Direct inhibition of the activated Stat3 dimer or the Stat3-DNA interaction is an attractive approach for cancer therapeutics, and does not require knowledge of possible upstream activators of Stat3 in any given tumor type. However, disrupting protein-protein or protein-DNA interactions with small molecules is challenging and also an emerging therapeutic approach. We used molecular dynamics (MD) and modeling studies to identify potential leads that were experimentally tested using electrophoretic mobility shift assay (EMSA) studies. NCI compound NSC-368262 (C48) was found to block DNA-binding of Stat3, but not of Stat1. Additional analogs were tested, but these showed little or no improvement in efficacy of Stat3-DNA inhibition. While no structure-activity relationship could be identified, closer examination of the chemical properties of the parental fragment suggested that C48 and the selected analogs alkylate Stat3. Comparison of the Stat3 and Stat1 structure showed that Stat3 possesses a unique surface cysteine, Cys468, that is located in the DNA-binding region of Stat3. The equivalent residue in Stat1 is a serine. Through mutagenesis, we demonstrate that alkylation of Cys468 inhibits Stat3 DNA-binding. We further show that alkylation of Stat3 by C48 inhibits proliferation and survival of tumor cell lines harboring constitutive Stat3 DNA-binding activity. In addition, C48 shows anti-tumor efficacy in xenograft and syngeneic mouse models using human and mouse breast cancer cells, respectively. Therefore, we have identified C48 as a potential small-molecule inhibitor of aberrant Stat3 signaling. Moreover, we provide evidence that selective alkylation of Stat3 at Cys468 constitutes a viable site for further development of selective Stat3 inhibitors for cancer treatment.</p><!><p>To identify potential Stat3 inhibitors, virtual ligand screening (VLS) using a commercial chemical library was performed using a snapshot of Stat3 from a previous MD simulation (12). Regions in Stat3 that showed low sequence similarity to Stat1 were identified as docking sites for VLS to enhance subtype selectivity. Of the 7000 ligands that docked to a distinct site, 437 compounds were selected based on ligand-buried surface area (>75%) and favorable van der Waals and hydrogen bond energies. Side chain rotamers for residues within 5 Å for each docked ligand were minimized. The ligands were sorted by the calculated binding energies (including desolvation energy of the ligand) and clustered by similarity in chemical functionalities, producing 52 compounds representative of the structural diversity.</p><p>Each compound was tested by EMSA experimentally and AP-906/42850375 (C36) was identified as a potential Stat3 inhibitor (Fig. 1A). C36 inhibited Stat3 DNA-binding activity with an IC50 of 30–50 μM (not shown). Next, using this compound as a template to search a publicly available 85,563 chemical library, as well as the National Cancer Institute (NCI) chemical library (about 300,000 compounds), we selected 48 new compounds that structurally resembled C36. EMSA identified three additional compounds that inhibited Stat3 DNA-binding activity at concentrations of 100 μM or lower: AP-906/42850385 (C29), AP-906/42850377 (C30) and NSC-368262 (C48) (Fig. 1A). All four compounds inhibited Stat3 DNA-binding activity with IC50 values of 10–50 μM (not shown). Further analysis, however, revealed that compounds C29, C30 and C36 lost their Stat3 inhibitory activity when dissolved and stored in aqueous solutions for 24 h or longer prior to EMSA analysis. In contrast, in EMSA, C48 did not show any loss of activity when stored in aqueous solution for 7 days or longer, even through repeated freeze-thaw cycles.</p><p>To assay for Stat3 subtype selectivity of compound C48, we performed EMSA using nuclear extract derived from Sk-Mel-5 human melanoma cells stimulated with IFN-γ. This extract contains activated Stat3/Stat3 and Stat1/Stat1 homodimers, as well as activated Stat1/Stat3 heterodimers, all of which bind to the same radiolabeled DNA probe. C48 blocked the DNA-binding activity of phosphorylated Stat3 homodimers (IC50 10–50 μM), without inhibiting the DNA-binding of phosphorylated Stat1 homodimers, even at concentrations as high as 500 μM (Fig. 1B). Moreover, C48 also disrupted the DNA-binding activity of Stat1/Stat3 heterodimers, although at a slightly higher concentration (IC50 50–100 μM). These data show that C48 specifically blocks DNA-binding activity of phosphorylated Stat3 homodimers and Stat1/Stat3 heterodimers, and suggest that C48 possesses subtype selectivity for Stat3 over Stat1.</p><!><p>To further examine the mode of action of C48 on Stat3 DNA-binding, we performed EMSA analysis as described above, while varying the order-of-addition of the interaction components. Pre-incubation of C48 with nuclear extract that contained activated Stat3 and Stat1, followed by addition of the oligonucleotide probe, prevented de novo binding of Stat3 homodimers and Stat1/Stat3 heterodimers to DNA (Fig. 1C, lanes 3-6). In contrast, pre-incubation of the nuclear extract with DNA, followed by addition of C48, did not disrupt pre-existing Stat3 DNA-binding (Fig. 1C, lanes 7-10). Similarly, pre-incubation of C48 with DNA, followed by addition of the nuclear extract, did not markedly affect STAT DNA-binding activity (Fig. 1C, lanes 11-14). These results demonstrate that compound C48 inhibits Stat3 DNA-binding activity only when allowed to interact with Stat3 protein before Stat3 protein encounters its DNA-binding site. Although these data suggest that compound C48 directly interacts with activated Stat3 protein, the order-of-addition requirement to inhibit the Stat3-DNA interaction suggests that compound C48 binds to a Stat3 site that is only accessible before Stat3 binds to DNA.</p><!><p>Examination of compound C48, as well as C36 and the other compounds, indicated that the benzylic carbon is potentially reactive, suggesting that these compounds might alkylate cysteine residues. Furthermore, examination of the Stat3 crystal structure indicated several surface exposed cysteines that could be modified (Fig. 2A) (19). Significantly, Cys468 of Stat3 is at the Stat3-DNA interface and its modification would sterically block DNA binding (Fig. 2B). Moreover, serine occupies the equivalent position in Stat1 and Stat5 suggesting a potential mechanism for the isoform specificity of C48 (Fig. 2C). To test whether Cys468 is susceptible to alkylation by C48, this residue was mutated to serine resulting in the generation of phosphorylated C468S Stat3 (e.g., phosphorylated Tyr705) mutant. As a control, phosphorylated wildtype Stat3 was utilized. We first demonstrated that both phosphorylated recombinant proteins, wildtype and C468S Stat3 mutant, bound to DNA (Fig. 3). Next, the wildtype and C468S phospho-Stat3 proteins were treated with increasing concentrations of compound C48. EMSA studies revealed no significant effect of C48 on DNA binding of C468S Stat3 (Fig. 3A, second row).</p><p>To further establish the importance of Cys468 alkylation in Stat3 as a modality for disrupting STAT/DNA interactions, a similar approach was studied using Stat1, which possesses a serine instead of a cysteine in the equivalent position (Fig. 2C). We reasoned that mutating Ser462 in Stat1 to Cys, would cause the mutant Stat1 to become sensitive to C48. In the absence of C48, both recombinant, phosphorylated Stat1 and recombinant, phosphorylated S462C Stat1 bound DNA with similar affinity (Fig. 3A, rows 3 and 4, respectively). However, as concentrations of C48 increased, the S462C mutant lost its ability to interact with DNA (Fig. 3A, row 4). As expected, DNA-binding of wildtype Stat1 was not affected by C48 (Fig. 3A, row 3). Finally, the DNA-binding property of phosphorylated Stat5 (nuclear extract isolated from K562 chronic myelogenous leukemia cells) was also insensitive to C48 exposure (Fig. 3A, row 5), whereas DNA-binding of phosphorylated Stat3 (nuclear extract isolated from Sk-Mel-5 melanoma cells) was inhibited by C48 (Fig. 3A, row 6). Just as for wildtype Stat1, in wildtype Stat5 serine is the equivalent residue to Cys468 in Stat3 (Fig. 2C).</p><p>In addition to EMSA, size exclusion chromatography (SEC) was used to further confirm that C48 blocks DNA binding. In these experiments, DNA was fluorescently labeled, which enabled direct monitoring of its hydrodynamic properties at 495 nm (e.g., eliminate spectral overlap of the DNA and protein signal at either 260 nm or 280 nm). SEC analysis of only the fluorescently-labeled DNA produced a peak that eluted at 16 mL (Fig. 3B, trace 3), whereas the admixture of wildtype Stat3 and DNA eluted at 12.5 mL (Fig. 3B, trace 2). SEC analysis of only wildtype phosphorylated Stat3 produced a peak at 12.5 mL when monitored at 280 nm (Fig. 3B, trace 4). Also no signal from Stat3 was observed when monitored at 495 nm (Fig. 3B, trace 5). Finally, treatment of wildtype Stat3 with C48 for 30 min followed by addition of the fluorescent DNA eliminated the peak that eluted at 12.5 mL (Fig. 3B, trace 1). Likewise, in similar SEC experiments, the DNA binding properties of the C468S phospho-Stat3 mutant treated with compound C48 were unaffected (Fig. 3C), whereas wildtype phospho-Stat3 treated with compound C48 failed to bind DNA (Fig. 3A and B). In fact, it appears that the C48 treated C468S phospho-Stat3 mutant binds slightly better than the non-treated mutant. While the precise mechanism for this enhancement in not immediately clear, it further underscores modification of Cys468 in Stat3 abrogates DNA binding. Collectively, this analysis indicates that C48 blocks Stat3 DNA binding.</p><p>In addition, mass spectrometry was used to monitor the alkylation of Stat3. The exposure of phosphorylated Stat3 (10 μM) with C48 (600 μM) for 30 minutes produced an 'envelope' of peaks, each differing by 317 Dal (Fig. 4A). The most intense peak of the envelope corresponds to six C48 adducts whereas the first peak corresponds to the addition of four C48 adducts. Attempts to identify which cysteine residues in Stat3 are modified by C48 by MS were not successful, possibly due to the nature C48's benzylic reactivity which may reverse during digestion and work-up. Thus, to confirm that one of these modification of Stat3 is Cys468 and understanding that prolonged exposure to C48 produces an envelope of multiple modifications, we exposed wildtype Stat3 and the C468S Stat3 mutant to C48 for one minute under the same conditions. In the mass spectra, three peaks were observed for wildtype Stat3 that corresponds to the alkylation of three, four, and five C48 residues (Fig. 4B, blue trace). Likewise, three peaks were also observed for mutant Stat3, but these correspond to the formation of two, three and four C48 adducts(Fig. 4B, pink trace). This shift of the envelope to one less adduct suggests that Cys468 is one of the first residues to be modified by C48. As further evidence of the reactive nature of C48 towards sulfhydryl groups, glutathione was used as a model compound. Mixing glutathione with C48 produced a fragment ion spectrum (MS/MS), which confirms alkylation of the glutathione thiol group by C48 (Supplemental Fig. 1).</p><p>Finally, C48 does not affect the dimerization state of phospho-Stat3, as evidenced by SEC and sedimentation equilibrium experiments using analytical ultracentrifugation (Supplemental Fig. 2). These data also point to modification and steric occlusion of the Stat3 DNA-binding site. Collectively, these biochemical studies strongly suggest that the specificity of C48 is due to its ability to alkylate Cys468, which is unique to Stat3.</p><!><p>Stattic has been reported as a selective inhibitor of Stat3 that is commercially available. This inhibitor possesses a potentially reactive vinyl sulfone moiety. To investigate whether Stattic alkylates Stat3, similar experiments were performed using SEC and mass spectrometry. With SEC, we observe that phosphorylated Stat3 treated with Stattic remains dimeric, but does not bind to DNA (data not shown), which is consistent with C48. Upon mass spectral analysis, we observe a mass increase of 1690 Dal for phosphorylated Stat3 (10 μM) treated with of Stattic (800 μM) after 30 minutes at 37 °C (Fig. 4A). This mass difference corresponds to the addition of eight Stattic molecules which suggests that the mechanism of action by Stattic is through alkylation.</p><!><p>Since C48 is selective for Stat3, we asked whether it would selectively block Stat3-mediated transcription in cell-based assays. An established, stably transfected murine embryonic fibroblast cell line that expressed a Stat3-YFP fusion protein (MEF-Stat3-YFP) (20) was used. This cell line allows for monitoring Stat3 nuclear translocation after stimulation with Oncostatin M (OSM), a known activator of the JAK/STAT pathway (21). Confocal laser scanning microscopy showed that in unstimulated, serum-starved MEF-Stat3-YFP cells, Stat3 is present in nucleus and cytosol and that the majority of Stat3 is located in the cytosol (Fig. 5, left panel). Our observed distribution pattern of Stat3 in unstimulated cells is in agreement with previous findings (22). Upon stimulation with OSM, receptor-associated Janus kinases phosphorylate Stat3 protein at Tyr705, which caused the activated Stat3 dimers to translocate into and accumulate in the nuclei (Fig. 5, second panel from left). Pre-incubation of MEF-Stat3-YFP cells with compound C48 (40 μM) completely prevented OSM-induced nuclear accumulation of Stat3-YFP, as compared to control-treated cells (Fig. 5, fourth panel from left). These data suggest that C48 inhibits Stat3 nuclear accumulation primarily by abrogating its interaction with DNA, permitting its relocation to the cytoplasm. In other words, the productive interaction of phospho-Stat3 with DNA is essential in sequestering Stat3 in the nucleus. In support of this notion, we note that others have observed that Stat3 in non-stimulated cells or Stat3 Y705F mutant continuously shuttles between the cytoplasm and nucleus (22).</p><p>To confirm these results, HeLa cells genetically engineered to carry a chromosomal integration of a luciferase reporter gene for which expression is regulated by either activated Stat3 or activated Stat1 was used. These cell lines allow for monitoring Stat1 or Stat3 transcriptional activity through measurement of luciferase activity (i.e., luminescence emission). When compared to OSM-induced Stat3-dependent luciferase activity, pre-treatment of cells with compound C48 prior to stimulation with OSM resulted in dose-dependent inhibition of luciferase activity with an IC50 of 3–10 μM (Fig. 6A). At 20 μM C48, the luminescence emission was significantly lower than that of cells that were not treated with OSM, suggesting there is basal Stat3 activity in unstimulated cells. These results indicate that the effect of C48 on Stat3 DNA-binding activity and Stat3-induced gene-expression in cells can be detected at C48 concentrations as low as 3–10 μM; however, a higher concentration (up to 20 μM) is required to completely abolish Stat3-dependent transcriptional activity. In contrast, pre-treatment of cells with C48 did not dramatically reduce IFN-γ-induced Stat1-mediated luciferase activity as compared to the luciferase activity of untreated IFN-γ-stimulated cells (Fig. 6B). Taken together, the results obtained from these cell-based assays, combined with the in vitro EMSA results (Fig. 1B), suggest that compound C48 disrupts DNA-binding of activated Stat3, but does not significantly affect Stat1-induced signaling.</p><!><p>The human breast cancer cell lines MDA-MB-468 and MDA-MB-231 harbor constitutive phosphorylation of Stat3 Tyr705, and thus, constitutive Stat3 DNA-binding activity. These cell lines have been shown to undergo apoptosis upon inhibition of Stat3 signaling using small-molecule inhibitors (23). In contrast, the human prostate cancer cell line LNCaP does not exhibit constitutive Stat3 DNA-binding activity, and therefore, does not rely on Stat3 signaling for survival. We treated these cell lines with C48 and measured cell viability by AnnexinV/PI staining as assessed by flow cytometry. MDA-MB-468 and MDA-MB-231, but not LNCaP cells, underwent apoptosis following a 48 h treatment with 1–20 μM C48 (Fig. 7A, B and C), and the IC50 for induction of apoptosis was 10–20 μM C48, a concentration range similar to that needed to block Stat3 DNA-binding activity in vitro and to inhibit Stat3 transcriptional activity in vivo. Western blot analysis of lysates from MDA-MB-468 cells treated for 48 h with C48 revealed much less Stat3 Tyr705 phosphorylation in cells treated with 10 μM C48 compared to untreated cells, and complete inhibition of phosphorylation with 20 μM C48 (Fig. 7D). In contrast, the activation state of p44/p42 MAP kinases was not significantly inhibited by C48, even when used at 20 μM. In addition, expression of Mcl-1, a pro-survival gene and known downstream target of Stat3, was fully blocked in cells treated with 20 μM C48. In addition, when treated with 20 μM C48, the majority of MDA-MB-468 cells are undergoing apoptosis, as indicated by the appearance of cleaved PARP fragment, which is comparable to the Annexin/PI results (Fig. 7A). These data suggest that compound C48 exerts its ability to kill human tumor cells at least in part through inhibition of Stat3 signaling.</p><p>Since we observed a decrease in the phosphorylation level of Stat3 upon treatment with C48, the question whether compound C48 could inhibit upstream activators of STAT proteins was addressed. Western blot analysis of lysates from MDA-MB-468 cells treated for 4 h with 50 μM C48 revealed no inhibition of phospho-Jak2 (Tyr1007/1008) and phospho-Src family (Tyr416) proteins (Fig. 7E). In addition, in vitro kinase activity assays were performed on Jak1 and Jak2 kinases. As shown in Supplemental Figure 3, the IC50 of compound C48 for inhibition of Jak1 and Jak2 kinases > 400 μM, the highest concentration used in this assay. Staurosporine was used as a positive control and inhibited the activity of both Jak1 and Jak2 kinase with an IC50 of < 1.0 nM. These data suggest that compound C48 does not inhibit the STAT upstream activators Jak1 and Jak2.</p><p>To test whether blocking the Stat3-DNA interaction would make phosphorylated Stat3 more susceptible to phosphatase activity, MDA-MB-468 cells were treated with compound C48 in the presence and absence of sodium orthovanadate, a phosphatase inhibitor. We observe that phosphorylation levels of Stat3 in MDA-MB-468 cells pre-treated for 30 min with 1 mM sodium orthovanadate prior to the addition of C48 for 2 is similar to that of untreated cells. These data suggest that treatment of cells with compound C48 facilitates the dephosphorylation of Stat3.</p><!><p>Several studies have shown that use of small molecule inhibitors to interfere with Stat3 signaling results in growth inhibition of human tumor xenografts (23). We extended these studies to evaluate the antitumor efficacy of C48 in a nude mouse model with subcutaneously established MDA-MB-468 human breast tumor xenografts with constitutively active Stat3. Tumor-bearing mice were given i.p. injections of C48 (200 mg kg-1) or vehicle control (ddH20 supplemented with 2% Tween-80) daily for 5 days per week, 8 weeks total, and tumor measurements were taken twice a week. As compared with tumors from control-treated mice, which continued to grow, tumors from C48-treated mice displayed significant growth-inhibition (Fig. 8A).</p><!><p>We also tested the effect of C48 in a syngeneic mouse model of breast cancer. C3L5 murine breast adenocarcinoma cells that possess constitutively active Stat3 were injected subcutaneously in C3H/HeJ mice. Tumor-bearing mice were i.p. injected with C48 (100 mg kg-1 or 200 mg kg-1) or vehicle control (ddH20 supplemented with 2% Tween-80) every day for 5 days per week. The tumors were measured every other day for 26 days. Similar to the xenograft model, tumors from control-treated mice continued to grow, while tumors from mice treated with 200 mg kg-1 C48 showed significant growth-inhibition (Fig. 8B).</p><!><p>As a central point to integrate and amplify oncogenic signals, transcription factors represent attractive targets to develop therapies broadly applicable to different forms of cancer and other diseases. However, successfully blocking the activity of transcription factors remains challenging. More specifically, protein-protein or protein-DNA interfaces are generally spread over large, relatively flat surfaces, and often do not provide sufficient cavities to develop high affinity, small molecule antagonists. Targeted, chemical modification of a residue located at or in the immediate vicinity of a protein-protein or protein-DNA interface, on the other hand, offers a potential solution to antagonize interactions characterized by extensive interfaces.</p><p>C48 was originally identified by VLS to bind to a novel site near the SH2 domain. While this cannot be ruled out, the main mechanism of action is through alkylation of Cys468. Furthermore, C48 abrogates Stat3 DNA-binding activity in vitro and induces apoptosis in two human tumor cell lines with constitutively active Stat3 signaling, but not in a human tumor cell line that lacks Stat3 activation. Finally, C48 was found to possess significant in vivo anti-tumor activity in both a human breast cancer xenograft mouse model and a syngeneic mouse model of breast cancer that harbored persistent Stat3 activation. Collectively, these data provide compelling evidence that C48 is a potential lead compound to antagonize Stat3 activity and that Cys468 is a viable site for further development of irreversible Stat3 inhibitors.</p><p>The results of this study may have broader implications to other known Stat3 inhibitors as well. Recently, a number of small molecule inhibitors of Stat3 have appeared in the literature. These include stattic, curcubitacins, withacnistin, and S3I-201, some of which were also identified via molecular modeling and virtual screening. What these molecules have in common is a reactive chemical moiety. Stattic, withacnisitin, and curbubitacins can be classified as Michael acceptors possessing vinyl sulfone and α,β unsaturated ketone functionalites. Withacnistin also contains a potentially reactive epoxide. S3I-201 possesses an α-keto tosylate group which is a potent alkylating agent in general. While we have shown that stattic readily alkylates Stat3 (Fig 4A), all of these reagents are capable of modifying cysteine residues even though they were not reported as such nor has this mode of action been ruled out. Despite being identified as SH2 inhibitors through VLS, we suspect that some of these previously reported Stat3 inhibitors are most likely acting through alkylation of Cys468.</p><p>While irreversible inhibitors are necessarily reactive and may potentially react with other targets, the clinical application of irreversible inhibitors and their continued development is promising. For instance, exemestane is a potent, irreversible aromatase inhibitor used for the treatment of estrogen-dependent breast cancer (24, 25). Likewise, the irreversible EGFR/HER2 inhibitor, BIBW 2992, developed by Boehringer-Ingelheim, is in phase III clinical trials for non-small cell lung cancer (26, 27). Recently, the modification of Cys179 in IKKβ by CDDO (2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid) was shown to inhibit IKKβ kinase activity, which in turn fails to activate NF-κB (28, 29). As such, CDDO sensitizes cells to apoptotic pathways and is currently in clinical trails to treat lymphoma and solid tumors (trial ID: NCT00352040). Our data strongly suggest that Stat3 is very sensitive towards alkylation resulting in potent inhibition of tumor growth in animal models. There are other cysteines in Stat3 for potential targeting as well, including Cys712. This cysteine is located in the SH2 domain of the tyrosine binding site and provides an excellent opportunity for developing selective Cys712 alkylators by way of SH2 binding. Studies along these lines are currently in progress. The successful application and continued development of "irreversible" inhibitors for Stat3 offers a promising approach to a new class of therapeutic agents for the treatment of cancer.</p><!><p>Virtual ligand screening (VLS) analysis was performed on the Stat3 dimer by docking 85,563 compounds from a City of Hope database of commercially available chemical libraries using the GLIDE-HTVS procedure from Schrodinger (30). A box size of 34 Å × 34 Å × 34 Å that covered the entire SH2 domain of the Stat3 dimer was used for the VLS procedure. We used a snapshot of the DNA-bound Stat3 dimer extracted from the MD simulations (12). The van der Waals radii were scaled for the ligand atoms by 0.5. Poses with sum of Coulomb and van der Waals energy greater than 100.0 kcal/mol were rejected. The docked poses from the VLS procedure were first sorted and filtered by buried surface area of the ligands calculated using Connolly surface area calculations (31). Ligand poses greater than 75% buried surface were retained and were further filtered by van der Waals and hydrogen bonding energies. Side chain rotamers of residues within 5 Å of the ligand in the binding site were reassigned using Prime module from Schrodinger Inc. (30). The binding energies of the resulting optimized conformations were calculated as Ebinding = Potential energy (ligand in protein) − Potential energy (ligand in water). The potential energy of the ligand was calculated by fixing the protein and using all atom forcefield OPLS using the Macromodel module (30). Finally, the ligand docked conformations were sorted by binding energies and then clustered by structural similarity.</p><!><p>Stat3β (residues 127-722) and Stat1β (residues 132-712) were amplified from murine and human cDNA, respectively. The corresponding genes were inserted between BamH1 and Xho1 sites of a modified pET28b vector encoding an N-terminal 6-His tagged SMT3 protein. The construct was verified by DNA sequencing (City of Hope DNA Sequencing Core). The Stat3 C468S and Stat1 S462C mutants were generated using the QuikChange Site-Directed Mutagenesis protocol (Stratagene) and verified by DNA sequencing.</p><p>To express recombinant phospho-Stat3 and phospho-Stat1 (wildtype and mutants), each plasmid was transformed into Escherichia coli (BL21(DE3) TK [TKB1] strain) (Stratagene), which harbors an inducible tyrosine kinase that can phosphorylate STAT proteins. A two-step induction protocol was followed during the expression procedure. Cells were grown to a density of A600 = 0.5−0.7, at which point IPTG was added to a final concentration of 0.25 mM. After 3 h of induction, cells expressing Stat3 or Stat1 were harvested and resuspended in TK induction medium containing 53 μM indoleacrylic acid. The tyrosine kinase-expressing culture was harvested after 4 h of incubation, resuspended in 1X PBS and stored at -80 °C.</p><p>Protein constructs were purified using standard methods. Briefly, cells were lysed by French press, clarified by centrifugation and loaded onto a Ni-NTA column (Qiagen). After an extensive wash (20 column volumes), the protein was eluted with an imidazole gradient. The SMT3 fusion was cleaved by Ulp1 protease, precipitated with 40% ammonium sulfate, dialyzed, and applied to a Superdex G200 preparative column (GE Healthcare). We observed that phospho-Stat3 eluted earlier than purified non-phospho-Stat3. The fractions were analyzed by a 10% SDS-polyacrylamide gel and fractions about the center of the peak were flash frozen at -80 °C. Each construct was purified in a similar manner.</p><!><p>6-FAM™– (Fluorescein, Ex: 495 nm, Em: 520 nm) labeled DNA oligonucleotides (Fl-DNA) were ordered from Integrated DNA Technologies with the following sequences: 5'TCATTTCCCGTAAATCCCTA3' and 5'TAGGGATTTACGGGAAATGA3'. The two strands were annealed and stored at -20 °C. Phospho-Stat3 (wildtype or C468S mutant; 10 mM) was incubated with Fl-DNA (30 min, 37 °C) before being loaded onto an analytical Superdex G200 column (GE Healthcare). The elution profile was monitored at 290 and 495 nm. To determine the effect of C48, C48 (100 μM) was incubated (30 min, 37 °C) with the phospho-Stat3-Fl-DNA complex and analyzed as above.</p><!><p>Samples were analyzed using a Synapt G2 quadrupole time of flight mass spectrometer and a NanoAcquity HPLC (Waters). Samples were separated using a 25mm × 150 μm ID column home-packed with Intrada WP-RP silica (Imtakt, Philadelphia, PA). The samples were loaded in a trapping only mode at 4 μl/min, then eluted into the mass spectrometer at 600 nl/min. Samples were eluted using a gradient from 3% B to 40% B in 9 minutes, followed by a ramp from 40% B to 95% B over 2 minutes. Buffer A was 0.1% Formic acid in water. Buffer B was 0.1% Formic acid in acetonitrile. Protein spectra were averaged over the width of the elution peak and deconvoluted using the Waters provided Maximum Entropy deconvolution software.</p><!><p>The human breast cancer cell lines MDA-MB-468 and MDA-MB-231, the human prostate cancer cell line LNCaP, the human melanoma cell line Sk-Mel-5 and the human chronic myelogenous leukemia cell line K562 were purchased from the American Type Culture Collection. The murine breast adenocarcinoma cell line C3L5 is maintained in the laboratory of Dr. John Yim (City of Hope). The Stat1 and Stat3 human cervical cancer reporter cell lines HeLa-Stat1-Luc and HeLa-Stat3-Luc were obtained from Promega. MEF-Stat3-YFP mouse embryonic fibroblast cells that express YFP-labeled, but not wildtype Stat3, were produced by Dr. Andreas Herrmann (City of Hope). All cell lines were maintained in DMEM supplemented with 5% FBS, 50 U mL-1 Penicillin and 50 μg mL-1 Streptomycin. Hygromycin B (500 μg mL-1) was used as selection marker for the reporter cell lines and MEF cells.</p><!><p>For Western blotting, cells were washed in 1X PBS containing 1 mM Na-ortho-vanadate and lysed in TGH buffer (1% Triton X-100, 10% glycerol, 50 mM NaCl, 50 mM HEPES, 1 mM EGTA, 1% Na-deoxycholate, 1 mM Na-ortho-vanadate, 2 μg/mL aprotinin, 0.5 μg/mL leupeptin, 50 μg/mL antipain and 1 mM PMSF). Total protein amount was determined using the Bio-Rad Protein Assay reagent, and equal amounts of total protein were loaded in each lane of a 10% SDS-polyacrylamide gel. Following electrophoresis, the proteins were transferred to PVDF membrane, washed with PBS/0.1% Tween-20, blocked with PBS/5% milk and incubated in 1X PBS/5% BSA at 4 °C overnight with the first antibody. The membrane was then washed with PBS/0.1% Tween-20, incubated for 1 h at room temperature with alkaline phosphatase-linked anti-rabbit or anti-mouse secondary antibodies and visualized using SuperSignal West Pico Reagent (Pierce). For detection of total Stat3, total Src, total Jak2 and total p44/p42, the corresponding phospho blots were incubated with stripping buffer (2% SDS, 64 mM Tris, pH 6.7, 0.7% β-Mercaptoethanol) and reblotted. Primary antibodies: total-p44/p42 MAP Kinase (Erk1 and Erk2, Cat#9102), phospho-p44/p42 MAP Kinase (Thr202/Tyr204, Cat#9101), phospho-Stat3 (Tyr705, Cat#9131), Mcl-1 (Cat#4572), total Jak2 (D2E12, Cat#3230), phospho-Jak2 (Tyr1007/1008, Cat#3771S), phospho-Src (Tyr416, Cat# 2101S), and PARP (Cat#9542) were from Cell Signaling. Total Stat3 antibody (C-20, Cat#sc-482) was from Santa Cruz Biotechnology. Anti-Src (GD11, Cat# 05-184) was from Upstate. Anti-β-actin antibody was from Sigma (Cat#A5441).</p><!><p>MEF-Stat3-YFP cells were plated on poly-L-Lysine-coated coverslips (24 well). After 24 h, the cells were pre-incubated for 2 h with up to 50 μM compound C48 in serum-free medium, followed by stimulation with Oncostatin M (25 ng/mL) for 30 min. Cells were fixed in formaldehyde and analyzed by confocal laser scanning microscopy as described (32). Briefly, slides were mounted with Vectashield hardSet mounting medium containing DAPI (Vector laboratories). Confocal imaging was carried out on a Zeiss LSM 510Meta confocal microscope equipped with a 63X 1.2 NA Zeiss water immersion objective. YFP signals were detected as previously described (22). DAPI was visualized using a two photon laser exciting at 435–485 nm. The images shown represent confocal slices of approximately 1 μm thickness.</p><!><p>Hela-Stat3-Luc and Hela-Stat1-Luc cells express the luciferase protein under the control of activated Stat3 and Stat1 protein, respectively. To measure the effect of C48 on STAT-mediated transcriptional activity, 10,000 Hela-STAT-Luc cells in DMEM containing 5% FBS were seeded in each well of a 96-well plate. The next day, cells were incubated for 2 h with 1 - 20 μM C48. Oncostatin M (OSM; 25 ng mL-1; Sigma) or Interferon-γ (IFN-γ; 25 ng mL-1; Sigma) was then added to each well to induce Stat3 (OSM) and Stat1 (IFN-γ) activation and luciferase expression. Eight hours later, cells were lysed and luciferase activity was measured as per the manufacturer's instructions (Promega).</p><!><p>To detect the DNA-binding activity of Stat1, Stat3, and Stat5 by EMSA, nuclear protein extracts were prepared using high-salt extraction as previously described (33). To test potential inhibitors of Stat3 DNA-binding, nuclear protein (5 μg) from Sk-Mel-5 human melanoma cells (source of activated Stat3/Stat3 homodimers) or Sk-Mel-5 cells stimulated with interferon-γ for 15 min (source of activated Stat1/Stat1 and Stat3/Stat3 homodimers, as well as Stat1/Stat3 heterodimers) was incubated (30 min, 37 °C) with 3-500 μM C48 at the concentrations indicated. Then a 32P-radiolabeled, double-stranded DNA oligonucleotide that binds Stat3/Stat3 and Stat1/Stat1 homo- and Stat1/Stat3 heterodimeric proteins was added to the nuclear protein and incubated for another 30 min. This oligonucleotide is a high-affinity variant of the sis-inducible element (hSIE; sense strand, 5'-AgC-TTC-ATT-TCC-CTG-AAA-TCC-CTA-3') derived from the c-fos gene promoter, which binds activated Stat3 and Stat1 proteins (34, 35). For Stat5 EMSA, 5 μg of nuclear protein from K562 CML cells was incubated with C48 as indicated above and a 32P-radiolabeled, double-stranded DNA oligonucleotide that binds Stat5/Stat5 homodimers (MGFE probe, mammary gland factor element, derived from the bovine β-casein gene promoter, 5'-AgA-TTT-CTA-ggA-ATT-CAA-3') was used to detect DNA-binding of Stat5. Anti-Stat3, anti-Stat1, and anti-Stat5 polyclonal antibodies (C20X, Santa Cruz Biotechnology) were used to identify Stat3, Stat1, and Stat5 in "super-shift" assays. For super-shift assays, concentrated antibody (1 μL) was pre-incubated with nuclear protein 20 min prior to the addition of radiolabeled probe (30 min, 37 °C) and separation by non-denaturing polyacrylamide gel-electrophoresis and autoradiographic detection.</p><!><p>Xenograft tumor studies were performed as previously described (36). Briefly, six-week-old athymic mice were purchased from Taconic Laboratories and acclimated for 3+ days prior to tumor implantation. All mice were maintained under specific pathogen-free conditions and were used in compliance with protocols approved by the City of Hope Institutional Animal Care and Use Committee. MDA-MB-468 human breast cancer cells (5 × 107) in a 1:1 mixture of Matrigel (BD Biosciences) and culture medium were subcutaneously implanted in the left flanks of nude mice. Tumor-bearing mice were randomized (8 mice per group) based on tumor volume prior to the initiation of treatment, and treatment was initiated when average tumor volume was at least 65 mm3. C48 (200 mg kg-1) was given intraperitoneally (i.p.) once daily for 5 days per week until termination of the experiment. C48 was dissolved in ddH20 supplemented with 2% Tween-80. At this dose, no lethal toxicity, or weight loss (greater than 10% body weight) was observed among treated animals. Tumors were measured twice per week with vernier calipers, and tumor volumes were calculated by the formula 0.5 * (larger diameter) * (smaller diameter) 2.</p><p>In the syngeneic mouse model, 7–9-week-old female C3H/HeJ syngeneic mice were subcutaneously injected with 5 × 105 C3L5 murine breast adenocarcinoma cells as previously described (37). Animals were monitored for tumor growth. Once the tumors were palpable (day 12 after tumor cell injection), animals were grouped (8 mice per group) to receive either i.p. injection of C48 (100 mg kg-1 or 200 mg kg-1) or vehicle in a 5 days on/2 days off cycle for 2 cycles. Tumors were measured every other day, by serial measurements of perpendicular diameters using digital calipers. Two-tailed, paired t test was used to calculate p values.</p><!><p>MDA-MB-468, MDA-MB-231 and LNCaP cells (1 × 107 each) were seeded into 10 cm dishes and allowed to attach overnight. The next day, medium was replaced with fresh medium (DMEM/5% FBS) containing either DMSO or 1-20 μM C48. Forty-eight hours later, cells were washed twice with cold PBS and harvested in PBS supplemented with Trypsin/EDTA. Cells were washed twice in PBS and stained using the Annexin-V-FITC apoptosis detection kit according to the manufacturer's instructions (BD Biosciences). Data acquisition and analysis was performed by the Flow Cytometry Core Facility at City of Hope using FACScalibur.</p><!><p>While this manuscript was under revision, a study reported identified six cysteine residues in Stat3 including Cys468 that are sensitive to hydrogen peroxide (38). The authors showed that 3 mM hydrogen peroxide blocked Stat3 from binding DNA, but not Stat1. These studies further confirm the importance of Cys468 and its modification on the Stat3-DNA interaction.</p><!><p>A) Structure of the initial lead Stat3 inhibitor compound (C36) and structures of Stat3 inhibitor compounds identified through a similarity search (C29, C30 and C48). B) Dose-response effect of compound C48 on the DNA-binding activity of Stat1 and Stat3 homodimers, as well as Stat1/3 heterodimers in vitro, as assessed by EMSA. C48 and nuclear extract (NE) that contained activated STAT proteins were pre-incubated for 30 min prior to addition of DNA for 30 min. SS, Super-shifted STAT proteins. C) Mode of action of C48 on STAT DNA-binding. As a control, NE was incubated with DNA for 30 minutes (lanes 1-2), C48 and NE that contained activated STAT proteins were pre-incubated prior to addition of DNA (lanes 3-6), NE and DNA were pre-incubated prior to addition of C48 (lanes 7-10), or C48 and DNA were pre-incubated prior to addition of NE (lanes 11-14).</p><!><p>A) The crystal structure of phosphorylated Stat3-β showing the Stat3 homodimer (cyan and lime) bound to DNA (pink) (PDB:2BG1). All 11 cysteine residues in Stat3-β are colored orange. B) Cys468 at the DNA interface is within 4.2 Å of direct contact with DNA. C) Sequence alignment of all human STAT family members showing that the cysteine at position 468 is unique to Stat3 (orange highlight).</p><!><p>A) Dose-response effect of compound C48 on DNA-binding activity of wildtype and mutant STAT homologs in vitro, as assessed by EMSA. Recombinant STATs or nuclear extract that contained activated STAT proteins was pre-incubated with C48 for 30 min prior to addition of DNA for 30 min. DNA-binding properties of STATs as assessed by size exclusion chromatography: B) Wildtype Stat3 and C) C468S mutant Stat3 in the presence and absence of 100 μM C48. Unlike the C468S Stat3 mutant (trace 1 in panel C), formation of wildtype Stat3-DNA complex was inhibited upon incubation with C48 (trace 1 in B). Each chromatogram was monitored at 495 nm except trace 4 in both panels.</p><!><p>A) Mass spectra of phosphorylated Stat3 treated with C48 and Stattic. Quadrupole time of flight mass spectra of phosphorylated Stat3 before treatment (green trace) and after treatment with C48 (blue trace) and Stattic (red trace). Multiple modifications are observed for Stat3. Based on the expected mass of the C48 fragment, more than 6 modifications were observed corresponding to 4 to 10 C48 adducts. Phosphorylated Stat3 treated with Stattic under the same conditions produce a predominant peak at 69954, which corresponds to the modification of precisely 8 residues.</p><p>B) Mutation of Cys468 reduces the number of C48 modifications. Wildtype Stat3 (blue trace) and C468S Stat3 mutant (pink trace), both unphosphorylated, were treated with C48 for one minute at 37 °C and analyzed by mass spectrometry. For wildtype Stat3, the mass spectrum indicates 3 adducts comprising 3, 4 and 5 modifications. For the C468S mutant, the mass spectrum also indicates 3 adducts, but consists of 2, 3 and 4 modifications. The green arrows indicate absence of modifications on wildtype Stat3 (wt) and the mutant Stat3 (mut). The inset shows the mass difference between the wildtype and mutant is precisely 16 daltons, as expected.</p><!><p>Effect of C48 on Oncostatin M (OSM)-induced activation and nuclear localization of Stat3-YFP protein (yellow) in serum-starved, MEF-Stat3-YFP expressing cells. The nuclei (blue) are stained with DAPI. Not all cells are transfected with the Stat3-YFP construct. Upper panel: 100x, lower panel 10x magnification. One representative result from 2 independent experiments is shown.</p><!><p>A) Effect of C48 on Oncostatin M (OSM)-induced, Stat3-mediated expression of luciferase as a measure of transcription activity. Serum-starved HeLa-Stat3-Luc cells were pre-incubated with C48 1 h prior to stimulation with OSM. Luminescence was measured 8 h post stimulation. B) Effect of C48 on IFNγ-induced, Stat1-mediated expression of luciferase as a measure of transcription activity. Serum-starved HeLa-Stat1-Luc cells were pre-incubated with C48 1 h prior to stimulation with IFNγ. Luminescence was measured 8 h post stimulation. One representative result is shown (n=3, in triplicate).</p><!><p>A-C) Effect of C48 on viability of the Stat3-pTyr705-positive cell lines MDA-MB-468 (A) and MDA-MB-231 (B) and the Stat3-pTyr705-negative cell line LNCaP (C). Cells were treated for 48 h with C48 or DMSO control and analyzed for Annexin V/PI staining. One representative result per cell line is shown (n=2, in triplicate). D-F) Western blot of total protein lysate from MDA-MB-468 cells treated with C48. D) Cells were treated for 48 h with C48 in the concentrations indicated prior to analysis for signaling proteins. E) Cells were treated for 4 h with 50 μM C48 prior to analysis of Jak2 and Src family phosphorylation. F) Cells were pre-treated for 30 min with Na3VO4 in the concentrations indicated, followed by addition of 50 μM C48 for 2 h. Cells were then analyzed for Stat3-pTyr705 levels.</p><!><p>A) Breast tumor xenograft mouse model. Tumor volume of MDA-MB-468 tumor-bearing mice treated once daily for 5 days a week with C48 (white circle) at 200 mg kg-1 (delivered i.p.) or vehicle (black circle) until termination of the experiment (day 66). B) Syngeneic mouse model. Tumor volume of C3L5 tumor-bearing mice treated (i.p.) once daily for 5 days a week with C48 at 100 mg kg-1 (black circle) or 200 mg kg-1 (black diamond), or vehicle control (white circle) until termination of the experiment (day 26). Results are presented as mean tumor volume (n=8). Bars represent standard error. *, P< 0.05 when vehicle-treated control group is compared to 200 mg kg-1 C48-treated group.</p>

PubMed Author Manuscript

Modulation of Folding Internal Friction by Local and Global Barrier Heights

Recent experiments have revealed an unexpected deviation from a first power dependence of protein relaxation times on solvent viscosity, an effect which has been attributed to \xe2\x80\x9cinternal friction\xe2\x80\x9d. One clear source of internal friction in protein dynamics is the isomerization of dihedral angles. A key outstanding question is whether the global folding barrier height influences the measured internal friction, based on the observation that the folding rates of fast-folding proteins, with smaller folding free energy barriers, tend to exhibit larger internal friction. Here, by studying two alanine-based peptides we find that systematic variation of global folding barrier heights has little effect on the internal friction for folding rates. On the other hand, increasing local torsion angle barriers leads to increased internal friction, which is consistent with solvent memory effects being the origin of the viscosity dependence. Thus, it appears that local torsion transitions determine the viscosity dependence of the diffusion coefficient on the global coordinate, and in turn internal friction effects on the folding rate.

modulation_of_folding_internal_friction_by_local_and_global_barrier_heights

Introduction<!>Local barrier of alanine dipeptide<!>Local barriers of alanine-8<!>Global barrier of alanine-8

<p>Protein folding has been successfully characterized using a funneled energy landscape, in which native interactions are the main driving force towards the folded, functional, state due to the evolutionary requirement for efficient folding.1–4 In this framework, the projection of the folding dynamics onto a well-chosen reaction coordinate (e.g. the fraction of native contacts, Q5) can be described as diffusion on a one-dimensional free energy surface, in the same picture as the description of chemical reactions in solution using Kramers theory.6 The required (position-dependent) diffusion coefficient for such a model will be influenced by the local features of the energy landscape.1 Most experiments only give direct information on the relative free energy of the stable states, or changes in barrier height, while probing contributions from local barriers on the energy landscape is much more difficult. One type of experiment which can potentially yield insights into the local dynamics on the free energy landscape involves measuring the dependence of protein relaxation times on solvent viscosity.7–16 Although the translational diffusion coefficients of small solutes have a simple negative first power (Stokes-like) dependence on solvent viscosity,17 protein relaxation times often deviate from a simple linear dependence on solvent viscosity with zero intercept. This phenomenon has been termed "internal friction" in the literature, in reference to the idea that the protein itself may act as a source of friction. As we discuss below, the actual origin of this effect may be different, but we adopt the experimental terminology of internal friction to describe this anomalous viscosity dependence. Such internal friction has been observed in native state dynamics,7 protein folding rates,8–14,16 folding transition-path times14,16 and unfolded state dynamics.15 In addition, internal friction has been inferred from the molecular phase observed in temperature-jump experiments, which is related to transition-state transit times.18</p><p>In theoretical work, the isomerization of local torsion barriers has been proposed to be one cause of internal friction.19–22 Recent explicit solvent simulations have demonstrated the link between torsion transitions and internal friction in both protein folding17 and unfolded dynamics.23 It has also been suggested that hydrogen bonding24,25 and salt bridges14 may play a role. In earlier work, we have found that the insensitivity of torsional transitions to solvent viscosity is most likely due to solvent memory effects: there is not a clear separation between the time scales for crossing a torsion barrier and the time over which the solvent loses memory of its previous configuration, and so the solvent friction is effectively lowered;17 a similar mechanism probably explains the role of salt-bridges in internal friction.14,26 While still related to friction, these possible origins of the observed viscosity dependence do differ somewhat from the intention of the original "internal friction" terminology,7 but we retain the term for consistency with the literature.</p><p>We have recently extended our work on the origin of internal friction to explaining the variation of internal friction effects from protein to protein and across the energy landscape of the same protein.27 Specifically, we showed that internal friction arises when torsion angle changes dominate the folding mechanism near the folding free energy barrier. In that work, however, it was not possible to separate clearly the effects of local and global barriers on the results. Changing the height of a local torsion barrier can change the importance of solvent memory effects on the rate of barrier crossing, and hence internal friction. But changing the height of the global folding barrier may also affect internal friction. One might expect that lower barriers, where a larger region of the energy landscape effectively contributes to the viscosity dependence of the rate, would be more likely to show internal friction effects. Indeed, internal friction is more often seen in fast-folding proteins8,10,11,14,18,28 than in slower-folding ones.29,30</p><p>Here, we set out to address the relative effect of local and global barriers on internal friction in protein folding dynamics by using two simple alanine-based peptides, alanine dipeptide and alanine-8. We choose alanine dipeptide because it is the smallest molecule exhibiting internal friction effects, and alanine-8 because it can form a small population of α-helix, a simple prototype for protein folding. An important consideration is computational cost, because we need to obtain accurate rates in a two-dimensional space of barrier height and solvent viscosity. While it would have been desirable to study model two-state folders such as the GB1 hairpin and Trp cage as in our earlier work,27 the GB1 hairpin does not exhibit any internal friction effect, and internal friction is only observed for the first barrier of Trp cage, which essentially corresponds to formation of the native helix, similar to the process we study here. We show a schematic of the local and global barriers we try to vary in Figure 1. Varying these two types of barriers independently is much more challenging in experiment, whereas in simulation we are able to vary the barriers systematically to cover a wide range of folding time scales in these two peptides. We find that the internal friction is reduced when lowering the torsion barriers, consistent with the reduction of barrier curvature (and vice versa for increasing barrier height). On the other hand, for variations of the global free energy barrier, we find no changes (within statistical uncertainty) of the internal friction.</p><!><p>The simplest example for which internal friction has been observed is the isomerization of alanine dipeptide between αR and extended minima.17 Here we systematically test the dependence of internal friction on barrier height, by introducing five different dihedral barrier heights covering a wide range of time scales, The method of varying the dihedral barrier is similar to our previous work,17 and to recent work23 showing that internal friction associated with unfolded chain end-end reconfiguration time is weaker when rescaling the dihedral barrier by a factor of 0.5.</p><p>The local barrier we vary is that for the Ψ torsion angle, which separates the extended (β and polyproline II) and α-helical states of the backbone. Changing the height of the barrier has little effect on the helical propensity of the peptide, but a large effect on the torsion angle relaxation time scale. We implement the variation of barrier height by introducing an extra torsion term (1)U(Ψ)=kΨ(1+cos(2Ψ−Ψs)) in which Ψs = 150° and kΨ = −5, −2.5, 0, 2.5, and 5 kJ/mol, noting that a positive kΨ would increase the torsion barrier and vice versa. Details of molecular dynamics methods can be found in Supporting Methods 1.1.</p><p>The resulting free energy profile along Ψ is shown in Figure 2A. The torsion barrier height ΔGΨ is estimated from the free energy difference between the extended states and the top of the barrier at Ψ = 70°. It varies roughly from 2.5 to 21.2 kJ/mol, introducing a variation of torsion relaxation times from 5.5 ps to 1.6 ns at normal water viscosity (Fig. 2D). We study the torsion relaxation time in different solvent viscosities via rescaling of the solvent mass31 in explicit solvent simulations, so that full solvent dynamics are included, as done in previous work on internal friction.17,23,24,27 We note that in simulations with implicit solvent, friction can also be varied directly but usually via a simple δ-correlated friction model.32,33 Finally, we calculate the relaxation time τ of torsion transitions by integrating the cosine autocorrelation function (ACF) for Ψ (Figure S1 and Supporting Methods 1.2), similar to previous work.23 As a cross validation on the results, we have also built a two-state Markov-state model (MSM)34–36 using the discretization described in Supporting Methods 1.3.</p><p>The relaxation time as a function of solvent viscosity and barrier height is shown in Figure 2D. We have obtained essentially identical results from the Ψ correlation time and the two-state MSM for all but the lowest barriers of alanine dipeptide; that discrepancy is most likely due to the break-down, for these low barriers, of the two-state approximation implicit in the MSM construction performed here. The deviation of the relaxation times from simple proportionality to viscosity has been quantified in experiments and simulations using two procedures. The first is a fit to a power law dependence on viscosity, τ(η) = τ0(η/η)β, where η is the viscosity, τ the relaxation time, and τ0 and η0 the relaxation time and viscosity corresponding to pure water under the conditions chosen. A power law exponent less than unity indicates internal friction. This relation fits well data at low viscosities that are primarily accessible in simulation. Internal friction can also be quantified from the τ-intercept of a linear fit to the data, known as the internal friction time τi.15 The linear fit describes the data at higher solvent viscosities better than a power law in both experiments15 and simulations.17,23 To characterize the internal friction, we report both the power law exponent β, as well as a parameter α, namely the intercept normalized by the relaxation time at normal viscosity (α = τi/τ0), to correct for the contribution to the relaxation time coming from varying the barrier height (a similar approach has been used previously23). The fitting methods and the applicability of β and α are discussed in Supporting methods 1.4. Here, we note only that both fits are phenomenological, but that they yield similar conclusions. A value of β = 1 or α = 0 corresponds to a linear relation between the relaxation time and solvent friction and therefore no internal friction, while smaller β values or larger α values suggest a strong internal friction.</p><p>There is a clear trend of increasing internal friction (larger β, or smaller α) in alanine dipeptide with increasing local torsion barrier height, whether the relaxation time is estimated from the ACF or the MSM (Figure 3). This emphasizes the major contribution of the torsion barrier itself to the internal friction of this simple peptide, consistent with the argument that solvent memory effects are the cause of the internal friction: since barrier curvature increases with barrier height, while the solvent friction kernel should remain the same, Grote-Hynes theory would predict a stronger deviation from a Kramers-like dependence on friction, as is indeed observed.37,38 To quantify the dependence of α or β on barrier height in Figure 3, we have determined the slope m of a linear fit to the dependence of α or β on barrier height, with errors δm estimated by a bootstrapping method. In Table 1, we report error bounds on the slope (m − δm, m + δm), as well as the overall probability of the true slope being positive.</p><p>An interesting feature of the results is that the exponent β does not reach a limiting value of unity for a vanishing barrier in alanine dipeptide. However, this limit is very much an exception in the data set, because the absence of a barrier means that barrier curvature in reaction rate theory cannot be used to illuminate the result. Some of the insensitivity to viscosity in this case may arise because alanine dipeptide is small enough to make some torsion transitions without displacing any water molecules, limiting the contribution from solvent viscosity to the torsion relaxation time. To test this hypothesis, we have frozen all water molecules by applying an harmonic position restraint to each cartesian component of the position of the oxygen atom of the water molecules, with a spring constant of 1000 kJ/mol/nm2 (corresponding to a mean-square displacement of 0.05 nm at 300 K). We show the Ramachandran plot and the ACF in both the non-restrained and restrained cases in the normal solvent viscosity in Figure S2. Both αR and extended minima are still present in the free energy surface with the restraints (although with slightly different energies, as expected).</p><!><p>In order to compare the dependence of internal friction on local barriers with the dependence on the global folding barrier, we need a minimal folding model. For this purpose, we have chosen alanine-8, which has a weak propensity for helix formation,39,40 and no clear global barriers in the force field we use. Therefore it serves as a good model for testing the effect of varying both the local and global barriers on the internal friction. First we introduce the same additional torsion term (Eq. 1) as used in alanine dipeptide to adjust the local torsion barrier, with kΨ = −3, −1.5, 0, 1, and 2 kJ/mol, and Ψs = 130° (note that Ψs is different from that used for alanine dipeptide, to capture a slight shift of the free energy surface F(Ψ) in the context of the longer peptide). As shown in the free energy for Ψ (averaged over all eight Ψ angles) in Figure 2B, the torsion barrier height ΔGΨ, defined as the free energy difference between the extended states and the top of the barrier at Ψ = 70°, varies from 6.0 to 15.5 kJ/mol (Figure 2B).</p><p>We probe the viscosity dependence of both local relaxation times, from the cosine ACF of Ψ averaged over all Ψ angles (individual torsion ACF is shown in Figure S3, S4, S5, S6 and S7), and of the global relaxation time, from the correlation time of the fraction of native contacts Q (Figure S8), similar to a previous study using the ACF of end-end reconfiguration time to study the global relaxation of an unfolded protein.23 The definition of Q is given in the Supporting Methods 1.5. Native contacts are defined relative to a fully helical structure for alanine-8. We obtain qualitatively similar results for the correlation time of the radius of gyration (Figure S9). We find about one order of magnitude variation of the global relaxation time when changing the local torsion barriers. Again, there is a clear correlation (Figure 3 and S1) between the local barrier height and the internal friction of global relaxation time, with βQ changing from 1.00 to 0.71 and αQ changing from 0.03 to 0.35 from the smallest to the largest torsion barriers. The internal friction of local relaxation time (βΨACF and αΨACF) become less sensitive to the barrier height for barriers above the normal dihedral barrier height. However a decreasing trend of internal friction from local relaxation time when increasing barrier height is still statistically most likely (Table 1). These results demonstrate that torsion barriers are a major contribution to internal friction effects, as probed by both local and global relaxation times.</p><p>We have also estimated the global relaxation time by using the torsion-discretized MSM. The relaxation time from the slowest mode in the MSM is consistent with that of the ACF, except for the three largest viscosities, for the highest barrier height (Figure 2E). This is probably because of sampling limitations for the highest barrier, in which the relaxation time is only one order of magnitude smaller than the trajectory length. This is also seen in the large error bar of βΨMSM and αΨMSM for the highest barrier using the MSM (Figure 3). However for the first four barrier heights, there is still a clear correlation between local barrier height and internal friction of relaxation time from the slowest mode of MSM. The discrepancy for the highest barrier may arise from limited sampling of transitions for such large barrier heights.</p><p>Qualitatively, our result of decreasing internal friction for lower barrier heights is consistent with earlier work showing a decrease of internal friction in unfolded proteins when the barrier height was scaled by a factor of 0.5.23 However, our interpretation is different in that we relate the internal friction to the intrinsic viscosity dependence of torsion isomerization, rather than to the internal friction inferred from the fit of a suitable polymer model.23</p><p>The internal friction of alanine-8 almost vanishes for the lowest torsion barrier we have tried, different from alanine dipeptide. This is presumably because, unlike alanine dipeptide, significant solvent displacements are required for torsion isomerization in the larger alanine-8 molecule. Thus, if the water is restrained in a similar fashion to what was done for alanine dipeptide, no global transitions are possible (in Q) and torsional transitions are only possible for terminal residues, as might be anticipated.</p><!><p>Having established the strong impact of local dihedral barriers on the internal friction of local and global relaxation time, the remaining question we want to address is whether there is an impact of the height of the global folding barrier on internal friction observed in the global relaxation time. The reason that some variation might be expected is that higher global barriers will make the global dynamics sensitive to a smaller region of the energy landscape, and hence may reduce the apparent internal friction. We revisit alanine-8 by varying the global barrier along the fraction of native contacts Q, which is known to be a reasonable coordinate for protein folding.41,42 The global barrier is tuned by introducing an external Gaussian potential (2)U=kQexp(−(Q−0.695)2/0.02), in which kQ is varied to be 6.27, 12.54 and 18.81 kJ/mol. This is a convenient, although slightly contrived, way of tuning the global barrier whilst exactly preserving the local features of the energy landscape. Changing the barrier height as in real proteins, by varying sequence, (or by varying contact strength in a computational model) would invariably change both the local and global barriers, making their effects on internal friction hard to disentangle.</p><p>When increasing global barrier height, sampling becomes challenging. Here we use a variant43 of transition-path sampling (TPS),44 in which transition paths are obtained by running trajectories from a dividing surface on a reaction coordinate, close to the top of the barrier on that reaction coordinate (Q here). It has been successfully used to study internal friction in the folding of the GB1 hairpin and Trp cage mini protein.27 TPS focuses on sampling rare transitions between stable states (here: unfolded and folded), avoiding wandering in each of these free energy basins between transitions, and therefore tremendously reduces the statistical error in the rate estimate, with orders of magnitude more folding events than in conventional brute force simulation.</p><p>In Figure 2C, we show four different barrier heights, including the case without an external bias on Q. Since the unbiased potential does not have a clear folding barrier on the free energy along Q, TPS would not give any advantage relative to brute force simulations. Therefore we still use the relaxation time from the ACF along Q as in the previous section. In the other cases, where TPS was performed, we have estimated β in two ways: firstly, from the folding relaxation rate computed from TPS, and secondly from the transition-path times obtained from the same calculation. It is clear that in all the four cases we have tested, the exponential fitting exponent β for the relaxation of Q is close to 0.75 (Figure 3), suggesting no significant change in the internal friction when changing global folding barriers. The internal friction for the transition path time has a similar trend, albeit with larger errors in the largest barrier. Consideration of statistical errors suggests no evidence of increasing internal friction when increasing global barrier height for both folding relaxation time and transition-path time (Table 1). Albeit for a simple peptide folding case, this suggests that the lower internal friction observed in slow folders is not intrinsic to their large folding barrier height.</p><p>The simplest interpretation of the results of the global barrier variation is by applying Kramers rate theory to the global transition. At least in the systems we have studied, local torsion barriers determine the viscosity dependence of the diffusion coefficient on Q and therefore the internal friction of the folding rate. Varying the global barrier on Q via an external potential scales the folding rate without varying the diffusion coefficient viscosity dependence. This is a non-trivial result because the folding diffusion coefficient is position-dependent and therefore its viscosity dependence will be too; varying the global barrier height could in principle have altered the sensitivity of rate to viscosity. A caveat is the somewhat contrived way in which the folding barrier has been varied, however alternative methods would not have yielded a clear conclusion. In all cases we have tested, including our previous work on two mini-proteins,27 torsion transitions remain the key factor in determining the viscosity dependence of the diffusion coefficient, and therefore the internal friction of folding.</p><p>In summary, our results from variation of local (dihedral) barrier heights confirm the importance of these barriers in determining internal friction, in both alanine dipeptide and alanine-8. Furthermore, the strong variation of solvent viscosity dependence with barrier curvature in alanine dipeptide strongly suggests solvent memory effects to be an origin of internal friction. Other effects may of course be important in different systems. Variation of the global folding barrier on Q, however, results in no change in internal friction of alanine-8. Though the current study is on short peptides, it still suggests that the larger internal friction of fast-folding proteins is related to the fact that many of them are α-helical and that their folding mechanisms are intrinsically more likely to exhibit internal friction,27 rather than to their low folding barrier heights.</p><p>A subject not addressed by the present work is the origin of internal friction in unfolded chains. In that case, the variation of solvent quality (by changing denaturant concentration) has been shown to strongly influence the degree of internal friction. Both torsional transitions23 as well as other factors such as solvent exclusion may play a role here, but further investigation on internal friction in disordered states is needed to address this.</p>

PubMed Author Manuscript

A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function.

a_dictyostelium_discoideum_mitochondrial_fluorescent_tagging_vector_that_does_not_affect_respiratory

<!>Introduction<!><!>Fluorescence and live-cell microscopy<!>Western blot analysis to monitor mitochondrial loading<!>Mitochondrial respirometry function<!>Statistical analysis<!><!>Results and discussion<!>Supplementary Movie 1<!>Supplementary Movie 2<!>Supplementary Movie 3<!>Supplementary Movie 4<!>Supplementary Movie 5<!><!>Results and discussion<!>Author statement<!>Declaration of competing interest<!>Supplementary Fig. 1

<p>Live-cell visualization of mitochondria dynamics is currently difficult in Dictyostelium discoideum.</p><p>We provide an easy and rapid procedure for visualizing mitochondria.</p><p>This technology employs a plasmid, REMIT-mRFPmars, expressing a mitochondrial targeting sequence linked to mRFPmars.</p><p>Expression of REMIT-mRFPmars does not alter mitochondrial load or function, providing a suitable tag for live-cell studies.</p><!><p>Mitochondria play important roles, most notably cellular energy production by oxidative phosphorylation [1], hence the aptly coined phrase "the powerhouse of the cell" [2]. Mitochondria are also involved in Ca2+ management [3], production of ROS [4], redox signalling [5,6] and apoptosis [7]. Many studies investigating mitochondrial function observe mitochondrial morphology and their dynamics within the cell [8]. Such observations can be achieved by the use of various fluorescent dyes such as Rhodamine 123 (R123) [9], tetramethylrhodamine-methyl-ester (TMRM) [10] and JC-1 (tetraethylbenzimi-dazolylcarbocyanine iodide) [11]. However, these dyes rely upon a mitochondrial membrane potential and can be washed out if the mitochondria experience depolarisation [12]. Furthermore, these dyes are unsuitable for use with aldehyde fixation due to resulting changes in mitochondrial metabolic state [12]. Other fluorescent dyes developed for visualizing mitochondria include the Mitotracker Red and Green dyes. Mitotracker Red binding depends on both the presence of a mitochondrial membrane potential while Mitotracker Green binding does not. These dyes can be used in combination with a number of cell fixation methods, however, they may cause cytotoxic effects following prolonged use. Other methods of real time mitochondrial imaging include the use of fluorescently tagged mitochondrial localised proteins, where the tagged protein is recognised by the mitochondrial 'Translocase of the outer/inner membrane' (TOM/TIM) protein complexes and transported into the mitochondria [13]. The transport of mitochondrial proteins into the mitochondrial matrix is facilitated by an N-terminal pre-sequence [14]. This pre-sequence can consist of 10–80 amino acid residues and is usually cleaved off by the matrix processing peptidase following localization [15]. However, these fluorescent proteins can interfere with the function of the native protein and impede mitochondrial function. As such, non-cytotoxic mitochondrial markers are required.</p><p>Dictyostelium discoideum is a tractable model system widely used for research in a range of fields including cell and developmental biology, evolutionary biology, as well as in immunology and molecular pharmacology studies. In cell and developmental biology, D. discoideum is often used to improve our understanding of cell motility [16,17]. In molecular pharmacology research, D. discoideum has been used to investigate mechanisms of action of pharmaceutical drugs including treatments for epilepsy [[18], [19], [20]] and neurodegenerative disorders [21,22]. Other studies investigate mechanisms of action of bioactive natural products such as curcumin, naringenin and a range of bitter tastants [[23], [24], [25]]. One recent study, identifying a mitochondrial protein, GcvH1, involved in the cellular function of cannabinoids on the glycine cleavage system [26], highlights the presence of an N-terminal mitochondrial localization sequence and thus raises the possibility of using this sequence for mitochondrial tagging in D. discoideum. To further these studies, a non-cytotoxic mitochondrial marker is required that can be used in D. discoideum without affecting cellular respiratory function.</p><p>In this study we created a novel expression plasmid (REMIT; red mitochondria) for real time visualization of mitochondria in D. discoideum. The REMIT plasmid allows the expression of an enhanced RFP protein (mRFPmars) [27] with a mitochondrial localization sequence situated at its N-terminus. We show that transfection of REMIT into D. discoideum cells facilitates the localization of mRFPmars to the mitochondrial matrix. We also show that the presence of mRFPmars within the mitochondria has no effect on mitophagy or cellular respiratory function. We therefore present a method that allows the real time visualization of mitochondria within D. discoideum cells with no deleterious effects.</p><!><p>REMIT plasmid construction. The mitochondrial localization sequence was ligated into the pDXA-389-2 plasmid 5' of the mRFPmars gene using EcoRI and BamHI restriction sites. Actin 6 promoter, A6P; actin promoter 15, AP15; Ampicillin resistance cassette, APr; monomeric red fluorescence protein, mRFPmars; actin terminator 8, A8T; Geneticin resistance cassette, G418r. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p><!><p>For immunolabeling, cells expressing REMIT were plated on round 12-mm glass coverslips, and after 20 min were fixed with 15% picric acid/2% paraformaldehyde in 10 mM PIPES, pH 6.0, for 20 min and post-fixed with 70% ethanol for 10 min [31]. Cells were then washed three times in PBS, once with 10 mM PIPES, and twice with PBS/1% glycine, and incubated in blocking buffer (PBS plus 2% bovine serum albumin) for 1 h at room temperature (RT). After blocking, the cells were washed three times with PBS and incubated with primary antibodies (2 μg/ml mouse monoclonal anti-porin antibody (Developmental studies hybridoma bank (DSHB); 70-100-1) [32], and 1:1000 rat anti-RFP (6G6 anti-red rat mAb, Chromatek) for 2 h, followed by the incubation with secondary antibodies (1:1000 Alexa 488-conjugated goat anti-mouse IgG (Invitrogen; A28175) and 1:1000 rabbit anti-rat (Alexa fluor® 488 rabbit anti-rat IgG, Life technologies), for 1 h. After immunostaining, samples were washed three times in PBS and embedded using Fluoromount-GTM, with DAPI (1:1000 of 1 mg/ml DAPI dissolved in methanol; Invitrogen, 00-4959) to stain DNA. For live-cell microscopy, cells were seeded in μ-dishes (Ibidi, 80606), or open chambers as described previously [28].</p><p>Confocal microscopy was performed at the Bioimaging core facility of the Biomedical Center (LMU Munich) using an inverted Leica TCS SP8 equipped with lasers for 405, 488, 552, and 638 nm excitation. Images were acquired with a HC PL APO 63x/1.40 oil PH3 objective. Recording was sequential to avoid bleed-through. Alexa-488, and RFP were recorded with the hybrid photo detectors, DAPI with the conventional photomultiplier tube.</p><p>High resolution live-cell imaging was performed using an inverted Zeiss LSM 900 Airyscan 2 microscope [33]. Images were acquired with a Plan-Apochromat 63x/1.40 oil DIC objective with a GaASP-PMT detector (450–700 nm) in the MPCX SR-4y modus at an excitation of 558 nm. Z-stacks (25 corresponding to 4.32 μm) were recorded over time (2.55 s per z-stack). 3D reconstructions of single z-stacks were performed using the Imaris software package (Bitplane, Zurich, Switzerland).</p><!><p>Cell lysates (30 μg) were separated by gel electrophoresis, transferred to nitrocellulose membranes (Merck Millipore, IPFL00010), and analysed by Western blotting. A mouse anti-porin primary antibody (0.2 μg/ml, DSHB, 70-100-1) and a goat anti-mouse secondary antibody (1:10000, Li-Cor, 926–32210) were used to confirm the presence of porin. A streptavidin conjugate (1:5000, Invitrogen, S21378) which binds to the mitochondrial protein MCCC1 (mitochondrial 3-methylcrotonyl-CoA carboxylase α [34]) was used to measure the levels of this mitochondrial protein. Blots were analysed using Odyssey software. Total protein loaded was stained with Revert 700 Total Protein Stain (Li-Cor, 926–11010) and imaged and quantified on the Odyssey CLx.</p><!><p>The effects that REMIT expression may have on mitochondrial stress were investigated in real time [35]. In these experiments, a Seahorse XFe24 Extracellular Flux Analyzer was used to measure mitochondrial respirometry within REMIT expressing cells, wildtype cells and cells expressing mRFPmars lacking the mitochondrial localization sequence. Mitochondrial respirometry was measured in terms of the basal O2 consumption rate, the O2 consumption rate devoted to the synthesis of ATP, the maximum O2 consumption rate, the contribution of Complex I to the maximum O2 consumption rate, the contribution of Complex II to the maximum O2 consumption rate, and the O2 consumption rate devoted to mitochondrial function other than ATP synthesis, i.e. "proton leak".</p><!><p>The distribution of all experimental data was tested using the Anderson-Darling test for normality. All data that showed a Gaussian distribution were analysed using parametric tests. Data from two groups not showing a Gaussian distribution were analysed using a Mann-Whitney T-test. The one-way analysis of variance (ANOVA) statistical test was used to test for significance between the means of three or more independent groups of normally distributed data.</p><!><p>Fixed-cell imaging of REMIT localizing to the mitochondria. (A) D. discoideum cells expressing REMIT-mRFPmars (red), were fixed and immunolabelled using an anti-porin antibody (green), and stained with DAPI to visualize DNA (blue). Confocal single plane imaging showed red fluorescent protein localizing to mitochondria in D. discoideum. Scale bar correspond to 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p><p>Live-cell imaging of REMIT localizing to the mitochondria. (A) Time lapse single plane imaging on a confocal microscope showing D. discoideum cells transfected with REMIT-mRFPmars. The upper panel shows phase contrast images, the lower panels the intensity of the mRFPmars signal according to grey levels depicted in colour-mode fire [38], scale bar 5 μm. Images correspond to Supplementary movie 1. Similar live cell imaging using red fluorescence are provided in Supplementary movie 2, using under agar inverted fluorescence in Supplementary movie 3, Z stack imaging in Supplementary movie 4, and 3D reconstruction of live-cell imaging in Supplementary movie 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p><!><p>Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrep.2020.100751</p><!><p>Confocal single plane imaging of live REMIT-expressing wild-type Dictyostelium cells, with intensity indicated by colour. Imaging corresponds to that shown in Fig. 3, and the frame rate is 5 images per second. Scale bar, 5 μm.</p><!><p>Confocal single plane imaging of live REMIT-expressing wild-type Dictyostelium cells, showing red flourescence. Imaging corresponds to that shown in Fig. 3, and the frame rate is 5 images per second. Scale bar, 5 μm.</p><!><p>REMIT-expressing WT Dictyostelium cells, imaged with live cells using inverted fluorescence microscopy under agar (Olympus IX71). The frame rate is 1 image per second. Scale bar correspond to 5 μm.</p><!><p>Z-stacks of live REMIT-expressing WT Dictyostelium cells were imaged by high-resolution microscopy (Zeiss LSM 900 Airyscan). Imaging is recorded at the frame rate of 5 images per second.</p><!><p>3D reconstruction of a z-scan recorded by high-resolution microscopy (Zeiss LSM 900 Airyscan 2) of live REMIT-expressing WT Dictyostelium cells. The first stack from the data set of Supplementary Movie 4 was used for the 3D reconstruction.</p><!><p>Expression of REMIT within D. discoideum has no effect on mitochondrial load or respiratory function. Mitochondrial load and respiratory function was evaluated in D. discoideum cells transfected with the REMIT plasmid. (A) Western blot analysis was used to assess levels of a mitochondrial-specific marker protein (MCCC1) and total protein levels. Data represent the mean and SEM (P > 0.05), n = 6 (Mann-WhitneyU test). (B) Mitochondrial respiratory function was evaluated in cells transfected with REMIT, with wildtype cells, and with the REMIT plasmid lacking the localization sequence (RFP). Respiratory function was quantified in terms of basal O2 consumption rate, O2 consumption rate devoted to the synthesis of ATP, maximum O2 consumption rate, contribution of complex I to the maximum O2 consumption rate, contribution of complex II to the maximum O2 consumption rate, O2 consumption rate devoted to mitochondrial function other than ATP synthesis, i.e. "proton leak". Data represent the mean and SEM (P > 0.05), n = 6 (One way ANOVA).</p><!><p>The requirement of mitochondria to carry out normal respiratory function is fundamental to maintaining a healthy cell. Any deleterious effect on respiratory function as a result of REMIT transfection would result in downstream processes being disrupted. Thus, it is necessary to confirm that mitochondrial respiratory function is not disrupted. Mitochondrial respirometry function was therefore measured in terms of the basal O2 consumption rate, the O2 consumption rate devoted to the synthesis of ATP, the maximum O2 consumption rate, the contribution of complex I to the maximum O2 consumption rate, the contribution of complex II to the maximum O2 consumption rate, and the O2 consumption rate devoted to mitochondrial function other than ATP synthesis, i.e. "proton leak" (Fig. 4B). These data were obtained from cells transfected with REMIT, cells transfected with REMIT lacking the MLS, and untransfected wildtype cells. For all six conditions no significant difference (P > 0.05) was found between the three cell lines. This shows that mitochondrial function is not affected despite the presence of mRFPmars localised within the mitochondrial matrix.</p><p>These experiments thus show that transfection of D. discoideum cells with REMIT provides a quick, cheap and convenient method to visualize mitochondria in real time. The use of REMIT would be advantageous in studies involving the need for both visualization and normal respiratory function. These studies can include investigation into mitochondrial fission and fusion events, mitochondrial dynamics and mitochondrial morphology.</p><!><p>CJP and RSBW conceived the research and wrote the paper. ECW and JD-O contributed to imaging and measuring mitochondria load. CS, LMF, SJA, PRF analysed mitochondrial function. AM-T provided confocal microscopy and 3D modelling. All authors contributed to editing the paper.</p><!><p>The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</p><!><p>GREMIT-expressing WT Dictyostelium cells, imaged with live cells using inverted fluorescence microscopy under agar (Olympus IX71). Scale bar correspond to 5 μm.</p>

PubMed Open Access

Nanofiber network with adjustable nanostructure controlled by PVP content for an excellent microwave absorption

Carbon nanofibers were widely utilized to improve microwave absorption properties since they are a promising lightweight candidate. Adjustable conductive nanostructures of carbon nanofibers were synthesized by electrospinning technique. The conductive network is controlled by the polyvinyl pyrrolidone (PVP) content due to the special hygroscopicity of PVP. The increased adhesive contacts of nanofibers provide more transmission paths for electrons to reduce the effect of air dielectric. Satisfactorily, the carbon nanofibers that carbonized from the polyacrylonitrile (PAN) and PVP (the mass ratio is 6:4) show excellent microwave absorption performance. The minimum reflection loss (RL) value is −51.3 dB at 15.2 GHz and the maximum effective absorption frequency width (<−10 dB) is 5.1 GHz with the matching thickness of only 1.8 mm. Thereby, we believe that this research may offer an effective way to synthesize lightweight carbon nanofibers microwave absorbents.

nanofiber_network_with_adjustable_nanostructure_controlled_by_pvp_content_for_an_excellent_microwave

<!>Results<!>Conclusion<!>Method

<p>In modern society, electromagnetic (EM) absorbents have become an indispensable part of information equipment, which can reduce the harm to the surroundings and the humans. The principle of EM absorbers is making microwave transform into other types of energy to attenuate EM wave [1][2][3] . Traditional microwave absorption materials like metal as well as alloys, ferrites and all varieties of these composites with strong microwave absorption abilities and board effective frequency width develop rapidly. However, it is also significant for actual demand to reduce the density and weight of microwave absorbers at the same time [4][5][6] .</p><p>As famous lightweight materials, carbon nanomaterials have been achieved tremendous attention. Various classifies of carbon nanoparticles such as carbon nanotubes, carbon cloths and so on are of many wonderful characters such as large areas, low cost, good stability and great electrical conductivity [7][8][9][10] . Thereby, carbon nanomaterials are quite befitting for EM wave absorption field 11,12 . Among numerous carbon nanomaterials, carbon nanofibers are of huge interest for EM wave absorption due to their one-dimensional nanostructure that can form conductive network [13][14][15] . For instance, Liu et al. successfully fabricated a kind of helical CNFs coated-carbon fibers through catalytic chemical vapor deposition. The minimum RL value was −32 dB at 9.0 GHz and the widest effective frequency width was 9.8 GHz with only 15% filler ratio 16 . Porous carbon nanotubes decorated carbon nanofibers were also achieved with the minimum RL value of −44.5 dB at 10.7 GHz as well as the broad effective frequency width of 7.1 GHz 17 . Chu et al. compared the microwave absorption abilities of different diameters, they believed that complex permittivity improved along with the decreasing diameters since it contributed to the conductive network 18 . Based on their researches, we can discover that the microwave absorption performance closely depends on their design of one-dimensional nanostructures on carbon nanofibers. In principle, morphology change of carbon nanofibers could dramatically influence the transferring path of the electrons as well as the construction of the conductive network 19 . Accordingly, the permittivity and polarization process would be controlled in the range of testing frequency artificially. Furthermore, electrospinning fiber technique can be used accurately to obtain diversiform nanofibers, which is a good choice to synthesize carbon nanofibers 20,21 .</p><p>In this paper, we added PVP as a structural adjuster to modify PAN based carbon nanofibers by changing the PVP content, followed by electrospinning and next annealing process. PVP, as a high-molecular compound, it is liable to dissolve in water. In addition, the more average molecular weight of PVP, the more chance of it to be agglutinating 22 . Considering with these intrinsic qualities of PVP, we easily gain nanofiber network with adjustable nanostructure controlled by PVP content. Gratifyingly, the microwave absorption performance is enhanced, too. For the carbon nanofibers carbonized from the PAN and PVP (the mass ratio is 6:4 and the filler ratio is 20%), the RL value is −51.3 dB at 15.2 GHz and the maximum effective absorption frequency width (<−10 dB) is 5.1 GHz with only 1.8 mm. This work provides a novel strategy to build the conductive network of carbon nanofibers through adjusting the content of PVP, which may be impulse the development of the lightweight microwave absorbers.</p><!><p>Figure 1 is the entire synthesis schematic diagram of our nanofibers conductive network. The precursor solution of PAN and PVP was transformed into nanofibers via a representative electrospinning technology. After being treated at 800 °C for 3 h under N 2 atmosphere inside tube furnace, carbon nanofibers with adjustable nanostructure were finally fabricated. Detailed nanostructures of different nanofibers are detected using SEM and TEM methods. Obviously, nanofibers of PAN/PVP samples show typical one-dimensional morphology of electrospun fibers in Fig. 2(a-c). There surfaces are smooth. In addition, we can easily see that the nanofibers become more and more thicker since the quality of PAN is constant and the quality of PVP is larger. The average diameters of PAN/PVP-7/3 sample, PAN/ PVP-6/4 sample and PAN/PVP-5/5 sample are 167 nm, 277 nm and 444 nm, respectively. After calcined at 800 °C under N 2 atmosphere, it can be seen that some of nanofibers are bent and bonded in Fig. 2(d-f). Notably, this phenomenon is relatively clearer with the increasing content of PVP. Due to the strong hygroscopicity of PVP, the solvent doesn't volatilize very well so that the nanofibers change into a sticky one. However, nanofibers of PVP (500) sample present a fracture situation. It is very difficult to identify the one-dimensional nanofibers in Fig. 1(f). Moreover, Fig. 2(g,h) displays the TEM pictures of PVP (333) sample. It should be noticed that there is typical carbon margin in Fig. 2(h). Also, physical photos of the white PAN/PVP-6/4 sample and the black PVP (333) sample are provided in Fig. 2(i).</p><p>The PAN/PVP nanofibers were also heated at 200 °C, 400 °C and 600 °C. As can be seen from Fig. 3, all of these samples present a curved one-dimensional nanofibers morphology. The nanofibers are getting thicker with the increasing content of PVP. Besides, the fusing phenomenon become serious when we added more PVP. The RL values of different carbon nanofibers are provided in Fig. 5(a-f) using the following formulas [23][24][25] : www.nature.com/scientificreports www.nature.com/scientificreports/</p><p>where Z in is the input impedance, Z 0 is the impedance of free space, f is the frequency, d and c represent the thickness and the velocity of light, respectively. When the thickness increases, the RL values shifts to lower frequency, indicating that EM wave absorption frequency and the thickness of absorbers can be modulated. Among PVP (214), PVP (333) and PVP (500) samples, PVP (214) sample shows the poorest microwave absorption abilities. Different microwave absorption performances have something to do with the content of PVP. As the RL values below −10 dB, effective microwave absorbers will make 90% microwave attenuated 26 . Thereby, when matching thickness are 1. 6(a,b). As seen in Fig. 6, PVP (333) sample has more boarder effective frequency width while the microwave absorption ability of PVP (500) sample is stronger. The electromagnetic parameters (complex permittivity: ε = ε′ − jε″; the complex permeability: μ = μ′ − jμ″) of carbon nanofibers are tested by a network analyzer. Herein, magnetic properties of carbon nanofibers can be neglected since the dielectric loss more critical. Obviously, permittivity shows a sustainable growth with the increasing content of PVP. The ε′ of PVP (214) sample is the lowest among these carbon nanofibers. The ε′ of PVP (333) sample is from 13.0 to 7.2 while PVP (500) sample descends from 14.6 to 8.2. The ε″ values of PVP (333) sample and PVP (500) sample are higher than that of PVP (214) sample. In Fig. 7, some small fluctuations of ε″ curves arise from the multiple nature resonances. According to pervious reports, it was found that the www.nature.com/scientificreports www.nature.com/scientificreports/ complex permittivity would improve with the increasing average diameters of the PAN based carbon nanofibers 18 . Nevertheless, our nanofibers do not suit this situation. This phenomenon may refer to their special adhesive nanostructures. Namely, average diameters could not be the main reason that effects the complex permittivity. As we all know, if there are some gaps between nanofibers, air will reduce the dielectric of nanofibers, bringing about lower permittivity values. Therefore, the adhesive carbon nanofibers would be the dominant reason that increases the dielectric constant. Thanks to the function of PVP, the more contact carbon nanofibers, the electrical conductivity is higher. As studied by other groups, dielectric loss can be caused by conductive loss as well as by polarization loss involved in the relaxation process 27 . Moreover, different polarization process including ionic polarization, electronic polarization and dipole polarization can be considered in this cause 28 . However, ionic polarization and electronic polarization always happen at high frequency such as 10 3 -10 6 GHz, dipole polarization should be the main reason that leads to the polarization process. Using the Eq. ( 3), dielectric loss degree can be evaluated in Fig. 7(d) [29][30][31] :</p><p>e</p><p>The results manifest that the dielectric loss tangent of PVP (214) sample is around 0.1. The dielectric loss tangent curve of PVP (333) sample show a rising trend followed by a drop condition. PVP (500) sample show a raising trend. So, we suggest that both PVP (333) sample and PVP (500) are of higher dielectric loss.</p><p>The RL values and frequency dependence on electromagnetic parameters of different sample filler ratios are displayed in Figs 8 and 9. Accordingly, the permittivity goes up with the increasing sample filler ratio. When the sample filler ratios are 30% and 40%, the microwave absorption abilities of PVP (333) sample are much better than that of other two samples. Interestingly, RL values decreases with the increasing content of PVP when the Impedance matching is an important factor to the microwave absorbers. If the nanofibers are of good impedance matching properties, more microwave will go into the nanofibers and will be attenuated 32,33 . To synthesize a satisfactory microwave absorber, it is the key to improve the microwave absorption performance or board the effective frequency width. Figure 10 www.nature.com/scientificreports www.nature.com/scientificreports/ In Fig. 11, PVP (214) sample shows the lowest average conductivity while the average conductivities of PVP (333) sample and PVP (500) can be greatly improved at S, C, X and Ku frequency range with the increasing content of PVP. The electronic impedance spectrum (EIS) for PVP (214), PVP (333) and PVP (500) were measured to evaluate their conductivity. As can be seen in the Nyquist plot in Fig. 11(b), the theoretical prediction of electrical conductivity in the following order PVP (214) < PVP (333) < PVP (500), indicating the higher conductivity with the increasing content of PVP. In this case, more contact positions of nanofibers will make electrons have multiple paths to transfer, which creates the increased conductivity. The carbon conductive network is fabricated simultaneously.</p><p>Table 1 gives some data of similar carbon nanofibers about their microwave absorption performance. In Table 1, we can easily conclude that all kinds of carbon nanofibers show enhanced microwave absorption abilities. Compared with carbon fibers that heap up one by one, nanofibers with some bonded places are more beneficial to make electrons transfer. When this type of carbon nanofiber is put in EM field, electron is likely to transfer with many paths. Hence, among these different carbon nanofibers, our adjustable shaped carbon nanofibers display the minimum RL value and broader effective bandwidth with the thinnest thickness, which implies that it is very potential to be used as lightweight microwave absorber.</p><!><p>Carbon nanofibers with adjustable nanostructure were gained by electrospinning method. When calcined at the same temperature (800 °C), the PVP content could be the main reason that influence the microwave absorption abilities. The increased adhesive contacts of nanofibers create the more paths for electrons to transfer and can reduce the effect of air dielectric. Furthermore, appropriate impedance matching is also responsible for the excellent microwave absorption abilities. In detail, the minimum RL value is −51.3 dB at 15.2 GHz and the maximum effective absorption frequency width (<−10 dB) is 5.1 GHz both with 1.8 mm. Our work may be helpful for the exploitation of lightweight microwave absorbents in near future.</p><!><p>Materials. All chemicals and reagents are supplied by business supporters and they are all without pretreated. These chemicals are polyacrylonitrile (PAN, M w = 150 000) and polyvinylpyrrolidone (PVP, M w = 5 800). N, N-dimethylformamide (DMF) is needed as well.</p><p>Synthesis of carbon nanofibers. Carbon nanofibers were obtained by electrospinning technology and following high-temperature carbonization. First, 0.5 g of PAN was added into 5 mL DMF with continuous magnetic stirring. Then, a certain amount of PVP was also introduced to this system with stirring for 24 h to achieve a transparent and syrupy liquid. The mass ratio of PAN and PVP is 7:3, 6:4 and 5:5, respectively. The precursor nanofibers are called PAN/PVP-7/3, PAN/PVP-6/4 and PAN/PVP-5/5, respectively. Second, this liquid was sucked into a 5 mL plastic syringe equipped with a stainless needle. The next process was the electrospinning. Specifically, the voltage parameters were 20 kV, the collection distance was 15 cm and the pushing speed was 0.5 mL/h. After that, the precursor PAN/PVP nanofibers were dried in a vacuum oven for a day and the PAN/PVP nanofibers were calcined at 800 °C for 3 h surrounded with N 2 atmosphere. Herein, the average rate was of 2 °C per minute. And the carbon nanofibers are marked as PVP (214), PVP (333) and PVP (500), respectively. When these PAN/PVP nanofibers were heated at 200 °C, 400 °C and 600 °C, these samples are named as PVP (214-200), PVP (333-200), PVP (500-200), PVP (214-400), PVP (333-400), PVP (500-400), PVP (214-600), PVP (333-600) and PVP (500-600), respectively.</p><p>Characterization. The crystal structure was measured by X-ray diffraction (XRD) under Cu Kα radiation.</p><p>The SEM (Hitachi-S4800) as well as TEM (Tecnai G2 F30) were used to observe the microtopography of these nanofibers. A vector network analyzer (Agilent, N5244A) was applied to test the electromagnetic parameters in the range of 2-18 GHz. The samples (20% filler ratio) and paraffin (80% filler ratio) were crushed into a cylinder. The inner diameter and the outer diameter were 3.0 mm and 7.0 mm. When changing the filler ratio, the samples (30%, 40% and 50% filler ratio) are called as PVP (214, 30), PVP (333, 30), PVP (500, 30), PVP (214, 40), PVP (333, 40), PVP (500, 40), PVP (214, 50), PVP (333, 50) and PVP (500, 50), respectively.</p>

Scientific Reports - Nature

A core–brush 3D DNA nanostructure: the next generation of DNA nanomachine for ultrasensitive sensing and imaging of intracellular microRNA with rapid kinetics

A highly loaded and integrated core-brush three-dimensional (3D) DNA nanostructure is constructed by programmatically assembling a locked DNA walking arm (DA) and hairpin substrate (HS) into a repetitive array along a well-designed DNA track generated by rolling circle amplification (RCA) and is applied as a 3D DNA nanomachine for rapid and sensitive intracellular microRNA (miRNA) imaging and sensing.Impressively, the homogeneous distribution of the DA and HS at a ratio of 1 : 3 on the DNA track provides a specific walking range for the DA to avoid invalid and random self-walking and notably improve the executive ability of the core-brush 3D DNA nanomachine, which easily solves the major technical challenges of traditional Au-based 3D DNA nanomachines: low loading capacity and low executive efficiency. As a proof of concept, the interaction of miRNA with the 3D DNA nanomachine could initiate the autonomous and progressive operation of the DA to cleave the HS for ultrasensitive ECL detection of target miRNA-21 with a detection limit as low as 3.57 aM and rapid imaging in living cells within 15 min. Therefore, the proposed core-brush 3D DNA nanomachine could not only provide convincing evidence for sensitive detection and rapid visual imaging of biomarkers with tiny change, but also assist researchers in investigating the formation mechanism of tumors, improving their recovery rates and reducing correlative complications. This strategy might enrich the method to design a new generation of 3D DNA nanomachine and promote the development of clinical diagnosis, targeted therapy and prognosis monitoring.

a_core–brush_3d_dna_nanostructure:_the_next_generation_of_dna_nanomachine_for_ultrasensitive_sensing

Introduction<!>Results and discussion<!>Execution efficiency of the designed core-brush 3D DNA nanomachine<!>Feasibility investigation of the core-brush 3D DNA nanomachine<!>Application of the 3D DNA nanomachine for sensitive detection of miRNA-21<!>Rapid and sensitive imaging of miRNA-21 in living cells<!>Conclusions

<p>In living systems, some highly complicated and hierarchical machines have evolved to perform signicant biological processes with remarkable precision and efficiency. [1][2][3] Inspired by natural ingenuity, diverse DNA-based nanodevices have been created to convert chemical energy into mechanical motion, [4][5][6][7][8] holding great promise for intelligent drug delivery, [9][10][11][12] disease diagnosis [13][14][15][16] and biosensing analysis. [17][18][19][20] Recently, threedimensional (3D) DNA nanomachines based on gold nanoparticles (AuNPs) [21][22][23][24] have attracted extensive attention due to their improved abilities in simulating complex biological operation compared with one-dimensional (1D) 25,26 or twodimensional (2D) DNA nanomachines. 27,28 However, the limited amount of track and the disordered immobilization of the DNA components (DNA walker and substrate) with a heterogeneous nano (Au)-bio (DNA) interface resulted in the low loading capacity of DNA components and the invalid movement of the DNA walker, further restricting the whole executive ability and efficiency of the Au-based 3D DNA nanomachines. On account of these disadvantages, it has long been a challenging goal to develop a new type of 3D DNA nanomachine that could not only greatly enhance the loading capacity of the DNA components in an orderly manner, but also signicantly improve the executive ability and efficiency.</p><p>Herein, a core-brush 3D DNA nanomachine with highly loaded and integrated properties was developed for intracellular microRNA (miRNA) imaging and ultrasensitive sensing with rapid kinetics (Scheme 1). Firstly, the extremely long DNA track cross-linked on a magnetic nanobead (MNB) was generated by rolling circle amplication (RCA), in which the locked DNAzyme walking arm (DA) and electrochemiluminescent signal tag Rubpy (Ru) labeled hairpin substrate (HS) at a ratio of 1 : 3 were orderly assembled into the DNA track with a repetitive array to construct the core-brush 3D DNA nanomachine (PART A). According to PART B, once the target miRNA-21 was introduced into the 3D DNA nanomachine, the hybridization of miRNA-21 with a locking strand (L) via strand-displacement reaction could release the active DA from the locked DA (L-DA) to hybridize with the adjacent HS. Fueled by the DNAzymeinduced ribonucleotide hydrolysis, the HS was then cleaved by the active DA in the presence of cofactor Mn 2+ to release the Rulabelled segment (S). When S was captured by the DNA hairpin (H) onto a gold nanoparticle-assembled glassy carbon electrode (depAu/GCE) through the Au-S bond, a highly intense ECL signal was generated for ultrasensitive detection of miRNA-21 with a detection limit as low as 3.57 aM (PART C). Impressively, when the DA and HS-1 labelled with a uorophore (FAM) and quencher (BHQ) were used to assemble our core-brush 3D DNA nanomachine (PART D), the intracellular miRNA-21 could activate autonomous motions of the DA to cleave the HS and release the FAM-labelled segment away from BHQ in the presence of the cofactor Mn 2+ , leading to the quick uorescence recovery of FAM within 15 min for sensitive imaging of lowabundance target miRNA-21. Compared with the limited amount of track in the Au-based 3D DNA nanomachine, the core-brush 3D DNA nanomachine not only provided a specic walking range for the DA to avoid inactive and stochastic selfwalking, but also greatly increased the loading capacity and local concentrations of DNA components to signicantly enhance the executive efficiency of the core-brush 3D DNA nanomachine. Thus, this strategy gives impetus to exploit highperformance 3D DNA nanomachines for specic biological applications in complex cellular environments, such as biodiagnostics and bioanalysis.</p><!><p>Characterization analysis of the core-brush 3D DNA nanomachine Polyacrylamide gel electrophoresis (PAGE) was employed to estimate the construction of the core-brush 3D DNA nanomachine. According to Fig. 1A at the bottom band of lane 11, but not in lane 12, which demonstrated that miRNA-21 could active the proposed 3D DNA nanomachine to generate numerous S.</p><p>Then, we also employed atomic force microscopy (AFM), UVvis spectroscopy, zeta potential analysis and dynamic light scattering (DLS) to verify the formation of the core-brush 3D DNA nanomachine. According to Fig. 1B and S1A, † the AFM characterization of the MNB core exhibited relatively smooth surfaces with a height of 66 nm approximately. Aer the accomplishment of the core-brush 3D DNA nanomachine, the apparent DNA brushes were observed on the surface of the core (Fig. 1C and S1B †) with an obvious increase in the height (70 nm). Likewise, the MNB core did not have any peak in the UV-vis absorption spectra (Fig. 1D, black line). Accompanied by the successful construction of the core-brush 3D DNA nanomachine, a very distinct characteristic deoxynucleotide absorption peak located at 260 nm was observed (Fig. 1D, red line). Moreover, the corebrush 3D DNA nanomachine possessed a decreased zeta potential of À26.41 mV compared with the MNB core of À10.57 mV owing to the negative charges of the DNA (Fig. 1E). As illustrated in Fig. 1F, the hydrodynamic diameter of the MNB core and corebrush 3D DNA nanomachine measured by DLS were around 68.5 nm and 233 nm, respectively. The hydrodynamic diameter of the core-brush 3D DNA nanomachine was bigger than the actual size owing to the formation of a hydrogen bond between the hydroxyl of H 2 O and DNA. The remarkable comparisons of the above results suggested successful formation of the proposed core-brush 3D DNA nanomachine.</p><!><p>To study the execution efficiency of the designed core-brush 3D DNA nanomachine (Fig. 2A) in contrast with the conventional 3D DNA nanomachine based gold nanoparticles (AuNPs) (Fig. 2B), we used uorophore (FAM) and quencher (BHQ)labeled HS-1 or FAM-labeled HS-2 to construct the proposed 3D DNA nanomachine and Au-based DNA nanomachine, respectively. In the presence of the cofactor Mn 2+ , the target miRNA-21 could activate the DA to hybridize and cleave the HS for releasing the FAM-labelled segment (S) away from the quencher BHQ or AuNPs and obtaining the prominent uorescence recovery of FAM. The real-time uorescence of FAM for the proposed core-brush 3D DNA nanomachine showed a speedy uorescence intensity growth and reached saturation about 900 s (Fig. 2C, curve a). In contrast, the typical Au-based 3D DNA nanomachine demonstrated a very slow uorescence signal growth and the uorescence saturation plateau did not appear within 2500 s (Fig. 2C, curve b). To calculate the initial rate of the DNA nanomachine, we veried the relationship between the uorescence intensities and reaction time of the proposed 3D DNA nanomachine and the Au-based 3D DNA nanomachine. According to Fig. 3D, the uorescence intensities shown the desirable linear relationships with the reaction time in the rst 250 s. The regression equations of the proposed core-brush 3D DNA nanomachine (Fig. 2D, curve a and linear t for curve a) and the Au-based 3D DNA nanomachine (Fig. 2D, curve b) were I ¼ 6.064t + 77.98 and I ¼ 0.7522t + 311.3, respectively. Based on the above results and related calculations, we found that the initial rate of our 3D DNA nanomachine (8.03 Â 10 À11 M s À1 ) was more than 7-fold higher than that of the control Au-based 3D DNA nanomachine (1.11 Â 10 À11 M s À1 ). Moreover, by rst-order derivation of the real-time uorescence increase curves of Fig. 2C, we could obtain the reaction rate curves of the DNA nanomachine as shown in Fig. 2E. The reaction rate of our designed 3D DNA nanomachine was extraordinarily high in the rst 500 s and then became very low or even went to zero (Fig. 2E, curve a). In comparison, the reaction rate and the overall efficiency of the Au-based 3D DNA nanomachine were relatively low (Fig. 2E, curve b). Collectively, these results demonstrated that the core-brush 3D DNA nanomachine possessed obviously higher execution efficiency than the Au-based 3D DNA nanomachine on account of the high loading and integration of the DNA components in an orderly manner on the programmable DNA track.</p><!><p>The core-brush 3D DNA nanomachine was composed of multiple components, and thus each component might have a potential impact on its overall executive efficiency. Firstly, we designed a control unassembled nanomachine that only contained a free DA and FAM and BHQ-labeled HS-1 at a ratio of 1 : 3 to conrm that the DA tended to walk along the HS-1 immobilized on the DNA track rather than the scattered HS-1 in the solution (Fig. 3A). Then, to verify that the highly executive efficiency of the proposed 3D DNA nanomachine was due to the ordered array of the DA and HS-1 on the programmed DNA track, the second control by mixing scattered DA with HS-1@MNB was designed to construct the DA-unset nanomachine in Fig. 3B. In this manner, the specic walking range for each DA was uncertain. Moreover, to ensure that the intense growth of uorescence intensity was due to the highly loaded and integrated mode of our designed 3D DNA nanomachine, a control unintegrated nanomachine that only included a single DNA track assembled with a DA and HS-1 at 1 : 3 was further designed as shown in Fig. 3C. According to Fig. 3D, the uorescence signal increase of the unassembled nanomachine (blue curve a) and the DA-unset nanomachine (black curve b) did not reach the saturation state in 2500 s. According to red curve c, although the unintegrated nanomachine reached reaction equilibrium quickly, its saturated uorescence intensity was relatively weak. As depicted in Fig. 3E, the reaction rate of the unassembled nanomachine (curve a) and the DA-unset nanomachine (curve b) was extraordinarily low and even went to zero. Besides, the initial rate of the unintegrated nanomachine (8.60 Â 10 À11 M s À1 ) was roughly close to that of our proposed 3D DNA nanomachine (Fig. 3F).</p><p>These results might be explained by effective collision theory that the reactants have to collide effectively with each other to make the reaction occur, and the concentrations of the reactants DA and HS-1 are proportional to the collision frequency. In the control unassembled nanomachine, the effective local concentrations of the DA and HS-1 were lower than that of being assembled together on the programmable DNA track, leading to low collision frequency and further decreasing the reaction efficiency. This result suggested that the programmable DNA track was a critical component to improve the effective local concentrations of reactants and further enhance the executive ability of the DNA walking nanomachine. Then, we thought that the chaotic reaction of scattered DA and HS-1 in the DA-unset nanomachine would reduce the utilization of DNA molecular components due to the uncertain walking range for each DA. In this manner, some DA-unset nanomachines might not hybridized with scattered DA, so these machines would not be in the operating state. Therefore, orderly assembling the DA and HS-1 into the programmable DNA track was the key to improving the utilization of DNA components. Furthermore, we also found that the saturated uorescence intensity of the unintegrated nanomachine was weaker than that of our core-brush 3D DNA nanomachine. This result suggested that the highly integrated mode might greatly increase the effective local concentrations to further enhance the total uorescence intensity. In principle, by comparing with the above control DNA nanomachines, our 3D DNA nanomachine possessed higher loading capacity and excellent executive efficiency, which gave impetus to ultrasensitive detection and rapid imaging of specic miRNA in cancer cells.</p><!><p>The proposed core-brush 3D DNA nanomachine was employed to construct an electrochemiluminescence (ECL) biosensing system for sensitive detection of miRNA-21. To verify the analytical performance of the sensing platform based on the proposed 3D DNA nanomachine, diverse concentrations of miRNA-21 were tested under the optimized experimental conditions (Fig. S2 and S3 †). According to Fig. 4A, the ECL intensity gradually increased with incremental concentrations of miRNA-21 throughout the tested range (10 aM to 100 pM). As presented in Fig. 4B, the calibration plot demonstrated an excellent linear relationship between the ECL response and the logarithm of the miRNA-21 concentration. And its regression equation was I ¼ 1749.29 lg c miRNA-21 + 6078.51 with a correlation coefficient of 0.9990 and detection limit of 3.57 aM (S/N ¼ 3). Moreover, compared with other methods for miRNA detection (Table S2 †), the proposed strategy possessed a remarkably wider linear range and extremely lower detection limit, which manifested that the proposed core-brush 3D DNA nanomachine has potential applications in ultrasensitive biomarker detection and metrology.</p><p>To validate the specicity of our proposed 3D DNA nanomachine for sensitive detection of miRNA-21, a series of interfering agents containing miRNA-126, miRNA-141, miRNA-203a, miRNA-155 and miRNA-182-5p were introduced as interference tests. According to Fig. 4C and D, the ECL intensity of interfering agents (10 pM) was almost negligible as the blank one. In contrast, the mixture containing target miRNA-21 was similar to that of miRNA-21 (100 fM), exhibiting an obvious ECL intensity. Meanwhile, the proposed biosensor also showed excellent selectivity for mismatched miRNA-21 (Fig. S7 †). Next, the selectivity of our strategy based on Mn 2+ -dependent DNAzyme was also monitored by PAGE. As demonstrated in Fig. 5A, lane 1 represents the HS, lane 2 represents the DA, and lanes 3-8 represent the cleavage of the HS by the DA in the presence of Mg 2+ , Mn 2+ , Zn 2+ , Ca 2+ , Cu 2+ and Pb 2+ as alternative cofactors, respectively. As expected, the most obvious band of cleavage products (S) appeared in lane 4, indicating that Mn 2+ was the best cofactor to truncate S from the DA. The above results clearly demonstrate the high selectivity of our proposed 3D DNA nanomachine. Meanwhile, the gray scale intensity of the PAGE bands was analyzed by Image J. The highest gray scale value of S and the most obvious band of S were observed in lane 4 of Fig. 5B and C with the cofactor Mn 2+ , which were consistent with the results of gel electrophoresis. Furthermore, the proposed biosensing platform also presented outstanding stability and repeatability selectivity (Fig. S8 †).</p><!><p>At rst, endocytosis inhibition experiments and MTT assays were implemented to reveal the uptake mechanism of our nanomachine (Fig. S9 †) and demonstrate the low cytotoxicity of our strategy (Fig. S10 †), respectively. Then, MCF-7 cells and HeLa cells were used to evaluate the imaging ability of the developed core-brush 3D DNA nanomachine for intracellular miRNA-21. As presented in Fig. 6A, MCF-7 cells, with high miRNA-21 expression, showed remarkably strong uorescence with the cultivation of the core-brush 3D DNA nanomachine. Meanwhile, uorescence that was obviously distinguishable from the background could be observed inside HeLa cells with a low miRNA-21 expression level (Fig. 6C). By contrast, the intracellular uorescence intensity of MCF-7 cells cultured with the conventional Au-based 3D DNA nanomachine was distinctly weaker than that of with the proposed 3D DNA nanomachine (Fig. 6B). Especially for HeLa cells, only a low intracellular uorescence intensity was noticed with the cultivation of the traditional Au-based 3D DNA nanomachine (Fig. 6D). Thus, compared with the conventional Au-based 3D DNA nanomachine, the proposed core-brush 3D DNA nanomachine could Fig. 5 (A) The specificity of the proposed strategy toward cofactor Mn 2+ (15 mM) against other cofactors (each at 150 mM). The quantitative gray scale value (B) and the total gray scale analysis (C) of part A. not only make it possible to image low-expressed miRNA-21 with higher sensitivity, but also offer a general strategy for accurate imaging in living cells with higher specicity. Moreover, we also used the miRNA-21 mimic and inhibitor to further verify the specic discrimination of intracellular miRNA-21 by the core-brush 3D DNA nanomachine (Fig. S11 †). We believe that the proposed core-brush 3D DNA nanomachine could provide an attractive way for sensitive imaging of intracellular specic miRNA in low abundance, which could contribute to study the biological processes and mechanisms within the cell system and its application in early diagnosis and treatment of diseases.</p><p>Furthermore, real-time imaging of MCF-7 cells cultured with the proposed 3D DNA nanomachine at different incubation time points within 60 min is tracked in Fig. 7. Impressively, the brightest uorescence was observed in about 15 min and no obvious uorescence growth was noticed within 60 min, indicating that the visualization of intracellular miRNA-21 was achieved within 15 min with the proposed core-brush 3D DNA nanomachine, which was noticeably faster than those of previously reported nanomachines (Table 1). These results conrmed that the rapid imaging strategy based on the proposed 3D DNA nanomachine might offer great potential for rapid point-of-care medical devices and early detection of cancer in clinical practices.</p><!><p>In summary, we presented a core-brush 3D DNA nanomachine by innovatively assembling DNA components (DA and HS) into a repetitive array on a programmable DNA track. Compared with traditional Au-based 3D DNA nanomachines, our strategy had the following combined advantages. First, the DNA track generated by a RCA reaction was well-designed to homogeneously arrange DNA components, which not only ensured a specic walking range for the DA to prevent stochastic and invalid movement, but also avoided the discorded nano-bio interface of Au/DNA in the Au-based 3D DNA nanomachine. Second, the highly integrated 3D DNA nanomachine possessed an enhanced loading capacity and movement efficiency due to the organized and high local concentration of DNA components. Third, as practical applications, the proposed 3D DNA nanomachine was successfully applied for rapid and sensitive detection and imaging of intracellular specic miRNA, which could help us explore its biological functions in tumor differentiation and proliferation as well as potential drug screening in early disease diagnosis.</p>

Royal Society of Chemistry (RSC)

Substrate preferences establish the order of cell wall assembly in Staphylococcus aureus

The Gram-positive bacterial cell wall is a large supramolecular structure and its assembly requires coordination of complex biosynthetic pathways. In the step that merges the two major biosynthetic pathways in Staphylococcus aureus cell wall assembly, conserved protein ligases attach wall teichoic acids to peptidoglycan, but the order of biosynthetic events is a longstanding question. Here, we use a chemical approach to define which of the possible peptidoglycan intermediates are substrates for wallteichoic acid ligases, thereby establishing the order of cell wall assembly. We have developed a strategy to make defined glycan chain-length polymers of either uncrosslinked or crosslinked peptidoglycan, and we find that wall teichoic acid ligases cannot transfer wall teichoic acid precursors to the crosslinked substrates. A 1.9\xc3\x85 crystal structure of a LytR-CpsA-Psr (LCP) family ligase in complex with a wall teichoic acid precursor defines the location of the peptidoglycan binding site as a long, narrow groove, and suggests that the basis for selectivity is steric exclusion of crosslinked peptidoglycan. Consistent with this hypothesis, we have found that chitin oligomers are good substrates for transfer, showing that LCPs do not discriminate crosslinked from uncrosslinked peptidoglycan substrates by recognizing features of the uncrosslinked stem peptide. We conclude that wall teichoic acids are coupled to uncrosslinked peptidoglycan chains at an early stage of peptidoglycan synthesis and may create marks that define the proper spacing of subsequent crosslinks.

substrate_preferences_establish_the_order_of_cell_wall_assembly_in_staphylococcus_aureus

<p>The bacterial cell wall is a complex macromolecule and the target of many antibiotics. Peptidoglycan (PG), strands of glycan polymers joined by peptide crossbridges, forms the foundation of the cell wall in all bacteria.1 In Gram-positive bacteria the peptidoglycan is covalently modified with additional glycopolymers that comprise up to 50% of the cell wall by mass.2 These glycopolymers are frequently wall teichoic acid (WTA) glycopolymers, which play important roles in cell growth and division.2 In pathogens such as Staphylococcus aureus (S. aureus), wall teichoic acid is essential for host colonization and virulence.3 Importantly, inhibiting wall teichoic acid biosynthesis re-sensitizes methicillin-resistant S. aureus (MRSA) to beta-lactams.4 Underlying all of these effects of wall teichoic acids on bacterial physiology is their central role in regulating bacterial cell wall biosynthesis.2,5 Understanding how and when wall teichoic acids are attached to peptidoglycan is fundamental to understanding cell physiology and has implications for developing novel antimicrobials.</p><p>In the final steps of peptidoglycan biosynthesis, S. aureus translocates the monomer building block Lipid II to the cell surface6 where it is polymerized into linear glycan strands that are crosslinked by penicillin-binding proteins (PBPs) to adjacent glycan strands via their stem peptides.7 Concurrently, WTA precursors are biosynthesized in the cytoplasm,2 translocated to the cell surface,8 and transferred to a peptidoglycan substrate.9 Studies in cells have tried to address whether WTA attachment to peptidoglycan occurs before or after glycan strands are crosslinked (Figure 1 and Figure S1), but have not reached a definitive conclusion.10 Here, using a chemical approach, we show that WTA precursors can only be attached to uncrosslinked peptidoglycan, implying that transfer occurs at an early stage of cell wall biosynthesis.</p><p>To compare WTA transfer to crosslinked and uncrosslinked peptidoglycan, we needed defined peptidoglycan fragments that could be resolved by polyacrylamide gel electrophoresis (PAGE). We previously reported that all three S. aureus LCP (LytR-CpsA-Psr) proteins can transfer a truncated radiolabeled wall teichoic acid precursor, LIIAWTA, to synthetic, uncrosslinked peptidoglycan oligomers,12b,13 resulting in a radiolabeled ladder of modified peptidoglycan fragments, but we were unable to test transfer to the corresponding crosslinked oligomers due to lack of access to substrates containing the pentaglycine branch required for crosslinking by S. aureus penicillin-binding proteins. We recently developed a strategy to obtain native Lipid II from S. aureus, making the preparation of crosslinked S. aureus peptidoglycan possible (Figure S2).11 To prepare short peptidoglycan oligomers, we incubated this Lipid II with a mutant transglycosylase, SgtBY181D (SgtB*), that releases peptidoglycan prematurely during polymerization (Figure 2a).12 The oligomers produced range from approximately two to ten disaccharide units and can be separated to disaccharide resolution. Here, we prepared the required crosslinked substrates by incubating native Lipid II with SgtB* to make peptidoglycan oligomers that were then crosslinked using S. aureus PBP4 (Figure 2a). We next tested (1) whether WTA-modified peptidoglycan could be crosslinked by a PBP, and (2) whether crosslinked peptidoglycan could be modified with WTA (Figure 2a). When WTA-modified, uncrosslinked peptidoglycan was treated with S. aureus PBP4, we observed disappearance of the radiolabeled ladder of modified fragments because the polymers became too large to enter the gel11a,14 (compare lanes 1 and 2, Figure 2b). Consistent with this, treatment with lysostaphin, which cleaves the pentaglycine-bridged crosslinks, restored the radiolabeled ladder (lane 3).11b,13b,15 In contrast, when crosslinked peptidoglycan (25% crosslinked; Table S1) was first incubated with the WTA ligase LcpB and then treated with lysostaphin, we did not observe the appearance of a radiolabeled ladder (lanes 4 and 5; compare lane 5 to lane 3, Figure 2b). These results showed that uncrosslinked PG modified with WTA is a good substrate for crosslinking, whereas crosslinked PG is a poor substrate for WTA transfer.</p><p>To quantitatively compare WTA transfer to uncrosslinked and crosslinked peptidoglycan polymers, we developed a paper chromatography assay that separates PG polymers from the radiolabeled WTA substrate, LIIAWTA (Figure 2c). Using wild-type SgtB, we prepared long, uncrosslinked peptidoglycan, split the reaction mixture, and incubated part of it with PBP4 to produce crosslinked peptidoglycan.16 The uncrosslinked and crosslinked substrates were then incubated with radiolabeled LIIAWTA and LcpB, and the reaction mixtures were separated.17 Strips were cut to isolate WTA-modified peptidoglycan polymers (retained at the baseline) from radiolabeled LIIAWTA starting material (Figure 2c, schematic). The WTA substrate was readily incorporated into uncrosslinked polymer, but minimal transfer to crosslinked polymer was detected. To test whether LcpB's preference for uncrosslinked peptidoglycan depends on the penicillin-binding protein used, we also tested the essential S. aureus enzyme, PBP2, which both polymerizes Lipid II and crosslinks the resulting polymers. To prepare uncrosslinked polymers for the comparison, we used PBP2 variant, PBP2S398G, which contains a mutation in its transpeptidase domain that prevents crosslinking (Figure S4).11a,11b LC/MS analysis showed that 17% of available sites were crosslinked by wild-type PBP2 (Table S1). We again found that only uncrosslinked peptidoglycan was a substrate for transfer (Figure S4), showing that the results do not depend on the peptidoglycan polymerase or transpeptidase used.</p><p>We took a structural approach to better understand the substrate preferences of LCP proteins. Efforts to crystallize the S. aureus LCP proteins were unsuccessful, so we focused on the B. subtilis TagT, which was previously crystallized with octaprenyl pyrophosphate.18 We first verified that TagT ligates LIIAWTA to synthetic, uncrosslinked peptidoglycan oligomers (Figure S5) and then crystallized it with two WTA precursors, LIWTA (containing a monosaccharide) or LIIAWTA (containing a disaccharide), yielding 1.8Å and 1.9Å structures, respectively (Figure 3a; Table S3). Most features of the structures are similar, but the orientations of the saccharide moieties and pyrophosphate are different, and only the structure with the disaccharide (LIIAWTA) contains a divalent cation in the active site (Figure S6). This finding is notable because LCPs are metal ion-dependent transferases (Figure S7)12b,18–19 and we have previously shown that WTA precursors must contain at least a disaccharide to serve as substrates for transfer.12b Although the N-acetyl glucosamine sugar of the LIWTA substrate makes several contacts with TagT, the corresponding GlcNAc of LIIAWTA is oriented differently due to changes in the glycosidic linkage and pyrophosphate bonds (Figure S6). These changes evidently prevent steric clashes that would otherwise arise between the ManNAc sugar and the protein. Therefore, the second sugar in the WTA precursor orients the substrate so that the pyrophosphate is in a conformation that can bind a divalent cation (Figure 3), making it competent for reaction.</p><p>The orientation of the metal-bound pyrophosphate defines the trajectory of nucleophilic attack by the peptidoglycan substrate (Figure 3b). The Mg2+ ion is coordinated by two oxygens on the adjacent LIIAWTA pyrophosphate, two waters, and one carboxylate oxygen from each of two strictly conserved aspartate residues (D97 and D82, Figure 3a). Aspartate to alanine substitutions at these positions either greatly (D82A) or completely (D97A) inhibited TagT ligase activity (Figure 3c), as did alanine substitutions at analogous positions in LcpB (Figure S8). Because the nucleophile approaches a phosphate in-line with the leaving group in a phosphoryl transfer reaction,20 and the bonds formed in this phosphoryl transfer reaction are known, we can infer that the peptidoglycan substrate binds in a long, narrow groove proximal to the anomeric phosphate of LIIAWTA (Figure 3b). This groove and the adjacent WTA binding pocket contain a number of strictly conserved residues, which were all found to be important for ligase activity (Figure 3c, Figure S9).21 Notably, three of these residues are arginines. R227 appears to play a role in stabilizing the pyrophosphoryl-oxygens of the WTA substrate (Figure S9). R219 makes no polar contacts to the WTA substrate, and its guanidinium nitrogens are positioned above the proposed trajectory of the peptidoglycan nucleophile. Likewise, R118 is adjacent to the proposed nucleophile, with one guanidinium nitrogen 3.0Å from the anomeric phosphate and the second nitrogen proximal to the proposed trajectory of the nucleophilic MurNAc hydroxyl (Figure 3b). Although uncommon, arginines can act as general bases in catalytic reactions.22 Catalytic arginines in solvent-exposed pockets are often near other arginines, which may tune the pKa, and may also be adjacent to a carboxylate that facilitates proton transfer. These features are found in the LCP structure.23 Based on our analysis, we propose that R118 acts as a base to deprotonate the C6-hydroxyl of MurNAc, while R219 plays a supporting role in coordinating the nucleophile (Figure 3d).</p><p>The peptidoglycan binding groove identified in the TagT-LIIAWTA structure is narrow, suggesting that crosslinked peptidoglycan is sterically excluded. Nonetheless, we considered the possibility that LCP enzymes might recognize specific chemical features in the stem peptides of uncrosslinked peptidoglycan, which differ in crosslinked peptidoglycan. To assess whether any feature of the stem peptides found in uncrosslinked PG is required, we tested chitin oligosaccharides of increasing length. These oligosaccharides have the same linkage stereochemistry as peptidoglycan, and also contain N-acetyl groups on every monosaccharide, but they lack the stem peptide and the lactic acid moiety present on the C3 position of every other sugar in peptidoglycan. We found that LcpB and TagT transferred WTA onto chitin oligosaccharides containing five or more sugars; shorter oligosaccharides reacted slowly or not at all (Figure 4; Figures S10–12). The corresponding deacetylated oligosaccharides could not be labelled with LIIAWTA, nor could cellulose-based oligomers (β(1,4)-linked glucose). Hence, the stem peptide of peptidoglycan is not required for WTA ligation, but the C2-N-acetyl groups play crucial roles in substrate recognition.</p><p>Taken together, the results presented here establish the order of the final steps of S. aureus cell wall assembly, and likely other Gram-positive organisms that contain LCPs, and also provide a mechanistic rationale for the sequence of events. Using comparable uncrosslinked and crosslinked peptidoglycan fragments assembled in vitro from native S. aureus Lipid II, we have shown that WTA precursors can only be transferred to uncrosslinked strands. Because Lipid II itself is not a substrate for transfer,12b these findings imply that uncrosslinked PG is first made and then modified with WTA before crosslinking. As the stem peptide is not required for recognition, WTA ligases do not discriminate between uncrosslinked and crosslinked substrates by recognizing a feature found only in the former. Instead, the basis for selectivity appears to be steric exclusion of crosslinked peptidoglycan from the long, narrow peptidoglycan binding groove. The ability to use chitin as an alternative substrate to uncrosslinked peptidoglycan will facilitate development of assays for LCP inhibitors, which may serve as beta-lactam potentiators to treat MRSA.4,11a</p><p>The order of cell wall assembly, with wall teichoic acid attachment occurring prior to crosslinking, suggests a role for these peptidoglycan modifications in regulating crosslinking. It has been observed previously that S. aureus PBP4 is mislocalized in the absence of wall teichoic acid,24 resulting in decreased crosslinking; a scaffolding model in which wall teichoic acids anchor PBPs has been proposed.4a It is also possible that wall teichoic acid marks have physical effects on peptidoglycan polymer conformation that affect crosslinking rates. Access to defined peptidoglycan substrates with and without WTA modifications now makes it possible to address these models.</p><p> ASSOCIATED CONTENT </p><p> Supporting Information </p><p>Supplemental figures and tables, experimental procedures, compound analysis, protein purification and crystallization protocols. This material is available free of charge on the ACS Publications website.</p><p>The authors declare no competing financial interests.</p>

PubMed Author Manuscript

A flexible copper sulfide composite membrane with tunable plasmonic resonance absorption for near-infrared light-driven seawater desalination

Near-infrared light driven devices for water evaporation are strictly limited by their inflexibility, high cost, complicated fabrication processes, and low energy-conversion efficiency. Here, a flexible copper sulfide composite membrane with tunable plasmonic resonance absorption for an efficient near-infrared light photothermal conversion is proposed. Both the uniformity of the morphology and the proportion of Cu + in the flower-like copper sulfide (CuS) superstructure are easily controlled by adjusting the amount of polyvinylpyrrolidone (PVP), which effectively improves the absorption of the CuS superstructure in the near-infrared region. Furthermore, the flexible CuS/Matrimid composite membrane constructed by combining CuS and polyimide membranes exhibits highly flexible properties, strong NIR absorption, fast heating (10 s), and good thermal stability. A highly efficient photothermal conversion is achieved by nearinfrared light-driven water evaporation. Under 808 nm light irradiation, the water evaporation conversion efficiency is ca. 80% and has excellent evaporation stability. The flexible CuS/Matrimid composite membrane developed in this study could have promising practical applications in near-infrared lightdriven devices for seawater desalination. Environmental signicanceThe seawater desalination driven by solar steam has emerged as one of the most promising ways to address this problem, due to its low energy input, high evaporation efficiency, and easy operation. NIR photothermal conversion materials, which can convert most of the NIR light energy into heat energy, can greatly improve the efficiency of NIR light utilization. Copper sulde, as an easy to fabricate doped semiconductor, shows excellent photostability, adjustable NIR absorption, and outstanding energy transfer efficiency. A NIR light-driven exible hot plate by incorporating doped semiconductors with a exible polymer membrane realizes the potential utility of the NIR light and doped semiconductors in seawater desalination.

a_flexible_copper_sulfide_composite_membrane_with_tunable_plasmonic_resonance_absorption_for_near-in

Introduction<!>Experimental<!>Preparation of the ower-like CuS nanostructure<!>Preparation and characterization of the CuS/Matrimid composite membrane<!>Characterization<!>Vapor generation by the CuS/Matrimid composite membrane hot plate<!>Tuning the NIR absorption of the CuS nanocrystals<!>Preparation and characterization of the exible hot plate based on the CuS/Matrimid composite membrane<!>Conclusion

<p>Water is the source of human life and the foundation of all things. It is because of moist water that the earth appears to be full of vitality, thriving. However, the rapid development of industrialization and modernization has led to the gradual reduction of available clean water resources. 1,2 Seawater desalination driven by solar steam emerges as one of the most promising ways to address this problem, due to its low energy input, high evaporation efficiency, and easy operation. 3 Generally, photothermal materials oating on the water surface or adhered to the container wall absorb sunlight, so as to carry out heat conversion and transfer, and generate water vapor through interface heating. However, the water evaporation efficiency was still inhibited by the high cost, poor stability, complicated fabrication processes, and low efficiencies of photothermal materials for solar light absorption, especially broadband nearinfrared (NIR, 780-2526 nm) light. 4,5 Therefore, it is imperative that novel NIR-driven materials or devices are devised, which have a high NIR utilization efficiency, so as to broaden the possible scope of application.</p><p>NIR photothermal conversion materials, which can convert most of the NIR light energy into heat energy, can greatly improve the efficiency of NIR light utilization. Doped semiconductors, noble metal nanostructures, organic polymers and carbon materials are four typical kinds of NIR photothermal conversion materials that are widely used in the eld of NIR light conversion. [6][7][8][9] In order to effectively use NIR light, several devices have been built recently based on NIR photothermal conversion materials. For example, Sun et al. constructed NIR light-induced shape memory polymers and silver nanoparticles for healing mechanical damage; 10,11 a carbon nanotube composite 12 and organic polymer 13,14 were used as NIR lightdriven photothermal-electrical and photo-magneto-thermoelectric devices by Wang et al. and Kim et al., respectively. Doped semiconductors, mainly including copper chalcogenide compounds (Cu 2Àx E, E ¼ S, Se, Te) 15 and transition metal oxides (WO 3Àx , MoO 3Àx , Mn x O y , etc.), 16,17 are a class of defect semiconductors with a local surface plasmon resonance (LSPR) effect. Compared with noble metal nanostructures, organic polymers, and carbon materials, doped semiconductors feature the advantages of low cost and stability. 18 Song et al. developed Cs x WO 3 nanoparticles to enhance the upconversion luminescence of monolayer upconversion nanoparticles as a high performance narrowband NIR photodetector, demonstrating the potential advantages of doped semiconductors. 19 Hence, it is meaningful to further explore NIR light-driven devices constructed from doped semiconductors, such as a NIR light-driven exible hot plate by incorporating doped semiconductors with a exible polymer membrane, to realize the potential utility of NIR light and doped semiconductors in seawater desalination.</p><p>Copper sulde, one of the most easily prepared doped semiconductors, shows excellent photostability, adjustable NIR absorption, and outstanding energy transfer efficiency, 15 and has been broadly applied to solar cells, catalysts, pollutant degradation, supercapacitors, and biomedicine. [20][21][22][23] Matrimid® 5218, a thermoplastic polyimide based on 5(6)-amino-1-(4 0aminophenyl)-1,3-trimethylindane, is soluble in a variety of common solvents and will leave a strong, durable, and high temperature-resistant exible coating. 24,25 More importantly, it is easy to cut into any shape and can be placed in any location. Thus, once inorganic materials are introduced into the polymer matrix, a combined effect of its polymeric and inorganic components' properties is thought to ensue. Therefore, here we propose a NIR light-driven exible hot plate based on the introduction of copper sulde into Matrimid® 5218, whereby copper sulde can absorb NIR light and Matrimid® 5218 provides exibility and endows the polymer matrix with high temperature resistance. This integrated device may show potential applications in seawater desalination with the synergy of the two components.</p><p>The main challenge for this proof-of-concept experiment is to fabricate a uniform copper sulde-Matrimid® 5218 exible membrane with strong NIR absorption. To do this, we relied on several steps. Firstly, N-methyl-2-pyrrolidinone (NMP), which is normally used to dissolve Matrimid® 5218, was used as a solvent to prepare the copper sulde nanocrystals. Thus, both the obtained copper sulde nanocrystals and Matrimid® 5218 are soluble in the same solvent, uniformly. Aer the evaporation of NMP, a strong, durable, uniform, and exible copper sulde-Matrimid® 5218 membrane was obtained. Secondly, to attain adequate absorption of NIR light, the doping state and morphology of copper sulde nanocrystals were tuned, accordingly, by simply controlling the PVP content. Finally, once irradiated with NIR light, the copper sulde-Matrimid® 5218 membrane's temperature rises rapidly, enabling its use as a NIR light-driven exible hot plate. The applications of this devised NIR light-driven exible hot plate in photothermal evaporation water were also explored. Due to its impressive photothermal effect and exible cutting advantages, this novel hot plate can be placed on either the outer or the inner surface of a given device, to prevent salt precipitation from accumulating on the surface, thereby enhancing the overall evaporation efficiency.</p><!><p>PVP (M w ¼ 29 000) was purchased from Sigma Aldrich. Copper(II) sulfate pentahydrate (CuSO 4 $5H 2 O), N-methyl-2-pyrrolidinone (NMP), sulfur, and polyimide were all purchased from Sinopharm Chemical Reagent Co., Ltd.</p><!><p>First, 0.5 g of PVP was dissolved in 15 mL of NMP, and then 0.5 mM (0.125 g) CuSO 4 $5H 2 O was added to the PVP solution under magnetic stirring. This stirring was continued until a bright green transparent solution was obtained, and then 1 mM sulfur powder (0.032 g) was added to the mixed solution. Aer 10 min of stirring, the above solution was transferred into a 25 mL Teon autoclave for the reaction at 180 C for 4 h. The blackgreen CuS was centrifuged and washed twice with NMP, and dissolved in 1 mL of NMP for its later use.</p><!><p>Polyimide (0.2 g) was mixed with 1 mL of CuS in differing amounts (i.e., 1, 2, 5, and 10 mg mL À1 ). Aer undergoing thorough mixing, an appropriate amount of a given sample was added dropwise onto a glass pane and we manually scraped the 500 mm surface with a membrane coater. Then it was quickly placed in a vacuumdrying oven and dried with a gradient temperature series: 80 C for 12 h, 140 C for 1 h, and 200 C for 1 h.</p><p>For absorption measurements, a blank PI membrane without CuS served as the baseline. The absorbance of the CuS/ Matrimid composite membrane generated with different amounts of CuS was measured accordingly.</p><p>For testing its photothermal properties, the blank membrane and CuS/Matrimid composite membrane were cut into small disks (2 cm diameter). An 808 nm laser (1 W cm À2 ) was used to irradiate the top of each membrane, whose temperature change was recorded with a thermal imaging device.</p><!><p>The structure of the CuS nanostructure was conrmed by X-ray diffraction (XRD, Rigaku DMAX2000), and its morphology was quantitatively examined by SEM (JEOL JEM-6460A) and TEM (JEOL TEM-2100). The electronic state of Cu in the ower-like CuS was determined by X-ray photoelectron spectroscopy (ESCALAB 250Xi), and the absorbance of the CuS nanostructure and CuS/Matrimid composite membrane was determined by UV-Vis-NIR (Beckman Coulter DU730) and UV-Vis diffuse reectance spectroscopy (Shimadzu UV-2450). The heating effect was tracked by using a FLIR A300 thermal imaging device.</p><!><p>The saltwater sample (26.5 g L À1 NaCl, 0.2 g L À1 NaHCO 3 , 0.28 g L À1 NaBr, 24 g L À1 MgCl 2 , 3.3 g L À1 MgSO 4 , 0.73 g L À1 KCl, and 1.1 g L À1 CaCl 2 ) and sewage sample (3.5 wt% NaCl, 10 ppm pchlorophenol) were respectively prepared according to previous reports. 4,26 The seawater was collected from the East China Sea, in Fengxian (Shanghai).</p><p>The CuS/Matrimid composite membrane was cut into a square (1 cm  1 cm) and this was stuck to the outside wall of a cuvette lled with water. The composite membrane was irradiated with an 808 nm laser (2 W cm À2 ) for 900 s, and water temperature was recorded with a thermal imaging device. Two key parameters, the water evaporation rate (n) and water evaporation efficiency (h), were calculated using eqn ( 1) and ( 2), respectively.</p><p>where H e denotes the total enthalpy of the liquid water-to-vapor phase transition (J g À1 ), n is the water evaporation rate, and Q s is the light density of solar illumination. 28 For the solar water desalination test, the CuS/Matrimid composite membrane was pasted on the inside of a 100 mL aluminum cup to better absorb solar energy. The aluminum cup Environmental Science: Advances Paper was then lled with seawater and irradiated with a xenon lamp at a power density of 1 W cm À2 . Both photothermal imaging and water temperature were recorded with a thermal imaging device (FLIR A310).</p><p>3 Results and discussion</p><!><p>To obtain copper sulde nanocrystals with high photothermal conversion efficiency, some rational designs have been adopted to enhance their absorption of NIR light, for example, by tuning the structure, copper deciency, and hybrid composite. Nearly all of these methods are used in hydrothermal or thermal decomposition processes. But in order to prepare a uniform copper sulde-Matrimid® 5218 exible membrane, a solvothermal method must be implemented that is able to synthesize the copper sulde by using NMP as the solvent. In this study, the copper sulde was synthesized by a solvothermal method that uses PVP as the surfactant and NMP as the solvent (Fig. 1a). The effects of PVP content on the crystal phase, shape, copper valence and NIR absorption of the copper sulde were analyzed.</p><p>Previous reports have demonstrated that the crystal phase is critical for tuning the localized surface plasmon resonance (LSPR) absorption for copper sulde. 29,30 Thus, the effect of PVP content on the crystal phase was rst investigated. The copper sulde was prepared solvothermally by adding sulfur and CuSO 4 into NMP with differing amounts of PVP (0-1 g). The structural changes of the obtained copper sulde samples were then investigated by studying the X-ray diffraction (XRD) patterns (Fig. 1b). The main diffraction peaks situated at the 2q angles of 29.28, 31.81, 32.95, 47.93, 52.71, and 59.34 of the copper sulde prepared without PVP (i.e., 0 g added) agreed well with the (102), (103), (006), (110), (108), and (116) lattice planes of covellite CuS (JACPDS card no. 06-464). No characteristic peak can be indexed to any other phase of copper sulde, except covellite CuS. When PVP was added in amounts of 0.125 to 1 g, evidently the peak patterns maintained the same structure as that of covellite CuS, while the intensity of characteristic peaks at the 2q angle of 32.95 decreased a little. This change in peak intensity may be attributed to the growth of the (006) lattice plane being inhibited by the strong coordination of PVP and Cu 2+ . The XRD patterns for copper sulde obtained in the absence and presence of PVP suggest that PVP shows no apparent effects on the crystal phase of copper sulde.</p><p>Next, the morphology of the copper sulde obtained with and without PVP was observed by scanning electron microscopy (SEM). The CuS material prepared without PVP is composed of a large-sized ower-like superstructure and irregular nanoparticles (Fig. 1c), while a more uniform ower-like superstructure smaller in size was obtained when PVP was introduced (Fig. 1d-h). PVP is competent to stabilize the CuS superstructure. In the chemical sintering process, the small particles will form compact solids due to the coalescence and Ostwald ripening behaviors triggered by the detachment of PVP. 31 With more PVP added, the growth of the CuS superstructure can be reliably controlled, and the ower-like superstructures of CuS appear uniform (Fig. S1 †), which ought to improve the photon reection capacity and the NIR absorption and further enhance the photothermal conversion effect. In addition, the size of the CuS superstructure can also be tuned by changing the PVP content. As the SEM images show, the size of the prepared CuS superstructure decreased 10-fold, from 10 to 1 mm, when the PVP content is increased from 0 to 1 g; this indicated that the higher the PVP content used, the smaller is the size of the CuS superstructure. These results demonstrated that the uniformity and size of the superstructure, which are benecial to promote the photothermal conversion effect, are governed by the PVP content.</p><p>Given that the electronic state of Cu is related to the hole density of the nanomaterials, which is crucial for NIR absorption, XPS measurements were conducted to conrm the electronic state of Cu in the prepared CuS nanostructures. The Cu 2p peaks of these CuS featured the typical asymmetric tail of covellite. 32 Without PVP present, the binding energy intensity values of Cu 2p 3/2 and Cu 2p 1/2 were slightly le-shied to 932.24 and 952.12 eV, with weak satellite peaks at 942.07 observed, indicating the presence of typical Cu 2+ but little Cu + in the obtained CuS nanostructures. [33][34][35] As more PVP was used, the two binding energy intensity values of Cu 2p 3/2 and Cu 2p 1/2 were increasingly right-shied and their satellite peaks gradually decreased, suggesting a greater proportion of Cu + in the CuS nanostructures (Fig. 2a). Moreover, the S 2p band of these CuS also corresponded to the typical "three peaks" of covellite, and the disuldes eventually disappeared as the amount of PVP increased, thus indicating a gradual shi to chalcocite (Fig. S2 †). To clarify the ratio of Cu 2+ to Cu + in the CuS nanostructures, their Cu 2p bands were adequately tted to four curve-tting bands. Using the area of the four curve-tting bands, the ratio of Cu 2+ to Cu + was calculated accordingly, yielding values of 0.71, 0.34, 0.32, 0.30, 0.27, and 0.28 (Fig. 2b and c). The results suggest that the ratio of Cu + in the CuS nanostructures increases as the amount of PVP increases until it's reduced to saturation, which can be ascribed to the effect of precursor reducibility by PVP. 36,37 The optical properties of CuS prepared by introducing various amounts of PVP were examined by Vis-NIR spectroscopy. To fairly compare the absorption intensity of these differently prepared CuS samples, their absorption at 650 nm was assigned the same value. With more PVP added, the normalized absorption intensity of the obtained CuS nanostructure in the NIR region (700-1000 nm) clearly increased (Fig. 2d). Moreover, absorption at 808 nm is linearly enhanced with increasing PVP content, until it reaches saturation when 0.75 g of PVP is added (Fig. 2e). The NIR absorption of CuS is the LSPR absorption of electrons and holes, which is like the LSPR absorption of electrons in the noble metal. The hole arises from the copper vacancy in the CuS nanoparticles, such that CuS nanoparticles with a higher hole density will exhibit stronger NIR absorption. 38,39 The copper vacancy can be tuned by modulating the proportion of Cu + in the CuS nanostructure, yet the Cu + in the CuS nanostructure can be controlled by the applied amount of PVP in the synthesized system. In addition, the superstructure facilitates the NIR absorption of CuS nanoparticles, likely because photo-absorption can be augmented by the faceted end planes of well-shaped crystals that serve as good light-cavity mirrors. 40,41 This inference is also corroborated by our nding of the NIR absorption of the CuS superstructure decreasing aer undergoing ball milling (Fig. S3 †). Therefore, the strong LSPR absorption of CuS in the NIR region can be simply tuned by the amount of PVP used in the synthesis system.</p><!><p>Materials with exibility, ease of cutting, and ease of construction are now receiving extensive attention from researchers. To broaden the application scope of the CuS nanostructure with strong NIR absorption, it would be prudent to develop a CuS exible membrane with excellent photothermal properties. Matrimid® 5218 is a widely used polymer matrix for various inorganic nanoparticles in microelectronics and the gas separation industry. More importantly, the Matrimid® 5218 membrane has good heat-resistance. Here, we developed a exible hot plate by incorporating the CuS nanostructure into Matrimid® 5218 uniformly. The CuS nanostructure was prepared in NMP and it can be dispersed in NMP very well, while the NMP performs well as a solvent for the Matrimid® 5218 polymer. Thus, a uniformly dispersed liquid of the CuS nanostructure and Matrimid® 5218 was easily obtained when they were added to NMP with ultrasonic dispersion. Following this step, the CuS/Matrimid membrane was prepared by applying a scrape coating/drying technique (Fig. 3a). The doped The absorption performance of the fabricated membranes with increased levels of CuS doping (0.5 wt%, 1.0 wt%, 2.5 wt%, 5.0 wt%, and 10 wt%) was evaluated from their Vis-NIR spectra (Fig. 3b). The absorption values rose considerably by increasing the CuS doping from 0.5 wt% to 10 wt% for the CuS/Matrimid composite membrane. Its good absorption properties in the NIR region will confer an excellent photothermal performance. We then tested its photothermal performance by recording the temperature change under 808 nm laser (1 W cm À2 ) irradiation for 60 s (Fig. 3c). Compared with the blank Matrimid membrane, the CuS-doped Matrimid membrane exhibited outstanding photothermal performance, in which the temperature is capable of rising from 84.5 C to 145.5 C when the doped CuS content is increased from 0.5 wt% to 10 wt%. Furthermore, the temperature increase of the CuS/Matrimid composite membrane is particularly fast under the laser irradiation, attaining its maximum in just 10 s. Aer that, the temperature can maintain its maximal value without undergoing signicant change, which is attributable to the generated heat from the CuS/Matrimid composite membrane under the laser irradiation being equal to the heat diffused into air. These results suggest that the CuS/Matrimid composite membrane has the property of fast heating under laser irradiation.</p><p>Next, the relevance of light density for the photothermal properties of the CuS/Matrimid composite membrane was investigated (Fig. 3d). For the CuS/Matrimid composite membrane with 2.5 wt% CuS, the maximum temperature reachable by the CuS/Matrimid composite membrane is 55 C to 145 C under irradiation of a laser with a light density from 0.1 to 1.5 W cm 2 ; this implies that light density exerts important effects. Thermal stability is a key parameter that determines whether the CuS/Matrimid composite membrane can be used as a hot plate. To evaluate it, the photothermal circle test was used. Aer 8 'on/ off' cycles of the laser-each cycle consisting of a laser on time of 1.5 min and a laser off time of 1.5 min-the CuS/Matrimid composite membrane still exhibited similar photothermal properties, indicating very good thermal stability (Fig. 3e), which can be attributed to its structural stability aer laser irradiation (Fig. S5 †). Moreover, the prepared CuS/Matrimid composite membrane still possessed its highly exible properties on par with those of the Matrimid® 5218 membrane without doping the CuS nanostructure, as demonstrated by the photographed Matrimid® 5218 membrane before (upper panel) and aer (lower panel) doping the CuS nanostructure (Fig. S6 †). In addition, the as-prepared membrane could be carved into many different shapes (Fig. 3f), without any inuence on its impressive photothermal performance (Fig. 3g), endowing it with more potential for realistic applications. The excellent performance of the CuS/ Matrimid composite membrane, characterized by its highly exible properties, strong NIR absorption, fast heating, good thermal stability and easy cutting, makes it a promising candidate for use in a NIR light-driven hot plate. highly efficient vapor generation by heat localization at the evaporation surface (Fig. 4a). Evidently, with the CuS membrane present, the temperature rose rapidly under continuous laser irradiation and remained stable for the entire 900 s duration. In stark contrast, the temperature in the blank panel showed only a slight difference aer its irradiation (Fig. 4b and c). Moreover, the cumulative weight loss was positively correlated with the irradiation time (Fig. 4d). Under 808 nm irradiation, the weight loss over the 900 s period was 5.86 kg m À2 , and the steady-state evaporation rate was calculated to be 23.4 kg m À2 h À1 . This is much higher than the evaporation rate of water in the absence of CuS, which was 3.06 kg m À2 h À1 . Hence, the photothermal evaporation conversion efficiency of the CuS/Matrimid composite membrane (z80%) is nearly 8 times higher than that of the blank membrane (Fig. 4e). Crucially, the steady-state evaporation rate and cumulative weight loss did not change signicantly over eight cycles of reuse (Fig. 4f). This suggests that the CuS/Matrimid composite membrane is highly stable and can be reused multiple times without a pronounced decrease in its evaporation capacity.</p><p>The NIR light-driven photothermal evaporation of water was also investigated in stimulated saltwater and wastewater. As expected, under continuous irradiation of the laser, both salt water and wastewater covered by the CuS/Matrimid composite membrane incurred a rapid heating effect (Fig. 5a). Due to the relatively complex nature and high concentration of ions in salt water, its temperature rise is affected to some extent (Fig. 5b). Accordingly, the weight change of salt water aer evaporation is not as great as that of wastewater or pure water (Fig. 5c). Nonetheless, much water was still evaporated within 15 min, and the evaporation rate and efficiency of salt water and wastewater are still good (Fig. 5d). More importantly, aer water evaporation, the concentration of ions in the collected distilled water had decreased substantially (Fig. 5e). In addition, the evaporation of the composite membrane is relatively stable (Fig. S7 †). Solar seawater evaporation and desalination tests showed similar heating and desalination effects, thus indicating that the CuS/Matrimid composite membrane may be useful for light-driven photothermal evaporation of water (Fig. S8 †). Overall, because of its strong NIR absorption, high photothermal conversion, exible cutting and localization, the CuS/Matrimid composite membrane shows great photothermal efficiency, which we anticipate will be applied in actual water evaporation and seawater desalination projects.</p><!><p>In summary, a kind of ower-like, self-doped CuS superstructure with tunable plasmonic resonance absorption and photothermal effects was designed, for which PVP was the surfactant and NMP is the solvent. The results show that with a greater amount of added PVP, there is an increased degree of Cu 2+ reduction, generating more copper defects that enhance the absorption ability of the CuS superstructure in the near infrared region, until Cu 2+ is no longer reduced. Furthermore, CuS membranes featuring high-temperature resistance and good exibility were prepared by combining CuS with polyimide membranes via coating and gradient high-temperature curing. Photothermal performance testing shows that the temperature of the CuS/ Matrimid composite membrane can rise to more than 100 C within just a few seconds under the irradiation of an 808 nm laser, suggesting that it functions as a robust photothermal conversion membrane. The responsive properties of the CuS/ Matrimid composite membrane to vapor evaporation driven by NIR light were explored. Compared with a blank membrane, the composite membrane evinced a heating effect and better evaporation efficiency, both in stimulated saltwater and sewage. This proves that the CuS/Matrimid composite membrane has promising application prospects. This work provides the possibility for further development of an NIR light-driven exible and tunable absorption semiconductor, which we anticipate will broaden the further application of this kind of device.</p>

Royal Society of Chemistry (RSC)

New Highly Active Antiplatelet Agents with Dual Specificity for Platelet P2Y1 and P2Y12 Adenosine Diphosphate Receptors

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P1,P4-di(adenosine-5\xe2\x80\xb2) tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.

new_highly_active_antiplatelet_agents_with_dual_specificity_for_platelet_p2y1_and_p2y12_adenosine_di

1. INTRODUCTION<!>2.1. Selection of Modifications<!>2.2 Chemistry<!>2.3. Platelet aggregation inhibition<!>2.4.1. Effect on P2Y1<!>2.4.2. Effect on P2Y12<!>2.4.3. Effect on P2X1<!>2.5. Inhibitor pre-incubation effect and reversibility of platelet aggregation inhibition<!>2.6. Platelet aggregation inhibition by individual stereoisomers of compound 5<!>2.7. Stability and metabolism in plasma<!>3. CONCLUSION<!>4.1. Chemistry<!>4.1.1. General procedure for synthesis of modified P1,P4-di-(5\xe2\x80\xb2-adenosine) tetraphosphates, sodium salts (compounds 2-15)<!>4.1.2. P1,P4-di-(2-Methylthio-5\xe2\x80\xb2-adenosine) tetraphosphate, sodium salt (2)<!>4.1.3. P1,P4-di-(2-methylthio-5\xe2\x80\xb2-adenosine) P1,P4-dithiotetraphosphate, sodium salt (3)<!>4.1.4. P1,P4-di-(2-methylthio-5\xe2\x80\xb2-adenosine) P2,P3-chloromethylenetetraphosphate, sodium salt (4)<!>4.1.5. P1,P4-di-(2-methylthio-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (5)<!>4.1.6. P1,P4-di-(2-ethylthio-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (6)<!>4.1.7. P1,P4-di-(2-propylthio-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (7)<!>4.1.8. P1,P4-di-(2-(3,3,3-trifluoropropylthylthio)-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (8)<!>4.1.9. P1,P4-di-(2-pentylthio-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (9)<!>4.1.10. P1,P4-di-(2-chloro-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetra-phosphate, sodium salt (10)<!>4.1.11. P1,P4-di-(2-bromo-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetra-phosphate, sodium salt (11)<!>4.1.12. P1,P4-di-(2-iodo-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetra-phosphate, sodium salt (12)<!>4.1.13. P1,P4-di-(N6-methyl-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (13)<!>4.1.14. P1,P4-di-(N6-propyl-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (14)<!>4.1.15. P1,P4-di-(2-methylthio-N6-methyl-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, sodium salt (15)<!>4.1.16. P1,P4-di-(2-methylthio-5\xe2\x80\xb2-adenosine) P1,P4-dithio-P2,P3-dichloromethylenetetraphosphate, sodium salt (16)<!>4.1.17. Separation of the diastereomers of 5<!>Diastereomer D1<!>Diastereomer D2<!>Diastereomer D3<!>Diastereomer D4<!>4.2. Stability and metabolism in rat and human plasma<!>4.3.1. General<!>4.3.2. Ethics Statement<!>4.3.3. Blood collection and sample preparation<!>4.3.4. ADP-induced platelet aggregation<!>4.3.5. P2Y12-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay<!>4.3.6. P2Y1-mediated cytosolic Ca2+ increase assay<!>4.3.7. P2X1-mediated entry of extracellular Ca2+<!>4.3.8. Statistical analysis

<p>Platelets play critical roles in hemostasis and its pathophysiology [1]. Undesired platelet activation can be a result of many common pathologies or interventions e.g. atherosclerotic plaque rupture, surgeries, percutaneous interventions, or major traumas, and may lead to excessive platelet aggregation and generation of occlusive thrombi. The ischemic events that follow, such as acute myocardial infarction and stroke, are leading causes of death and incapacitation in the developed world and are major contributors to health care costs. It is also increasingly recognized that platelets are an essential and integral part of the immune system [2–4], and abnormal platelet activation can be a contributing factor to vascular inflammation and associated vascular injury and atherosclerosis [5]. As a result, therapeutics that control platelet reactivity have achieved significant use in clinical practice, and the development of new drugs of the class has been a major focus of the research community and the pharmaceutical industry [6,7].</p><p>ADP plays a major role in the process of platelet activation [8]. It is released by activated platelets [9], and is an agonist at two platelet purinergic G-protein coupled transmembrane receptors – the Gq coupled P2Y1 and the Gi coupled P2Y12. A third platelet P2 receptor, P2X1, is an ATP-activated ion channel. P2Y1 activation initiates ADP-induced platelet aggregation, and is responsible for platelet shape change [10]. However, without P2Y12 activation, the result is a small and reversible platelet aggregation. P2Y12 activation results in amplification and stabilization of the aggregation response. There is a complex interplay between P2Y1 and P2Y12 receptors [11], and co-activation of both is necessary for full platelet aggregation [12]. The role of P2X1 may be associated with platelet shape change in response to ATP, and may contribute to the activation of platelets by low collagen concentrations and high shear stress, thus playing a role in localized thrombus formation in small arteries [13].</p><p>The P2Y12 receptor is the most important platelet drug target. It is irreversibly inhibited by the major class of antiplatelet agents – the thienopyridines [14,15]. It is also the target of the newer reversible antiplatelet drugs ticagrelor [16], cangrelor [17], elinogrel [18], and other drug candidates [19] in various stages of development. P2Y1-selective antagonists have been identified [20,21], but the lack of clinical candidates contrasts with the essential role of this receptor in platelet aggregation [22]. By using selective inhibitors, Nylander et al. [23] found that simultaneous targeting of P2Y1 and P2Y12 is highly synergistic. We [24,25] have reported that Ap4A and its phosphonate analogs inhibit both human platelet P2Y1 and P2Y12 receptors and that the IC50s for inhibition of ADP-induced human platelet aggregation were lower than the IC50s for each of the receptors.</p><p>Both ADP and ATP scaffolds have been heavily modified in search of new P2 receptor agonists and antagonists (for reviews, see [22,26–29]), and the efforts resulted in the discovery of the highly potent P2Y12 antagonists cangrelor [17] and ticagrelor [16], and highly potent P2Y1 agonists and antagonists [30,31].</p><p>P1,P4-Di(adenosine-5′) tetraphosphate (Ap4A) is the most important member of the group of dinucleoside polyphosphates. It is found in a variety of cells, is secreted extracellularly, and is involved in the regulation of variety of intra- and extracellular physiological functions [32]. In platelets Ap4A is stored in dense granules and is therefore released along with ADP and ATP upon platelet activation [33]. It is well known that Ap4A inhibits ADP-induced platelet activation [34], and modifications of Ap4A's tetraphosphate chain have been shown to improve on this effect and to increase the biological stability [35–37]. We recently reported that Ap4A and its tetraphosphate chain modified (P1- and/or P4-thio, and P2,P3-chloromethylene) analogs inhibit platelet aggregation by targeting both P2Y1 and P2Y12 receptors [24,25].</p><p>While the modifications of the tetraphosphate chain of the Ap4A scaffold have been extensively explored [24, 25, 35–37], and some modifications of the ribose moiety, namely, the 2′,3′-O-benzylidene derivative have been made [38], no exploration of the base modifications has been attempted. This is due, in part, to the lack of a chemical method allowing rapid synthesis in good yield of new Ap4A analogs. We have recently reported synthesis and properties of new reagents, diimidazolyl derivatives of diphosphoric and (methylene)bisphosphonic acids, which allow rapid synthesis of dinucleoside tetraphosphates in high yields [39]. We explored this method to prepare a number of new Ap4A analogs with modifications in the adenosine base and the tetraphosphate moiety, and now report on their synthesis, their properties as platelet aggregation inhibitors and their activities toward platelet purinergic (P2) receptors. This SAR effort resulted in the discovery of highly potent platelet aggregation inhibitors which selectively target both platelet ADP receptors – P2Y1 and P2Y12, and have potential as novel antiplatelet drugs distinct from current antiplatelet agents, including the ATP analog cangrelor, which target only P2Y12.</p><!><p>Introduction of certain substituents at position 2 of the adenine greatly enhances the agonist potency of ADP, and the antagonist potency of ATP toward platelet ADP receptors [19]. For instance, the EC50 of ADP and 2-MeSADP toward P2Y1 in a functional assay are 8000 nM and 6 nM, respectively, and toward P2Y12 are 69 nM and 0.3 nM, respectively. In development of ATP type inhibitors of P2Y12, Ingall et al. [40] synthesized a homologous series of 2-substituted ATP analogs. According to the authors, any substitution at this position increases receptor affinity, but the effect is most pronounced when the substituent is a non-polar group attached through a sulfur atom. For instance, going from adenosine 5′-(P2,P3-dichloromethylenetriphosphate) to its 2-ethylthio analog decreased the IC50 1000 fold. Replacement of EtS group with n-PrS increased the inhibitory potency another 100 fold. Further increase of the chain length or introduction of additional substitution did not provide additional advantage [40].</p><p>In development of P2Y1 antagonists, the group of Jacobson prepared a series of C-2, and N6 substituted carbocyclic adenosine-3′,5′-diphosphate analogs (the so called "methanocarba" analogs) [41]. The effect of C-2 substitution at the adenine base on the antagonist potency was in the order I>Br>Me>Cl>H>F. In contrast to P2Y12 (see above), increasing the size of a C-2 alkyl group or attaching it through a sulfur atom decreased the potency.</p><p>Substitutions at the N6 amino group of the adenine moiety also proved to be beneficial in development of P2Y1 and P2Y12 agonist or antagonists. In Ingall's SAR study [40] mono-alkylation of N6 improved the antagonist potency of the ATP analogs toward P2Y12, although the effect was less pronounced than that of the substitution at C-2 position. The optimal length of the alkyl chain was 3–4 carbon atoms, and dialkylation or acylation of N6 significantly reduced the activity. The effect of the N6 alkylation proved to be additive with the effect of the C-2 substitution, and led to the discovery of the ATP based antiplatelet drug candidate cangrelor. In contrast to P2Y12, the P2Y1 receptor is less tolerant to bulky substituents at N6, and the optimal modification appears to be mono-methylation [42]. Both P2Y1 and P2Y12 poorly tolerate substitutions at C-8 of adenine [43].</p><p>Modifications in the polyphosphate groups of ADP and ATP have also been extensively explored. Replacement of a phosphate oxygen by sulfur is well tolerated by both P2Y1 and P2Y12, and leads in many cases to more potent analogs [26]. Actually, P2-thioADP is a better agonist of P2Y1 than ADP [30], and almost as effective as ADP toward P2Y12 receptor [26]. An additional benefit of this modification is the improved stability of the thiophosphates toward enzymatic degradation. Replacement of the oxygen atom between P2 and P3 by halomethylene groups improves the inhibitory potency of the ATP scaffold in respect to P2Y12, as well as the degradation stability [40]. All these observations are summarized in Fig. 1.</p><p>Assuming a related mode of binding of Ap4A and ADP/ATP to P2Y12/P2Y1 receptors we chose to investigate the effects of substitutions at C2 and N6 of the adenine base, with or without modifications in the tetraphosphate chain of the Ap4A scaffold, on platelet aggregation and on platelet P1Y1, P2Y12, and P2X1 receptors. To this end the new Ap4A analogs 2-16 shown in Fig. 2 have been synthesized and studied. The known, base un-substituted compound 1 has been prepared and studied for reference purposes, too [24].</p><!><p>The C-2 and N6 substituted adenosine derivatives were prepared as follows: 2-alkylthioadenosines, precursors for compounds 2-9 and 16, were prepared by S-alkylation of 2-thioadenosine [44]. N6-Methyladenosine, precursor of compound 13, was prepared from commercially available 6-chloropurine riboside and methylamine. N6-Propyladenosine, the precursor of 14, was prepared from inosine by adaptation of the general method of Wan et al.[45]. 2-Methylthio-N6-methyladenosine, the precursor of compound 15, was synthesized in three steps from 2-methylthioadenosine by adaptation of a method described by Ingall et al.[40].</p><p>The precursor nucleosides for compounds 10 and 12, 2-chloro-, and 2-iodoadenosine, are commercially available. 2-Bromoadenosine [43], needed for compound 11, was prepared in 3 steps from guanosine through 2′,3′,5′-tri-O-acetylguanosine, 2-amino-6-chloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine [46], and 2-bromo-6-chloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine [47], as described.</p><p>The nucleosides were directly phosphorylated to 5′-monophosphates with phosphoryl chloride in trimethylphosphate [48], or to 5′-thiomonophosphates with thiophosphoryl chloride in pyridine [49]. Coupling of the mono-, or thiomonophosphates with the diimidazolides of diphosphoric or (monochloromethylene)bisphosphonic acids in the presence of zinc chloride (Scheme 1) afforded the corresponding dinucleoside tetraphosphates [39].</p><p>The attempt to prepare compound 16 by the diimidazolide method was not successful. The diimidazolide of (dichloromethylene)bisphosphonic acid was prepared and characterized successfully, yet it failed to react with 2-methylthioadenosine 5′-thiomonophosphate (MeSAMP(S)) under all conditions, and catalytic systems we tried. This is probably due to the unexpectedly high steric hindrance by the dichloromethylene group on the phosphorous atoms. Compound 16 was prepared, albeit in low yield, by zinc chloride catalyzed coupling of the cyclic trimetaphosphate 17 with MeSAMP(S) (Scheme 2) [50].</p><!><p>All new Ap4A analogs (compounds 2-16) inhibited ADP-stimulated human platelet shape change (by optical aggregometry, data not shown) and both primary and secondary platelet aggregation (Fig. 3). We studied the concentration effect of this inhibition using the 96-well microplate method [51]. This is a simple, rapid, low volume method to simultaneously measure platelet aggregation of multiple samples, thereby avoiding the problem of platelet aging and inter-patient variability. Typical antagonist concentration – optical transmission curves for different concentrations of compound 5 are presented in Fig. 3.</p><p>To compare the ability of different Ap4A derivatives to rapidly and competitively inhibit human platelet activation by ADP, in one set of experiments platelets were exposed to a mixture of ADP (3 μM) and different concentrations of each Ap4A derivative (i.e. platelets were not pre-incubated with the drugs). In other set of experiments, platelets were exposed to the Ap4A derivatives for 15 min to achieve steady-state inhibition, and then challenged with 5 or 20 μM ADP. The corresponding IC50s for each set of experiments are summarized in Table 1. Typical antagonist concentration – aggregation inhibition curves are presented in Fig. 4.</p><p>Introduction of small thioalkyl substituents at position 2 of the base significantly increased platelet aggregation inhibition activity, and the effect gradually diminished with the increase of the length of the alkyl substituent, the optimal size being between methyl and ethyl. A halogen substituent at this position also increased the inhibitory activity in the order I > Br > Cl. Contrarily, N6-methyl and N6-propyl substituents significantly reduced the inhibitory activity. Similarly to our previous finding for base unsubstituted Ap4A analogs [24], introduction of P1,P4-dithio, and P2,P3-monochloromethylene substitutions on the polyphosphate chain of the base substituted (2-MeS) analog (compounds 3, 4, and 5) improved the inhibitory activity, albeit the effect of both substituents was not clearly additive. In contrast to the monochloromethylene group, the P2,P3-dichloromethylene group decreased the antagonist activity (2-MeS base substitution, compound 16 vs. compounds 3 and 5). These observations clearly differ from the effect of analogous modifications in the ATP scaffold on platelet aggregation inhibition, where bulky alkyl substitutents at C2 and N6 of the adenine base and the P2,P3-dichloromethylene group were well tolerated and resulted in significant increase of activity, indicating a distinct mode of interaction of Ap4A analogs with their respective platelet targets.</p><!><p>The effect of the new Ap4A analogs on human platelet P2Y1 receptors was determined by measuring the P2Y1-mediated cytosolic Ca2+ increase from intraplatelet stores after stimulation with ADP (3 μM) as previously described [24]. All of the compounds reduced the effect of ADP on the cytosolic Ca2+, i.e. inhibited P2Y1. None of the compounds caused increase of the cytosolic Ca2+ in absence of ADP, i.e. none showed agonist effect toward P2Y1. Typical concentration/inhibition effect curves are presented in Fig. 5A, and the corresponding IC50s are listed in Table 2. The 2-methythio base substitution increased the antagonist properties of Ap4A, from 32.5 μM (IC50 of Ap4A [24]) to 4.9 μM (IC50 of the di-2-MeS analog of Ap4A, compound 2, Table 2). The same effect of the 2-MeS group was observed for the analogs with modifications in the polyphosphate chain. For instance, the P1,P4-dithio-P2,P3-monochloromethylene derivative (1, base unsubstituted) had IC50 of 10.2 μM [24] and compound 5 (2-MeS base substitution) had IC50 of 2.15 μM. Further increase of the size of the thioalkyl group appeared to reduce the inhibitory potency, yet the trend was not clearly defined, and the di-n-pentylthio derivative (9) had IC50 of 4.21 ± 1.98 μM. The N6-methyl substitution (13) also resulted in an increase in the P2Y1 inhibitory properties, with IC50 of 1.97 μM, but this effect decreased with the increase of the alkyl chain size to n-propyl (14, 8.8 μM). The positive effect of 2-MeS and N6-Me groups on P2Y1 inhibition was additive (compound 15, IC50 of 0.36 μM). The effect of the polyphosphate chain substitutions follows previously observed effects on the base-unsubstituted Ap4A analogs [24]: the P1,P4-dithio substitution significantly increased P2Y1 inhibition, and the P2,P3-monochloromethylene substitution decreased it (compound 2 vs. compounds 3, and 4). Interestingly, the P2,P3-dichloromethylene group appeared to be better tolerated than the monochloromethylene (5 vs. 16).</p><!><p>The effect of Ap4A analogs on human platelet P2Y12 receptors was determined by measuring P2Y12-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP) [52]. VASP phosphorylation was measured by flow cytometry using a kit (BioCytex, Marseilles, France) after P2Y12 activation with 3 μM ADP. None of the test compounds caused decrease of VASP phosphorylation in absence of ADP, i.e. none showed agonist effect toward P2Y12, and all of them reduced the effect of ADP on VASP phosphorylation, i.e. showed antagonist effects. Typical concentration-inhibition curves are presented in Fig. 5B, and the corresponding IC50s are listed in Table 2. Methylthio substitution at C2 of the base had a significant effect on inhibitory activity: while Ap4A is only a partial antagonist of P2Y12 with a IC50 above 250 μM [25], compound 2 (2-MeS Ap4A analog) was a full antagonist with IC50 of 1.4 μM. In contrast to the ATP scaffold, increase of the alkyl chain above ethyl did not result in increase of the inhibitory potency. Also, in contrast with the ATP scaffold, the N6-alkyl substituents were poorly tolerated, and both N6-methyl and N6-propyl substitutions (compounds 13 and 14) significantly reduced the inhibitory activity. Interestingly, introduction of a 2-MeS group together with N6-Me group almost completely abolished the negative effect of the latter, and resulted in relatively potent P2Y12 inhibitor (compound 15). Introduction of a monochloro-methyene group between P2 and P3 had a significantly increased inhibitory activity (4 vs 2 and 5 vs. 3, Table 2). The dichloromethylene group between P2 and P3 was less effective than the monochloromethylene group (compound 16 vs. compound 5).</p><!><p>Ap4A is an effective P2X1 receptor agonist [25]. Since P2X1 plays a role in the process of platelet activation [53] and in vasoconstriction [54], such an agonist effect would be undesirable for potential antithrombotic agents. Therefore, we tested the effect of the new base substituted Ap4A analogs on the activation of platelet P2X1 receptors. Because P2X1 is an ion channel whose activation causes an influx of extracellular Ca2+, this was done by flow cytometric measurement of the cytosolic Ca2+ increase after P2X1 stimulation. At the same time control experiments with a selective P2Y1 inhibitor were done to control for possible P2Y1 mediated Ca2+ increase. The effects of the test compounds were compared with that of 20 μM of a standard stable P2X1 agonist, P2,P3-methyleneATP, and are presented as percent of that of P2,P3-methyleneATP in Table 2. Methylthio substitution at C2 decreased the agonist properties (198% for 1 μM of Ap4A [24] vs. 48% for compound 2). P1,P2-Dithio, and especially P2,P3-monochloromethylene substitutions reduced the Ap4A scaffold agonist properties toward P2X1, and compound 5 was practically devoid of agonist activity. This activity re-appeared, however, with further increase of the alkyl chain length. The 2-halo, and N6-alkyl substitutions effectively reduced the P2X1 agonist activity. Interestingly, the doubly base substituted 2-MeS and N6-Me analog (compound 15) was an effective P2X1 agonist.</p><p>Examining the data in Tables 1 and 2 shows that human platelet aggregation is inhibited with IC50s lower that the IC50s for inhibition of both P2Y1 and P2Y12. A synergistic effect from the simultaneous inhibition of both ADP receptors was reported [23] after studying the combined use of selective P2Y1 and P2Y12 receptors antagonists on platelet aggregation, and can be explained by the complex interplay between the two receptors and the signaling pathways they trigger [11,56]. We still cannot, however, completely exclude the contribution of additional, yet unknown, mechanism(s) of ADP induced platelet aggregation inhibition by the test compounds.</p><!><p>Comparing the IC50 data from Table 1 for ADP induced human platelet aggregation inhibition in which the inhibitor is pre-mixed with the activator (3 μM ADP) with that in which the inhibitor is added to the platelets 15 min before their activation (5 μM ADP) reveals that in many cases, but not all, the IC50s in the second case are lower, even though the platelets are activated at higher ADP concentration, indicating that pre-incubation of the platelets with the inhibitor contributes, in some cases, to its activity. To shed light on this phenomenon, in a separate experiments we examined by optical aggregometry the inhibition of ADP induced human platelet aggregation by three of the compounds (5, 6, and 11) at 20 μM ADP concentration. In Fig. 6 are presented the corresponding concentration-inhibition curves and IC50s for an experiment without pre-incubation (panel A) and with 15 min pre-incubation of the platelets with the inhibitor (panel B). While the pre-incubation of the platelets with compounds 5 and 6 (2-MeS, and 2-EtS substitutions, resp.) resulted in an almost 50-fold decrease of their IC50s (from 2.56 μM to 63.6 nM for 5, and from 3.32 μM to 54.3 nM for 6), it had little effect with compound 11 (2-Br substitution, 6.22 μM to 4.52 μM).</p><p>In a separate experiment we tested the effect of the varied pre-incubation times (0, 5 min, 15 min) on the aggregation inhibition of compound 5 (at 25 and 100 nM concentration), and found that full pre-incubation effect develops within 15 min of pre-incubation (Data not shown).</p><p>In order to confirm that this effect was not due to irreversible receptor inhibition, we tested the reversibility of platelet aggregation inhibition after their exposure to compound 5. Expression of the activation markers GPIIb-IIIa and P-selectin on the surface of human platelets was used to determine the reversibility of their activation by 20 μM ADP. Compound 5 inhibited the expression of both markers with remarkable potency (IC50s of 1.1 nM for GPIIb-IIIa, and 0.6 nM for P-selectin, Fig. 7A), especially considering the high activator (ADP) concentration used in this experiment. Figure 7B shows that, after pre-incubation with 5 and dilution (100x) with platelet poor plasma, the platelets recovered ca. 80–90% of their ability to express the activation markers after stimulation indicating that the inhibitory effect of 5, and presumably the rest of the series, is reversible.</p><!><p>Thio substitution at P1 and P4 renders the corresponding phosphorous atoms chiral. Therefore, compounds 3 and 16 exist as 3 stereoisomers with absolute configurations at P1 and P4 phosphorous atoms RPRP, SPSP, and RPSP = SPRP (the subscript "P" indicates that this absolute configuration applies to an asymmetric phosphorous and not to a carbon atom). Those three stereoisomers are diastereomeric to each other because of the chiral character of the ribose moiety. The stereochemical character of the P2,P3-monochloromethylene group in compounds 1 and 5-15 depends on the absolute configuration at P1 and P4. When P1 and P4 have the same configuration (RPRP, or SPSP) the carbon atom of the chloromethylene group has two identical substituents, and is not asymmetric. However, when those two atoms are in different absolute configurations (RPSP = SPRP,) the carbon atom of the monochloromethylene group becomes pseudo-asymmetric and can exist in two absolute configurations designated with r and s (the prefix "pseudo" indicates that this carbon contains two substituents that differ only in their stereo-configuration, and lower case r and s, instead of upper case R and S are used for the same reason) [60]. Therefore compounds 1 and 5-15 can have 4 different stereoisomers with configurations RPRP, SPSP, RPrSP = SPrRP, and RPsSP = SPsRP. In the past we isolated the four diastereomers of base unsubstituted compound 1 and found a statistically significant difference in their inhibition of ADP induced human platelet aggregation [61]. Using the same technique (preparative reverse phase HPLC) we separated the four diastereomers of compound 5 (Fig. 8) and studied their individual inhibition of ADP induced human platelet aggregation by optical aggregometry. The concentration dependence of the aggregation inhibition for each diastereomer is plotted in Fig. 9, and the corresponding IC50s are listed in Table 3. There was no statistical significant difference in the IC50 values of the individual diastereomers and the diastereomeric mixture (compound 5). One possible explanation of this difference between compounds 1 and 5 is an increased contribution of the substituted base to the inhibitor–receptor interaction, with corresponding reduction of the relative contribution of the polyphosphate chain.</p><!><p>Unmodified dinucleoside polyphosphates are quickly hydrolyzed by plasma ecto-nucleotide pyrophosphatase/phosphodiesterase activity (NPP1, PC-1; NPP2, autotaxin; NPP3, Gp130) [62]. For instance, Ap4A is degraded in human plasma and whole blood with half-lives of 2.0 and 4.4 min, respectively [63]. The hydrolysis is non-symmetric between P1 and P2 (or P3 and P4), generating one molecule of mono- and one molecule of triphosphate [63,64]. Modification of P1 and P4 phosphate groups to thiophosphates significantly increases the plasma stability of dinucleoside tetraphosphates [35]. For instance, in rat plasma, Ap4A had a half-life of 2.99 min, whereas its P1,P4-dithio analog had a half-life of 154 min [65]. While the P2,P3-chloromethylene substitution did not have a significant impact on plasma stability (the half-life of P2,P3-chloromethylene Ap4A analog was 6.29 min) [65], it significantly improved the chemical stability. The combination of P1,P4-dithio, and P2,P3-chloromethylene modifications on Ap4A resulted in derivatives that had remarkable enzymatic and chemical stability in plasma [35].</p><p>By using very long incubation times we were able to observe enzymatic degradation of compound 5 in rat and human plasmas (the absence of chemical degradation was confirmed by control incubations with heat denaturated plasma). A representative chromatogram of HPLC analysis after incubation in human plasma for 77 h at 37 °C is shown in Fig. 10. The four diastereomers of 5 (D1–D4) are indicated on the chromatogram. Also indicated in the chromatogram are the two detected metabolites (2-methylthioadenosine 5′-thiomonophosphate, MeSAMP(S) and 2-methylthoadenosine, MeSAdo), which were identified by analysis of synthetic reference compounds, and confirmed by LCMS analysis of representative incubation mixtures. The internally normalized peak areas of the four diastereomers of 5 (by the order of their elution, D1, D2, D3, and D4) and the two detected metabolites (MeSAMP(S) was not detected in human plasma) are plotted in Fig. 11 panel A (rat plasma) and panel B (human plasma).</p><p>The kinetics of degradation of 5 in both plasmas did not follow 1st order, most likely due to loss of plasma activity during the long incubation times. Indeed, in a control experiment the rate of degradation of 5 in rat plasma which was aged for 24 h at 37 °C was the same as in fresh plasma after 24 h incubation with the test compound (data not shown). The first three time points (0, 1 and 4 hours) were used to estimate the initial first order elimination constants and the associated half-lives (by non-linear regression using GraphPad Prism) and the results are presented in Table 4.</p><p>Compound 5 was degraded much faster in rat than in human plasma. Also the stability of the individual diastereomers was markedly different – D1 being most and D4 least stable. D2 and D3 had close and intermediate stability. This is in agreement with our previous studies [61] of the plasma stability of the individual diastereomers of the base-unsubstituted compound 1, and confirms our previous conclusion that phosphorothioates in the SP absolute configuration resist hydrolysis by plasma pyrophosphatase/phospho-diesterases. The final metabolite in both rat and human plasma was 2-methylthioadenosine. Also, the intermediate 2-methylthioadenosine 5-monothiophosphate was observed, albeit at lower levels (it was below the limit of quantification in human plasma). The intermediate triphosphate analog was not detected.</p><!><p>In conclusion, we have prepared for the first time a series of base-substituted Ap4A analogs (with and without polyphosphate chain substitution) and studied their platelet related properties. We used this information to establish structure-activity relations for platelet aggregation and purinoreceptor inhibition by this class of compounds. Some of the compounds showed very potent (nanomolar level) inhibition of ADP induced human platelet aggregation (Table 1) and simultaneous inhibition of both P2Y1 and P2Y12 receptors (Table 2). Compound 5 reversibly inhibited ADP-stimulated expression of platelet activation markers GPIIb-IIIa and P-selectin with remarkably low IC50 values of 1.1 and 0.6 nM, respectively. Also this compound did not have an effect on human platelet P2X1 receptors (Table 2) and on human P2Y2, P2Y4, and P2Y6 receptors (data not shown). Simultaneous targeting of platelet P2Y1 and P2Y12 receptors might have a synergistic effect, and may provide an antithrombotic drug with superior efficacy and safety profiles. The only rapidly reversible, injectable antiplatelet drug currently available for clinical use in patients who are in need of immediate antiplatelet therapy and may develop bleeding complication, or be in need of urgent surgical intervention is the ATP derivative cangrelor. Taking into account the rapid elimination from circulation of the nucleoside polyphosphates, the present class of compounds may serve as a basis for the development of antiplatelet agents with rapid onset and offset of the pharmacological effect, and with a unique mechanism of action to fill an important unmet clinical need. Compound 5 is currently undergoing preclinical testing for this purpose.</p><!><p>All solvents and reagents used were obtained commercially and used as such unless noted otherwise. 1H and 31P NMR spectra were recorded in CDCl3, DMSO-d6, or D2O solutions at 298 °K using a Bruker Avance 300 instrument. Chemical shifts are reported as parts per million (ppm) relative to tetramethylsilane (TMS) for 1H and phosphoric acid for 31P NMR. All 31P spectra were proton decoupled unless otherwise noted. Spin multiplicities are given as s (singlet), br s (broad singlet), ms (multiple singlets), d (doublet), t (triplet), q (quartet), or m (multiplet). All chemical reactions and the purity of the intermediates were analyzed using TLC (silica gel 60 F254 plates, UV or I2 visualization) and LCMS (ThermoFinnigan ICQ Advantage with Surveyor LC system) with a XBridge C18 3.5 μm 2×50 mm column and a gradient from 0.1 to 100% acetonitrile in 0.1% formic acid (unless otherwise indicated in procedure) at a flow rate of 0.2 ml/min. Detection was at 210–420 nm for UV and electrospray ionization in the positive and the negative ionization modes (ESI+, ESI−) for MS. Preparative silica gel chromatography was performed using an ISCO Flash chromatography system and RediSep flash cartridges (particle size: 35–70 μm, Teledyne ISCO) or PuriFlash cartridges (particle size: 50 μm, spherical, Interchim). Preparative reverse phase HPLC was carried out using Waters XBridge C18 5 μm OBD 19×250 mm column on a Varian ProStar instrument. All nucleotides were purified by preparative ion-exchange HPLC using a Varian Load&Lock 50×300 mm column packed with TSKgel SuperQ-5PW (20 μm, Tosoh). The elution was carried out with a gradient from 20 mM to 500 mM (monophosphates), or from 20 mM to 2 M (tetraphosphates) triethylammonium bicarbonate (TEAB, pH 8) containing 10% acetonitrile (MeCN) for 180 min, at a flow rate of 35 ml/min. TEAB was prepared by saturation of water/triethylamine mixture with carbon dioxide. All final compounds (1–16) were determined to be greater than 95% pure via analysis by ion-exchange (IE) HPLC on a DNAPac PA200 4×250 mm column (Dionex) eluted with a gradient from 10% MeCN in water to 1 M ammonium bicarbonate and 2% MeCN in water for 15 min at a flow rate of 1 ml/min and UV detection at 260 nm (273 nm for the 2-alkylthio analogs), and by reverse phase (RP) HPLC on a XBridge C18 3.5 μm 2×50 mm column, eluted with a gradient of MeCN in 20 mM TEAB at a flow rate of 0.2 ml/min, and UV detection (190–420 nm). The exact gradient parameters are indicated in each procedure.</p><p>Compounds 3 and 16 exist as mixtures of 3 diastereomers, and compounds 5-15 as mixtures of 4 diastereoisomers. Where those diastereomers are separated the individual retention times are given. 2-Thioadenosine [66], 2-bromoadenosine [43], (monochloromethylene)bisphosphonic acid [67], the P1,P2-diimidazolide of (monochloromethylene)bisphosphonic acid [39], and 2-methylthioadenosine 5′-monophosphate [68] were prepared as previously described (See Supporting Information for details). The procedures for synthesis of all other nucleoside, and nucleoside monophosphate analogs, and their characterization information are presented in the Supporting Information.</p><!><p>Bis-triethylammonium salt of modified adenosine 5′-monophosphate or 5′-monothiophosphate (0.30 mmol) was dissolved in anhydrous DMF (5 ml), and the solvent was evaporated under vacuum (0.2 mm Hg) at 35 °C to produce a foam. The residue was dissolved in anhydrous DMF (5 ml) under Ar. Disodium di-(1-imidazolyl)pyrophosphate or disodium di-(1-imidazolyl)chloromethylene-bis-phosphonate [39] (0.1 mmol) was added to this solution, followed by anhydrous zinc chloride (408.9 mg, 3.0 mmol). The mixture was stirred protected from moisture for 30 min, and then added to a stirred mixture of Chelex® 100 resin in the sodium form (10 ml; Sigma-Aldrich) and 0.1 M triethylammonium bicarbonate buffer (20 ml, pH 8). After stirring for 15 min the mixture was filtered, and the resin was washed twice with water (10 ml each). The combined filtrate and washings were loaded on a preparative ion-exchange HPLC column (5 × 30 cm, Load&Lock®, Varian) packed with SuperQ-5PW resin (Tosoh Inc.) in the triethylammonium form, which was pre-equilibrated with 2 column volumes of 0.02 M TEAB buffer, pH 8, containing 10% (v/v) MeCN. A linear gradient of TEAB containing 10% (v/v) MeCN, from 0.02 to 2 M for 180 min, was passed through the column at a rate of 35 ml/min. The fractions containing the product were combined and evaporated under vacuum. The residue was re-evaporated three times from methanol (50 ml each), dissolved in methanol (0.5 ml), and mixed with a 2.0 M solution of sodium perchlorate in acetone (5 ml). The mixture was diluted with acetone (15 ml) and stirred for 2 h. The colorless solid was collected by centrifugation, and washed by suspending in acetone (20 ml), centrifugation, and decanting. This acetone washing was repeated two more times, and the colorless solid was dried first under a stream of nitrogen, and then for 6 h under high vacuum.</p><!><p>Prepared by the above procedure from 2-methylthioadenosine 5′-monophosphate bis-triethylammonium salt (179 mg, 0.30 mmol) and disodium di-(1-imidazolyl)pyrophosphate (32.2 mg, 0.1 mmol). Yield: 68 mg, 67%; 1H NMR (300 MHz, D2O) δ: 8.15 (s, 2H, H-8), 5.94 (d, J = 5.8 Hz, 2H, H-1′), 4.72 (m, partially overlaps with solvent peak, H-2′), 4.49 (dd, J1 = 3.7 Hz, J2 = 5.0 Hz, 2H), 4.29 (m, 2H, H-3′), 4.20 (m, 4H, H-5′,5″), 2.46 (s, 6H, SCH3); 31P NMR (121 MHz, D2O) δ: −11.2 (m, P1+P4), −22.6 (m, P2+P3); MS (ESI−), observed, m/z: 927.2 (100.0%), 928.1 (26.3%), 929.1 (15.9%), 930.1 (3.9%), 931.1 (0.9%); calculated for [M–H]−, C22H31N10O19P4S2−, m/z: 927.0 (100.0%), 928.0 (30.2%), 929.0 (17.4%), 930.0 (4.2%), 931.0 (1.2%); purity 97.8%, RP HPLC RT: 9.30 min (gradient from 0 to 30% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 8.8 min.</p><!><p>Prepared by the above procedure from 2-methylthioadenosine 5′-monothiophosphate bis-triethylammonium salt (183 mg, 0.30 mmol) and disodium di-(1-imidazolyl)pyrophosphate (32.2 mg, 0.1 mmol). This compound was isolated as a mixture of 3 diastereomers in ratio 1:2:1 due to the different stereo configurations at P1 and P4. Yield: 66 mg, 63%; 1H NMR (300 MHz, D2O): δ 8.24 (s, 0.5H, diast. 1 H-8), 8.19 (s, 0.5H, diast. 2 H-8), 8.18 (s, 1H, diast. 3 H-8), 5.90 (d, 2H, J = 5.8 Hz, H-1′), 4.77 – 4.64 (m, 2H, H-2′), 4.52 – 4.44 (m, 2H, H-3′), 4.30 – 4.24 (m, 2H, H-4′), 4.24 – 4.15 (m, 4H, H-5′), 2.445 (s, 3H, diast. 3 SCH3), 2.435 (s, 1.5H, diast. 1 or 2 SCH3), 2.427 (s, 1.5H, diast. 2 or 1 SCH3); 31P NMR (121 MHz, D2O), ppm: 43.53 – 42.94 (m, P1+P4), −23.93 – −24.39 (m, P2+P3); MS (ESI−), observed, m/z: 958.9 (100.0), 959.9 (27.3), 960.9 (23.9), 961.9 (6.6), 962.9 (2.2); calculated for [M–H]−, C22H31N10O17P4S4−, m/z: 959.0 (100.0%), 960.0 (31.7%), 961.0 (26.4%), 962.0 (7.2%), 963.0 (3.0%); purity 97.1%, RT RP HPLC: 9.75, 9.95, 10.41 min (gradient from 0 to 30% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 9.1 min.</p><!><p>Prepared by the above procedure from 2-methylthioadenosine 5′-monophosphate bis-triethylammonium salt (179 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 83 mg, 79%; 1H NMR (300 MHz, D2O): δ 8.14, 8.12 (2H, s, H-8), 5.95 (2H, d, J = 5.5 Hz, H-1′), 4.73 – 4.67 (2H, m, H-2′), 4.51 – 4.47 (2H, m, H-3′), 4.28 – 4.23 (2H, m, H-4′), 4.18 (1H, t, 2JP-H = 15.0 Hz, CHCl), 4.21 – 4.09 (4H, m, H-5′), 2.449, 2.445 (6H, s, SCH3). 31P NMR (121 MHz, D2O), ppm: 2.30 – 1.72 (m, P2+P3), −10.55 – −11.05 (m, P1+P4); MS (ESI−), observed, m/z: 959.2 (100.0%), 960.2 (33.1%), 961.1 (50.5%), 962.1 (13.5%), 963.1 (6.6%), 964.1 (1.4%); calculated for [M–H]−, C23H32ClN10O18P4S2−, m/z: 959.0 (100.0%), 960.0 (31.2%), 961.0 (49.4%), 962.0 (14.3%), 963.0 (6.8%), 964.0 (1.6%); purity 97.4%, RT RP HPLC: 9.47 min (gradient from 0 to 30% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 11.85, 12.12, 12.38 min.</p><!><p>Prepared by the above procedure from 2-methylthioadenosine 5′-monothiophosphate bis-triethylammonium salt (183 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 88 mg, 81%; 1H NMR (300 MHz, D2O) δ 8.35 – 8.11 (ms, 2H, H-8), 6.09 – 5.94 (m, 2H, H-1′), 4.90 – 4.35 (1H, multiple t, CHCl), 4.82 – 4.70 (m, 2H, H-2′), 4.60 – 4.50 (m, 2H, H-3′), 4.37 – 4.13 (m, 6H, H-4′+H′5′), 2.53 – 2.47 (ms, 6H, SCH3). 31P NMR (121 MHz, D2O), ppm: 43.15 – 42.02 (m, P1+P4), 2.00 – 1.22 (m, P2+P3); MS (ESI−), observed, m/z: 990.9 (100.0%), 991.9 (29.5%) 992.9 (56.7%), 993.9 (16.6%), 994.8, (10.0%); calculated for [M–H]−, C23H32ClN10O16P4S4, m/z: 990.95 (100.0%), 991.96 (32.8%), 992.95 (57.5%), 993.95 (17.1%), 994.94 (11.5%); purity 99.2%, RT RP HPLC: 3.55, 4.07, 4.88, 5.58 min (gradient from 0 to 12% MeCN in 20 mM triethylammonium acetate for 12 min); IE HPLC: 8.9, 9.1 min.</p><!><p>Prepared by the above procedure from 2-ethylthioadenosine 5′-monothiophosphate bis-triethylammonium salt (188 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 60 mg, 54%; 1H NMR (300 MHz, D2O) δ 8.15 – 8.02 (multiple s, 2H, H-8), 5.88 – 5.83 (m, 2H, H-1′), 4.78 – 4.35 (1H, multiple t, CHCl), 4.67 – 4.55 (m, 2H, H-2′), 4.42 – 4.35 (m, 2H, H-3′), 4.22 – 4.13 (m, 2H, H-4′), 4.13 – 3.98 (m, 4H, H-5′), 3.00 – 2.86 (m, 4H, SCH2), 1.21 – 1.12 (mt, 6H, CH3); 31P NMR (121 MHz, D2O), ppm: 44.32 – 42.78 (m, P1+P4), 2.79 – 2.12 (m, P2+P3); MS (ESI−), observed, m/z: 1019.1 (100%), 1020.1 (38.0%), 1021.0 (53.8%), 1022.0 (19.5%), 1022.9 (11.8%), 1023.9 (3.1%); calculated for [M–H]−, C25H36ClN10O16P4S4−, m/z: 1018.98 (100.0%), 1019.99 (33.0%), 1020.98 (59.3%), 1021.98 (19.1%), 1022.98 (12.0%), 1023.98 (2.3%); purity 99.2%, RT RP HPLC: 11.05, 11.98 min (gradient from 0 to 30% MeCN in 20 mM triethylammonium acetate for 15 min); IE HPLC: 8.4, 8.7 min.</p><!><p>Prepared by the above procedure from 2-propylthioadenosine 5′-monothiophosphate bis-triethylammonium salt (192 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 80 mg, 70%; 1H NMR (300 MHz, D2O) δ 8.29 – 7.95 (ms, 2H, H-8), 5.89 – 5.82 (m, 2H, H-1′), 4.81 – 4.31 (1H, multiple t, CH-Cl), 4.67 – 4.55 (m, 2H, H-2′), 4.43 – 4.33 (m, 2H, H-3′), 4.24 – 4.16 (m, 2H, H-4′), 4.16 – 4.00 (m, 4H, H-5′), 2.98 – 2.81 (m, 4H, SCH2), 1.63 – 1.42 (m, 4H, SCH2CH2), 0.87 – 0.78 (multiple t, 6H, CH3); 31P NMR (121 MHz, D2O), ppm: 43.8 – 42.5 (m, P1+P4), 3.1 – 1.8 (m, P2+P3); MS (ESI−), observed, m/z: 1047.0 (100%), 1048.0 (36.7%), 1048.9 (55.3%), 1049.9, (18.4%), 1050.9, (10.8%), 1051.9 (4.6%); calculated for [M–H]−, C27H40ClN10O16P4S4−, m/z: 1047.0 (100.0%), 1048.0 (37.2%), 1049.0 (60.0%), 1050.0 (20.5%), 1051.0 (12.4%), 1052.0 (3.6%); purity, 98.6%, RT RP HPLC: 9.99, 10:25 min (gradient from 0 to 50% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 8.4, 8.6 min.</p><!><p>Prepared by the above procedure from 2-(3,3,3-trifluoropropylthio)adenosine 5′-monothiophosphate bis-triethylammonium salt (208 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloromethylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 76 mg, 61%; 1H NMR (300 MHz, D2O) δ 8.28 – 8.10 (ms, 2H, H-8), 5.98 – 5.91 (m, 2H, H-1′), 4.95 – 4.40 (1H, multiple t, CHCl), 4.67 – 4.60 (m, 2H, H-2′), 4.53 – 4.43 (m, 2H, H-3′), 4.33 – 4.25 (m, 2H, H-4′), 4.25 – 4.11 (m, 4H, H-5′), 3.24 – 3.12 (m, 4H, CF3CH2), 2.70 – 2.50 (m, 4H, SCH2); 31P NMR (121 MHz, D2O), ppm: 44.2 – 42.6 (m, P1+P4), 3.0 – 2.1 (m, P2+P3); MS (ESI−), observed, m/z: 1155.1 (100%), 1156.1 (35.2%), 1157.0 (54.9%), 1158.0 (18.4%), 1159.0 (10.2%), 1159.9 (3.2%); calculated for [M–H]−, C27H34ClF6N10O16P4S4−, m/z: 1155.0 (100.0%), 1156.0 (37.1%), 1157.0 (60.0%), 1158.0 (20.4%), 1159.0 (11.1%), 1160.0 (3.3%); purity, 96.1%, RT RP HPLC: 11.59, 11.87 min (gradient from 0 to 50% MeCN in 20 mM triethylammonium acetate for 15 min); IE HPLC: 8.2, 8.5 min.</p><!><p>Prepared by the above procedure from 2-pentylthioadenosine 5′-monothiophosphate bis-triethylammonium salt (200 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 75 mg, 64%; 1H NMR (300 MHz, D2O) δ 8.26 – 8.12 (multiple s, 2H, H-8), 5.98 – 5.93 (m, 2H, H-1′), 4.89 – 4.39 (1H, multiple t, CH-Cl), 4.73 – 4.64 (m, 2H, H-2′), 4.52 – 4.45 (m, 2H, H-3′), 4.32 – 4.23 (m, 2H, H-4′), 4.23 – 4.11 (m, 4H, H-5′), 3.03 – 2.92 (m, 4H, SCH2), 1.63 - 1.50 (m, 4H, SCH2CH2), 1.34 – 1.12 (m, 8H, CH2CH2CH3), 0.82 – 0.72 (m, 6H, CH3); 31P NMR (121 MHz, D2O), ppm: 44.05 – 42.83 (m, P1+P4), 2.96 – 2.13 (m, P2+P3); MS (ESI−), observed, m/z: 1103.3 (100%), 1104.3 (43.1%), 1105.1 (61.8%), 1106.1 (21.8%), 1107.0 (11.0%), 1108.0 (3.2%); calculated for [M–H]−, C31H48ClN10O16P4S4−, m/z: 1103.1 (100.0%), 1104.1 (41.6%), 1105.1 (61.8%), 1106.1 (23.1%), 1107.1 (13.3%), 1108.1 (4.1%); purity, 95.2%, RT RP HPLC: 12.08, 12.50, 12.67 min (gradient from 0 to 50% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 8.4, 8.6 min.</p><!><p>Prepared by the above procedure from 2-chloroadenosine 5′-monothiophosphate bis-triethylammonium salt (180 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 71.5 mg, 69%; 1H NMR (300 MHz, D2O) δ 8.39 – 8.26 (multiple s, 2H, H-8), 5.91 – 5.86 (m, 2H, H-1′), 4.90 – 4.39 (1H, multiple t, CH-Cl), 4.70 – 4.61 (m, 2H, H-2′), 4.53 – 4.46 (m, 2H, H-3′), 4.36 – 4.27 (m, 2H, H-4′), 4.27 – 4.11 (m, 4H, H-5′); 31P NMR (121 MHz, D2O), ppm: 43.75 – 42.42 (m, P1+P4), 2.63 – 1.95 (m, P2+P3); MS (ESI−), observed, m/z: 967.0 (92.2%), 968.0 (27.0%), 969.0 (100.0%), 970.0 (28.9%), 971.0 (42.0%), 972.0 (10.3%), 973.0 (8.6%), 973.9 (1.8%); calculated for [M–H]−, C21H26Cl3N10O16P4S2−, m/z: 966.9 (89.1%), 967.9 (25.8%), 968.9 (100.0%), 969.9 (28.1%), 970.9 (42.3%), 971.9 (11.4%), 972.9 (8.3%), 973.9 (2.1%); purity, 99.3%, RT RP HPLC: 7.60, 7.88, 8.12, 8.33 min (gradient from 0 to 30% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 6.7, 6.9 min.</p><!><p>Prepared by the above procedure from 2-bromoadenosine 5′-monothiophosphate bis-triethylammonium salt (193 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 76.7 mg, 66%; 1H NMR (300 MHz, D2O) δ 8.48 – 8.33 (multiple s, 2H, H-8), 6.02 – 5.96 (m, 2H, H-1′), 5.01 – 4.50 (1H, multiple t, CHCl), 4.78 – 4.72 (m, 2H, H-2′), 4.63 – 4.57 (m, 2H, H-3′), 4.46 – 4.37 (m, 2H, H-4′), 4.37 – 4.22 (m, 4H, H-5′); 31P NMR (121 MHz, D2O), ppm: 44.05 – 42.58 (m, P1+P4), 2.92 – 2.02 (m, P2+P3); MS (ESI−), observed, m/z: 1055.1 (42.0%), 1056.2 (10.0%), 1057.0 (100.0%), 1058.0 (29.6%), 1058.9 (77.3%), 1059.9 (20.6%), 1060.8 (24.4%), 1061.8 (6.8%), 1062.8 (2.0%); calculated for [M–H]−, C21H26Br2ClN10O16P4S2−, m/z: 1054.8 (41.2%), 1055.8 (11.9%), 1056.8 (100.0%), 1057.8 (28.6%), 1058.8 (80.3%), 1059.8 (22.4%), 1060.8 (24.1%), 1061.8 (6.3%), 1062.8 (2.8%); purity, 97.8%, RT RP HPLC: 7.05, 7.29 min (gradient from 0 to 50% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 6.8, 7.1 min.</p><!><p>Prepared by the above procedure from 2-iodoadenosine 5′-monothiophosphate bis-triethylammonium salt (208 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 90.6 mg, 73%; 1H NMR (300 MHz, D2O) δ 8.38 – 8.16 (multiple s, 2H, H-8), 5.89 – 5.84 (m, 2H, H-1′), 4.93 – 4.39 (1H, multiple t, CH-Cl), 4.64 – 4.57 (m, 2H, H-2′), 4.52 – 4.45 (m, 2H, H-3′), 4.34 – 4.27 (m, 2H, H-4′), 4.27 – 4.14 (m, 4H, H-5′); 31P NMR (121 MHz, D2O), ppm: 43.95 – 42.45 (m, P1+P4), 3.13 – 2.02 (m, P2+P3); MS (ESI−), observed, m/z: 1151.0 (100.0%), 1152.0 (29.7%), 1152.9 (46.2%), 1153.9 (13.7%), 1154.9 (5.8%), 1155.9 (1.3, %); calculated for [M–H]−, C21H26ClI2N10O16P4S2−, m/z: 1150.8 (100.0%), 1151.8 (28.9%), 1152.8 (48.3%), 1153.8 (13.1%), 1154.8 (6.3%), 1155.8 (1.4%); purity, 96.4%, RT RP HPLC: 9.32, 9.75, 9.95, 10.12 min (gradient from 0 to 30% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 6.8, 7.1 min.</p><!><p>Prepared by the above procedure from N6-methyladenosine 5′-monothiophosphate bis-triethylammonium salt (174 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 67.1 mg, 66%; 1H NMR (300 MHz, D2O) δ 8.38 – 8.16 (2H, ms, H-8), 7.98 – 7.92 (ms, 2H, H-2), 5.98 – 5.91 (2H, m, H-1′), 5.03 – 4.42 (1H, mt, CH-Cl), 4.77 – 4.68 (2H, m, H-2′), 4.55 – 4.47 (2H, m, H-3′), 4.37 – 4.29 (2H, m, H-4′), 4.29 – 4.08 (4H, m, H-5′), 2.90 – 2.82 (6H, ms, NCH3); 31P NMR (121 MHz, D2O), ppm: 43.97 – 42.43 (m, P1+P4), 3.03 – 1.92 (m, P2+P3); MS (ESI−), observed, m/z: 927.1 (100.0%), 928.1 (31.9%), 929.1 (47.0%), 930.1 (19.3%), 931.0 (8.1%); calculated for [M–H]−, C23H32ClN10O16P4S2−, m/z: 927.0 (100.0%), 929.0 (49.0%), 928.0 (31.1%), 930.0 (14.2%), 931.0 (6.6%); purity, 99.2%, RT RP HPLC: 11.13, 11.63, 11.84, 12.28 min (gradient from 0 to 15% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 6.6, 6.8 min.</p><!><p>Prepared by the above procedure from N6-propyladenosine 5′-monothiophosphate bis-triethylammonium salt (182 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloro-methylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 74.0 mg, 69%; 1H NMR (300 MHz, D2O) δ 8.38 – 8.19 (2H, multiple s, H-8), 7.95 – 7.90 (multiple s, 2H, H-2), 5.98 – 5.91 (2H, m, H-1′), 4.98 – 4.42 (1H, multiple t, CH-Cl), 4.77 – 4.64 (2H, m, H-2′), 4.55 – 4.47 (2H, m, H-3′), 4.36 – 4.27 (2H, m, H-4′), 4.27 – 4.09 (4H, m, H-5′), 3.30 – 3.11 (4H, multiple b s, NCH2), 1.61 – 1.46 (m, 4H, NCH2CH2), 0.92 – 0.83 (m, 6H, CH3); 31P NMR (121 MHz, D2O), ppm: 44.02 – 42.41 (m, P1+P4), 2.97 – 2.03 (m, P2+P3); MS (ESI−), observed, m/z: 983.1 (100.0%), 984.1 (33.6%), 985.1 (48.3%), 986.1 (15.0%), 987.0 (6.9%), 988.1 (1.5%); calculated for [M–H]−, C27H40ClN10O16P4S2−, m/z: 983.1 (100.0%), 984.1 (35.6%), 985.1 (50.4%), 986.1 (16.4%), 987.1 (7.3%), 988.1 (1.9%); purity, 98.8%, RT RP HPLC: 10.95 min (gradient from 0 to 30% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 6.7, 6.9 min.</p><!><p>Prepared by the above procedure from 2-methylthio-N6-methyladenosine 5′-monothiophosphate bis-triethylammonium salt (188 mg, 0.30 mmol) and disodium di-(1-imidazolyl)chloromethylene-bis-phosphonate (35.5 mg, 0.1 mmol). Yield: 79.9 mg, 72%; 1H NMR (300 MHz, D2O) δ 8.10 – 7.90 (2H, ms, H-8), 5.90 – 5.83 (2H, md, H-1′), 5.03 – 4.35 (1H, mt, CH-Cl), 4.75 – 4.63 (2H, m, H-2′), 4.52 – 4.43 (2H, m, H-3′), 4.29 – 4.21 (2H, m, H-4′), 4.21 – 4.02 (4H, m, H-5′), 2.88 – 2.71 (6H, ms, SCH3), 2.40 – 2.25 (6H, ms, NCH3); 31P NMR (121 MHz, D2O), ppm: 41.96 – 40.32 (m, P1+P4), 0.69 – −0.16 (m, P2+P3); MS (ESI−), observed, m/z: 1019.0 (100.0%), 1020.0 (36.6%), 1020.9 (53.8%), 1021.9 (18.0%), 1022.9 (11.3%); calculated for [M–H]−, C25H36ClN10O16P4S4−: 1019.0 (100.0%), 1020.0 (35.0%), 1021.0 (59.3%), 1022.0 (19.2%), 1023.0 (12.0%); purity, 95.8%, RT RP HPLC: 8.89, 9.19, 9.56 min (gradient from 0 to 50% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 8.3, 8.6 min.</p><!><p>2-Methylthoadenosine (1.88 g, 6 mmol) was dissolved in dry DMF (24 ml) under Ar. p-Toluenesulfonic acid (2.30 g) was added on stirring, followed by trimethyl orthoformate (31.3 g, 32.3 ml, 300 mmol). The resulting solution was kept at rt under Ar for 18 h. The p-toluenesulfonic acid was removed by addition of Dowex MWA-1 anion exchange resin in the OH− form (10.3 g). The resin was filtered and washed twice with DMF (10 ml each). The combined filtrate and washings were evaporated under vacuum, and the residue was dried under high vacuum at rt for 6 h to give 2-methylthio-2′,3′-(methoxymethylene)adenosine as a colorless foam. Yield: 1.90 g, 89% as two diastereomers; 1H NMR (300 MHz, DMSO-d6) δ 8.13, (0.5H, s, diast. 1 H-8), 8.09 (0.5H, s, diast. 2 H-8), 7.28 (2H, bs, NH2), 6.10 (0.5H, d, J = 2.9 Hz, diast. 1 H-1′), 6.06 (0.5H, s, diast. 2 CHOMe), 6.01 (0.5H, d, J = 2.5 Hz, diast. 2 H-1′), 5.96 (0.5H, s, diast. 1 CHOMe), 5.41 (0.5H, dd, J = 2.5, 6.3 Hz, diast. 2 2′), 5.37 (0.5H, dd, J = 2.9, 7.1 Hz, diast. 1 2′), 5.00 – 4.94 (1.5H, m, diast. 2 H-3′ + OH), 4.87 (0.5H, dd, diast. 1 H-3′), 4.15 (0.5H, m, diast. 2 H-4′), 4.05 (0.5H, m, diast. 1 H-4′), 3.41 (2H, m, H-5′), 3.25 (1.5H, s, diast. 1 OCH3), 3.13 (1.5H, s, diast. 2 OCH3), 2.36 (1.5H, s, diast. 2 SCH3), 2.35 (1.5H, s, diast. 1 SCH3); MS (ESI−): 354.3 [M–H]−; purity, 95.3%, RT RP HPLC: 20.53 min (gradient from 0 to 50% MeCN in 20 mM aqueous triethylammonium acetate for 15 min).</p><p>2-Methylthio-2′,3′-(methoxymethylene)adenosine (178 mg, 0.5 mmol) was evaporated twice from dry DMF (5 ml each). The flask was equipped with a stir bar, sealed and flushed with Ar. Dry THF (5 ml) was added via a syringe followed by diisopropylethylamine (155 mg, 209 μl, 1.2 mmol). The contents were stirred until solids dissolved and cooled to –10 °C. 2-Chloro-1,3,2-benzodioxaphosphorin-4-one (salicyl chlorophosphite, 122 mg, 0.6 mmol) was dissolved in dry THF (3 ml) under Ar, and the solution was added by a syringe dropwise and with stirring to the cooled nucleoside solution at –10 – 0 °C. The cooling bath was removed and the reaction mixture was left for 10 min at rt.</p><p>(Dichloromethylene)bisphosphonic acid (147 mg, 0.6 mmol) was dissolved in methanol (4 ml). Tributylamine (334 mg, 428 μl, 1.8 mmol) was added and the mixture was evaporated under vacuum. The residue was rendered anhydrous by repeated evaporation from dry DMF (3×10 ml) under vacuum. The residue was dissolved under Ar in 3 ml anhydrous DMF and was added by a syringe dropwise with stirring to the cooled (–10 °C) reaction mixture at –10 – 0 °C. The cooling bath was removed and the reaction mixture was left for 30 min at rt under Ar. A suspension of sulfur (fine powder, 32 mg, 1 mmol) in 2 ml dry DMF was added by a syringe. After another 30 min stirring at rt under Ar a solution of 2-methylthioadenosine 5′-monothiophosphate bis-triethyammonium salt (459 mg, 0.75 mmol, rendered anhydrous by repeated evaporation from dry DMF under vacuum) and tributylamine (370 mg, 475 μl, 2 mmol) in 4 ml dry DMF were added by a syringe with stirring, followed by a solution of anhydrous zinc chloride (340 mg, 2.5 mmol) in 3 ml dry DMF. The reaction mixture was concentrated under vacuum at rt to half of its volume and stirred under Ar overnight. TEAB (0.1 M, 50 ml) and Chelex® ion exchange resin in the sodium form (20 ml) were added, and the mixture was stirred for 2 h and then filtered. The filtrate was extracted with ether containing 1% triethylamine (100 ml) and then evaporated under vacuum to dryness. The residue was treated with 20 ml methanol and filtered. (The filtrate contained mostly unreacted monothiophosphate.) The solid was dissolved in 8 ml water and subjected to preparative ion-exchange chromatography on TSKgel SuperQ-5PW as described in the general section, and the fractions were analyzed by LCMS. During the reaction partial loss of the 2′,3′-methoxymethylene protecting group was observed (most likely caused by the ZnCl2 catalyst). The major products were the corresponding protected and unprotected triphosphates. The peaks containing the products (P1-(2-methylthio-2′,3′-(methoxymethylene)-5′-adenosine)-P4-(2-methylthio-5′-adenosine)-P1,P4-dithio-P2,P3-dichloromethylenetetraphosphate and the deprotected 16) eluted at 143 and 152 min, respectively, were combined, evaporated under vacuum, and stripped from the residual buffer by repeated evaporation from methanol. The residual 2′,3′-methoxymethylidene protection was removed by treatment with 10% hydrochloric acid (1 ml) for 3 h. The acid was neutralized with triethylamine to pH 9, and the solution was loaded on a XBridge Prep C18 5 μm 20×250 mm column and eluted with a gradient from 50 mM TEAB to 50 mM TEAB in 50% aqueous MeCN for 30 min at 15 ml/min and 273 nm UV detection. The fractions containing the product were evaporated under vacuum, and repeatedly evaporated from methanol (3×30 ml). The product was converted to the sodium form by dissolving in water (1 ml) and passing through a column of Dowex 50WX2 in the sodium form (30 × 10 mm), elution with water (5 ml), concentration under vacuum to 1 ml and lyophilization. Yield: 33.5 mg, 6%; 1H NMR (300 MHz, D2O) δ 8.23, 8.20, 8.17 (2H, ms, H-8), 5.99 – 5.94 (2H, md, H-1′), 4.79 – 4.71 (2H, m, H-2′), 4.55 – 4.46 (2H, m, H-3′), 4.34 – 4.27 (2H, m, H-4′), 4.27 – 4.11 (4H, m, H-5′), 2.48, 2.47 (6H, two s, SCH3); 31P NMR (121 MHz, D2O), ppm: 43.79 – 42.89 (m, P1+P4), 1.53 – −1.26 (m, P2+P3); MS (ESI−), observed, m/z: 1025.1 (100%), 1026.1 31.7%), 1027.0 (91.7%), 1028.0 (28.5%), 1028.9 (31.4%), 1030.0 (8.6%), 1030.9 (3.8%), 1032.0 (0.9%); calculated for [M–H]−, C23H31Cl2N10O16P4S4-: 1024.9 (100.0%), 1026.9 (90.5%), 1025.9 (32.7%), 1028.9 (30.3%), 1027.9 (28.3%), 1029.9 (8.8%), 1030.9 (4.9%), 1031.9 (1.3%); purity, 96.5%, RT RP HPLC: 8.53 min (gradient from 0 to 50% MeCN in 20 mM aqueous triethylammonium acetate for 15 min); IE HPLC: 9.3 min.</p><!><p>Analytically the diastereomers of 5 were separated by reverse phase HPLC on a XBridge RP C18 column, 3.5 μm, 4.6×150 mm (Waters) at 30 °C with isocratic elution with 15% methanol in 20 mM potassium phosphate buffer pH 8.5 containing 10 mM EDTA, at a flow rate of 1 ml/min and UV detection at 273 nm (Fig. 8). Preparative separations (12.5 mg per injection) were done on a 20 × 250 mm XBridge C18 column 5 μm column at rt, eluted isocratically for 60 min with the same mobile phase, and then a linear gradient to 30% methanol for 60 min, at a flow rate of 10 ml/min. The fractions, after concentration to 1/5 of their original volume under vacuum, were desalted by loading on the same column equilibrated with 0.2 M TEAB buffer, pH 8, and then eluted with a gradient from the equilibration buffer to 50% methanol in the equilibration buffer at 15 ml/min for 30 min. After desalting, the fractions were evaporated on a rotary evaporator, and the residual TEAB was removed by repeated evaporation from methanol. Finally the diastereomers were converted to the sodium salts by passing through a column of Dowex W50X2 (20 × 5 mm) in the sodium form, elution with two column volumes of water, and lyophilization. The purity of each diastereomer was above 95% as judged by analytical RP HPLC.</p><!><p>Yield: 1.7 mg, 15%; 1H NMR (300 MHz, D2O) δ 8.29 (1H, s, H-8), 8.18 (1H, s, H-8), 6.00 (1H, d, J = 5.1 Hz, H-1′), 5.99 (1H, d, J = 6.1 Hz, H-1′), 4.80 – 4.71 (2H, m, H-2′), 4.67 (1H, t, J = 17.6 Hz, CH-Cl), 4.56 – 4.51 (2H, m, H-3′), 4.33 – 4.25 (2H, m, H-4′), 4.25 – 4.08 (4H, m, H-5′), 2.495, (3H, s, SCH3), 2.477 (3H, s, SCH3); 31P NMR (121 MHz, D2O), ppm: 43.84 – 42.23 (m, P1+P4), 2.29 – 1.52 (m, P2+P3); RT RP HPLC: 6.76 min; IE HPLC: 8.9 min.</p><!><p>Yield: 3.3 mg, 29%; 1H NMR (300 MHz, D2O) δ 8.26 (1H, s, H-8), 8.15 (1H, s, H-8), 5.96 (1H, d, J = 5.7 Hz, H-1′), 4.76 – 4.70 (2H, m, H-2′), 4.54 – 4.47 (2H, m, H-3′), 4.45 (1H, t, J = 17.4 Hz, CH-Cl), 4.32 – 4.27 (2H, m, H-4′), 4.27 – 4.12 (4H, m, H-5′), 2.475, (3H, s, SCH3), 2.461 (3H, s, SCH3); 31P NMR (121 MHz, D2O), ppm: 43.58 – 42.97 (m, P1+P4), 2.31 – 1.69 (m, P2+P3); RT RP HPLC: 7.52 min; IE HPLC: 9.1 min.</p><!><p>Yield: 3.8 mg, 34%; 1H NMR (300 MHz, D2O) δ 8.56 (1H, s, H-8), 8.11 (1H, s, H-8), 5.99 (1H, d, J = 5.1 Hz, H-1′), 5.98 (1H, d, J = 6.1 Hz, H-1′), 4.82 (1H, t, J = 17.6 Hz, CH-Cl), 4.80 – 4.72 (2H, m, H-2′), 4.55 – 4.50 (2H, m, H-3′), 4.34 – 4.23 (2H, m, H-4′), 4.23 – 4.09 (4H, m, H-5′), 2.485, (3H, s, SCH3), 2.473 (3H, s, SCH3); 31P NMR (121 MHz, D2O), ppm: 43.14 – 42.46 (m, P1+P4), 2.53 – 1.70 (m, P2+P3); RT RP HPLC: 10.62 min; IE HPLC: 8.9 min.</p><!><p>Yield: 2.4 mg, 21%; 1H NMR (300 MHz, D2O) δ 8.20 (1H, s, H-8), 8.17 (1H, s, H-8), 5.97 (1H, d, J = 5.8 Hz, H-1′), 4.77 – 4.71 (2H, m, H-2′), 4.52 (1H, t, J = 17.5 Hz, CH-Cl), 4.55 – 4.49 (2H, m, H-3′), 4.32 – 4.26 (2H, m, H-4′), 4.26 – 4.10 (4H, m, H-5′), 2.479, (3H, s, SCH3), 2.473 (3H, s, SCH3); 31P NMR (121 MHz, D2O), ppm: 43.51 – 42.62 (m, P1+P4), 2.32 – 1.71 (m, P2+P3); RT RP HPLC: 12.41 min; IE HPLC: 9.1 min.</p><!><p>Commercial frozen, pooled, heparin-anticoagulated rat or human plasma (Bioreclamation, Westbury, NY) was thawed upon arrival, aliquoted (3 ml) in sterile polypropylene vials, re-frozen in dry ice, and stored at –45 °C. At the time of testing the plasma aliquots were thawed and incubated at 37 °C for 5 min. Compound 5 (15 μl of 10 mM solution in physiological saline, 50 μM final concentration) was added, the sample was mixed briefly, and incubated at 37 °C. Aliquots (250 μl) were taken at the specified times, mixed with 250 μl MeCN, vortexed briefly, and then centrifuged at 15000 xG for 15 min. The supernatant was filtered through centrifugal ultrafiltration device (Nanosep®, Pall, 10K membrane, 15 min at 14000 xG). The filtrates (250 μl) were evaporated on a Speed-Vac (4 h, 3 mm Hg, 35 °C), reconstituted in 50 μL of 50 mM potassium phosphate 10 mM EDTA buffer pH 8.5, centrifuged (5 min, 15000 xG) and the supernatants were transferred into micro-insert equipped HPLC vials for analysis. Blank rat or human plasma (3 ml) was incubated for 5 min or for 24 h at 37 °C, spiked with water (15 μl), and then processed as above to prepare blank samples. The processed samples were analyzed by HPLC on a XBridge Shield C18, 3.5 μm, 150×4.6 mm column (Waters) with 5 μl injection and isocratic elution with 20% methanol in 50 mM potassium phosphate buffer pH 8.4 containing 10 mM EDTA at a flow rate of 1 ml/min and UV detection at 273 nm. The quantification was done by internal normalization of the peak areas of the four diastereomers and the metabolites. The rate constants for degradation in plasma and the associated half-lives were estimated by a non-linear fitting of a first order elimination model to the internally normalized chromatographic peak areas of the first three time points.</p><!><p>MRS2179, probenecid, adenosine 5′-(P2,P3-methylene)triphosphate (β, γ-CH2-ATP), EGTA and apyrase (grade VII) were from Sigma-Aldrich, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) was from Calbiochem, FLUO-4 was from Invitrogen, ADP was from Bio/Data, and CD41–phycoerythrin–Cy5 was from Beckman Coulter.</p><!><p>The studies involving human volunteers were approved by the University of Massachusetts Medical School and Boston Children's Hospital Institutional Review Boards (IRB). Written IRB-approved informed consent was obtained prior to blood collection.</p><!><p>Human blood samples were taken from healthy volunteer donors free from aspirin or other non-steroidal anti-inflammatory drugs for more than 7 days. Blood was drawn from antecubital veins into tubes containing 3.2% sodium citrate. For platelet aggregation assays the blood was centrifuged at 110 × g for 12 minutes, and platelet rich plasma (PRP) was immediately removed. Centrifugation 1 at 1650 × g for 10 minutes was to obtain platelet-poor plasma (PPP).</p><!><p>The 96-well microplate method for the detection of ADP-induced platelet aggregation and the concentration dependence of its inhibition by the tested compounds was used as previously described [24,25], thereby avoiding the problem of platelet aging [51]. In brief, PRP at 37° C was added to a pre-warmed 96-well microplate containing ADP (3 μM final concentration) and test compounds (various concentrations) or vehicle (10 mM Hepes, 0.15 M NaCl, pH 7.4). Light transmission at 580 nm was recorded immediately and at 11 second intervals for 6 min at 37° C with intermittent programmed shaking of the plate in a Molecular Devices microplate reader. Within each experiment all samples were run in duplicate and each experiment was repeated 3 – 5 times with PRP from different donors.</p><!><p>VASP phosphorylation was measured by flow cytometry using a BioCytex kit, essentially according to the manufacturer's recommendations, except that a small volume of the test compound solution or vehicle (HEPES-saline) was added to each assay tube as previously described [24,25]. Analysis was performed in a FACSCalibur (Becton Dickinson) flow cytometer.</p><!><p>ADP-dependent, P2Y1-mediated increase in platelet cytosolic Ca2+ was measured by detecting changes in FLUO-4 fluorescence as previously described [24,25]. In brief, citrated whole blood was added to a loading solution consisting of 5 μM FLUO-4, CD41-PE-Cy5 and 1 mM probenecid, and the mixture was incubated for 30 minutes at room temperature. Samples were diluted 36-fold in 10 mM HEPES, 0.15 M NaCl, pH 7.4 and analyzed in a FACSCalibur flow cytometer. After obtaining a 30 second baseline recording, the acquisition was paused, and 60 μL of ADP (3 μM final concentration) and test compound solutions at various concentrations or ADP plus vehicle (HEPES-saline) were quickly added, the sample mixed, and the acquisition resumed (total pause time less than 10 seconds). FLUO-4 fluorescence before and after addition of ADP (3 μM final concentration) and test compound solutions was monitored. The mean FLUO-4 fluorescence of the baseline 30-second interval and of 10-second post-stimulant intervals was calculated. The cytosolic Ca2+ increase was calculated as the ratio of the maximal post-stimulant FLUO-4 fluorescence to the baseline FLUO-4 fluorescence. The percent inhibition of ADP-induced Ca2+ increase due to the addition of the test compounds was calculated relative to 3 μM ADP plus vehicle (HEPES-saline).</p><!><p>Measurement of P2X1-mediated entry of extracellular Ca2+ based on changes in FLUO-4 fluorescence was performed as previously described [25]. The non-hydrolyzable ATP analog β,γ-CH2-ATP (20 μM) was used as a positive control. To confirm that any increases in intracellular Ca2+ observed were unrelated to P2Y1 activation, experiments were repeated with 100 μM MRS2179, a selective P2Y1 inhibitor, in the ambient buffer. The ability of high concentrations of the test compounds to antagonize P2X1 activation by 20 μM β,γ-CH2-ATP was also tested.</p><!><p>The results were analyzed using GraphPAD Prism software, version 4.00 for Windows (GraphPad Software, San Diego, CA). All data are expressed as mean followed by 95% confidence interval (95% CI). Student's t-test was used to determine statistical significance when two groups of data were compared. One-way ANOVA and Bonferroni's multiple comparison tests were used when three or more groups of data were compared.</p>

PubMed Author Manuscript

Concerted Nucleophilic Aromatic Substitution Reactions

AbstractRecent developments in experimental and computational chemistry have identified a rapidly growing class of nucleophilic aromatic substitutions that proceed by concerted (cSNAr) rather than classical, two‐step, SNAr mechanisms. Whereas traditional SNAr reactions require substantial activation of the aromatic ring by electron‐withdrawing substituents, such activating groups are not mandatory in the concerted pathways.

concerted_nucleophilic_aromatic_substitution_reactions

<!>Aromatic Substitution Reactions<!>Classical Nucleophilic Aromatic Substitution<!><!>Classical Nucleophilic Aromatic Substitution<!><!>Classical Nucleophilic Aromatic Substitution<!><!>Classical Nucleophilic Aromatic Substitution<!><!>Concerted Nucleophilic Aromatic Substitution (cSNAr)—Early Developments<!><!>Concerted Nucleophilic Aromatic Substitution (cSNAr)—Early Developments<!><!>Concerted Nucleophilic Aromatic Substitution (cSNAr)—Early Developments<!><!>Concerted Nucleophilic Aromatic Substitution (cSNAr)—Early Developments<!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Some Contributions by Computational Studies<!><!>Fluorodeoxygenation of Phenols and Derivatives<!><!>Fluorodeoxygenation of Phenols and Derivatives<!><!>Fluorodeoxygenation of Phenols and Derivatives<!><!>Aminodeoxygenation of Phenol Derivatives<!><!>Aminodeoxygenation of Phenol Derivatives<!><!>Aminodeoxygenation of Phenol Derivatives<!><!>Aminodeoxygenation of Phenol Derivatives<!>Hydrides as Nucleophiles<!><!>Hydrides as Nucleophiles<!><!>Hydrides as Nucleophiles<!><!>Hydrides as Nucleophiles<!><!>Hydrides as Nucleophiles<!><!>Hydrides as Nucleophiles<!><!>Hydrides as Nucleophiles<!>P, N, Si, C Nucleophiles<!><!>P, N, Si, C Nucleophiles<!><!>P, N, Si, C Nucleophiles<!><!>P, N, Si, C Nucleophiles<!><!>P, N, Si, C Nucleophiles<!><!>P, N, Si, C Nucleophiles<!>Organic Rearrangements via Spiro Species: Intermediates or Transition States?<!><!>Organic Rearrangements via Spiro Species: Intermediates or Transition States?<!><!>Organic Rearrangements via Spiro Species: Intermediates or Transition States?<!><!>Organic Rearrangements via Spiro Species: Intermediates or Transition States?<!><!>Organic Rearrangements via Spiro Species: Intermediates or Transition States?<!><!>Newman–Kwart and Related Rearrangements<!><!>Newman–Kwart and Related Rearrangements<!>Sulfur Nucleophiles<!><!>Sulfur Nucleophiles<!><!>Sulfur Nucleophiles<!><!>Sulfur Nucleophiles<!><!>Hypervalent Iodine Substrates<!><!>Hypervalent Iodine Substrates<!><!>Hypervalent Iodine Substrates<!><!>Hypervalent Iodine Substrates<!><!>Reactions of Arenediazonium Salts<!><!>Reactions of Arenediazonium Salts<!>Reactions of Metal Nucleophiles with Fluorinated Arenes<!><!>Reactions of Metal Nucleophiles with Fluorinated Arenes<!>An Updated Perspective Emerges on the Prevalence of cSNAr Reactions.<!><!>An Updated Perspective Emerges on the Prevalence of cSNAr Reactions.<!>Summary and Outlook<!>Conflict of interest<!>Biographical Information

<p>S. Rohrbach, A. J. Smith, J. H. Pang, D. L. Poole, T. Tuttle, S. Chiba, J. A. Murphy, Angew. Chem. Int. Ed. 2019, 58, 16368.</p><p>Dedicated to Professor Koichi Narasaka on the occasion of his 75th birthday</p><!><p>Substitution reactions on aromatic rings are central to organic chemistry. Besides the commonly encountered electrophilic aromatic substitution,1 other mechanisms include SNAr nucleophilic aromatic substitutions2, 3 and the distinct but related SNArH and vicarious nucleophilic substitutions,4 substitutions brought about through benzyne intermediates,5, 6 radical mechanisms including electron transfer‐based SRN1 reactions7 and base‐promoted homolytic aromatic substitution (BHAS) couplings,8 sigmatropic rearrangements,9 substitutions arising from deprotonation of arenes (directed metalations),10 the vast array of organometallic mechanisms11, 12 and SN1 reactions.13 All of these areas of chemistry are too vast to reference comprehensively, and so are simply represented here by one or two key reviews or recent references. Among these various reaction types, SNAr reactions have attracted a lot of recent attention, because of a recognition that many such reactions may proceed by concerted (cSNAr),14, 15 rather than classical two‐step mechanisms.</p><!><p>Nucleophilic aromatic substitutions have been studied at least since the 1870s.16, 17, 18 The long‐accepted mechanism,4, 5 exemplified in Scheme 1 for dinitroarene 1, involved a two‐stage process that featured a Meisenheimer intermediate 2. In these substitutions, the arene is significantly activated for substitution by the presence of one or more electron‐withdrawing substituents in the positions that are ortho or para to the site of substitution to provide resonance stabilisation, and with nitro as a favoured substituent. In Terrier's excellent book on SNAr reactions in 2013,3 he wrote that "concerted reactions are the exception rather than the rule" and "there is little doubt that most of the activated SNAr substitutions must proceed through the early‐recognised addition‐elimination mechanism".</p><!><p>Classical two‐step mechanism for SNAr reactions.</p><!><p>Evidence in favour of a two‐stage substitution was cited when intermediates were isolated. Thus, as reviewed by Bunnett and Zahler2 in 1951, a number of reactions gave rise to isolated intermediate adducts (Scheme 2). Key studies were performed by Meisenheimer,19 who isolated a common intermediate 5 from reaction of methyl ether 4 with NaOEt, and from reaction of NaOMe with the ethyl ether 6. This intermediate was then decomposed into a mixture of the parent ethers on acidification. Adduct intermediates of this sort, for example, 7–11, which are routinely called Meisenheimer intermediates, are widespread in organic chemistry, and are well reviewed.20</p><!><p>Some known Meisenheimer intermediates.</p><!><p>Nucleophilic aromatic substitutions are often carried out on pyridines, pyrimidines and related heterocycles, and indeed these substitutions are commonplace and important in medicinal chemistry and agrochemistry. Although intermediates from these substitutions have not been isolated where good leaving groups are present, we are familiar with isolation of intermediates where poor leaving groups are in play. Examples of intermediates at the extreme of this scale that can be isolated are the salts resulting from addition of organolithium compounds to pyridines, such as 12, that is, compound 13 which, on heating, gives the substituted pyridine 14 with elimination of LiH (Scheme 3).21, 22, 23 Generation and isolation of such intermediates will be affected by the power of the ring substituents in stabilising negative charge, as well as by the pK a values of the conjugate acids of the incoming and departing groups.</p><!><p>Organolithium additions to pyridine, and re‐aromatisation.</p><!><p>Supportive evidence in favour of the nucleophilic nature of the substitution mechanisms arises from Hammett studies, where significant positive ρ values are associated with the rate‐determining step. It must be remembered, when comparing ρ values, that they vary with the temperature of the experiments.</p><p>Examples reported by Miller24, 25 (Scheme 4) indicate that there is extensive negative charge build‐up in the rate‐determining step. Although the cases below in Scheme 4 have particularly high ρ values, it is recognised that many SNAr reactions have values between +3 and +5. Looking at the substrates chosen by Miller is revealing. His series of substrates 17 a–d consisted of four examples, where R=NO2, Ac, CF3 and Cl. Whereas NO2 and Ac are substituents that can delocalise a negative charge by resonance, clearly CF3 and Cl cannot, although they can contribute inductive stabilisation to different extents. Miller's Hammett analysis showed25 that the four substrates had an excellent correlation with σ* for these substituents,26 suggesting a common mechanism for them.</p><!><p>Miller's studies of Hammett correlations.</p><!><p>Although the literature adopted two‐stage SNAr reactions as the norm, despite the reactivity of substrates like 17 d studied by Miller, noted above, further anomalies began to appear. These studies have culminated in the recent paper by Jacobsen et al.,14 which transforms our perception of the prevalence of cSNAr reactions. This will be discussed later in Section 13 of this review. Papers referenced below are cited for their relevance to cSNAr reactions.</p><p>A very early example was the work of Pierre et al.27 who, in just a single paper that was published in 1980, studied the reaction of KH with aryl halides. This report simply involved hydrodehalogenation of substrates 19 in tetrahydrofuran (THF) as solvent (Scheme 5). The reactions were not pursued with detailed mechanistic investigations, but the observations made were illuminating.</p><!><p>Proposal for concerted SNAr reactions by Pierre et al.27</p><!><p>By conducting the experiments with KH in [D8]THF, Pierre et al. were able to show that the substituting hydrogen indeed came from KH. They were able to dismiss any idea of a benzyne mechanism, since no H2 was evolved. The order of reactivity was: ArI > ArBr > ArCl > ArF, which is the reverse of the order often found in classical SNAr reactions. Since the reactions proceeded in the absence of activating substituents like nitro groups on the ring undergoing substitution, they proposed a concerted reaction mechanism with a four‐centred transition state but, at that time, no computational methods were available to support these ideas. Perhaps because this reaction seemed so anomalous, but most likely because it was both a single paper in this area by the authors and also was not written in English, the paper received very little attention. Nevertheless, it heralded a lot of subsequent developments. We will return to this example later in this review.</p><p>An early study pointing to concerted nucleophilic substitution was conducted by Fry and Pienta who, in 1985,28 provided mechanistic evidence through Hammett correlations. When studying rate constants for nucleophilic aromatic substitution of arenesulfonate groups in 22 by halide anions in dodecyltributylphosphonium salts 23 (Scheme 6), using a range of different R1 substituents, Hammett plots gave reasonable fits to straight lines, with ρ values of +1.5 and +1.1 for σ and σ− respectively (Scheme 6 A). The effect of the R1‐group on the rate of the reactions was therefore substantially lower than for many literature SNAr reactions. Indeed, the substrates that were trialled included 22 d (R1=OMe), which can clearly not provide credible stabilisation for a developing negative charge on the ring in a Meisenheimer intermediate. Importantly, the reaction series also showed some sensitivity of the transition state to the R2‐substituent on the leaving group (Scheme 6 B, ρ=+0.22). The similarity of rates regardless of the halide identity (Scheme 6 C) ruled out an SRN1 mechanism, as the differences in halide redox properties would require a much more substantial rate difference between the different halides. However, in their conclusion, the authors postponed speculation on the precise mechanism of their reactions.</p><!><p>Some SNAr reactions provided Hammett correlations with low ρ‐values.</p><!><p>On the other hand, Williams et al. reported a number of nucleophilic aromatic substitution reactions with concerted mechanisms on substituted 1,3,5‐triazines 26–29.29, 30, 31, 32 They found that the reaction of various phenolate ions with 26 (Scheme 7),29, 30 followed a linear relationship on a Brønsted plot over a range of pK ArOH values above and below that of the conjugate acid of the leaving group (4‐nitrophenol). The lack of curvature in the free energy relationship suggested that there was no change in mechanism when moving from strongly electron‐withdrawing groups to weakly electron‐donating groups, which is consistent with a concerted mechanism.</p><!><p>Substitutions of aryloxy‐substituted triazines.</p><!><p>The same 1,3,5‐triazine core, with aryloxy and pyridine leaving groups, was also studied in aminolysis reactions with various amines.29, 30 Hammett plots for the reaction of morpholine (ρ=+1.65) and N,N‐dimethylaminopyridine (ρ=+0.82) were recorded. Detailed arguments allowed the authors to conclude that a concerted substitution was occurring. These rigorous papers were important in raising awareness of concerted nucleophilic aromatic displacements.</p><!><p>Related to these studies, computational methods were employed33 to examine the hydrolysis of protonated chlorotriazines for example, 46, (Scheme 8) which are of interest in agrochemistry. In both gas phase and in water, Meisenheimer intermediates could not be located, suggesting that these reactions instead proceed in a concerted manner, albeit with high kinetic barriers, at least when a neutral water molecule was the nucleophile.</p><!><p>Hydrolysis of protonated triazines.</p><!><p>In fact, computational studies played a significant part in providing credibility for the concerted nature of cSNAr reactions over the past 30 years. In all the computational studies cited here, the geometries were optimised with density functional theory (DFT) methods unless otherwise stated. We now cluster some of the computational results that suggested the cSNAr mechanism, although further cases will also be referenced at appropriate places later in this review.</p><p>Nucleophilic aromatic halogen identity‐substitution reactions were investigated computationally in the gas phase (Scheme 9) by Glukhovtsev et al.34 The exchange reactions of 49 with the corresponding halide anion X− (for Cl, Br, I) all proceed via a Meisenheimer‐like transition state structure 50. No intermediate was found. However, the authors observed Meisenheimer intermediates for the fluoride addition to fluorobenzene and for the chloride addition to 2,4‐dinitrochlorobenzene and picryl chloride (2,4,6‐trinitrochlorobenzene). This study was expanded by Uggerud et al.35 with second‐row (NH2 −, OH−, F−), third‐row (PH2 −, SH− Cl−) and fourth‐row (AsH2 −, SeH−, Br−) nucleophiles. Additionally, a more diverse array of substituents, R, was considered. A Meisenheimer intermediate was observed for all three second‐row nucleophiles with substituents as different as ‐NH2 and ‐NO2 (for both NH2 − and F− as the nucleophile) and for substituents ‐H and ‐NO2 with OH− as the nucleophile. For the third‐ and fourth‐row nucleophiles, concerted mechanisms were calculated in several instances. In general, a concerted mechanism was predicted for more electron‐rich aromatic systems. A stepwise mechanism with a Meisenheimer intermediate would become more favourable as electron‐withdrawing groups are attached to the aromatic ring.</p><!><p>Computational investigations of identity substitutions.</p><!><p>Building on the halogen‐exchange reactions mentioned above, fluorodechlorination reactions and fluorodenitration reactions of aryl chlorides and nitroaryls in dimethyl sulfoxide (DMSO), were reported by Sun and DiMagno.36 Computational studies were performed for the fluorodenitration reactions. para‐Substituted nitroaryls were analysed and grouped according to the Hammett parameter of the substituents. It was observed that for substituents with a Hammett constant σ− ≤0 (H and more electron‐donating substituents), the reaction proceeds via a concerted mechanism with a Meisenheimer‐like transition state.</p><p>Nucleophilic displacement of nitro groups, in 5,7‐dinitroquinazoline‐4‐one 51, by methylamine as nucleophile, was reported by Goel et al.37 Their computational study built upon a previous experimental study38 that had shown that the nitro group in the peri‐position to the carbonyl was regioselectively displaced over the nitro group in the para‐position, affording 52 in 85 % yield (Scheme 10). In that experimental paper, the authors had proposed the reaction to occur via a σ‐intermediate, but evidence for this complex was not presented. Goel et al. studied the formation of the σ‐complex, but no stable complex could be found by DFT calculations.37 The activation energy for a concerted nitro group substitution was found to be 33.8 and 18.1 kcal mol−1 for para‐ and peri‐substitution via transition states 53 and 54, respectively. The reason for the regioselectivity is given by the hydrogen‐bonding stabilisation between the amine and the carbonyl in the transition state for peri‐substitution, which is strong enough to divert the methylamine away from the less sterically hindered para‐position.</p><!><p>Concerted substitutions in 5,7‐dinitroquinazolin‐4‐one 51.</p><!><p>The effect of the medium on substitution reactions has also been investigated widely for SNAr reactions. The displacement reaction (Scheme 11) of the nitro group from nitrobenzene 55 with fluoride in the gas phase has been studied experimentally and computationally in the gas‐phase by Riveros et al.39 The DFT model predicts that the reaction follows a concerted pathway with a very low activation energy.</p><!><p>Low activation energy predicted in gas‐phase substitution.</p><!><p>The effect of explicit solvation and counter‐cations on the displacement of a nitro group in nitrobenzene by a fluoride anion has been reported by Park and Lee40 through a computational approach. Including explicit solvation (two molecules of water) and different counter‐cations led to the same concerted mechanism as predicted by Riveros et al.39 for the gas phase, as discussed above.</p><p>The regiochemistry of displacement of halide leaving groups from poly‐halogenated substrates has been widely studied by computational methods. In 1999, Tanaka et al. reported their studies41 on the regiochemistry of substitution of pentafluoronitrobenzene with ammonia as nucleophile, as the solvent changed from hexane to nitromethane. These studies predicted (and provided a mechanistic proposal to explain) concerted substitution in the para‐position, but two‐step substitution in the ortho‐position.</p><p>In subsequent years, computation‐based studies on regioselectivity have been widely undertaken. Perfluoroarenes, and perhaloarenes more generally, have been the subject of a number of studies of selective substitution reactions, representing their importance in materials chemistry and in ligand generation as well as in detoxification programmes. Experimental and computational approaches have been combined by Paleta et al. in their study of pentafluorobiphenyl.42 With a range of N‐, O‐ and S‐nucleophiles, the regioselectivity of substitution of 2,3,4,5,6‐pentafluorobiphenyl was explored and showed significant regioselectivity for substitution of the fluorine that was para‐ to the phenyl group. The computational studies which used the nucleophiles i) ammonia, ii) solvated lithium fluoride [as LiF.(Me2O)2] and iii) solvated lithium hydroxide [as LiOH.(Me2O)2], mirrored the experimentally observed regioselectivity but showed that, in all cases, a concerted one‐step displacement reaction was occurring.</p><p>In a combined computational and experimental study, the substitution reactions of pentafluoropyridine by phenolates evidenced predominant displacement of the 4‐substituent on the pyridine.43, 44 For the resulting phenoxypyridines, extensive experimental analysis led the authors to understand that the displacement of 4‐pentafluorophenoxide (as opposed to other leaving groups) by fluoride anion from 58 (Scheme 12) was anomalous, and semi‐empirical computational studies (PM3) supported a concerted mechanism.</p><!><p>Concerted mechanism proposed in displacements of 4‐pentafluorophenoxides.</p><!><p>Following an earlier model for determining the site of substitution in aromatic perfluorocarbons,45 predictions of the regioselectivity of SNAr reactions were made by Brinck and an AstraZeneca team including Svensson, Liljenberg et al.46, 47 based on the relative stability of Meisenheimer intermediates. As such, their model addressed the classical two‐stage mechanism. However, their computational studies could not locate these intermediates in cases where the leaving group was chloride or bromide (such as in 61, Scheme 13), suggesting concerted reaction mechanisms in those cases.</p><!><p>No Meisenheimer intermediates were found in computational studies on displacements on pentachloropyridine.</p><!><p>A descriptor‐based model to predict relative reactivity and regioselectivity in SNAr reactions was introduced by Stenlid and Brinck.48 In contrast to the selectivity models presented above, this descriptor solely relies on the ground‐state electronic structure of the aromatic substrate. Consequently, it can also be applied to SNAr reactions that do not proceed by a stepwise mechanism via a Meisenheimer intermediate, such as the reaction between 64 and piperidine (65) (Scheme 14). The series spanned examples from R=NH2 to R=NO2. The rate constants for all these examples had been reported previously. A satisfactory correlation between these constants and the newly introduced descriptor was found. The observation48 that, according to the computational model, reactions of 68 with secondary amines do proceed via a concerted SNAr reaction was related to an extensive experimental study of 1‐X‐2,4‐dinitrobenzene with a series of secondary amines.49</p><!><p>Concerted substitution reactions studied by Stenlid and Brinck.48</p><!><p>Pliego and Piló‐Veloso50 investigated the effect of ion‐pairing, explicit hydration and solvent polarity on the fluorodechlorination reaction of 4‐chlorobenzonitrile (70) (Scheme 15). This computational model predicts the reaction to follow a concerted mechanism. By varying the solvent polarity, it was found that for a given fluoride salt MF, there is a solvent with ideal polarity which just allows for the dissociation of the ion pair but does not solvate the fluoride ion too strongly.</p><!><p>Studies on the effect of ion‐pairing, explicit hydration and solvent polarity on the fluorodechlorination reaction.</p><!><p>In a more recent contribution, Silva and Pliego investigated51 SNAr reactions on bromobenzene and (ortho‐, meta‐, or para‐) methoxybromobenzenes with different nucleophiles in the gas phase and in solution phase by computational methods (Scheme 16). A concerted mechanism was observed with hydroxide, cyanide, and methoxide nucleophiles attacking bromobenzene in the gas phase (albeit the transition state energy for the reaction with cyanide was high (ΔG*=27.2 kcal mol−1). Including solvent effects in their computations made all three reactions kinetically less favourable (e.g. hydroxide in DMSO: ΔG*=29.3 kcal mol−1; in MeOH: ΔG*=37.8 kcal mol−1. These barriers are markedly higher than in the gas phase ΔG*=1.6 kcal mol−1).</p><!><p>Substitution reactions of bromomethoxybenzenes.</p><!><p>No change in mechanism is mentioned when going from gas‐phase to solution‐phase models. Interestingly, when the authors investigated meta‐methoxybromobenzene with hydroxide, methoxide and cyanide as the nucleophile, they obtained lower activation barriers (e.g. ΔG*=25.8 kcal mol−1 for m‐methoxybromobenzene with methoxide in DMSO vs. ΔG*=27.1 kcal mol−1 for bromobenzene with the same nucleophile in the same solvent).</p><p>The effects of solvation on SNAr reactions in liquid ammonia and in the gas phase by a combination of metadynamics and committor analysis methods have been studied by Moors et al.52 They found that for 4‐nitrofluorobenzene (74), the reaction proceeded via a concerted mechanism in the gas phase via transition state 77, but via an intermediate Meisenheimer complex 83 in solution (Schemes 17 and 18). For 4‐nitrochlorobenzene (75), the reaction proceeded via a concerted mechanism via transition state 78 in both the gas phase and in solution, and for 2,4‐dinitrofluorobenzene (76), the reaction occurs via a Meisenheimer intermediate 79 in both solution and the gas phase.</p><!><p>Gas‐phase reactivity with ammonia as nucleophile.</p><p>Reactivity in solution with ammonia as nucleophile.</p><!><p>Fluoride played a major role in the important studies53, 54, 55, 56 by Ritter et al. who had already reported53 the deoxyfluorination reaction of phenols 84 with PhenoFluor 85 (Scheme 19).54 Intermediate 87 (Ar=Ph) was independently synthesised and treated under the reaction conditions, and afforded the corresponding aryl fluoride 89.</p><!><p>Ritter's studies53, 54, 55, 56 on the deoxyfluorination of phenols.</p><!><p>When DFT studies were carried out, a single transition state, 88, was observed, which is characteristic of a concerted mechanism. A large primary 16O/18O kinetic isotope effect (KIE) (KIE=1.08±0.02) was observed, showing that the cleavage of the C−O bond is involved in the rate‐determining step. A Hammett plot also shows that there is no change in mechanism when moving from electron‐deficient phenols to electron‐rich phenols (ρ=+1.8) indicating that there is not a build‐up of full negative charge in the ring at the transition state.56 The formation of the urea by‐product is also highly exergonic, which contributes to the driving force for this reaction. These reactions feature spiro transition states, further examples of which will appear later in this review (Sections 7 and 8).</p><p>On a related theme, Sanford et al. reported a mild deoxyfluorination of phenols 97 via aryl fluorosulfonate intermediates 98.57 This transformation was found to be compatible with ortho‐, meta‐, or para‐electron‐withdrawing groups, and could also be applied to electron‐neutral and moderately electron‐rich substrates to provide fluorinated products 100–111 (Scheme 20).</p><!><p>Examples of deoxyfluorination by Sanford et al.</p><!><p>Computational data suggest that the binding of fluoride in 112 to sulfur to form pentacoordinate sulfonate 113 is enthalpically favourable and the activation enthalpy to the transition state (ΔH*) was found to be feasible at room temperature (Table 1). The transition state 114 was shown to involve concerted formation of the C−F bond and cleavage of the C−O bond without the formation of a Meisenheimer intermediate (Figure 1).</p><!><p>Energy profile for conversion of 112 to fluoroarene 115.</p><p>Enthalpy changes associated with Figure 1</p><p>Entry</p><p>R (structure 112)</p><p>ΔH bind</p><p>[kcal mol−1]</p><p>ΔH*</p><p>[kcal mol−1]</p><p>1</p><p>CN</p><p>−4.1</p><p>13.2</p><p>2</p><p>CF3</p><p>−3.5</p><p>15.6</p><p>3</p><p>H</p><p>−1.7</p><p>20.8</p><p>4</p><p>Me</p><p>−1.3</p><p>22.2</p><p>5</p><p>OMe</p><p>−1.1</p><p>24.0</p><!><p>Chiba et al. have recently reported remarkable reactions of a sodium hydride–lithium iodide composite. One of the reaction types reported by that team promoted the nucleophilic amination of methoxyarenes 117, via intra‐ (Scheme 21) and intermolecular (Scheme 22) reactions.58 This methodology was compatible with electron‐donating and electron‐withdrawing substituents on the methoxyarene.</p><!><p>Intramolecular nucleophilic amination of methoxyarenes.</p><p>Intermolecular nucleophilic amination of methoxyarenes.</p><!><p>A Hammett plot with p‐substituted methoxyarenes 129 showed that the ρ‐value was low (ρ=+1.99). The proposal of a concerted mechanism was backed up by computational analysis, where a single transition state was observed for the conversion of 140→141 with formation of a partial negative charge, consistent with a cSNAr process (ΔG*=14.7 kcal mol−1, Figure 2). Chiba's demethoxylation studies feature deprotonated amines as nucleophiles. Demethoxylation by a hydroxycyclopentadienyl iridium hydride nucleophile has been proposed as a concerted nucleophilic aromatic substitution by Kusumoto and Nozaki,59 although no mechanistic evidence has yet been revealed to support this.</p><!><p>Free energy profile for the cyclisation of amide salt 142.</p><!><p>Chiba and co‐workers extended their chemistry with NaH and additive salts to perform further intermolecular displacements.60 For example, substitution of the methoxy group in 3‐methoxypyridine (145), (Scheme 23) by piperidine 146 was achieved in high yield using sodium hydride with LiI as additive. With NaI as alternative additive, the reaction proceeded in much poorer yields and with NaH alone, no reaction was seen.</p><!><p>Sequential substitutions of methoxyarenes by amines.</p><!><p>This reaction is quite flexible. In the dimethoxy case 148, substitution at the 2‐position occurs first to give 149, but the product can undergo a second substitution by a different amine to give 150. In 3,5‐dimethoxy case 151, iterative diamination can again be achieved. In these cases, no Hammett correlations have been published, but the displacement from the unactivated 3‐position of a pyridine identifies these reactions as prime candidates for cSNAr pathways.</p><!><p>Recent discoveries relating to concerted aromatic substitutions have seen several that feature hydride as nucleophile or base. Chiba et al. recently reported the hydrodehalogenation of haloarenes 154, (Scheme 24) by their sodium hydride–iodide composite.61 Without the addition of the iodide salt, sodium hydride cannot carry out this special function.</p><!><p>Hydrodehalogenations effected by sodium hydride‐lithium iodide complex.</p><!><p>Various aryl bromides were reduced under these conditions, with both electron‐rich and electron‐deficient substituents being equally tolerated. Computational studies show a highly exothermic reaction with a single transition state 167 for concerted nucleophilic aromatic substitution, with an energy barrier of 20.9 kcal mol−1 (Figure 3). The Hammett plot using NaH with NaI, converting iodoarenes to arenes in THF at 85 °C shows a linear correlation with ρ=+0.47, which is supportive of a cSNAr process.</p><!><p>Free Energy profile for reaction of bromobenzene with solvated monomeric sodium hydride.</p><!><p>As the cSNAr pathway is initiated by an interaction between the hydride donor and the π* orbital of the aromatic ring, it was reasoned that this methodology could also be applicable to the reduction of haloalkenes, upon treatment with the sodium hydride‐iodide composite.</p><p>This was indeed the case, with retention of configuration being observed as the major product for both (Z)‐ and (E)‐ alkenes 169 and 173 (Scheme 25).61, 62</p><!><p>Stereo‐retention prevails in hydrodehalogenations of vinyl bromides.</p><!><p>Murphy, Tuttle et al. recently reported on the solvent‐dependent role of potassium hydride in haloarene reduction.63 Pierre et al. had proposed a cSNAr mechanism for dehalogenation of haloarenes in 1980, as mentioned earlier.27 They had verified that the hydrogen atom delivered to the aryl halide had come from KH. They had ruled out a benzyne intermediate in their reactions, and presented their proposal based on the observed order of reactivity (ArI > ArBr > ArCl) which was in contrast to the normal order of reactivity in a standard SNAr reaction on iodobenzene 174 (Scheme 26). Pierre's proposal was therefore revolutionary, being made before computational methods became widely available. Murphy and Tuttle's investigation confirmed Pierre's proposed mechanism computationally, with a Gibbs free energy barrier of 22.4 kcal mol−1. Studies carried out in [D8]THF also reveal that the H‐atom in 178 comes from the KH rather than from the solvent, in line with Pierre's claim. Surprisingly, in benzene as solvent, Murphy and Tuttle showed that a quite different electron transfer mechanism played an important role in reduction of haloarenes with KH.</p><!><p>Support for four‐centred transition state in the Pierre reaction.</p><!><p>Computational studies on hydrodehalogenation of haloarenes by cSNAr have been reported by Cramer et al.64, 65 In all cases, the transition state for the addition of hydride to a substituted site led to concerted displacement of the halide anion via transition state 180 (Scheme 27).</p><!><p>Concerted displacements of halides from haloarenes by naked hydride ions are predicted from computation.</p><!><p>Ogoshi et al. recently reported66 a catalytic and regioselective hydrodefluorination of polyfluoroarenes (Scheme 28) and polyfluoroalkenes using silanes (Ph3SiH, MePh2SiH, Me2PhSiH or Et2SiH2) and catalytic tetrabutylammonium difluorotriphenylsilicate (TBAT 194, Scheme 29).</p><!><p>Products of hydrodefluorination from the study of Ogoshi et al. H indicates an H atom that has displaced F; only the major product isomer is shown in each case.</p><p>Proposed cycles for hydrodefluorination by TBAT (194) and a silane.</p><!><p>This hydrodefluorination process tolerated many other functional groups, including esters, nitriles and nitro groups. Two mechanistic cycles were proposed by the group, based on DFT studies, both of which proceed through a cSNAr displacement step.</p><p>The first cycle (Scheme 29 A) involves generation of 195 from TBAT (194) and the hydrosilane. This can then coordinate to the polyfluoroarene 193 via π–π stacking, affording 196. The cSNAr step can then occur with hydride from the silicate displacing a fluoride in the transition state 197. The eliminated fluoride can then either be trapped intramolecularly by a fluorosilane or intermolecularly by a hydrosilane to regenerate 194 or 195, respectively. The alternative mechanistic cycle involves dihydrosilicate 200 as an intermediate, which can be formed by disproportionation of 195 (Scheme 29 B). Hydride transfer within complex 202, displaces a fluoride ion from the polyfluoroarene. The displaced fluoride can then be trapped by the hydrosilane to regenerate 195. Computational data for the mechanisms in Scheme 29 show that the Gibbs free energy barriers for the key substitution steps are 19.0 kcal mol−1 and 10.8 kcal mol−1, respectively. Meisenheimer intermediates were not detected for either pathway, indicative of cSNAr reactivity.</p><!><p>The range of nucleophiles was further widened when Würthwein et al. reported the reaction of di‐ and trifluorobenzenes 204–206 with Me2EM (E=P, N; M=SiMe3, SnMe3 Li) (Scheme 30).67, 68 To illustrate the utility of the transformation, phosphane products were later used as ligands in polyfluorophosphane palladium dichloride complexes. Computational chemistry was again shown to be a useful tool in predicting the mechanism of this reaction, indicating a single transition state with no Meisenheimer adduct formation, that is, a cSNAr mechanism.</p><!><p>Phosphinodefluorination of aryl fluorides.</p><!><p>The single transition state corresponds to the simultaneous C−E bond formation, and in the case of Me2PSiMe3 with fluorobenzene, provided a barrier of ΔE*=30.3 kcal mol−1 for the formation of the C−P bond and the Si‐assisted loss of fluoride (Figure 4). Di‐ and trifluorobenzenes were also examined experimentally and by computation, and provided faster reactions and lower calculated barriers. Compounds 210 and 211 form a van der Waals complex (vWc) 212, followed by a cSNAr reaction through 213, forming van der Waals complex 214. Dissociation of this complex affords products 215 and 216 (Figure 4).</p><!><p>Energy profile of phosphinodefluorination reaction.</p><!><p>Aryl fluorides have recently been subjected to quite a different cSNAr reaction by Würthwein, Studer et al., using silyllithium reagents as nucleophiles.69 This was an interesting development, as previous reactions of aryl halides (notably iodides) with similar reagents had led to substitution directly on the halogen atom to give a silyl halide and an aryllithium as a reactive intermediate that then conducted an SN2 reaction on the silyl halide. In this case, however, aryl fluorides underwent cSNAr. Hammett studies gave a ρ value of +3.2, and computational investigation afforded Gibbs free energies of activation of 19–21 kcal mol−1 (Scheme 31). Very recently, two related studies have appeared from other groups.70, 71</p><!><p>Products arising from silyldefluorination reactions.</p><!><p>A flexible route to phenanthridinium cations 224 was published by Hartley et al. using an imine nucleophile to displace a halide (Scheme 32).72 The imine 223 which is formed in situ is not isolated, but directly converted to product 224 by heating. The mechanism of the ring‐forming SNAr reaction from 223 to 224 was investigated computationally with model compounds. The models were chosen to closely represent the synthesised molecules. It was found that for all examined model compounds, the reaction proceeds via a concerted SNAr pathway. In particular, a concerted mechanism was not only observed for examples with electron‐donating substituents (e.g. p‐MeO) but also for examples with electron‐withdrawing substituents (e.g. p‐NO2). All transition states were accessible (ΔG*=between 17 kcal mol−1 and 28 kcal mol−1) with lower energy barriers for examples with more electron‐withdrawing substituents, as expected.</p><!><p>Hartley's cSNAr route to phenanthridinium salts.</p><!><p>Carbon nucleophiles were used by Médebielle, Rossi et al. in the synthesis of tetracyclic indoles, for example, 227 (Scheme 33 A) and azaindoles.73 Computational studies were used to probe the mechanism of the reaction. Electron transfer was considered, but gave very high energy barriers. The most reasonable proposal was that the reactions proceeded by nucleophilic aromatic substitution. The calculated reaction profile showed no intermediates, that is, it was a cSNAr reaction.</p><!><p>Carbon nucleophiles in cSNAr displacements (DCM: dichloromethane; AcOH: acetic acid).</p><!><p>An unusual nucleophile 228 was employed by Tretyakov et al.74 (Scheme 33 B) to afford a new nitronyl nitroxide 229. DFT calculations supported the observed regioselectivity and indicated that the reaction follows a concerted pathway. The zwitterionic product was treated with sodium nitrite in acetic acid to yield the nitroxyl 230.</p><!><p>Spiro transition states appeared in the fluorodeoxygenation section of this review, but spiro species occur much more widely, as seen in this and the following section of this review. Tell‐tale signs of concerted nucleophilic substitutions arise when the arene at which substitution is occurring has no substituents to significantly stabilise a Meisenheimer intermediate. This is the case in Clayden's stereocontrolled arylation of amino acids.75, 76 Here, infrared spectroscopy was used to follow the conversion of alanine derivatives 231–235, via their enolates 236 into products 238 as shown in Scheme 34. Conformational control of anilides plays an important role here. In tertiary anilides (e.g. 231), the aryl group is aligned strictly anti to the carbonyl group.77</p><!><p>A) Stereocontrol in Clayden's aryl transfer reactions; B) earlier aryl reactions showing nucleophilic substitution at the meta‐ position of a pyridine; C) vinyl transfer reactions.</p><!><p>Formation of enolate 236 ensues, and transfer of the aryl group then occurs with control of stereochemistry to afford the anion 237, from which the amino acid product 238 was isolated. Plainly, without appropriate stabilising substituents on the arene, no Meisenheimer intermediate can be detected or envisaged, and yet the transformation occurs smoothly in high yield. A Hammett plot for substrates 231–234 revealed ρ=+4.5 in this case against σ−, indicating significant charge build‐up on the arene and showing that the arene‐transfer step is the rate‐determining step. The leaving groups in these cases are amide anions. The fact that they are not good leaving groups, is consistent with the need for significant charge build‐up on the ring in the transition state, before the departure of the leaving group is triggered.</p><p>On the other hand, for substituents on the arene where σ− > +0.2, the arene transfer is facilitated and is apparently no longer rate‐determining; in those cases, the enolate formation step takes over this role.</p><p>Prior to this most recent work, Clayden et al. had studied extensive alternative applications of these aryl transfer reactions, notably in ring‐expansion reactions,78, 79, 80, 81, 82, 83 for example, with substrates 240, 241.78 Although Hammett plots are not reported for these series, the analogy to the amino acid cases just discussed make it highly likely that they follow cSNAr pathways through transition states 244. In the presence of dimethylpropyleneurea (DMPU) and lithium diisopropylamide (LDA), ring‐expanding isomerisation was effected with excellent retention of stereochemistry. Thus, the initially generated benzyllithium is configurationally stable for the period needed to carry out the rearrangement.</p><p>Scheme 34 B and 34 C show additional applications of transfer reactions. The attack of the benzyllithium nucleophile 246 on the meta‐position of the pyridine ring is again a strong indicator of cSNAr reactivity. The stereoselectivity of the reaction is again noteworthy. Both this example and the vinyl transfer reaction with substrate 248 in Scheme 34 C bear resemblance to the results of Chiba et al. in Sections 4 and 5 of this review.</p><p>The substitution steps in Clayden's work are examples of Truce‐Smiles rearrangements,84 intramolecular substitution reactions that go through a spiro transition state or intermediate, with carbon nucleophiles and nitrogen leaving groups. A quite different example of cSNAr chemistry was reported by Coquerel85 in 2013 that also involved a Smiles‐type rearrangement, this time with an oxygen nucleophile and a nitrogen leaving group. In a study of the reactions of benzyne with pyridine, they isolated an unusual product of rearrangement, 253. This was rationalised through the pathway shown (Scheme 35) where generation of the zwitterion 257 leads to internal deprotonation to give pyridine carbene 258. This nucleophilic carbene then reacted with the reactive ketone carbonyl group of N‐protected isatin 259, and the resulting alkoxide then secured a phenyl transfer reaction to liberate a neutral pyridine nitrogen in 253.85 Computational studies revealed that the conversion of 260 to 253 was occurring by a concerted process. In the transition state 261, the carbon atom undergoing substitution adopts sp3‐like geometry as characterised by the computed bond angles and bond lengths. The phenyl group bearing the pyridinium substituent in 261 did not feature any activating substituent [other than the leaving group] and the activation barrier (ΔE*) was very accessible at 10.9 kcal mol−1.</p><!><p>Unexpected product 253, together with a proposal for its mechanism of formation.</p><!><p>The Julia‐Kocieński reaction86 (Scheme 36) also involves a Smiles‐type rearrangement step and has been studied in detail with computational methods. The effect of coordinating counter cations and different solvents on the Z/E selectivity of the product alkenes is rationalised. It was found that the rearrangement step through spiro species 265 (Scheme 36) follows a concerted mechanism in all examined cases (different solvents and counter‐ions). The authors note that at no point during this rearrangement is a significant amount of negative charge transferred onto the tetrazole ring. Instead the negative charge is directly transferred from the attacking alkoxide nucleophile to the sulfur atom of the leaving group. The transition state is asynchronous and early. The new carbon‐oxygen bond is formed to a significant extent while the carbon‐sulfur bond still is mainly intact.</p><!><p>The Julia–Kocieński reaction features concerted displacement at the tetrazole ring.</p><!><p>As mentioned above, the Smiles rearrangement is an intramolecular substitution reaction featuring a spiro species on the reaction path. Concerted pathways had been considered for other examples of the Smiles rearrangement early on.87 In contrast to the cases just cited, computational studies showed that several examples of the reaction proceed by a stepwise mechanism via a Meisenheimer intermediate.</p><p>The Smiles rearrangement of 269 was investigated computationally with a range of different functionals (Scheme 37).88 It was found that, depending on the functional, structure 270 can either be optimised as a local minimum or as a transition state. Benchmark models at Møller–Plesset MP2/6‐31+G(d,p) and MP4(SDQ)/6‐31+G(d,p) level of theory showed that 270 is an intermediate. In general, functionals with <10 % Hartree–Fock (HF) exchange were unable to correctly identify 270 as a local minimum and predicted a concerted mechanism instead. Notably, the popular B3LYP functional was found to fail to predict the correct stepwise mechanism despite having 20 % HF exchange. M06, M06‐2X and ωB97X were found to give results satisfactorily close to the Møller–Plesset results, that is, they all predicted a stepwise mechanism with reasonably accurate barrier heights.</p><!><p>A stepwise mechanism was predicted for this Smiles rearrangement from computational studies.</p><!><p>Further examples 272,89 275,90 278 91 of Smiles rearrangements were explored (Scheme 38) through computational methods89, 90, 91, 92 and each of these gave clear intermediates, rather than concerted ipso‐substitution reactions. So, overall, the studies on Smiles rearrangements indicate that there is a delicate balance between concerted and stepwise substitution reactions.</p><!><p>Further examples of Smiles rearrangements where computational research predicts stepwise mechanisms.</p><!><p>Closely related to the above reactions that featured five‐membered ring spiro species, four‐centred transient spiro rings are proposed for a number of other rearrangement reactions, namely the Chapman, Schönberg93 and Newman–Kwart rearrangements. Of these, the Schönberg rearrangement of diarylthionocarbonates 281 to diarylthiolcarbonates 282 (Scheme 39) was studied intensively first.</p><!><p>The Newman–Kwart and related rearrangement reactions.</p><!><p>Tarbell et al.94 proposed a four‐centred transition state to be at the heart of this rearrangement. The reactions are accelerated by electron‐withdrawing substituents in the aryloxy ring. The Newman–Kwart rearrangement, for example, 283→284, was excellently reviewed in 2008 by Lloyd‐Jones et al.95 Relles et al. found96 similarities between the Chapman and Newman–Kwart rearrangements on studying their properties separately through Hammett correlations ρ=+1.62 for the Newman–Kwart rearrangement and +1.63 for the Chapman rearrangement. A similar assessment by Miyazaki97 versus σ− gave ρ=+1.83 for the Newman–Kwart rearrangement and ρ=+1.06 for the Chapman rearrangement. Woodward, Lygo et al.98 (2003) conducted computational studies on the Newman–Kwart rearrangement of two analogous series of atropisomerically pure thionocarbamates, one derived from binol (288) and one from octahydrobinol (289) (Scheme 39). They observed experimentally that the octahydrobinol cases rearranged essentially without racemisation, while the binol case showed significant racemisation. Their computational studies at different levels of theory showed that the barrier for the rearrangement of the octahydrobinol case was notably lower than for the binol case, while the barrier for thermal racemisation of the substrates had the reverse order. Jacobsen and Donahue99 used DFT calculations to back the proposal for a four‐centred transition state.</p><p>More recently, a radical cation version of the Newman–Kwart rearrangement has been discovered100 that proceeds under mild conditions and that has quite a different response to substituents than in the thermal rearrangement. Cramer has reported recent studies that provide further computational characterisation of the thermal Newman–Kwart rearrangement as well as its radical cation counterpart; the radical cation variant is also viewed as being a concerted substitution reaction.101, 102, 103</p><!><p>Sulfur nucleophiles have also featured prominently in the recent literature. Tobisu, Chatani, et al. have just reported104 an unusual outcome to reaction of 2,2′‐bis(methythio)‐1,1′‐biaryls 290 (Scheme 40) with catalytic amounts of methanethiolate salts in dimethylformamide (DMF) as solvent.</p><!><p>cSNAr reactions in the formation of dibenzothiophenes.</p><!><p>Here, the reaction commences with demethylation of the ArS–Me bond to afford an arenethiolate 291, which then attacks the adjacent arene, displacing methanethiolate anion to complete a cycle by forming 292. Computational studies were unable to identify any intermediate in the latter step, which therefore appears to be concerted.</p><p>Hedrick, Alabugin et al. recently reported105 that the synthesis of fluorinated poly(arylthioethers) 295 proceeds via a concerted mechanism (Scheme 41). Through computational studies, it was shown that firstly triazabicyclodecene (TBD), 296, nucleophilically attacks the trimethylsilyl (TMS) group of MeSSiMe3, displacing a methanethiolate anion which hydrogen bonds to the TBD‐TMS cation forming 297. This then forms a complex 298 with hexafluorobenzene (293), before the methanethiolate anion displaces fluoride in a concerted manner in transition state 299, aided by hydrogen bonding between the fluorine and the amine catalyst.</p><!><p>Substitution of perfluorophenylbenzenes by methanthiolate occurring through highly ordered transition states.</p><!><p>Dissociation of Me3SiF occurs from 301, regenerating 296, followed by complexation of another MeSSiMe3 and the monothiolated arene 300, forming 302. A second concerted displacement occurs para to the first displacement, due to stabilisation from the first methanethiolate group acting as a σ‐acceptor (via transition state 303). Dissociation of fluorotrimethylsilane regenerates the catalyst 301 and affords dithiolated product 304.</p><p>Calfumán et al. carried out an experimental and computational study into the reaction of atrazine 305 with various bio‐thiols 307–310, and propose that these reactions occur on the borderline between concerted and stepwise mechanisms (Scheme 42).106 A Brønsted plot shows β=+0.5, which corresponds to a stepwise mechanism via a Meisenheimer intermediate, however, computational analysis of the intrinsic reaction coordinate reveals that no Meisenheimer intermediate can be found. The authors suggest that this may be because the loss of the chloride is extremely fast.</p><!><p>Thio‐dehalogenation of atrazine 305 occurs on the borderline between concerted and stepwise mechanisms.</p><!><p>Investigations107 of the nucleophilic aromatic displacement of chloride from a 4‐chlorobenzoyl CoA model compound 311 (Scheme 43) with the acetate ion suggest that this reaction proceeds via a concerted mechanism. In the same study the nucleophilic aromatic substitution of chloride from tetrachlorohydroquinone 313 with thiomethanolate was found to proceed via a concerted mechanism (with the semi‐empirical method, PM3). The authors point out that in solution phase (or on the enzyme) the accumulating negative charge in the transition state may be stabilized. Consequently, the reaction that proceeds via a concerted pathway in the gas phase could proceed via a stepwise pathway with a Meisenheimer intermediate in solution phase.</p><!><p>Bio‐inspired substitution reactions.</p><!><p>Olofsson et al. investigated108 O‐arylations with diaryliodonium salts through experimental and computational methods, using hydroxide ion, alcohols and phenols as nucleophiles. The iodonium salts are represented as covalent diaryliodine(III) triflates, for example, 315 (Scheme 44) that undergo displacement of the triflate (‐OTf) leaving group in the Ar2I‐OTf molecule by a nucleophile, before other chemistry transpires. The overall mechanistic picture is complex in that different mechanistic possibilities arose depending on the nucleophile and the iodine(III) substrate. However, in electron‐poor iodine(III) substrates such as (p‐NO2C6H4)I(Ph)OTf, 315, they propose a direct ipso displacement by hydroxide ion at the C−I bond of the nitroarene ring to lead to p‐nitrophenol. They similarly represent an attack of alkoxides on Ph2IOTf (318), as involving an initial conversion of the triflate complex to the dialkoxy "ate" complex that then undergoes concerted substitution at the ipso centre as shown. Additionally, they show oxidation of alcohols by the iodine(III) substrates as involving concerted delivery of hydride to the ipso carbon with loss of iodoarene.</p><!><p>cSNAr substitutions on arenes with a hypervalent iodine substituent.</p><!><p>Similar reactions were more recently carried out109 on cyclic secondary amines by Stuart et al., as well as primary amines,110 by Olofsson et al. Stuart describes the final step of his proposed reaction mechanism as a reductive elimination whereby Ar−N bonds were created in the same step as the Ar−I bond was being cleaved. No computational or Hammett or other analyses of these reactions are available at the time of writing this review, but the analogy to the reactions of Olofsson et al. with alcohols is clear.</p><p>Uchiyama et al.111 provided a route to ortho‐iodo diaryl ethers. They found that upon studying aryl‐exchange reactions of diaryl‐λ3‐iodanes with aryl iodides, the aryl exchange occurred via what they termed a cSNAr process (Scheme 45 A), but different from those encountered so far in this review. SN1 reactivity, benzyne pathways, and single electron transfer were all ruled out. SN1 was ruled out by the absence of any fluoroarene that would be expected to form if the reaction proceeded via an aryl cation, such as seen in the formation of fluorobenzene 327 from benzenediazonium 326 (Scheme 45 B). A benzyne pathway was ruled out by deuterating one aryl group on 331 and no D/H scrambling was observed (Scheme 45 C). Aryl radical intermediates were ruled out by the addition of a radical scavenger, 9,10‐dihydroanthracene, (Scheme 45 D) and by preparation of a radical clock substrate 335, which did not afford any cyclised products (Scheme 45 E).</p><!><p>Mechanistic studies on nucleophilic aromatic substitution reactions on hypervalent iodine substrates. (DCE: 1,2‐dichloroethane).</p><!><p>Kinetic data suggest that both reagents are involved in the transition state. Density Functional Theory (DFT) calculations suggest that the reactants 336 and 337 weakly coordinate through the BF4 ion before a concerted aryl group migration occurs via two I(II) species with some positive charge development at the ipso‐carbon (339, Scheme 46). Dissociation of the aryl iodide from the tetrafluoroborate affords the products 336 and 337. The reaction is reversible, and proceeds with thermodynamic control.</p><!><p>Free Energy profile for nucleophilic aromatic substitution reactions on hypervalent iodine substrates.</p><!><p>Bakalbassis et al. used computational methods to study the reaction of aryl migration in aryliodonium ylides 341 and 344, and found this to be a concerted process with a barrier of 17.7 kcal mol−1 and 6.4 kcal mol−1 for substrates 341 and 344, through transition states 342 and 345 respectively (Scheme 47).112</p><!><p>Aryl migration in iodonium ylides.</p><!><p>Computational studies into the reaction of the benzenediazonium ion 347 with water have been reported by Glaser et al.113 They considered three mechanisms (Scheme 48 A); i) a unimolecular SN1Ar mechanism with generation of an intermediate phenyl cation; ii) a bimolecular SNAr that proceeds without the pre‐ and post‐coordination of the water and the diazonium salt; and iii) a bimolecular SNAr that proceeds with pre‐ and post‐coordination of the water and diazonium salt.114</p><!><p>Substitution reactions of arendiazonium salts by water.</p><!><p>The authors propose that the transition state for the reaction features a phenyl cation which interacts loosely with both water and dinitrogen (350) via pathway (ii) in Scheme 48 A, despite the fact that pathway (i) has a lower ΔG *. This is explained by the fact that a phenyl cation 348 would not really exist in aqueous solutions, and the C−N bond cleavage could never evolve to completion without water binding to the developing phenyl cation. The transition state was shown to involve the "out‐of‐plane" attack 352 a rather than the "in‐plane" attack 352 b (Scheme 48 B).113</p><p>Singleton and Ussing have also studied the hydrolysis of arenediazonium cations in water, and do not agree with the results of Glaser, due to there being no consideration of entropic values in Glaser's work, and the fact that it does not agree with kinetic data.115 Kinetic isotope effects for 13C indicated that there is significant weakening not only of the C1−C2 bond in the rate‐determining step, but also of the C2−C3 bonds (see 353, Scheme 48 C). This is consistent with a structure resembling a distorted aryl cation in the transition state, as C1 gains some sp character as a cation. The authors point out that in the transition state, both N2 and water are distant from the forming cation, and that the mechanism lies somewhere between SN1Ar and SN2Ar.</p><!><p>The displacement of a fluoride atom from polyfluoroarenes with a magnesium(I) complex was studied experimentally and computationally by Crimmin et al.116 The mechanism was found to proceed via a concerted SNAr pathway (Scheme 49). The activation energy found by the DFT method (25.7 kcal mol−1) was in good agreement with the experimentally determined activation energy (21.3 kcal mol−1). A similar mechanism was found by DFT for the corresponding bimetallic Mg–Zn complex. In this complex the zinc centre acts as the nucleophile. In an earlier study on the Mg–Mg complex,117 experimental evidence speaking against single electron pathways was gathered. A SNAr mechanism was proposed and predicted to be concerted by DFT.</p><!><p>Recent remarkable displacements by magnesium nucleophiles.</p><!><p>In a very recent study with an analogous fluoride‐metal exchange reaction with a corresponding bimetallic Mg–Fe complex, a cSNAr pathway was identified by DFT.118 However, an alternative step‐wise SNAr mechanism was found to have a lower overall activation energy. With the Mg–Fe complex, the iron atom acts as the nucleophile.</p><!><p>Building on computational and experimental observations, notably from the Ritter group, Jacobsen et al. recently surveyed14 SNAr reactions by a combination of experimental and computational methods (Scheme 50). In advance, they based their expectations on the fact that isolated Meisenheimer intermediates can arise when i) substituents on the arene undergoing substitution provide good stabilisation of an intermediate anion, and ii) where the leaving group is relatively poor, so that the intermediate has some kinetic stability. Specifically they initially studied three reactions. Case A satisfies both of the above criteria, Case B features substituents that do not provide such good stabilisation of negative charge, and also boasts an excellent leaving group, bromide, while Case C features substituents that can provide excellent stabilisation while also bearing an excellent leaving group. As such, Case A would likely be a classical SNAr reaction, Case B would likely be concerted and Case C could be borderline between the two mechanistic extremes. Their experimental approach was based on studying kinetic isotope effects in substrates that involve fluoride as a leaving group or as a nucleophile in SNAr reactions. If a kinetic isotope effect is involved in the formation or cleavage of the C−F bond, then this will be reflected in a 13C/12C isotope effect for that carbon. NMR methods for determining isotope effects were greatly developed by Singleton and Thomas119 in 13C spectra, but the novel development of Jacobsen et al. is to make use of the NMR sensitivity of the 19F nucleus. Studying multiple quantum filtered (MQF) 19F{1H} spectra allowed clear observation and quantitation of the 13C–19F satellites to the 12C–19F peak with very accessible acquisition times for reasonable quantities of substrate (the MQF technique suppresses the appearance of the latter peak).</p><!><p>Three reactions studied in depth by Jacobsen et al.</p><!><p>With the isotope effects measured, the important point was to compare this figure with that calculated using benchmarked computational methods, which also indicate whether an intermediate or a transition state is present. A key indicator of the concerted or stepwise nature of the reaction involving C−F formation or rupture relates to a comparison of this KIE to the maximum computed KIE on the reaction energy surface. Strong bonds in the ground state can lead to loss of more vibrational energy in the TS and therefore to large KIEs.</p><p>The largest KIE values arise when the bonding to both nucleophile and leaving group are weak in the TS, that is, in concerted reactions. For example in Case A, a strong C−F bond is broken, leading to large maximum KIE (1.070). In contrast, in Case B, a weak C−Br bond is broken as reflected in the lower maximum KIE (1.045). The measured KIE in both cases was 1.035 but this represents 47 % of the maximum KIE for Case A, but 87 % of the maximum KIE for case B. This translates to a stepwise nature for Case A and a concerted reaction for Case B. They then extended their studies to 120 SNAr reactions with a variety of arene ring types, nucleophiles and leaving groups. Their calculations showed that 99 of the selected substitution reactions (83 %) proceed with concerted mechanisms.</p><!><p>In 2013, aromatic nucleophilic substitutions were reviewed, and the classical stepwise mechanism was deemed to be the usual mechanism, while concerted nucleophilic substitutions were very rare.3 The past six years have certainly built on the undercurrent that existed before 2013 and it is likely that a torrent of concerted examples will appear in the next few years. Investigations have been helped by computational techniques that shed light on the mechanisms. What is clear is that the concerted or stepwise nature of the reactions is strongly influenced by substrate nucleophile and leaving group, but also by the environment, and that some substitutions may present as concerted or stepwise depending on the conditions. We need to be careful about information from Hammett correlations for at least two reasons: i) Hammett ρ‐values depend on the temperature at which the experiments are performed and so comparisons need to bear this in mind; ii) if a particular reaction undergoes a transition from stepwise to concerted for a range of substituents on the substrate, this may present as a clear change in ρ‐value, but the two pathways could have similar ρ‐values, and this could mask the transition. With computational methods, the selection of the method and the basis set clearly influences the outcome of the calculations, and so continued study in this area will be crucial.</p><p>With these important changes in perception coming now for nucleophilic aromatic substitution and its implications for Meisenheimer intermediates, it is interesting to see that the counterpart in electrophilic aromatic substitution, that is, concerted electrophilic aromatic substitution, featuring Wheland transition states rather than intermediates, is also beginning to appear.120, 121, 122 We are thus at a time of exciting developments in mechanistic organic chemistry.</p><!><p>The authors declare no conflict of interest.</p><!><p>Simon Rohrbach obtained his Master's Degree in Organic Chemistry at the University of Bern, Switzerland, before joining the research group of John A. Murphy at the University of Strathclyde, UK. He is working on method development and elucidation of complex reaction mechanisms applying both experimental and computational approaches.</p><p></p><p>Andrew Smith obtained his MChem in Chemistry from the University of Strathclyde in 2015, before starting his PhD work under the supervision of Professor John Murphy at Strathclyde. He is currently studying the mechanistic pathways involved in reducing systems.</p><p></p><p>Jia Hao Pang completed his undergraduate studies at Nanyang Technological University (NTU) Singapore in 2016 before beginning his PhD work in the laboratory of Shunsuke Chiba at NTU. He is currently focusing on chemistry of main group metal hydrides for methodology development.</p><p></p><p>Darren Poole completed his DPhil in 2014 (University of Oxford, Prof Timothy Donohoe). He joined GSK as a synthetic chemist in 2014, and became a Scientific Leader and GSK Associate Fellow in 2018, with a particular interest in applying new technologies to drug discovery.</p><p></p><p>Tell Tuttle earned his PhD in 2004 under the supervision of Prof. Elfi Kraka and Prof. Dieter Cremer at Göteborg University, Sweden. He began his independent career in 2007 and is currently Professor of Theoretical Chemistry at the University of Strathclyde. His research is focused on the use of computational methods for the directed discovery of new reactivities and functional materials.</p><p></p><p>Shunsuke Chiba earned his Ph.D. in 2006 under supervision of Prof. Koichi Narasaka at the University of Tokyo. In 2007, he embarked on his independent career as the faculty of Nanyang Technological University (NTU) Singapore, where he is currently Professor of Chemistry. His research group focuses on methodology development in the area of synthetic chemistry and catalysis.</p><p></p><p>John Murphy was born in Dublin and educated at the University of Dublin (TCD) and the University of Cambridge. After Fellowships at Alberta and Oxford, he was appointed as Lecturer, then Reader, at the University of Nottingham. Since 1995, he has held the Merck–Pauson Professorship at the University of Strathclyde. His interests are in mechanism and synthesis.</p><p></p>

PubMed Open Access

Pharmacokinetics and safety of panitumumab in a patient with chronic kidney disease

PurposeData on panitumumab dosing in cancer patients with renal insufficiency are lacking. Here, we report a 63-year-old metastatic colorectal cancer patient with chronic kidney injury with a glomerular filtration rate of approximately 11 mL/min.MethodsPharmacokinetic parameters, including dose-normalized area under the curve, clearance and elimination half-life (T 1/2) after the 11th and 12th infusions were estimated using trapezoidal non-compartmental methods. Data were compared to previous reported pharmacokinetic data from studies in patients with normal renal function.ResultsThe results show that the pharmacokinetic data in this patient with kidney failure are comparable to those in patients with adequate renal function. Moreover the treatment was well tolerated in this patient.ConclusionThis study suggests that panitumumab can be safely used in cancer patients with renal impairment without dose adjustment.

pharmacokinetics_and_safety_of_panitumumab_in_a_patient_with_chronic_kidney_disease

Introduction<!>Case<!>Panitumumab sampling and measuring<!>Pharmacokinetic parameters<!>Toxicity<!>Case<!>Pharmacokinetics<!><!>Pharmacokinetics<!><!>Pharmacokinetics<!>Discussion

<p>Panitumumab is a fully humane monoclonal antibody targeting the epidermal growth factor receptor (EGFR) and is registered for the treatment of RAS wild-type metastatic colorectal cancer, either alone or combined with chemotherapy. As previously discussed elsewhere, clearance of panitumumab mainly occurs by an EGFR sink. In case of saturation of all receptors, panitumumab will be cleared by immunologic mechanisms, such as complement-dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity and apoptosis [1]. Therefore, theoretically renal insufficiency is not likely to influence the pharmacokinetics of panitumumab. The study of councilman et al. showed that nephrotic syndrome was associated with increased rituximab clearance, and therefore, decreased half-life. An possible explanation for the observed effect is loss of monoclonal antibody in the urine and not altered clearance [2].</p><p>The most recent summary of product characteristics (SmPc) of panitumumab states that a population pharmacokinetic analysis (among race, age, gender, hepatic function, concomitant chemotherapy and EGFR membrane-staining intensity in tumor cells) renal function does not influence the pharmacokinetics of panitumumab, however, it is not tested in patients. The only available clinical information concerns a case report showing safety and efficacy of panitumumab (combined with oxaliplatin, folic acid and 5-FU) in a hemodialysis patient [3]. However, to our knowledge, this is the first case study showing actual pharmacokinetic parameters in a patient with chronic kidney injury without dialysis (CKD).</p><!><p>A 63-year-old Caucasian male was diagnosed with colon cancer with hepatic metastases in November 2011. His medical history included diabetes type 2, congestive heart failure and CKD with unknown etiology. The estimated clearance according to the modification of diet in renal disease (MDRD) was 21 mL/min at this time. The primary tumor was resected because of its obstructive character. Histopathological analysis showed a poorly differentiated adenocarcinoma, KRAS wild type. A few weeks later, the patient started palliative chemotherapy, consisting of oxaliplatin, folic acid and 5-FU (FOLFOX). This therapy was discontinued after eight cycles since his renal function declined. After a period without treatment, he started with panitumumab in May 2013. By then, his renal function had further declined to an estimated clearance of 11 mL/min (MDRD). His weight was 106 kg. Panitumumab was dosed at the recommended full dose of 6 mg/kg diluted in 100 mL sodium chloride solution (0.9%) and administered in 60 min, without pre-medication following a standard procedure. Serum samples for pharmacokinetic analysis were collected after the 11th and 12th infusion of panitumumab and toxicity data were collected. The patient gave informed consent and the Medical Ethics Committee approved the study.</p><!><p>Serum samples were planned at 0.5, 1, 2, 4, 8, 24 h, 4 days, and 7 days after the 11th panitumumab infusion. Before the 12th infusion (day 15) and 30 min, 1 h and 14 days later the blood samples were drawn. The samples were allowed to clot for 30 min, followed by centrifuging at 3000 rounds per minutes. The serum was transferred to a tube and stored at − 80 °C until analysis.</p><p>Panitumumab serum drug concentrations were performed by PPD laboratories (Richmond, VA, USA) using a validated immunoassay with electrochemiluminescence as described before [1].</p><!><p>Pharmacokinetic parameters were estimated by trapezoidal noncompartmental methods using MW/PHARM 3.5 of Mediware (Groningen, The Netherlands). Pharmacokinetic parameters for panitumumab i.e., area under the serum concentration–time curve (AUC) maximum observed serum concentration (C max), and minimum observed serum concentration (C min)—were determined. Half-life (T 1/2), volume of distribution (V) and clearance (CL) were calculated.</p><p>For comparison, historical data from the summary of product characteristics (SPC) [2] and cohort 1 of Stephenson et al. were used [4]. From this study, the dose-normalized (for the dose of 6 mg per kg) AUC, clearance, elimination half-life, minimum and maximal concentrations were used. In case the value was within the reported serum level ± 1 standard deviation, the found value was considered not to be clinically relevant or clinically different.</p><!><p>Information on toxicities were collected at baseline, just before each course, at the day of infusion and 7 days after infusion. Information on toxicities were also collected during each unplanned hospital visit or contact. Toxicities were graded using CTCAE version 1.1.</p><!><p>A total of 12 infusions of panitumumab were administered. The patient experienced grade 2 skin toxicity, treated with topical agents (no minocycline because of the CKD). At the beginning his condition improved significantly and he had a mixed response with regression of liver metastases and new pleural metastases. However, after the 12th cycle of panitumumab in the end of October 2013, his lesions had clearly progressed. Treatment with regorafenib was considered in November 2013, however, by then his CKD had progressed further and at that point starting dialysis seemed inevitable (mostly because of electrolyte disturbances). Considering his poor prognosis, patient declined dialysis and soon after that he was admitted to a hospice. He died a few weeks later.</p><!><p>The maximal observed serum concentration of panitumumab was 125 μg/mL after the 11th and 12th infusion. The minimum concentration observed just before the 12th infusion was 37.0 and 48.0 μg/mL, 13 days after the 12th infusion. The reported serum concentrations were used to calculate the AUC, half-life and clearance (Table 1).</p><!><p>Pharmacokinetic parameters of the case and historical data from SPC and the study of Stephenson et al.</p><p>SD standard deviation</p><!><p>In Table 1, an overview of the pharmacokinetic parameters of panitumumab in study populations with normal renal functions and this case is shown. In this table, the pharmacokinetics of the 11th and 12th infusion of 6 mg/kg in the Stephensons cohort and data from the SPC are depicted and used for comparison. In Fig. 1 the time concentration curve after the first and second infusion of panitumumab are depicted.</p><!><p>Time curve of panitumumab concentration following 1 h infusion of 616 mg of panitumumab in a patient with a glomerular filtration rate (MDRD) of approximately 11 mL per minute</p><!><p>In this case, the calculated AUC was 1555 and 1752 μg day/mL after the 12th infusion. The calculated clearance was 3.4 and 3.8 mL/day/kg and the half-life was 9.3 and 10.8 days, respectively, after the 11th and 12th infusion. A comment should be made regarding the calculated half-life after the 12th infusion. This half-life may be biased due to limited sampling (at 30 min, 1 h and 14 days) because the distribution phase may not be terminated after one hour.</p><p>Compared to the historical data, the maximal concentration measured in our case was lower as compared to the reported maximum concentration in the SPC and the Stephenson's cohort. Furthermore, the AUC calculated after the 12th infusion was higher in our case compared to the historical data. The AUC calculated after the 11th infusion was within the earlier reported mean and standard deviation. The minimum concentration, half-life and clearance calculated in this study were all comparable to the historical data of the cohorts and SmPC.</p><!><p>Until now, no clinical studies have been conducted to examine the pharmacokinetics of panitumumab in patients with renal impairment. Previously, we have reported a similar case study with cetuximab in CKD [5] and hereby report one on the pharmacokinetics of panitumumab in a patient with CKD. During the registration of panitumumab, a population pharmacokinetic analysis was performed to explore the potential effects of selected covariates on panitumumab pharmacokinetics. These theoretical results showed that renal function had no apparent impact on the pharmacokinetics of panitumumab. However, recommendations on dosing in patients with kidney disease are lacking. Knowledge of the precise impact of CKD on panitumumab pharmacokinetics is highly relevant as the percentage of cancer patients over 75 years is expected to increase disproportionally [6] and glomerular filtration rate naturally declines during life [7]. In addition, most patients have previously been treated with oxaliplatin which may also negatively influence renal function. Clearly, CKD is not uncommon in colorectal cancer patients.</p><p>The pharmacokinetic parameters AUC and maximum concentrations of our case are different compared to earlier reported data [4]. The pharmacokinetic parameters, minimum concentration, clearance and half-life were similar to the results reported from population without renal failure. It is important to mention that all the reported half lives in the cohorts and in the SPC are low compared to the half-life reported in the study of Ma et al. [8]. They reported a half-life of 18.3 days for panitumumab. Thus, the here reported half lives of panitumumab (case and included studies) may be overestimated due to non-linear elimination shape.</p><p>The maximum concentration reported in our patient appeared to be lower. The weight of this patient, however, was 106 kg and the BMI was 30. In obese, the total blood volume is increased; this increase could be an explanation for the lower maximum concentrations of panitumumab [9].</p><p>The AUC after the 11th infusion was comparable with historical data. The AUC after the 12th infusion was slightly higher than the reported AUC's in former studies in patients without renal failure. The AUC during the 11th and 12th infusion were estimated in steady state so this could be due to inter-patient variability. It is unlikely to be caused by the decreased renal function in this patient because clearance of panitumumab occurs extensively by the EGFR sink and the reticuloendothelial system. Another factor which may influence the clearance is the tumor burden and the antigen density of the tumor. As a consequence a lower tumor burden or antigen density may lead to reduced clearance and thus a higher AUC.</p><p>Besides the slightly higher AUC after the 12th infusion, the pharmacokinetic parameters are in line with population without chronic kidney disease. Furthermore, during treatment, no additional toxicity was noted, except the expected skin toxicity commonly reported in EGFR antibody treatment. The absence of an effect of the renal function on the pharmacokinetics was as expected and in line with other studies. Due to their molecular size, monoclonal antibodies are not excreted by the urine. For panitumumab and other monoclonal antibodies, population pharmacokinetic studies used models to study the effect of the renal clearance as a covariate and did not find an effect on clearance [10]. In this study, we validated the lack of effect in a patient.</p><p>In conclusion, the pharmacokinetics of panitumumab between patients with decreased renal functions and patients with normal renal function seems similar.</p>

PubMed Open Access

Substrate Recognition by the Class II Lanthipeptide Synthetase HalM2

Class II lanthipeptides belong to a diverse group of natural products known as ribosomally synthesized and post-translationally modified peptides (RiPPs). Most RiPP precursor peptides contain an N-terminal recognition sequence known as the leader peptide, which is typically recognized by biosynthetic enzymes that catalyze modifications on a C-terminal core peptide. For class II lanthipeptides these are carried out by a bifunctional lanthipeptide synthetase (LanM) that catalyzes dehydration and cyclization reactions on peptidic substrates to generate thioether containing, macrocyclic molecules. Some lanthipeptide synthetases are extraordinarily substrate tolerant, making them promising candidates for biotechnological applications such as combinatorial biosynthesis and cyclic peptide library construction. In this study, we characterized the mode of leader peptide recognition by HalM2, the lanthipeptide synthetase responsible for the production of the antimicrobial peptide haloduracin \xc3\x9f. Using NMR spectroscopic techniques, in vitro binding assays, and enzyme activity assays, we identified substrate residues that are important for binding to HalM2 and for post-translational modification of the peptide substrates. Additionally, we provide evidence of the binding site on the enzyme using binding assays with truncated enzyme variants, hydrogen-deuterium exchange mass spectrometry, and photoaffinity labeling. Understanding the mechanism by which lanthipeptide synthetases recognize their substrate will facilitate their use in biotechnology, as well as further our general understanding of how RiPP enzymes recognize their substrates.

substrate_recognition_by_the_class_ii_lanthipeptide_synthetase_halm2

Introduction<!>NMR Spectroscopic Investigation of HalA2 binding to HalM2<!>Affinity of HalA2-LP and analogs for HalM2<!>Leader peptide perturbation affects core peptide processing<!>HalA2-LP binds to the capping helices of HalM2<!>Conclusions<!>Biological reagents and materials:<!>General high-performance liquid chromatography (HPLC)<!>General mass spectrometry<!>General solid-phase peptide synthesis<!>Cloning of HalM2 truncants<!>HalM2 overexpression and purification<!>HalA2LP overexpression and purification<!>Circular Dichroism Spectroscopy of HalA2-LP analogs<!>Enzymatic activity assays for HalM2 and HalM2 mutants<!>Hydrogen deuterium exchange mass spectrometry<!>Fmoc protection of L-photoleucine<!>Synthesis of HalA2LP and analogs<!>Selective N-terminal labeling of HalA2LP with Fluorescein<!>Saturation Transfer Difference NMR and 2D TOCSY/NOESY NMR<!>Fluorescence polarization (FP) and competition FP binding assays<!>Cloning of HalA2 and CylLS precursor peptide mutants<!>Co-expression of precursor peptide mutants<!>Photocrosslinking and enrichment of HalM2 variants with HalA2PAL analogs<!>Mass spectrometry of enriched photocrosslinked samples<!>Analysis of crosslinked data

<p>Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse family of natural products that exhibit a broad range of biological activities.1 RiPP precursor peptides are typically comprised of an N-terminal leader peptide region and a C-terminal core region.2 The leader peptide generally serves as a recognition motif, which allows the recruitment of a broad suite of modifying enzymes that catalyze chemical transformations in the core peptide.</p><p>By virtue of their genetically encoded substrates and the modular nature of the post-translational modifications, RiPP natural products are well suited for a wide variety of biotechnological applications. For example, various approaches may introduce non-proteogenic amino acids into the precursor peptide for subsequent modification, yielding novel molecules with interesting activities.3–16 Additionally, libraries of precursor peptides can be generated for testing of a large number of variants.17–31 The precursor peptide may also be manipulated to allow for combinatorial biosynthesis, in which enzymes from different RiPP systems are used in tandem to generate new scaffolds.32–36 The successful application of such approaches is facilitated by a clear understanding of how the modification machinery recognizes the leader peptide.</p><p>Lanthipeptides are one of the largest groups of RiPPs and are characterized by the presence of lanthionine or methyllanthionine residues ((Me)Lan). These structures are formed by crosslinking of cysteine residues to dehydrated serine/threonine residues.37 Thus far, four distinct enzyme architectures capable of performing these post-translational modifications have been characterized. Class I lanthipeptide biosynthesis requires two separate enzymes, a tRNAGlu-dependent LanB dehydratase, and a LanC cyclase that catalyzes the formation of the lanthionine moiety. Much work has contributed to the understanding of class I lanthipeptide biosynthesis, notably the identification of the LanB substrate binding domain and characterization of the precursor binding motif recognized by LanBs and many other RiPP biosynthetic enzymes.38–40 This common binding domain, termed the RiPP recognition element (RRE),39 shares structural homology with PqqD, a small protein involved in the biosynthesis of another RiPP-derived natural product, pyrroloquinoline quinone. Class I lanthipeptide precursor peptides are recognized through the FNLD amino acid motif on the leader peptide41–43 by the RRE of the cognate dehydratase.38</p><p>Unlike class I systems, class II, III and IV lanthipeptide synthetases are structurally distinct, multifunctional enzymes that catalyze both dehydration and cyclization reactions.44–46 These three classes of lanthipeptide synthetases utilize nucleoside triphosphates to phosphorylate the Ser/Thr targeted for dehydration followed by phosphate elimination.45, 47 Insights have been made into substrate recognition and catalysis of class III and IV lanthipeptides,47–50 but in comparison, little is known regarding the binding site on both the leader peptide and class II LanM enzymes, which lack the RRE.51 A recent comprehensive hydrogen-deuterium exchange mass spectrometry (HDX-MS) study along with biochemical follow-up experiments provided the first insights regarding potential binding sites on the class II lanthipeptide synthetase HalM2.52</p><p>Class II lanthipeptide synthetases phosphorylate serine and threonine residues on their substrates by using ATP, followed by phosphate elimination and cyclization (Figure 1a). Only one LanM enzyme has been structurally characterized. CylM, the class II synthetase involved in the biosynthesis of enterococcal cytolysin contains an N-terminal dehydratase domain that is structurally similar to eukaryotic lipid kinases, and a C-terminal cyclase domain that is structurally similar to the class I LanC proteins.51 However, no substrate-bound structures are available and, therefore, the mechanism by which LanMs recognize their cognate precursor peptides remains poorly understood. A better understanding of the recognition mechanism would be valuable, as several LanM's demonstrate remarkable catalytic flexibility towards the core peptide region. For example, a single LanM from Prochlorococcus MIT9313 (ProcM) has been shown to act on over 20 distinct precursor peptides to generate multiple unique (methyl)lanthionine ring topologies.53–54 As such, class II synthetases pose high potential for biotechnological applications.25–27</p><p>Herein, we report new insights into the mechanism of leader peptide recognition used by HalM2 involved in the maturation of the antimicrobial peptide haloduracin β.55–56 Using NMR techniques, binding studies and biochemical experiments, we characterize key residues on the leader peptide of HalA2 that are important for binding. Additionally, we show that changing these residues on a non-cognate substrate is sufficient to bestow on this peptide substrate recognition by HalM2. Using HDX-MS at two temperatures and photocrosslinking experiments, we provide evidence that HalM2 binds to the leader peptide on the dehydratase domain, specifically on the architectural motif known as the capping helices, which complements the aforementioned HDX studies.52 Identification of both leader peptide motifs and LanM binding elements will aid in genome-mining efforts and facilitate rational design and implementation of LanM enzymes to perform post-translational modifications for future engineering approaches.</p><!><p>Most class II lanthipeptide leader peptides have a variable N-terminal region, but a conserved C-terminal region (Figure 1b). To identify which residues are important for interaction between the leader peptide and lanthipeptide synthetase, we utilized saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy with the HalA2-HalM2 system. STD-NMR relies on the nuclear Overhauser effect via spin diffusion and allows the selective identification of protons on the ligand that are in proximity to the receptor site.57 The previously reported binding affinity and off-rate of HalA2 binding to HalM258–59 suggest this interaction should be amenable to STD NMR. Previous studies using STD-NMR on other RiPP biosynthetic enzymes have proven successful in characterizing important residues for substrate binding.60 We generated the leader peptide of HalA2 (HalA2-LP) by heterologous expression of the peptide in Escherichia coli. Likewise, His6-HalM2 was isolated by heterologous expression and purification by immobilized metal affinity chromatography.58 First, the proton chemical shifts of HalA2-LP were assigned by 1H-1H TOCSY and 1H-1H NOESY experiments (Supplemental Table S1). In RiPP nomenclature, peptide residues are numbered by positive residue numbers for amino acids in the core peptide counting forwards from the leader peptide cleavage site, and negative numbers are used for amino acids in the leader peptide counting backwards from the cleavage site. An STD-NMR experiment was then performed on a sample containing both HalA2-LP and HalM2 by irradiating the protein signal at 0.0 ppm and subtracting the off-resonance signal by irradiating at 30 ppm by phase cycling (Figure 2b).61 The STD signal intensity was normalized to account for the different number of protons associated with each signal.</p><p>Several key features were identified during analysis of the spectra. First, the N-terminal region of HalA2-LP displayed either weak or moderate saturation transfer (Figure 2c–d), whereas the C-terminal region of HalA2-LP contained the majority of signals that displayed moderate to strong signals. These findings are consistent with the lack of conservation of the N-terminal region in class II leader peptides, which may not be as important for substrate binding by the cognate LanM (Figure 1b). Interestingly, the signals with the strongest intensity were associated with protons of hydrophobic amino acids and not with the conserved acidic residues (Figure 2c–d). Specifically, the side chain protons of Leu−24, Phe−22, Val−18, Leu−13, and Leu−10 showed the strongest STD signals, indicating that they are likely interacting with HalM2. This extended stretch of hydrophobic residues suggests that the binding interface on HalM2 may be extended rather than a binding site that recognizes a short stretch of amino acids.</p><!><p>To corroborate the findings from the STD-NMR experiment, we performed competition fluorescence polarization (FP) assays using analogs of HalA2-LP. The initial binding isotherm for fluorescently labeled HalA2-LP (Fluor-HalA2-LP, Figure S1), yielded a dissociation constant (Kd) of 8.8 ± 1.2 μM (Figure 3, Figure S2a).58 A competition experiment in which Fluor-HalA2-LP was allowed to bind to HalM2, followed by titration with unlabeled HalA2-LP yielded an inhibition constant (Ki) of 2.1 ± 0.1 μM (Figure 3, Figure S2b). Assuming the inhibition is the result of competitive displacement, Ki values reported here are representative of the affinities of the peptides for the enzyme (for a discussion on Ki values, see the Supporting Information).</p><p>We next synthesized a set of peptides by solid phase peptide synthesis (SPPS) to interrogate regions of the HalA2-LP. The N-terminal region of HalA2-LP (HalA2-LP(−40 to −28)) was unable to compete off Fluor-HalA2-LP even at very high concentrations (Figure 3, Figure S2c). Surprisingly, the C-terminal region alone (N-acetyl-HalA2-LP(−27 to −8)) was also unable to displace Fluor-HalA2-LP, except at high concentrations (Ki > 250 μM) (Figure 3, Figure S2d). Because this region encompasses the entire sequence identified to bind strongly by STD-NMR spectroscopy, we had expected that it would be both necessary and sufficient for binding. Instead, neither the N- nor the C-terminal regions alone completely recapitulate the binding affinity of the full-length leader peptide. We next synthesized a nearly full length leader peptide, HalA2-LP(−40 to −8), which yielded a Ki of 13.7 ± 1.5 μM (Figure 3, Figure S2e). Although we cannot rule out that the LP binds to a highly extended binding surface, it is likely that the short C-terminal peptide in Figure 3 may not be able to adopt the necessary secondary structure to yield productive binding; therefore, it appears that both the N- and C-terminal regions contribute synergistically to the overall binding affinity. In support of this hypothesis. analysis by circular dichroism (CD) spectroscopy showed that in aqueous solution, HalA2-LP(−40 to −28) and HalA2-LP(−27 to −8) adopt random coil structures, whereas HalA2-LP(−40 to −8) displays partial alpha helical character (Figure S3). As a cautionary note, many class I leader peptides adopt helical conformations in solution,37, 62 but bind to the RRE as a β-strand.38</p><p>Next, we sought to systematically investigate the influence of the hydrophobic residues identified by STD-NMR on binding using mutagenesis. We focused our investigations on residues that not only exhibited strong STD-NMR signals, but also are within the conserved C-terminal region identified by sequence alignment (Figure 1b). The conserved acidic residues in this region were not mutated because of the weak STD-NMR signals as well as previous mutagenesis studies that suggest these residues do not play an important role in LanM catalysis.63–66 Replacement of Val−18 with alanine (HalA2-LP(−40 to −8/V−18A)) only modestly affected the binding affinity (Ki = 19.8 ± 1.3 μM) (Figure 3, Figure S2g). Likewise, replacement of Leu−10 with alanine (HalA2-LP(−40 to −8/L−10A)) also modestly affected the binding affinity (Ki = 25.4 ± 1.3 μM) (Figure 3, Figure S2f). Moving along the peptide backbone, replacing both Leu−10 and Leu−13 to alanine resulted in a stronger decrease in binding affinity (Ki = 137 ± 39 μM)) (Figure 3, Figure S2h). Continuing systematically replacement of bulky hydrophobic residues with Ala showed that simultaneous substitution of Val−18, Leu−13, and Leu−10 (HalA2-LP(−40 to −8/V−18A/L−13A/L−10A)) did not result in additional loss of affinity because this variant displayed a similar Ki as the double mutant (142 ± 41 μM; Figure 3; Figure S2i). However, replacement of four residues, Phe−22, Val−18, Leu−13, and Leu−10 resulted in additional losses in binding affinity (Ki = 220 ± 40 μM; Figure 3; Figure S2k). Replacement of all five hydrophobic residues identified by STD-NMR with alanine (HalA2-LP(−40 to −8/L−24A/F−22A/V−18A/L−13A/L−10A)), resulted in the weakest binding affinity (Ki > 250 μM; Figure 3; Figure S2l). It appears that the C-terminal residues contribute slightly more to the overall affinity because when we replaced Leu−24, Phe−22, and Val−18 with alanine (HalA2-LP(−40 to −8/L−24A/F−22A/V−18A)), we observed a ~6-fold decrease in binding affinity compared to the wild type sequence (Ki = 79 ± 12 μM; Figure 3; Figure S2j). The attenuation of binding affinity when changing the series of hydrophobic amino acids to alanine supports the hypothesis that the main driving force of HalA2−LP interaction with HalM2 is through hydrophobic interactions along an extended binding surface.</p><!><p>In addition to monitoring binding affinity, the effect of leader perturbation on HalM2's ability to carry out core peptide modification was examined. After co-expression with HalM2 in E. coli, His-tagged HalA2-L−24A/F−22A/V−18A/L−13A/L−10A (hereafter referred to as HalA2-A5) was purified and the extent of dehydration monitored using matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Whereas HalM2 is normally capable of dehydrating wild-type HalA2 seven times, mutation of these five leader peptide residues resulted in at most four dehydrations, with a majority of ions observed corresponding to unmodified peptide (Figure 4b,c). Previous studies have shown that HalM2 and other LanM enzymes have weak dehydration activity on the core peptide even when no leader peptide is present, suggesting some recognition of the core peptide by the synthetase.58, 67 Given the weak binding affinity of the HalA2-A5 leader peptide observed by our binding studies, it is likely that the modifications observed in vivo with the HalA2 variant in which these five residues were mutated is the result of weak recognition of both the mutant leader and core peptide regions, consistent with previous data demonstrating synergism in the binding of the leader and core peptides, with the former contributing the most.58</p><p>To probe the role in core peptide processing of the N-terminal region of the substrate, we also co-expressed HalM2 with HalA2Δ(−40 to −30) in which the first 10 residues were removed from the leader peptide. A mixture of incompletely dehydrated products was observed, suggesting inefficient processing of the core peptide (Figure S4b). Based on these data and the FP experiments described above, we surmised that the reduced efficiency of core peptide processing is likely due to decreased binding affinity of the substrate to HalM2. Further removal of residues from the leader peptide of HalA2 lead to loss of any observable peptides by MALDI-TOF MS, possibly due to degradation in E. coli.49</p><p>To investigate the generality of the findings with HalM2 to other class II systems, we performed a similar set of truncation experiments on two additional precursor peptides for the virulence factor enterococcal cytolysin, CylLL and CylLS.68–70 CylLL is dehydrated seven times by its cognate lanthipeptide synthetase CylM whereas the enzyme dehydrates CylLS four times. Removal of the first ten residues in CylLL [CylLL-Δ(−30 to −21)] again impaired processing, as evidenced by the formation of intermediate dehydration states (Figure 4e). However, removal of the first 10 residues in CylLS [CylLS-Δ(−42 to −31)] still resulted in the efficient production of 4-fold dehydrated product (Figure 4f). Notably, the leader peptide of CylLS is about 10 residues longer than that of CylLL. Removal of any further residues on CylLL or CylLS lead to a similar loss of observable peptide as seen with HalA2.</p><p>Typically, LanM enzymes are specific for their cognate leader peptides and do not act on peptides from unrelated gene clusters. Previous studies have demonstrated that complete replacement of a leader peptide of a non-cognate core peptide with a leader peptide from the substrate of a given LanM lanthipeptide synthetase will allow the enzyme to process the new core peptide.71–73 Similar results were obtained for class I lanthipeptides.36, 74–75 As we have identified select residues important for HalM2 binding to HalA2-LP, we hypothesized that selective introduction of these residues in a non-cognate substrate might endow successful HalM2 processing. Alignment of the leader peptides of CylLS with HalA2 identified four residues shown in this work to be important for HalM2 that differ in the two peptides (Figure 4d). Given that wild-type CylLS is not accepted by HalM2 as a substrate (Figure 4g), we changed these four residues in its leader region to the corresponding amino acids in HalA2 to generate the variant CylLS-P−23L/L−18V/M−13L/I−10L (termed CylLs-H2l for HalA2-like, Figure 4d). After co-expression of the variant peptide with HalM2 in E. coli and immobilized metal affinity purification, up to complete, four-fold dehydration was observed in the CylLS-P−23L/L−18V/M−13L/I−10L peptide (Figure 4h). Furthermore, substituting a single residue to generate CylLS-P−23L was not sufficient to allow for core peptide processing by HalM2 (Figure S5b). We note that three of these substitutions are rather conservative suggesting the possibility that functional LanA-LanM pairs utilize proper fitting of the hydrophobic side chains on the leader peptide into the corresponding binding region on the LanM.</p><!><p>LanM enzymes share structural similarity to eukaryotic lipid kinases, such as mTOR, but contain several unique structural insertions.51 One of these insertion regions was termed the capping helices, which are located on the N-terminal dehydratase domain (Figure S6). To investigate where on HalM2 the leader peptide binds, we performed HDX-MS studies using the HalA2 leader peptide to investigate regions of protection from exchange. Owing to the low binding affinity between HalM2 and HalA2-LP (8.8 ± 1.2 μM, Figure 3), we expected only small differences between HDX of the bound and unbound states. To accentuate the differences and increase the confidence in binding sites assignment, the HDX experiments were executed under two different exchange temperatures, 4 °C and 25 °C. As a result, we identified seven distinct peptides that exhibit protection upon ligand binding at both temperatures (Figure S6). Among the regions identified by HDX are parts of the capping helices, consistent with the recently reported detailed HDX experiments that suggest that a leader binding site may be present within the capping helices52 as well as results with photoaffinity crosslinking experiments (vide infra). We note that the previous HDX experiments and our study differ in experimental conditions, including the inclusion of AMP-PNP as ATP analog and the use of full length HalA2 (instead of just the leader peptide) in the recent report. Although both studies identified regions in the capping helices that are shielded upon ligand binding, the exact sequences differ. We did not observe deuterium shielding at both exchange temperatures in the H110-Y127 stretch of the capping helices that was shielded with full length HalA2.52</p><p>We sought to apply two additional approaches to interrogate where binding is occurring on HalM2. First, we expressed and purified several HalM2 truncants (Figure S8), wherein we serially deleted domains of the enzyme, and measured the HalA2 binding isotherm by FP. As determined by the initial fluorescence polarization experiments, full length HalM2(1–990) binds to Fluor-HalA2 with a Kd of 8.8 ± 1.2 μM (Figure 3). Removing the entire cyclase domain to generate HalM2(1–649) resulted in a Kd of 47.8 ± 5.7 μM (Figure 5a). This relatively modest decrease in affinity suggests the main HalA2 binding site is located in the dehydratase domain. These findings are consistent with previous activity data with LanM protein variants on which the dehydratase domains were expressed and reconstituted independently.51, 76–78 The stand-alone dehydratase domains in these studies catalyzed full dehydration of their substrates, suggesting that the leader peptide binding site exists within the dehydratase domain. Our findings are also supported by recent experiments in which loops on the capping helices were replaced with GlySer stretches.52 Further truncation into the dehydratase domain provided HalM2(1–348), and this small protein bound Fluor-HalA2-LP with a Kd of 54.4 ± 9.0 μM (Figure 5b) suggesting it contains the main LP binding site. Truncation from the N-terminus was less successful, as we were only able to express HalM2(36–990) whereas larger truncations did not result in isolated protein. Nevertheless, we observed that HalM2(36–990) had near wild-type binding affinity, confirming that at least one binding site exists between residues 36 and 348 of HalM2 (Figure 5c). However, it cannot be ruled out that the cyclase domain may provide either a secondary binding site, or confer structural stability that facilitates leader peptide binding, as the truncated enzymes do display a decrease in binding affinity.</p><p>We tested HalM2(1–649) for in vitro activity and determined that it was catalytically active and could perform up to seven-fold dehydration of HalA2 (Figure S9b). Previously, it was suggested that a ß-sheet motif between the dehydratase and cyclase domain may bind the leader peptide.51 We removed this ß-sheet motif to generate HalM2(1–625) and found that this enzyme also catalyzed up to seven-fold dehydration of HalA2 (Figure S9c). Thus, the ß-sheet motif does not appear to be important for catalysis.</p><p>In a third strategy to identify where leader peptide binding may be occurring, we turned to a photocrosslinking approach. Photoaffinity based identification of ligand binding sites has proven to be a powerful technique to obtain relatively high-resolution data of ligand contact sites, including in RiPP biosynthetic enzymes.79–80 We synthesized HalA2-LP analogs in which either Leu−13 or Leu−10 was replaced with photoleucine (L*). Photoleucine is a diazirine-containing leucine analog that upon photoirradiation will form a highly reactive carbene species that can crosslink to nearby atoms.81 Additionally, Gly−8 was replaced with propargylglycine (Pra), an alkyne containing glycine analog, to allow for click-chemistry based enrichment of crosslinked peptides.82 We performed photocrosslinking and streptavidin-based enrichment with three different HalM2 variants, HalM2(1–990), HalM2(1–649), and HalM2(36–990) in the presence or absence of AMP-PNP.</p><p>The crosslinked and affinity enriched peptides were analyzed by liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (LC-nESI-MS/MS) and characterized using KOJAK.83 This software application performs analysis of crosslinking mass spectrometry data to identify peptide-peptide crosslinks using spectral processing and database search algorithms.83 Analysis of the peptide fragments shared by all three HalM2 variants when crosslinked to HalA2PAL-L−13L* revealed multiple hot spots of leader peptide interaction. Residues 31–49 and 598–635 were captured in all samples (Figure 6c). Residues 31–49 belong to distinct loop regions of the capping helices (Figure 7b) and are one region observed to be protected in the HDX experiments in this work (Figure S7). The crosslinking data are also consistent with the fluorescence polarization results that indicated that the leader peptide binds to the N-terminal domain of HalM2. Intriguingly, the loop where crosslinking occurred is spatially adjacent to the loop previously identified by HDX-MS and biochemical studies.52 Notably, these loops lack electron density in the CylM crystal structure, suggesting that they are disordered and flexible.51 The crosslinks of Leu−13 to residues 598–635 map to regions of HalM2 located close to the dehydratase-cyclase interface. Although these regions are distal to the putative binding site on the capping helices, it is possible that upon ligand binding, the cyclase domain changes conformation, allowing for the substrate to access the cyclase active site. Indeed, the distance from the two loops of the capping helices to the cyclase active site is at least 60 Å (Figure 7). Because LanM proteins are monomers in solution,51 a significant conformational change would have to bring the cyclase active site closer to the capping helices for the core peptide to be able to reach it. The recent HDX-MS experiments with full length HalA2 substrate also suggest that ligand binding triggers conformational changes in the cyclase domain of HalM2.52 However, we cannot rule out that a secondary binding site exists on the interface of the domains that gave rise to the observed crosslinks to residues in the interface region. If so, the substrate would have to disengage from and re-engage with the enzyme when switching between dehydration and cyclization.</p><p>Photocrosslinking with HalA2PAL(L−10L*) further supports interactions with the capping helices, as crosslinks with the peptide spanning residues 31–49 were observed in all three enzyme variants tested and regardless of whether AMP-PNP was present or not (Figure 7). Analysis of each individual enzyme variant revealed crosslinked peptides to the additional regions also observed with HalA2PAL(L−13L*), but these peptides did not overlap to within five amino acids for each enzyme variant (Figure S10e–d). Based on these data, we propose that HalA2-LP likely engages the flexible loops on the capping helices of HalM2 for binding via hydrophobic interactions. The binding of HalA2-LP then triggers conformational changes to allow access of the substrate to both active sites. These movements are likely facilitated by the long, unstructured linkers between the two enzyme domains (Figure 7).</p><!><p>In this work, we provide new insights into the molecular mechanism by which class II lanthipeptide leader peptides are recognized. We identified the residues on the leader peptide of HalA2 that are involved in binding to its cognate lanthipeptide synthetase HalM2. As seen for the engagement of leader peptides to the RRE domain in class I lanthipeptide dehydratases, the interaction appears to be mediated by hydrophobic residues, but unlike class I these residues are in the C-terminal region of the leader peptide and appear to be part of a more extended binding interface. We used the identification of these residues to engineer processing of the non-cognate substrate CylLS by HalM2.</p><p>In terms of identification of the binding site on HalM2, we utilized in vitro binding assays of truncated proteins to determine that at least one binding site exists on the capping helices of the dehydratase domain. Furthermore, we performed HDX-MS and photocrosslinking to identify a loop on HalM2 that appears important for substrate recognition. These experiments are complementary to a recent study that came to similar conclusions based on HDX experiments but under different conditions from ours. Our photocrosslinking experiments also suggest that the cyclase domain undergoes conformational movement to become proximal to the site of substrate binding.</p><p>LanMs are substrate tolerant enzymes that have high potential for use in biotechnological applications. The tolerance with regards to variations in the core peptide of their substrates has been exploited for a variety of purposes, including understanding the mode of action of class II lanthipeptides19, 22, 30, 84 and generation of macrocyclic peptide libraries for selection against biological targets.25–27 The current work contributes to the understanding of the molecular aspects of leader peptide binding to the enzyme which is required to catalyze chemical transformations on the substrate. This knowledge may be applied to future work in developing these enzymes as biocatalysts for design of libraries of potentially therapeutic molecules.</p><!><p>Cloning and plasmid propagation were conducted in chemically competent NEBTurbo cells (New England Biolabs). Expression of recombinant peptides and proteins was performed in chemically competent BL21 Star (DE3) (Thermo Fisher Scientific). Primers and double-stranded DNA fragments were purchased from Integrated DNA Technologies. Media used for bacterial cultures were purchased from Fisher Scientific. Phusion DNA polymerase, and NEB HiFi Master Mix were purchased from New England Biolabs. Sanger sequencing of plasmids was performed by ACGT, Inc. Plasmid DNA was purified using QIAprep spin columns (Qiagen). HisTrap columns were purchased from GE Healthcare. Gels for SDS-PAGE were purchased from Bio-Rad Labs. Protein solutions were concentrated using Amicon Ultra Centrifugal filters (Sigma Millipore). Antibiotics were purchased from GoldBio. All other chemicals were purchased from Fisher Scientific unless otherwise noted. HalA2, HalM2, CylLS, and CylLL were expressed and purified as previously described.58, 69</p><!><p>All preparative HPLC was performed on an Agilent 1260 Infinity II preparative liquid chromatography system connected to a 1260 Infinity II VWD, and a 1260 Infinity II fraction collector. Peptides were purified on a Macherey-Nagel NUCLEODUR C18 HTec column (5 μm, 100 Å, 10 × 250 mm,) using 0.1% trifluroroacetic acid (TFA) in H2O (solvent A) and 0.1% TFA in CH3CN (solvent B) running at 4 mL/min. A gradient method was utilized consisting of the following steps: 2% B for 10 min, ramp to 100% B over 35 min, hold at 100% B for 5 min, and ramp to 2% B for 5 min.</p><!><p>Matrix-assisted laser desorption/ionization time of flight mass spectra (MALDI-TOF-MS) were recorded on a Bruker UltrafleXtreme instrument operating in positive-ion mode. Samples were prepared by desalting using C18 ZipTips (Millipore), and spotting with equal volume of a 25 mg/mL solution of Super-DHB matrix (Sigma) dissolved in 1:1 CH3CN/H2O. LC-ESI-qTOF MS and MS/MS spectra were recorded on a Waters Synapt G1 instrument with a Waters Acquity UPLC system equipped with a Waters BEH C18 (1.7 μm, 130 Å, 50 × 2.1 mm) column using 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in CH3CN (solvent B) as solvent system.</p><!><p>The general synthetic strategy for solid-phase peptide synthesis utilized NovaPEG Rink Amide LL (EMD Millipore) as the solid support. 1-Cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOxim; Chem Impex) was used as the activator for peptide coupling. Diisopropylethylamine (DIPEA; Sigma) was used as a base. 20% piperidine (Sigma) in dimethylformamide (DMF) with 0.1 M 1-hydroxybenzotriazole (HOBt; Chem Impex) was used as the deprotection solution. 0.5 M O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU; Chem Impex) in DMF was used as the activator for the initial amino acid loading. The first amino acid was coupled using the following procedure: 1 g of resin was swelled in 7.5 mL of DMF + 7.5 mL of dichloromethane (DCM) for 15 min. Solvent was removed by filtration, and 8 mL of deprotection solution was added to the resin followed by 1 h of N2 (g) agitation. The resin was washed 3 times with DMF, and 5 equiv. of Fmoc-protected amino acid were added with 5 equiv. of HCTU and 5 equiv. of DIPEA. Coupling was allowed to proceed at RT under N2 (g) agitation for 3 h. The resin was washed 3 times with DMF, and 5 mL of a 3:2 v/v solution of acetic anhydride/pyridine was added to the resin. Capping was allowed to proceed with N2(g) agitation for 30 min. The resin was washed three times with DMF, followed by three times with DCM. The resin was then used on a CEM Liberty Blue automated microwave-assisted peptide synthesizer (CEM Corporation). Unless otherwise noted, double coupling was performed for all amino acids. Peptides were cleaved and deprotected using ~12 mL of global cleavage cocktail (95:5:5 of TFA/H2O/triisopropylsilane). After 2 h of cleavage, the suspension was filtered through glass wool and solvent was evaporated under a N2 (g) stream. The concentrated solution was added dropwise to cold diethyl ether to precipitate the peptide. The peptide suspension was centrifuged at 4,000xg for 3 min, the ether was discarded, and the pellet was washed three times with fresh, cold diethyl ether. Solvent was removed in vacuo using a SpeedVac evaporator system. The crude peptide was dissolved in 25% CH3CN/H2O and subjected to further HPLC purification.</p><!><p>Genes encoding Hisx6-HalM2(36–990), Hisx6-HalM2(1–649), and Hisx6-HalM2(1–348) were generated using site-directed ligase-independent mutagenesis (SLIM) according to published protocols.85 Briefly, four primers were generated: forward base (FB) and reverse tail (RT) primers, and reverse base (RB) and forward tail (FT) primers. The tail primers have a 5' overhang sequence that shares homology to the protein region that is included in the truncant. Two 25 μL PCR reactions were performed with either the FB+RT primers or RB+FT primers and 5 ng of template pET15b-HalM2 using Phusion DNA polymerase. The PCR was checked by loading 5 μL of the reaction onto a 1% agarose gel for gel electrophoresis. To set up the hybridization: 10 μL of each PCR reaction was mixed with 10 μL of H2O and 10 μL of 5X hybridization buffer (750 mM NaCl, 125 mM Tris pH 8.0, 100 mM EDTA). The DNA mixture was hybridized by thermocycling using the following protocol: initial denaturing for 3 min at 99 °C, 3 cycles of 65 °C for 5 min and 30 °C for 40 min. NEBTurbo cells were transformed with 5 μL of the hybridized DNA, and positive colonies were selected for by plating on LB agar + ampicillin. All plasmids were confirmed by Sanger sequencing.</p><!><p>Terrific Broth (TB; 1.5 L) supplemented with 100 μg/mL ampicillin was inoculated at 1:100 dilution with overnight cultures of BL21 (Star) cells transformed with the desired plasmid. The cultures were grown at 37 °C with shaking to an OD600 of 0.8. The culture was cooled for 15 min by placing the flasks on ice. The cells were induced by addition of 0.3 mM IPTG and incubated at 18 °C for an additional 16 h. The cells were harvested by centrifugation (6,000xg for 12 min), and the pellets were stored at −70 °C. The frozen cell pellets were thawed and resuspended in 30 mL of Lysis Buffer (20 mM Tris pH 7.5, 0.5 M NaCl, 10% glycerol). The cells were lysed by sonication. The lysate was centrifuged at 25,000xg for 45 min. The clarified lysate was filtered through a 0.45 μm syringe filter and applied to a pre-equilibrated HisTrap 5 mL column. After loading the lysate, the column was attached to an ÄKTA Pure chromatography system using the following buffer system: Buffer A (20 mM Tris pH 7.5, 0.5 M NaCl) and Buffer B (20 mM Tris pH 7.5, 0.5 M NaCl, 0.5 M imidazole). The column was washed with 5 column volumes (CV) of 5% B, followed by a linear gradient to 100% B over 15 CV with a flow rate of 2.5 mL/min and monitoring protein elution at A280. The fractions were screened by 4–20% SDS-PAGE, and the fractions containing the purest protein were pooled. The pooled sample was concentrated using Amicon (100 kDa MWCO) and applied onto a Superdex 200 HiLoad 16/60 size exclusion column (GE Healthcare) pre-equilibrated in GF Buffer (50 mM HEPES pH 7.5, 30 mM KCl, 5% glycerol). The column was run at 1.0 mL/min, and A280 was monitored for protein elution. The fractions containing the highest purity protein as determined by SDS-PAGE were pooled, concentrated by Amicon, and the concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher) with bovine serum albumin standards. The protein solution was aliquoted, flash-frozen in N2 (l), and stored at −70 °C.</p><!><p>E. coli BL21 (Star) cells were transformed with pET15b-HalA2LP,58 and a single colony was used to inoculate a starter culture in LB + 100 μg/mL ampicillin. Terrific Broth (TB; 1.5L) supplemented with 100 μg/mL ampicillin was inoculated with 1:100 dilution of overnight starter culture. The cultures were grown at 37 °C with shaking to an OD600 of 0.8. The culture was cooled for 15 min by placing the flasks on ice. The cells were induced by addition of 1 mM IPTG and incubated at 18 °C for an additional 16 h. The cells were harvested by centrifugation (6,000xg, 12 min), and the pellets were stored at −70 °C. The frozen cell pellets were thawed and resuspended in 45 mL of Buffer B1 (20 mM NaH2PO4 pH 7.5, 0.5 M NaCl, 0.5 mM imidazole, 6 M guanidine HCl). The cells were lysed by sonication. The lysate was centrifuged at 25,000xg for 45 min. The clarified lysate was filtered through a 0.45 μm syringe filter and applied to a pre-equilibrated His60 Ni Superflow resin (3 mL bed volume; Clontech). The resin was washed with 15 mL of Buffer B2 (20 mM NaH2PO4 pH 7.5, 0.3 M NaCl, 30 mM imidazole, 4 M guanidine HCl). The resin was washed a second time with 15 mL of Wash Buffer (20 mM NaH2PO4 pH 7.5, 0.3 M NaCl, 30 mM imidazole). The peptide was eluted with 10 mL of Elution Buffer (20 mM NaH2PO4 pH 7.5, 0.3 M NaCl, 1 M imidazole). The peptide was desalted by ZipTip and analyzed by MALDI-TOF MS. The peptide was further purified by acidification to 2% TFA (v/v), filtration through a 0.22 μm syringe filter, and subjected to HPLC purification using the method described above. HPLC fractions were confirmed to have the proper mass by MALDI-TOF MS and were lyophilized to dryness. The dried peptides were dissolved to 10 mM in ddH2O.</p><!><p>The spectra were recorded using a J-715 Circular Dichroism Spectropolarimeter from JASCO Inc. (Easton, MD). A quartz cuvette (Starna Cells Inc., Atascadero, CA) with a path length of 0.1 cm was used for the measurements. The temperature was held at 20 °C using a Peltier element. The CD spectra were acquired using a 100 nm/min scan speed and averaged across three spectra. All peptides were dissolved in H2O at a concentration of 100 μM. The data was processed using CAPITO (https://capito.uni-jena.de/) and plotted in Origin 2019b.</p><!><p>HalM2 assay buffer was composed of 20 mM HEPES pH 7.5, 5.0 mM MgCl2, 1.0 mM ATP, 0.5 mM TCEP, and 100 μM of HalA2 precursor peptide. The reaction was initiated by addition of 5.0 μM enzyme. The reaction was incubated at 25 °C for 18 h, then quenched with addition of 1% TFA. The reaction mixture was desalted using C18 ZipTips and analyzed by MALDI-TOF MS.</p><!><p>HalM2(1–990) and HalA2-LP were used for HDX experiments. In the unbound state, HalM2 was diluted into Tris buffer (10 mM Tris, 100 mM KCl, pH = 7.4) to a final concentration of 40 μM. In the HalA2-LP bound state, a stock solution containing 40 μM HalM2 and 560 μM HalA2-LP (1:14 mixing ratio) was prepared 12 h before the HDX experiment to allow equilibration. The D2O solution (Cambridge Isotope Lab, Tewksbury, MA) was prepared to match the buffer condition of the protein stock solution. The quench solution contains 4 M urea (Sigma-Aldrich, St. Louis, MO) at a pH adjusted to 2.5. The HDX experiments were executed on a manual platform as previously.50 The HDX was initiated by diluting 2 μL of protein solution into 18 μL of D2O buffer (to achieve 90% D) for exchange. Exchanges at 4 °C were executed in triplicate with exchange times of 10 s, 30 s, 60 s, 900 s, and 7200 s, whereas HDX at 25 °C were in duplicate at 10 s, 30 s, 60 s, 900 s, 3600 s, and 7200 s. The HDX was quenched by adding 30 μL quenching solution and incubating at 25 °C for 10 s. The quenched solution was then submitted to a custom-packed column containing immobilized pepsin for digestion at a flow rate of 200 μL/min with water containing 0.1% trifluoroacetic acid. The digested peptides were captured by a ZORBAX Eclipse XDB C8 column (2.1 mm × 15 mm, Agilent Technologies, Santa Clara, CA). After 3 min digestion and desalting, the flow was switched to an LC gradient: 15 min, 5% phase B to 50% phase B in 11 min, followed by a sharper increase to 100% B at 11.5 min, kept at 100% until 13 min, and completed with 5% B for the last 2 min. Phase A was water (with 0.1% formic acid), and phase B was 80% acetonitrile (with 0.1% formic acid). The LC gradient transferred the digested peptides to a C-18 column (XSelect CSH C18 2.5 μm, 2.1 × 50 mm, Waters, Milford, MA) for separation and admission to the mass spectrometer for analysis. The data were collected on an LTQ-FT mass spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with a positive-ion electrospray ionization source. The valves, columns, tubing for desalting, and HPLC separation were submerged in an ice-water bath to minimize back-exchange. The pepsin column was at room temperature for effective digestion.</p><p>Prior to HDX, peptide mapping was by MS/MS, and peptides were identified by Byonic (Protein Metrics, San Carlos, CA) and processed with Byologic (Protein Metrics). Peptide lists together with the raw data were submitted to HDExaminer (Sierra Analytics, Modesto, CA) for analysis. The deuterium uptake for each peptide was manually inspected to insure correct assignment.</p><!><p>L-photoleucine (0.1 g, 0.7 mmol; Iris Biotech GmbH) was dissolved in 1.7 mL of H2O and 3.3 mL of 1,4-dioxane. NaHCO3 (1.9 equiv., 112 mg) was added to the reaction vessel. Fmoc-OSu (1.2 equiv., 283 mg; Chem Impex) was suspended in 0.5 mL of 1,4-dioxane and added dropwise to the reaction vessel. The reaction vessel was covered with foil, and the reaction was allowed to proceed overnight at RT. The reaction was quenched by adding a solution of 2 M aqueous HCl dropwise to adjust the pH ~ 3. The reaction mixture was extracted three times with ethyl acetate, the organic layer was washed with brine, and dried over anhydrous MgSO4. The solvent was removed in vacuo yielding a colorless oil, which was purified by flash chromatography using a 5% MeOH/DCM + 0.01% acetic acid solvent system. The solvent was removed under reduced pressure to yield Fmoc-L-photoleucine (0.24 g, 89% yield) as a colorless oil. 1H NMR (500 MHz, CD3OD): δ = 7.8 (d, 2H), 7.72 (m, 2H), 7.40 – 7.29 (m, 4H), 4.43 – 4.42 (m, 3H), 4.14 – 4.10 (m, 1 H), 2.02–1.98 (m, 2H), 1.05 (s, 3H).</p><!><p>Synthesis of HalA2LP(−40 to −8) and respective alanine replacement analogs were synthesized using NovaPEG Rink Amide LL resin loaded with Fmoc-Gly at 0.1 mmol scale. Each peptide was synthesized, cleaved, and purified by HPLC using the protocols listed above. Synthesis of HalA2PAL(L−13pL) and HalA2PAL(L−10pL) was conducted using a similar procedure with the following modifications: Fmoc-propargylglycine (Chem Impex) was loaded onto NovaPEG Rink Amide LL resin, as described in the general SPPS section, and Fmoc-photoleucine was coupled at a 0.05 mmol scale.</p><!><p>HalA2LP was subject to selective N-terminal labeling with fluorescein using the following protocol. NHS-Fluorescein (Thermo Fisher) was dissolved to 10 mg/mL in DMF. HalA2LP was diluted to 1 mg/mL in 1 mL in 0.1 M NaH2PO4 buffer pH 6.1, then 1 equiv. of NHS-Fluorescein solution was added to the reaction. The reaction was allowed to proceed at RT for 2 h in the dark. The reaction was acidified by adding 1% TFA (v/v), filtered through 0.45 μm filter, and purified by HPLC. Single-labeled product was isolated and confirmed to be N-terminally labeled by MS/MS fragmentation.</p><!><p>NMR spectra were acquired on an Agilent VNMRS 750 MHz narrow bore magnet spectrometer equipped with a 5 mm HCN triaxial gradient probe. The sample was prepared by initially buffer exchanging purified HalM2 into PBS pH 6.8 by Amicon. HalA2LP (1 mM) and HalM2 (10 μM) were mixed together in a final volume of 550 μL in 10% D2O / PBS. All acquisitions were performed at a fixed temperature of 25 °C. Standard Varian pulse sequences were used for 1H (8 scans, WATERGATE solvent suppression), 1H 1D Saturation Transfer Difference experiment with DPFGSE sculpted solvent suppression (dpfgse_satxfer, VNMRJ4.2A Biopack), 1H-1H TOCSY (70 ms mixing time, 16 nt per t1 with 300 t1 increments, WATERGATE solvent suppression), 1H-1H NOESY (400 ms mixing time, 32 nt per t1 with 300 t1 increments, WATERGATE solvent suppression). For STD-NMR, phase cycling was utilized to perform subtraction between on-resonance and off-resonance spectra. For on-resonance spectra, the selective saturation was set to 0.0 ppm via a train of Gaussian soft pulses with a 50 ms duration each followed by a delay of 100 μs. 60 selective Gaussian pulses were used for a total saturation time of 3 s, and off-resonance irradiation was set to 30 ppm, with a total of 2048 scans. 1H-1H TOCSY and 1H-1H NOESY data was processed in NMRPipe (NIST) and chemical shift assignment was performed using NMRViewJ 9.2.0 software (One Moon Scientific). The STD intensity was normalized to the number of protons per signal.</p><!><p>FP experiments were conducted as previously described58 with the following changes: HalM2 was serially diluted in GF buffer, then added to FP buffer (50 mM HEPES pH 7.5, 30 mM KCl, 5% glycerol, 1 mM MgCl2, 1 mM TCEP, 0.25 mM AMP-PNP, 0.0025% IGEPAL-CA630, 10 nM Fluor-HalA2LP). The sample was allowed to incubate at RT for 15 min, then the parallel and perpendicular fluorescence intensities were measured. All experiments were conducted in triplicate in 386-well solid black polystyrene microplates (Corning) on a Synergy H4 Hybrid plate reader (BioTek). Data analysis was performed using Origin 9.6. Polarization was calculated using the following formula: P = (I|| - I⊥) / (I|| + I⊥). Competition FP assays were conducted using serially diluted HalA2LP analogs in GF buffer. Each dilution was mixed with FP Buffer + HalM2 (14 μM). Curves were fit to either non-linear dose-response or hyperbolic function. Ki was calculated using the following equation: Ki = IC50 / (1 + [L]/Kd) where [L] = 10 nM Fluor-HalA2LP.</p><!><p>HalA2 leader peptide mutants: Δ(−44 to −34), Δ(−34 to −24), Δ(−24 to −14), and Δ(−14 to −4)were prepared using the SLIM methodology as described above using pRSFDuet-HalA2(MCSI)-HalM2(MCSII) as template. The numbering of the mutants corresponds to universal RiPP nomenclature, where the C-terminal residue of the leader peptide is termed the −1 position, and the numbering counts backwards towards the N-terminus (i.e. the residue directly N-terminal to the −1 position is −2). Similarly, the N-terminal residue of the core peptide is termed +1, and the numbering counts forwards towards the C-terminus.1 The gene encoding HalA2(L−24A/F−22A/V−18A/L−13A/L−10A) was ordered as a gBlock and was inserted into MCSI of pRSFDuet-HalM2(MCSII) using Gibson assembly. Similarly, CylLS(P−23L/L−18V/E−14L/M−13L/I−10L) was ordered as a gBlock and inserted into MCSI of pRSFDuet-HalM2(MCSII) by Gibson assembly.</p><!><p>E. coli BL21 (Star) cells were transformed with the respective co-expression plasmid, and a single colony was used to inoculate a starter culture in LB + 50 μg/mL kanamycin. A flask containing 50 mL of TB + 50 μg/mL kanamycin was inoculated using a 1:100 dilution of the overnight starter culture. The culture was grown at 37 °C with shaking until OD = 0.8, then the culture was cooled on ice for 15 min, induced with 0.3 mM IPTG and shaken for 24 h at 18 °C. The cells were harvested by centrifugation at 4500xg for 15 min. The pellet was resuspended in 3 mL of Buffer B1, lysed by sonication, and centrifuged at 25,000xg for 45 min. The clarified lysate was filtered through a 0.45 μm syringe filter and applied to a pre-equilibrated His60 Ni Superflow resin (1 mL bed volume; Clontech). The resin was washed with 5 mL of Buffer B2 4. The resin was washed a second time with 5 mL of Wash Buffer. The peptide was eluted with 1 mL of Elution Buffer. The peptides were desalted using C18 ZipTips and analyzed by MALDI-TOF MS.</p><!><p>HalM2(1–990), HalM2(36–990) and HalM2(1–649) were diluted to 2.0 mg/mL (17.3, 18.1, and 26.2 μM, respectively) solution in XL buffer (50 mM HEPES pH 7.5, 30 mM KCl, 1 mM MgCl2, 0.25 mM AMP-PNP) in a final volume of 50 μL. HalA2PAL(L−13pL) or HalA2PAL(L−10pL) were added in 5-fold molar excess to protein (86.7, 90.5, and 131 μM, respectively). The mixture was incubated on ice for 30 min. The solution was then irradiated with 365 nm light using a B-100AP/R light source (UVP) for 15 min on ice. Then, 15 μL of a 1 mg/mL solution of Glu-C (NEB) was added to each sample and the samples were incubated at 37 °C for 4 h. The samples were then heated to 95 °C for 10 min. After cooling to room temperature, 60 μL of the sample was transferred to a fresh 1.5 mL Eppendorf Protein LoBind microcentrifuge tube and subjected to copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). CuSO4 (0.2 mM), TCEP (5.0 mM), and BTTES (1.0 mM; Click Chemistry Tools) were added to the sample and incubated at 25 °C for 5 min. DADPS-Biotin-Azide (0.2 mM; Click Chemistry Tools) was added to each sample to initiate the CuAAC reaction. The reaction was allowed to proceed at 25 °C with shaking for 2 h. Then, 25 μL of pre-equilibrated Pierce Streptavidin Plus UltraLink Resin (Thermo Fisher) was added to each reaction, and the sample was incubated overnight at 4 °C. The resin was washed by centrifuging at 4,000xg for 1 min, discarding the supernatant, adding 200 μL of PBS, and repeating this procedure 10 times. The resin was further washed 2 times with ddH2O. The washed resin was then subjected to cleavage of the linker to biotin by addition of 100 μL of 5% formic acid / H2O, followed by incubation at 25 °C with shaking for 1 h. The elution was collected by centrifugation and the resin was incubated a second time with 100 μL of 5% formic acid / H2O. The eluants were combined and lyophilized prior to mass spectrometry analysis.</p><!><p>Dried peptide samples were resuspended in 0.1% formic acid (FA) in 5% acetonitrile (ACN) and injected into an Ultimate 3000 UHPLC system (Thermo Scientific). The peptides were subjected to reversed phase separation using a 15 cm Acclaim PepMap 100 C18 column (2 μm particle size) with a linear gradient of 5% ACN in 0.1% FA to 35% ACN in 0.1% FA; this 45 min gradient was followed by column washing and regeneration. Detection was accomplished using a Thermo Orbitrap Fusion mass spectrometer operated in data dependent mode with a cycle time of 3 s. Precursor scans from 350 to 1600 m/z (120 k resolution, 2e5 AGC) were acquired in the orbitrap; these scans were followed by collision induced dissociation (35% NCE) of the most abundant species and detection of fragment ions in the ion trap. The isolation window and dynamic exclusion time were set to 1.6 m/z and 60 s, respectively.</p><!><p>Thermo *.raw files were converted to *.mzML using MSConvert (Proteowizard). The *.fasta file was defined for HalM2 and HalA2PAL(L−13pL) or HalA2PAL(L−10pL) with the following custom amino acids: J = photoleucine, Z = propargylglycine-DADPSlinker after acidic cleavage. In addition, the C-terminal amidation of the synthetic photoaffinity-labeled peptide was defined. The crosslink analysis was performed using KOJAK (Institute for Systems Biology, Seattle, WA). The following parameters were defined to account for nitrogen loss from photoleucine J, C-terminal amidation, and the of structure Z: cross_link = J CDEFGHIKLMNPQRSTVWY −28.006148 PhLeu; modification_protC = c −0.98401558; enzyme = [ED] | {P} GluC; aa_mass = J 125.0589119; aa_mass = Z 256.1535405; decoy_filter = reverse; ppm_tolerance_pre = 10.0. Reversed sequences were used as input for the decoy database search. All samples were acquired in duplicate. Only intermolecular crosslinked peptides that were identified in both replicates were kept for further analysis. For each identified crosslink tandem MS analysis identified fragment ions for each of the two peptides (one from HalA2 and one from HalM2). The MS-MS analysis also identified on which amino acid of the peptide crosslinking occurred. See the Supplemental file on KOJAK analysis for the crosslinks observed. For binning of two crosslinked peptides within the HalM2 sequence (Figure 6 main text), when the position of the crosslink on peptide A was within ± 5 residues of peptide B the two peptides were combined in one bin. Additionally, only crosslinked peptides that contained at least three fragment ions in both peptide A and peptide B were used in the analysis. Peptides shared between HalM2(1–990), HalM2(36–990), and HalM2(1–649) were defined as a true crosslink.</p>

PubMed Author Manuscript

A Well-balanced Force Field ff03CMAP for Folded and Disordered Proteins

Molecular dynamics simulation as an important complement of experiment is widely used to study protein structures and functions. However, previous studies indicate that current force fields cannot, simultaneously, provide accurate descriptions of folded proteins and intrinsically disordered proteins (IDPs). Therefore, a CMAP optimized force field based on the Amber ff03 force field (termed ff03CMAP herein) was developed for balanced sampling of folded proteins and IDPs. Extensive validations of short peptides, folded proteins, disordered proteins, and fast-folding proteins show that simulated chemical shifts, J-coupling constants, order parameters, and residual dipolar couplings with the ff03CMAP force field are in very good agreement with NMR measurements and are more accurate than other ff03-series force fields. The influence of solvent models was also investigated. It was found that the combination of ff03CMAP/TIP4P-Ew is suitable for folded proteins and that of ff03CMAP/TIP4PD is better for disordered proteins. These findings confirm that the newly developed force field ff03CMAP can improve the balance of conformer sampling between folded proteins and IDPs.

a_well-balanced_force_field_ff03cmap_for_folded_and_disordered_proteins

Introduction<!>Molecular Dynamics Simulations.<!>Benchmark of PDB Coil Structures.<!>CMAP Method.<!>Quantification in the Evaluation for Force Fields.<!>Calculation of Experimental Observables.<!>CMAP Optimization.<!>Evaluation of ff03-series Force Fields.<!>Short Peptide Ala5.<!>Folded Proteins.<!>Intrinsically Disordered Proteins.<!>Ab Initio Folding of Fast-Folding Proteins.<!>Conclusion

<p>Proteins can exist in three states, folded, molten globule, and random coil.[1] Folded proteins are easier to study because they are ordered and stable. But disordered proteins also need exploring. In eukaryotes, more than 30 percent of proteins contain disordered regions with more than 50 consecutive residues.[1] Proteins with disordered regions or overall intrinsically disordered proteins (IDPs) have been proved to have important biological functions, such as molecular recognition, molecular assembly, protein modification, and so on.[2] Furthermore, IDPs are associated with many human diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, cancer, and cardiovascular disease, to name a few.[3–4] IDPs are more flexible and unstable with little secondary structures than structured or ordered proteins. "Intrinsically disordered" implies a sequence-dependent nature in IDPs that trend to be lack of ordered structures.[5] Many experimental methods have been utilized to study IDPs, such as electron paramagnetic resonance (EPR), X-ray diffraction, Nuclear Magnetic Resonance (NMR), and small-angle X-ray scattering (SAXS).[6–8]</p><p>Because of their important biological functions, IDPs have become common topics in molecular dynamics studies in recent years. Due to limited accuracy in standard protein force fields, a set of special-purpose force fields have been developed for simulating IDPs, such as ff99IDPs, ff14IDPs, ff14IDPSFF, ff03ws, RSFF2, a99SB-disp, CHARMM36IDPSFF and so on.[9–15] In addition, the D. E. Shaw group also modified the dispersion interaction of TIP4P water model (TIP4P-D) to improve the quality of IDPs simulations.[16] However, it remains elusive to reach a good balance between ordered and disordered states with either standard or special-purpose force fields.</p><p>ff03 is a new-generation Amber force field that has become widely used in biomolecular simulation studies.[17] Based on ff03, the Best group modified backbone dihedral potentials in the context of the TIP4P/2005 water model. Their efforts lead to three ff03 variants: ff03*, ff03w, and ff03ws.[12, 18–19] These modifications were shown to partially improve the performance of conformer sampling of IDPs and folded proteins. The effect of solvent model was found to be important in the sampling of IDPs in their studies.[12, 18–19] TIP3P is the most commonly used water model in earlier force fields.[20] Improvement of water models has always been concurrent with protein force field developments. Besides TIP4P-D, TIP4P-Ew and TIP4P/2005 are also two commonly used four-site water models and should be investigated in any force field improvement efforts.[21–22] These four-site water models have been found to reproduce well the hydrophobic effect and water density in a wide temperature range.</p><p>In this development, we systematically analyzed the original ff03 force field, its published variants, and explored a new variant based on the CMAP method, ff03CMAP. The combinations of force fields and corresponding solvent models are ff03 with TIP3P, ff03* with TIP3P, ff03w with TIP4P/2005, ff03ws with TIP4P/2005 as in the published efforts. Based on our analyses, we recommend the combinations of ff03CMAP with TIP4P-Ew and TIP4P-D, respectively, suitable for folded proteins and IDPs.[12, 17–19] In order to evaluate the performance of these force fields, 16 proteins were simulated to probe the quality of various force field/water model combinations in reproducing experimental measurables. The tested short peptides and proteins are shown in Figure 1.</p><!><p>Initial structures were built by the LEaP module in the AMBER 14 suite if not available,[38] which was also used to conduct MD simulations.[39] All systems were neutralized and solvated in boxes of different water models.[20] All bonds involving hydrogen atom were constrained with the SHAKE algorithm.[43] Particle Mesh Ewald (PME) algorithm was used to calculate long-range electrostatic interactions.[41] Initial structures were relaxed with 10000 steps of minimization, then subject to heating up for 20ps, and equilibration for 10ps in the NPT ensemble with PMEMD. The CUDA version of PMEMD was used to accelerate the simulations.[42] The simulation temperature and ion strength were set according to their respective experimental conditions. The number of replica-exchange molecular dynamics (REMD) replicas and temperatures were set by an online temperature predictor for parallel tempering simulations.[40] All simulation conditions are shown in Table S1.</p><p>Insert a table for all tested force field water combinations.</p><!><p>Coil database was built and extracted from PDB. The DSSP program was utilized to classify the secondary structures and extract main chain dihedrals from these structures.[44–45] 2,611,450 amino acids without secondary structure were collected. The counts of amino acids in the coiled database are shown in Figure S1. The Ramachandran plots for the database were used as the benchmark for the optimization of dihedral distribution.</p><!><p>Grid-based energy correction maps (CMAP) is a useful method for automatically correcting dihedral distribution for the additive force field, which is based on the backbone dihedral distribution and has been used to develop IDP-specific force fields.[10, 46] CMAP was first published to modify the CHARMM force field and was transferred into Amber software.[9–11, 47–50] We minimized the main-chain dihedral distribution differences between the MD simulation and the benchmark for each of the 20 amino acids. A 576-(24 × 24) grid was used to cover the phi/psi map. The tetrapeptide models (Nme-Ala-X-Ala-Ace, where X represents one of 20 naturally amino acids, Nme for aminomethyl, and Ace for acetyl) were utilized in the CMAP optimization via MD simulations in the TIP4PEw water model. Ten cycles of CMAP optimization were conducted to minimize the distribution differences between the MD simulation and the benchmark. In each cycle, the solvated tetrapeptides were simulated for 200 nanoseconds. After the CMAP optimization, we added an additional structural factor which is the partial dihedral energy distribution of 'S' fragments predicted by DSSP in the PDB coil database to avoid overestimation of disordered state when using the new force field. Root-mean-square deviations of population (RMSp) is calculated to compare the difference between the MD simulation and the benchmark with equation (1). (1)RMSp=∑i=1576(PiDB−PiMD)2576 where PiDB is the population of the i-th grid in database benchmark and PiMD is the population of the i-th grid in the MD simulation.</p><!><p>To quantitatively compare different ff03 variants for folded proteins and disordered proteins with experimental measurements, we utilized the normalized force-field score.[14] For folded proteins, equation (2) was used to calculate averaged normalized RMSD from each class of experimental data as (2)FoldedProteinFFScore=1N∑i=1NFFrmsdrmsdNorm where N is the number of classes of experimental measurements, FFrmsd is the RMSD of the i-th class for simulated and experimental values, and rmsdNorm is the lowest RMSD of i-th class in all force fields. According to this metric, FFScore is always greater than or equal to 1, and 1 is the best score theoretically which means this force field perfectly reproduced the experimental data. For disordered protein, we divided the experimental measurements into two groups because there are fewer experimental measurements than the folded proteins, for which there are chemical shifts and other NMR measurements. If experimental data for both chemical shifts and NMR measurements are available, FFScore is calculated with equation (3). (3)DisorderedProteinFFScore=CSScore+NMRScore2 and if there is only chemical shift, the FFScore is calculated with equation (4). (4)DisorderedProteinFFScore=CSScore Where CSScore and NMRScore are calculated as same as the score of class in folded protein FFScore.</p><!><p>Backbone chemical shifts were calculated by SHIFTX2 for Cα, Cβ, C, N, Hα and HN atom types.[51] Backbone scalar coupling constants were calculated using published Karplus relations for 3JHNHα, 3JHαC, 3JHNC, 3JCC, 3JHNCα, 3JHNCβ, 2JCαN, 1JCαN, 1JHαCα, and 1JCαCβ[52–60] and side-chain scalar coupling constants with Karplus relations for 3JCCγ and 3JNCγ.[61] Backbone RDCs were calculated using PALES with a local alignment window of 15 residues.[62–63] Backbone amide and side-chain methyl axis S2 order parameters were calculated with the direct method described in Trbovic et al.[64] Small angle X-ray scattering (SAXS) curves were calculated using the FoXS package.[65] Cα RMSD and radius of gyrations (Rg) were calculated using CPPTRAJ in AmberTools.[38] Conformational clustering was performed with the kClust program in the MMTSB tool.[66] MDTraj, a python package, was also used for miscillenous calculations.[67] The PyMOL molecular visualization system was used to show 3D structures for all proteins.[68] All experimental measurements are listed in Table S2.</p><p>Biphasic exponential decay model was used to analyze the IDPs sampling convergence for force fields. The equations (5) and (6) were used to calculate the fitting half-time.[71] (5)ΔCαChemicalShift(Nt)=A1e−(xτ1)+A2e−(xτ2)+N0 (6)t1/2=τln(2) where N0 is the plateau of an observable, t1/2 is the half-life time, A and τ are constants. In this model, the decay consists of two stages for fast stage and slow stage. The slower τ2 value was utilized to calculate the slow stage half-life and evaluate the convergence rate of IDPs simulation.</p><!><p>Ten cycles of CMAP optimization were performed for each amino acid. In the first cycle (CMAP0), the initial φ/ψ distribution was obtained from the standard ff03 force field, where the lowest RMSp is 0.234% among 20 amino acids. In contrast, after 10 cycles of optimization, the lowest RMSp is less than 0.064%, as shown in Figure S2. Comparison of the distributions of CMAP0 and the benchmark database, we found that there is almost no left-handed helix distribution except MET, GLY and LEU. In addition, an obviously energy barrier exists between the β-sheet region and the α-helix region, so it would be difficult to sample both types of structures. After optimization, these limitations no longer present. The parameters for the best RMSp for each amino acid were selected as the final CMAP values. These parameters and structural factors were integrated with standard ff03 force field to generate the new force field ff03CMAP.</p><!><p>We assessed the performance of ff03-series force fields in reproducing experimental data. The same conditions were used in all MD simulation among all tested force fields. The FF scores for short peptides, IDPs, and fold proteins are shown in Figure 2 and specific values are listed in supplementary Table S3. Figure 2 suggests that combination ff03CMAP/TIP4PD agrees the best with experiment for short peptides and IDPs. In addition, combination ff03CMAP/TIP4PEw leads to the best agreement with experiment for folded proteins. In summary, use of ff03CMAP can yield more accurate sampling the conformers for short peptide, IDPs and folded proteins.</p><!><p>Table 1 shows the RMSD's of secondary chemical shifts, J-coupling constants and force field score for Ala5. There are 6 types of secondary chemical shifts and 7 types of J-coupling constants. For the CS score, the performance of ff03CMAP/TIP4PEw are much better than all other force fields. However, the performance of ff03*/TIP3P is the best for the NMR score. If we combine CS and NMR scores (i.e. overall FF score), ff03-drived force fields are significantly improved over the origin ff03 and the ff03CMAP/TIP4PEw is the best. The detail RMSD's of secondary chemical shifts and J-coupling constants are shown in Figures S3–S4.</p><!><p>In order to evaluate the stability of folded proteins when modeled with ff03CMAP, three representative folded proteins were simulated: CspTm (all-β), ubiquitin (α/β) and SPR17 (all-α). The initial structures are extracted from PDB and the simulation time is 1 μs for each system.</p><p>Table 2 shows the FF scores of the three tested proteins for all ff03-series force fields. It is obviously that the FF score for combination ff03CMAP/TIP4P-Ew is the best among all tested force fields and the value close to 1. This suggests that ff03CMAP/TIP4PEw indeed can be used to simulate folded proteins. And we also found that the original ff03 force field performs better performance than other revised ff03 force fields. It is no surprise that the performance of ff03CMAP/TIP4PD is a little worse than that of ff03CMAP/TIP4PEw because TIP4P-D water model would destabilize the folded states of proteins as reported.[16] The details of the FF score composition for the three proteins are shown in Tables S4–S6.</p><p>To quantify the fluctuation in simulations, Cα RMSD's of the folded proteins are shown in Figure 3 [Can you only compute the RMSDs of the secondary structures/i.e. stable portions of the proteins?]. For CspTm and ubiquitin, the RMSDs in the ff03CMAP/TIP4PEw simulations are small and stable, which is consistent with the FF score. However, the RMSD's for SPR17 rise over 4Å after 700ns in the ff03CMAP/TIP4PEw simulation. The RMSDs of ff03ws for all three folded proteins fluctuate quite significantly, implying less stable folded states for the tested proteins.</p><p>More detailed analyses were conducted for ubiquitin to compare the performances of these force fields. Secondary chemical shifts and backbone scalar coupling constants of ubiquitin (Figures S6–S7) suggest that ff03CMAP/TIP4PEw simulation agree the best with experimental data. The same can be said for the side-chain scalar coupling constants as shown in Tables S16–S17. RDCs of backbone N-HN, Cα-Hα, Cα-C, C-N and C-HN were also calculated shown in Figure S8. Similar to chemical shifts and scalar coupling constants, the performance of ff03CMAP/TIP4PEw simulation also agrees among the best, along with the ff03 simulation, while the ff03ws simulation agrees the worse. The order parameters for backbone amide and side-chain methyl groups are shown in Figure 4 and Table S18, respectively. The order parameters in the ff03CMAP/TIP4PEw simulation are also in good agreement with experimental data. The figure further indicates that the order parameters of the loop regions in the ff03CMAP/TIP4PD simulation are much lower than those from other force fields. [What is the point of the following statement?]Only ff03CMAP/TIP4PEw exhibited similar behavior to experiment for side-chain order parameters (Table S18?).</p><p>The RMSDs of secondary chemical shift, J-coupling, order parameters, and RDCs are gathered in Table 3. The summary indicates that ff03CMAP/TIP4PEw performs excellently in in reproducing all available experimental measurements and its FF score is very close to 1. While the FF score of ff03CMAP/TIP4PD is more than 1.5, which suggests TIP4P-D water model unsuitable for the simulation of folded proteins.</p><p>In order to further evaluate the stability of ff03CMAP for folded proteins, the dominant conformers of ubiquitin from six ff03-series force fields are retrieved and shown in Figure 5. It was found that top 3 clusters in the ff03 simulation occupy 100.00% of the snapshots. All the conformers include high percentage of helical structures. Top 5 clusters in the ff03* simulation also occupy 100.00% of the snapshots with partially non-helical structures. In the ff03w simulation, only 2 clusters were found, and the conformers are highly structured. In the ff03ws simulation, top 8 clusters only occupy 78.70% of the snapshots. In the ff03CMAP/TIP4PEw simulation there is only one cluster and 4 clusters in the ff03CMAP/TIP4PD simulation. Additional conformation clustering was also conducted for the CspTm and SPR17 simulations (supplementary Figures S24–S25). These conformer clusters indicate that ff03CMAP/TIP4PEw, ff03, and ff03w may be the better ff03 choices for folded proteins MD simulation.</p><!><p>We tested 9 typical disordered proteins with 19 to 124 residues. The FF scores of the six ff03-series force fields are listed in Table 4. Except for HEWL19 and HIVRev, the FF scores of ff03CMAP/TIP4P-D simulations are the lowest, and most of them are very close to 1, indicating very good agreement with experiment. Although the FF scores of ff03CMAP/TIP4PD for HEWL19 and HIVRev are not the best, the differences with the best performing force fields are not significant. This suggests that ff03CMAP/TIP4P-D can reproduce well conformers of tested IDPs. The details of the FF score composition for each tested system are shown in Tables S7–S15.</p><p>It is interesting to note that all ff03 revisions improve over the original ff03 in IDP simulations, as they are all designed to reproduce the properties of IDPs. As expected, the CMAP method can provide accurate descriptions of IDPs as in previous developments.[9–11, 15, 72] In addition, the TIP4P-D water model is demonstrated again to be suitable for IDP simulations.[16] In order to understand the influence of solvent models, three IDPs were simulated with ff03/TIP4P-D (Tables S10, S12–S13). The results show that TIP4P-D water model indeed partly improves the performance of tested IDPs conformers. However, the results are still much worse than those of ff03CMAP/TIP4P-D, suggesting that the CMAP improvement in ff03CMAP/TIP4P-D simulations plays a key role in reproducing the IDPs conformers.</p><p>To further illustrate the property of ff03CMAP force fields, we calculated the average RMSDs for different experimental measurements of IDPs (Table 5). We found that the performance of ff03CMAP/TIP4PD is the best for all experimental observables, and ff03CMAP/TIP4PEw also performs reasonably well. It is noticeable that ff03CMAP significantly improved the quality of simulated Cα and N secondary chemical shifts and 3JHNHα scalar coupling constant, which have are closely related to backbone dihedrals. It is clear that the CMAP method can be used to correct the dihedral distributions and the TIP4P-D water model further refines the interactions between protein and water, leading to excellent observed performance in the ff03CMAP/TIP4PEw simulations for IDPs.</p><p>For IDP simulations, underestimation of Rg is a common limitation for generic protein force fields.[14, 16] We calculated Rg distributions for all tested IDPs (Figure S23). The force field with four-site water models [Which one, be specific] can sample a wider range of Rg distributions and larger Rg mean values, especially for ff03CMAP/TIP4P-D and ff03ws. Conformers are more compact in force fields in the TIP3P water model such as ff03 and ff03*. We compared the experimental Rg and simulated Rg of three IDPs. The analysis indicates that ff03CMAP force field and ff03ws overestimate the Rg of Aβ40 and RS. The average RMSDs[What is this RMSD? For a single Rg value?] of ff03CMAP/TIP4PEw are also very small and close to experimental value. For ACTR, only the Rg in the ff03CMAP/TIP4PD simulation is located within the experimental range and other force fields significantly underestimate it.</p><p>Besides the above overall assessments, we next use a classical example of IDPs, RS, in the following discussion to illustrate the performance of these force fields. To evaluate the backbone and side-chain sampling for RS, we compared secondary chemical shifts and scalar coupling constants. Figure 6 shows the secondary chemical shifts and backbone scalar coupling constants for six ff03-series force fields. Tables S19–S20 list detailed data used in analysis. The chemical shifts and scalar coupling constants calculated from all revised ff03 force fields are much closer to experimental data than those from the original ff03 force field, and the RMSDs between simulated and experimental values from ff03CMAP/TIP4P-Ew and ff03CMAP/TIP4P-D combinations are smaller than those from other ff03-series force fields (Table 4). We also calculated the backbone N-HN, Cα-Hα and Cα-C RDCs with a local alignment window of 15 residues (Figure 7), whose performance is similar to that of chemical shifts and coupling constants, with both ff03CMAP/TIP4PEw and ff03CMAP/TIP4PD giving lower RDC(Q, what does Q mean? Why lower value is better) with smaller standard deviations (why smaller sd is better?) than other tested force fields.</p><p>Next FF scores were used to compare the performance of all force fields (listed in Table 6) in RS simulations. The RMSDs of the original ff03 force field are the largest and FF score is larger than 4. ff03CMAP/TIP4PD combination gives the best agreement with all experimental measurements and the FF scores are around 1.1.</p><p>Finally we computed the ensemble-averaged SAXS curves for RS and fitted with the experimental curve (Figure 8). The χ2 value was used to evaluate the quality of the fitted result to the given experimental SAXS profile as shown in the literature.[73] Our analysis shows that the χ2 of ff03CMAP/TIP4PEw is the smallest among 6 tested ff03-series force fields. This suggests that ff03CMAP/TIP4PEw can reproduce the SAXS property for RS, while the ff03CMAP/TIP4PD combination leads to conformers that are too expansive.</p><p>To further illustrate the conformer sampling efficiency, kClust was used to cluster conformers according to φ angle and Cα RMSD. Representative conformers and their occupations are shown in Figures S26–S34. The results indicate that both ff03CMAP and ff03ws can sample more flexible and diverse disordered conformers, while the representative conformers in the ff03 simulation contain several short helixes with tight packing. Convergence of conformer sampling is another important issue for IDP simulations. We used biphasic decay model to evaluate the convergence time scales for IDP simulations. It is interesting to note that ff03CMAP simulations have smaller decay half times, which suggests ff03CMAP simulations converge earlier than other ff03-series simulations.</p><!><p>We performed REMD for 3 typical fast-folding proteins, such as 16-residue two β-sheets GB1, small β-hairpin-forming protein CLN025, and helical 15-mer AAQAA3.[34, 36–37] The melting curves in the ff03CMAP/TIP4PEw simulations are shown in Figure 9. The melting curves show that GB1 and CLN025 can be ab initio folded when modeled with ff03CMAP/TIP4PEw. However, a few folded structures were observed in the REMD simulation of AAQAA3. To study whether the ab initio folding of helical structures can be improved by modifying CMAP parameters, we updated a new set of CMAP parameters by only decreasing the parameters in αh region with the revised force field termed as ff03CMAP2. Our REMD simulation shows that ff03CMAP2/TIP4PEw performs significantly better in helix folding. In the meantime, ff03CMAP2/TIP4PEw can maintain almost same melting curves for sheet and hairpin fast-folding proteins. [You should say whether the IDP performance can still be maintained.]</p><!><p>The backbone dihedral term for all 20 amino acids was optimized to improve the performance of the current force field. TIP4P-Ew and TIP4P-D are combined with newly developed force field ff03CMAP to simulate different type proteins. Extensive tests of typical short peptide, folded proteins, disordered proteins, and fast-folding proteins show that the simulated chemical shifts, J-coupling, order parameters, and RDC with the ff03CMAP force fields are in quantitative agreement with those from NMR experiment and are more accurate than other ff03-series force fields. The influences of solvent models were also investigated. The results indicate that ff03CMAP/TIP4PEw for folded proteins and ff03CMAP combined with TIP4P-D was suitable for disordered proteins (ff03CMAP/TIP4PEw also shows good performance in IDPs). Therefore, these findings confirm that the newly developed force field ff03CMAP can improve the balance and efficiency of conformer sampling between intrinsically folded proteins and disordered proteins. Although ff03CMAP force field has the limitation of folding helix structures, this can be improved by adjusting CMAP parameters which is the ff03CMAP2.</p>

PubMed Author Manuscript

Isotope Depletion Mass Spectrometry (ID-MS) for Enhanced Top-Down Protein Fragmentation

Top-down mass spectrometry has become an important technique for the identification of proteins and characterisation of chemical and posttranslational modifications. However, as the molecular mass of proteins increases intact mass determination and top-down fragmentation efficiency become more challenging due to the partitioning of the mass spectral signal into many isotopic peaks. In large proteins, this results in reduced sensitivity and increased spectral complexity and signal overlap. This phenomenon is a consequence of the natural isotopic heterogeneity of the elements which comprise proteins (notably 13 C). Here we present a bacterial recombinant expression system for the production of proteins depleted in 13 C and 15 N and use this strategy to prepare a range of isotopically depleted proteins. High resolution MS of isotope depleted proteins reveal dramatically reduced isotope distributions, which results in increases in sensitivity and deceased spectral complexity. We demonstrate that the monoisotopic signal is observed in mass spectra of proteins up to ~50 kDa. This allows confident assignment of accurate molecular mass, and facile detection of low mass modifications (such as deamidation).We outline the benefits of this isotope depletion strategy for top-down fragmentation.The reduced spectral complexity alleviates problems of signal overlap; the presence of monoisotopic signals allow more accurate assignment of fragment ions; and the dramatic increase in single-to-noise ratio (up to 7-fold increases) permits vastly reduced data acquisition times. Together, these compounding benefits allow the assignment of ca. 3-fold more fragment ions than analysis of proteins with natural isotopic abundances. Thus, more comprehensive sequence coverage can be achieved; we demonstrate near single amino-acid resolution of the 29 kDa protein carbonic anhydrase from a single top-down MS experiment.Finally, we demonstrate that the ID-MS strategy allows far greater sequence coverage to be obtained in time limited top-down data acquisitions -highlighting potential advantages for topdown LC-MS/MS workflows and top-down proteomics.

isotope_depletion_mass_spectrometry_(id-ms)_for_enhanced_top-down_protein_fragmentation

Introduction<!>4<!>Results and Discussion.<!>ID-MS allows direct determination of monoisotopic mass of intact proteins up to 50 kDa.<!>6<!>ID-MS dramatically improves protein sequence coverage in top-down fragmentation.<!>11<!>13<!>ID-MS improves top-down ECD on an LC Timescale.<!>Conclusion<!>Experimental Section<!>Datasets

<p>Top-down mass spectrometry (MS) has emerged as a powerful technique for the analysis of protein sequence and the detailed characterisation of chemical modifications to protein side-chains. [1,2] Consequently, top-down MS is a powerful strategy for the comprehensive identification and characterisation of all proteoforms arising from genetic variation, alternative splicing, and post-translational modifications (PTMs). The technique consists of first measuring the intact molecular mass of a protein, followed by gas phase fragmentation of a selected proteoform ion by tandem mass spectrometry. The resulting fragment ions are assigned based on their observed accurate mass. If sufficient numbers of fragments can be assigned, top-down MS can provide a complete description of protein sequence and PTM state. [3] Over the last two decades several fragmentation techniques have been employed for top-down studies. However, electron-based fragmentation techniques such as electron capture dissociation (ECD) [4,5] and electron transfer dissociation (ETD) [6] have been the most widely applied and they offer improved diversity of backbone site cleaved when compared techniques which rely on vibrational excitation, such as collision induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). [7,8] Thus top-down ECD and ETD can provide comprehensive sequence coverage for the analysis of small proteins (<20 kDa). However, as protein mass increases, top-down fragmentation efficacy notably diminishes; spectra become increasingly complex, and a series of other compounding factors result in reduced sequence coverage (the challenges of top-down MS have been discussed in depth in several recent publications). [9][10][11] One fundamental factor which proves detrimental in top-down analysis is the increasing breadth of the isotopic distribution that accompanies increasing molecular mass.</p><p>For proteins, the isotopic heterogeneity of the organic elements (particularly the ~1.1% natural abundance of 13 C) results in the ion signal being spread over a distribution of discrete isotopologues (the isotope distribution); with each isotopologue differing in composition by a neutron. As protein mass increases, this isotope distribution widens, and so the overall signal gets more disperse. For example, a 10 kDa protein the isotope distribution will consist of 12 isotopologue signals; whereas for a 50 kDa protein, the number of isotopologues observed can be over 40. [12] This phenomenon reduces the signal to noise ratio (S/N) and can lead to the overlapping of signals for species which are close in mass (e.g. proteoforms of the same protein with similar masses). Furthermore, proteins, or protein fragment ions, over ~10 kDa commonly do not display a monoisotopic signal of sufficient ion abundance to accurately assign. In the context of a top-down fragmentation experiment, these compounding difficulties all reduce the number of fragment ions which can be confidently assigned as protein mass increases.</p><!><p>One solution to this problem is the production of isotopically enriched/depleted proteins, by recombinant production in hosts grown on carbon/nitrogen sources that are enriched or depleted in specific naturally occurring heavy isotopes. The feasibility of this strategy has been demonstrated by Marshall et al., in 1997. [13] They reported the production and intact mass analysis of the 12 kDa FK506-binding protein, in media depleted in 13 C and 15 N. Using this approach, [13] the authors demonstrated an increase in the mass spectral sensitivity and the detection limit. Despite the publication of this seminal report over twenty years ago, the use of this strategy has been limited; despite theoretical studies highlighting the potential benefits of the approach. [9,14] The reason for this may be the technical difficulty in producing isotopically depleted proteins, and currently the strategy has only been applied to produce a handful of small proteins (<15 kDa). [15][16][17] Herein we detail a robust method for the recombinant production of isotopically depleted protein in E.coli and demonstrate its benefits to top-down protein analysis by producing and characterizing a series of proteins up to 50 kDa. All isotopically depleted proteins displayed dramatically simplified isotope distributions and, as a consequence, we report a reduction in mass spectral complexity and dramatic S/N increases. Using this strategy, termed isotope depletion mass spectrometry (ID-MS), we show that the monoisotopic mass signals can be observed in isotopically depleted proteins up to 50 kDa. This allows direct and accurate determination of molecular mass for large proteins and protein fragment ions, for the first time. Finally, we perform top-down fragmentation of isotopically depleted proteins and demonstrate that the reduced spectral complexity and increased S/N allow assignment of fragment ions with increased confidence, and results in dramatically improved sequence coverage.</p><p>5</p><!><p>We chose three well-characterised proteins as model systems for this study -encapsulated ferritin (EncFtn, 13.2 kDa), [18] carbonic anhydrase (CA, 29.3 kDa), [19] and serine palmitoyltransferase (SPT, 47.3 kDa). [20] These proteins were recombinantly expressed in E. coli using M9 minimal growth media, containing glucose and ammonium sulfate as the sole carbon and nitrogen sources. This allowed isotopically doubly-depleted protein samples to be prepared by using isotopically-depleted glucose (99.9% 12 C 6 ) and ammonium sulfate (99.99% 14 N 2 ) in the cell culture preparation. Full details of the expression protocol can be found in the (Supporting Information, Figure S1).</p><!><p>After protein expression and purification, MS analysis of the intact proteins was performed using high resolution electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). ESI mass spectra of samples prepared in natural abundance cell culture and double-depleted cell culture exhibited identical charge state distributions (Figure S2). However, dramatically simplified isotope distributions were observed in the mass spectra of proteins produced in isotopically depleted media when compared to natural isotopic abundance proteins (Figure 1). (Top) The observed isotope distribution for samples prepared from natural isotope abundance cell culture and (bottom) the observed isotopic distribution for samples prepared from isotope depleted cell culture. The theoretical isotopic distributions are overlaid on the spectra as scatter plots (natural abundance: 98.89% 12 C, 99.63% 14 N; isotopically depleted abundances 99.90% 12 C, 99.99% 14 N). In each spectrum, the monoisotopic species is highlighted with an asterisk (*).</p><!><p>For EncFtn (monoisotopic molecular mass 13,186.4 Da, Figure 1A), the width of the isotopic distribution decreased from 14 Da in the natural isotopic abundance protein to 7 Da in the isotopically depleted protein (isotopologues with abundance greater than 0.5% of the base peak); and the monoisotopic peak increased from ~0.06% of the total signal (i.e. below the noise) to ~30% of the total signal, and was the highest peak in the distribution. For CA (29,294.8 Da) and SPT (47,201.8 Da) the monoisotopic peak was not visible in the natural isotopic abundance protein distribution. In contrast, in the corresponding mass spectra obtained from isotopically depleted proteins, the monoisotopic peak was easily identifiable.</p><p>For isotopically depleted CA, the monoisotopic peak accounted for ~13% of the total signal, and for the isotopically depleted SPT protein, the monoisotopic peak still accounts for ~2% of the total signal. Similar to EncFtn, the isotopically depleted variants of CA and SPT exhibited a reduction in isotopic distribution width of 20 Da to 10 Da and 26 Da to 16 Da respectively. In all case, the S/N also improved as the same number of proteins ions were partitioned between fewer isotopologue peaks.</p><p>As first proposed by Marshall in 1997, [13] we have demonstrated that 13 C and 15 N double depletion increases the proportion of the monoisotopic isotopologue and results in an observable monoisotopic signals for protein up to ~50 kDa. This allows the unambiguous and immediate assignment of the accurate molecular mass of intact proteins. In contrast, for accurate mass assignment of natural isotopic abundance proteins above ~10 kDa, it is necessary to infer the monoisotopic mass by matching the observed isotope distribution with the calculated theoretical isotope distribution of an 'average' protein -i.e. a repeating polymer of the model amino acid averagine. [21,22] This 'poly-averagine approximation' method relies on obtaining a statistically reliable experimental isotope distribution and often results in significant mass error (up to 3 ppm) and/or the misassignment of the monoisotopic mass by +/-1 Da, regardless of the resolution achieved in the data acquisition. [12,23] Consequently, this makes confident detection of low molecular mass PTMs, such as disulfide bond formation or deamidation, at the protein level, particularly challenging. [24][25][26][27] Therefore, the ID-MS strategy may be particularity powerful for the detection and characterisation of these low molecular mass PTMs. In order to demonstrate this, we produced an EncFtn 'deamidated' single-point variant (N58D), in an isotopically depleted form. MS analysis of isotopically depleted N58D EncFtn allowed confident detection of the deamidation at the protein level. Direct detection of protein deamidation was also possible from mixtures of WT and deamidated proteoforms (Supporting Information, Figure S3).</p><!><p>Typically, top-down fragmentation generates many hundreds of fragment ions, with each ion appearing in multiple charge states, and exhibiting its own isotopic distribution. [28] Thus, the resulting spectra are highly complex and consist of many thousands/ tens of thousands of individual peaks, over a wide dynamic range of ion-abundance. As the observed fragment ions fall in a comparatively narrow m/z range (typically m/z 500-2000), fragment ion isotope distributions often overlap; and, even with high resolving power instrumentation, superposition of peaks is common. Therefore, fragment ions can be overlooked or misassigned due to low signal and/or signal overlap.</p><p>In order to investigate the benefit of the isotope depletion strategy for top-down mass spectrometry, we analysed natural isotopic abundance and isotope depleted forms of both EncFtn and CA, using both CID and ECD fragmentation. Initially, CID was performed on the [M+16H] 16+ precursor ion of natural isotopic abundance and isotopically depleted EncFtn. Both fragmentation spectra were remarkably similar on initial inspection, displaying identical high abundance fragment ions at similar m/z (Figure S4A). However, all fragment ions derived from the isotopically depleted EncFtn exhibited reduced isotope distribution widths, which greatly reduces signal overlap of individual fragments. In addition, the S/N ratio displayed by isotopically depleted fragment ions was dramatically increased (for example, the complementary ion-pairs b 37 4+ and y 78 10+ exhibit S/N gains of 7.0-fold and 4.7-fold in the isotopically depleted spectrum when compared to the natural isotopic abundance spectrum).</p><p>It was also apparent that, for this 13 kDa isotopically depleted protein, the monoisotopic signal was the base-peak (i.e. the highest signal) in every isotopically depleted fragment ion's isotope distribution. This allowed direct determination of the accurate monoisotopic mass of every fragment ion (Figure S4C). Taken together, these three advantages led to confident assignment of substantially more CID product ions in the isotopically depleted EncFtn CID spectrum. For CID of the [M+16H] 16+ of EncFtn, 110 b and y fragment ions were assigned in the natural isotopic abundance spectrum (39 b-ions, 71 y-ions; 45.7% total sequence coverage); in comparison, 217 b and y fragment ions (84 b-ions, 133 y-ions; 64.7% total sequence coverage) were assigned in the natural isotopic abundance spectrum (Supporting Information, Figure S5). This increase in the observed fragment ion number is similar to that demonstrated by Akashi et al., [16] who reported an 63% increase in the number of assigned fragment ions when performing CID of an isotopically-depleted version of the 10 kDa protein cystatin. However, for CID of both natural isotopic abundance and isotopically depleted EncFtn, the assigned b-and y-ions only constitute only around 20-30% of the total number of observed fragments; and even employing an isotopically depleted strategy with top-down CID, it is clear that there are regions of the protein with limited sequence coverage. Further analysis of the unassigned fragment ions in both CID spectra revealed a substantial number of internal 8 fragments, and widespread neutral loss during fragmentation (-H 2 O, -CO, -NH 3 ). Taking these fragmentation channels into considerations allowed assignment of a total of 448 product ions (a, b, x, y, and y -H2O ions; 82% total sequence coverage) in the CID spectrum of isotopically depleted EncFtn (Supporting Information, Figure S5).</p><p>The lack of product ion specificity, and the biased nature of fragmentation with CID has been well documented, [8] and this limits the utility of the technique for top-down studies of proteins over 10-15 kDa. In contrast to CID, electron-driven dissociation techniques (such as ECD and ETD, together termed 'ExD') are thought to result in relatively unbiased fragmentation throughout the protein sequence. [29][30][31] Thus, potentially higher sequence coverage has been reported (especially in larger proteins) and ExD fragmentation is a far more attractive technique for top-down fragmentation as protein mass increases. However, one drawback of the ExD approach is its relatively inefficient precursor-to-product ion conversion and so ExD characteristically results in c-and z-type fragment ions of low ion abundance.</p><p>Therefore, we reasoned that the substantial increased S/N evident in top-down ID-MS may potentially be of more benefit when used in conjunction with ExD studies. 9 Figure 2 shows the spectrum obtained after ECD of EncFtn (spectral averaging of 150 acquired transients; magnitude mode). Post-acquisition, Autovectis was used to process the data in absorption mode and assign fragment ions (for details see Supporting Information, Figure S6). ECD of a single charge state of natural isotopic abundance EncFtn yielded 131 c-10 ions and 125 z-ions. This included fragment ions from throughout the protein sequence and represented a total sequence coverage of 84.5% (Figure 2C, left); in our hands, this result is entirely typical for top-down ECD fragmentation of a 13 kDa protein. In comparison to ECD of natural isotopic abundance EncFtn, fragment ions obtained from ECD of isotopically depleted EncFtn displayed reduced isotopic distribution widths with a dominant monoisotopic signal and increased signal abundance (typically ~2-fold to 7-fold S/N increases were observed; dependant on fragment ion molecular mass). These factors allow assignment of many more low abundance ECD fragment ions, and accurate assignment of fragment ions which overlap in the natural isotopic abundance spectrum (Figure 2B and further examples in Supporting Information, Figure S7). In addition, ECD of the isotopically depleted protein allowed accurate assignment of sidechain losses and revealed low abundance ions in 'congested' regions of the spectrum (see Supporting Information, Figure S8). ECD of the isotopically depleted EncFtn yielded 276 c-ions and 220 z-ions fragment ions from this single experimental condition; a total sequence coverage of 97.4% (Figure 2C, right). Cleavages N-terminal to proline are not generally observed in ECD. [32] Remarkably, if this is taken into account, of 114 peptide bonds in EncFtn only 2 possible cleavages were not observed. In addition, complementary c-and zion pairs cover over 85% of the protein sequence.</p><p>It is clear from our analysis of EncFtn that three characteristics of the isotope depletion MS strategy lead to dramatic improvements for top-down fragmentation -namely, (i) improved overall S/N, (ii) increased monoisotopic signal abundance, and (iii) decreased isotope distribution width.</p><p>These compounding benefits should be more evident as the precursor protein mass increases over 20 kDa. Therefore, we tested the utility of top-down isotopically depleted MS at higher mass, analysing bovine CA (29 kDa) by top-down ECD. CA has been used extensively to characterise top-down fragmentations technologies by multiple research groups and on multiple MS platforms; [11,23,[33][34][35] thus it constitutes an ideal model study.</p><p>Either the [M+32H] 32+ (m/z 916) or the [M+22] 22+ (m/z 1332) charge state of CA was isolated and subject to ECD (Figure 3). ECD of the [M+22H] 22+ charge state of natural isotopic abundance CA produced highly complex spectra (20,000 peaks with S/N > 2.5), which exhibit overlapping fragment ion isotope distributions throughout the spectrum (Figure 3A,3B, top). In addition, following substantial spectral averaging (300 averaged transients), even more fragment ions were observed with low S/N, this was especially evident as fragment ion mass increased. In total, from this single dataset, 229 c-and z-fragment ions could be assigned, representing 50.0% sequence coverage (Figure 3C, left). Low sequence coverage was especially evident in the central region of the protein.</p><!><p>As expected, ECD of the isotopically depleted CA resulted in significantly reduced spectral complexity and fragment ion distribution overlap. Fragment ions were observed with increased S/N (~2-to 8-fold increase -similar to previous results, vide infra; Figure 3B and further examples in Supporting Information, Figure S9). Interestingly, compared to the equivalent natural isotopic abundance spectrum, a similar number of individual peaks were observed in the ECD spectrum of isotopically depleted CA, suggesting that substantially more fragmentation channels should be evident. As a consequence, from the ECD spectrum of the [M+22H] 22+ of isotopically depleted CA, 593 fragment ions (377 c-ions, 216 z-ions) were assigned; i.e. approximately a three-fold increase in the number of fragment ions assigned from the natural isotopic abundance CA sample. These fragment ions yielded a sequence coverage of 82.6% for the isotopically depleted protein (Figure 3C, right). Comparable assignment rate increases were possible when analysing the ECD spectra of the [M+32H] 32+ charge state of isotopically depleted CA (Supporting information, Figure S10). If the sequence coverage observed for both charge states are combined, the overall sequence coverage obtained for isotopically depleted CA was over 90% (95.2% if bonds with adjacent proline residues were discounted); i.e. only 12 cleavages were not observed in this 263-amino acid protein -very close to the 'ideal' of single amino-acid level resolution throughout the protein sequence (Supporting Information, Figure S10). To our knowledge, represents the most comprehensive sequence coverage of CA observed to date, irrespective of fragmentation technique or MS platform.</p><p>12 The fragmentation maps (protein sequence coverage) achieved after ECD of the [M+22H] 22+ charge state of natural isotopic abundance CA (left; 50%) and isotopically depleted CA (right; 82.6%).</p><!><p>One striking characteristic of the ECD of isotopically depleted proteins is the ability to assign extended stretches of complimentary c-and z-ions, even in central regions of larger proteins.</p><p>In effect, allowing comprehensive fragment ion sequence coverage 'deeper' into the protein sequence. Comparison of the mass distributions of the fragment ions assigned after ECD of natural isotopic abundance and isotopically depleted CA show that more fragment ions are assigned in the isotopically depleted ECD spectrum from across all molecular mass ranges (Figure 4A). However, these histograms highlight that the ID MS strategy has the greatest benefit for the assignment of fragment ions of higher masses; where ECD of isotopically depleted CA consistently affords 3-to 8-fold more fragment ions than ECD of natural isotopic abundance CA. For example, in ECD of isotopically depleted CA resulted in 52 fragment ions in the mass range 15-18 kDa; whereas only 10 fragment ions of similar mass were assigned from natural isotopic abundance CA.</p><p>Not only is sequence coverage improved in isotopically depleted ECD, but fragment ions are also assigned with lower error in the isotopically depleted ECD spectrum. Figure 4B shows the distribution of errors for the assigned ECD spectra of natural isotopic abundance and isotopically depleted CA. Using a 'poly-averagine'-based approach for deconvolution of the ECD spectrum of natural isotopic abundance CA, the resulting fragment ions were assigned with a RMS error of 1.306 ppm. While AutoVectis analysis of the ECD spectrum of the same charge state of isotopically depleted CA allowed assignment of fragment ions with a RMS error of 0.800 ppm.</p><p>The ability to assign dramatically more fragment ions, especially fragment ions of mass >10 kDa, is a direct consequence of the inherent increase in the S/N which accompanies isotopically depleted MS. In addition, the ability to directly observe the monoisotopic signals in isotopically depleted fragment ions is also highly advantageous, as it removes the requirement to obtain isotopic distributions with sufficient S/N for precise poly-avergine based deconvolution methods. Furthermore, the mass error introduced using the poly-averagine approximation during deconvolution is removed; leading to assignment of fragment ions with lower overall mass error. Therefore, higher confidence in fragment ion assignment can be achieved, which is particularly important for the interpretation of highly complex spectra, such as top-down analysis of large proteins or assigning branched protein ions. [36]</p><!><p>One of the overarching goals of top-down mass spectrometry is to achieve comprehensive protein sequence coverage using spectral acquisition times that are compatible with front-end chromatography; potentially allowing top-down protein analysis to be used in an LC-MS/MS proteomics workflow. Significant advances have been made in this field of top-down proteomics in recent years; [37][38][39] however, it is particularly challenging to achieve extensive sequence coverage as protein molecular mass increases, and the sequence coverage achieved in top-down LC-MS/MS experiments is often restricted to limited regions at the Nand C-termini of the protein. Although this is often sufficient to provide a 'sequence-tag' and allow protein identification, low sequence coverage is insufficient for confidently mapping protein modifications and full characterisation at the proteoform level. These limitations are due to the time-constraints of the experiment and the inability to perform the extensive spectral averaging required to obtain fragment ion signals of sufficient abundance. In effect, there is a compromise that exists between the signal-to-noise level achieved and the spectral acquisition time.</p><p>Spectral averaging produces a gain in the S/N ratio that is approximately proportional to the square root of the number of scans averaged. [40] Because of this non-linear relationship, the increased S/N inherent in our isotopically depleted MS approach should be particularly effective for increasing the fragment ion sequence coverage obtainable with limited spectral averaging. In order to investigate this, ECD spectra were acquired in the same fashion for both the natural isotopic abundance and isotopically depleted forms of EncFtn (13 kDa) and CA (29 kDa) using both 20 or 5 spectral averages; which constituted total data collection times of ~25 and ~6 seconds respectively. The resulting spectra were analysed and fragment ions assigned (Supporting Information, Figure S11 and S12) and compared to the longer spectral acquisition time, described above (Figure 5). As expected, for natural isotopic abundance EncFtn reduction in the spectral averaging reduces the obtained protein sequence coverage significantly and with spectral averaging limited to 5 transients, only 48 ions could be assigned constituting 31% total sequence coverage. In contrast, for isotopically depleted EncFtn the reliance on extensive spectral averaging to obtain high sequence coverage is far less pronounced, and 86.2% protein sequence coverage was achieved with only 5 averaged spectra. For the larger protein, CA (29 kDa), it is clear that without extensive spectral averaging the sequence coverage obtained after ECD of the natural isotopic abundance protein is severely limited -28.4% sequence coverage is obtained with 20 averaged spectra and 14.4% sequence coverage is obtained upon averaging only 5 spectra. As discussed above, this phenomenon is well-documented in larger proteins, and is a current bottleneck in top-down proteomics. Dramatic improvements are observed using the isotopically depleted strategy and in-depth sequence coverage can still be assigned under time-limited data acquisitions. ECD of isotopically depleted CA using 20 and 5 spectral averages affords sequence coverage of 61.7% and 47% respectively. These initial findings demonstrate the potential benefit of applying isotopically depleted strategies in top-down proteomic workflows and highlight the possibility of achieving comprehensive sequence coverage of larger proteins on chromatographic timescales.</p><!><p>We have produced several isotopically depleted proteins with molecular masses up to ~50 kDa. We demonstrate that mass spectra of intact isotopically depleted proteins display decreased isotope distribution widths and increased S/N. In addition, direct observation of the monoisotopic signal of isotopically depleted proteins is possible; allowing accurate molecular mass to be directly determined, even in large proteins. Applying ID-MS in conjunction with topdown fragmentation affords reduced spectral complexity, increased S/N and increased mass accuracy; together this allows assignment of dramatically more fragment ions (typically 2-to 3-fold) and consequently increased protein sequence coverage. We also highlight the potential of applying ID-MS for performing top-down fragmentation on a chromatographic timescale for top-down proteomic applications.</p><p>Finally, we note that this isotope depletion strategy is analogous to the isotope enrichment techniques which have become integral to biomolecular nuclear magnetic resonance (NMR) spectroscopy. [41] Similarly, it is clear that ID-MS has huge promise for many biomolecular MS applications, particularly for proteins (or other biomolecules [42] ) of high molecular mass. Techniques such as hydrogen/deuterium exchange MS, native protein MS, and structural MS will all benefit greatly from the advantages which accompany isotopic depletion.</p><!><p>Isotopically-depleted proteins were produced by recombinant expression in E. coli using minimal media supplemented with 12 C(99.9%)-glucose and 14 N(99.99%)-ammonium sulfate as the sole carbon and nitrogen sources; see Supporting Information for detailed protocols.</p><p>MS experiments were performed on a 12T SolariX Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an Infinity ICR cell (Bruker Daltonics, Bremen, Germany). Ionisation was achieved with a TriVersa NanoMate nanoelectrospray robot (Advion Bioscience, Ithaca, NY). Data was processed in magnitude mode, using Data Analysis (Bruker Daltonics, Bremen, Germany); and, in the absorption mode, using an in-house developed, enhanced version of AutoVectis (Nottingham Trent University and Spectroswiss Sàrl, Lausanne, Switzerland). Full details are available in the Supporting Information.</p><!><p>All mass spectrometry datasets used in this study are available to download, in their original data formats, at Edinburgh DataShare (www.http://datashare.is.ed.ac.uk/handle/10283/760), using the following link: http://dx.doi.org/10.7488/ds/2446.</p>

ChemRxiv

The First Spectrum of Manganese, Mn I

In 1894, two short series of threefold spectral terms were discovered in the arc spectrum of manganese, and in 1922 other regularities involving fivefold and sixfold terms were discovered by Catalán who coined the word “multiplet” for the group of related lines resulting from combinations of such complex terms. Multiplet analyses of complex spectra promptly led to the present formal quantum interpretation of all such phenomena, but comparable progress in the analysis of the Mn I spectrum was handicapped by the paucity of experimental data.New observations of about 2500 wavelengths and intensities plus 440 Zeeman patterns made available in 1948–49 have now been completely exploited to derive additional atomic energy levels and thereby explain more of the observed Mn I lines. The result is that a total of 42 even terms with 125 levels and 60 g-values have now been designated and allocated to electron configurations, and 94 odd terms with 266 levels, 164 g-values, plus 13 miscellaneous levels. These terms are distributed among four multiplicities (doublets, quartets, sextets, octets), and transitions between even and odd terms account for more than 2030 lines ranging in wavelength from 1785 Å to 17608 Å.

the_first_spectrum_of_manganese,_mn_i

I. Introduction<!>2. Experiments<!>3. Results

<p>The spectra of manganese have interested and engaged scientists for nearly 120 years. In 1910 Kayser [1]4 listed 67 publications on this subject since 1845, and in 1934 Kayser and Konen [2] compiled a bibliography of 154 additional items through 1931. In the present paper only a few of the above will be referred to for historical background, but most of the papers published since 1931 that describe or interpret the Mn I spectrum will be mentioned to extend the bibliography.</p><p>In 1894 Kayser and Runge [3] reported finding the first regularities among lines in the arc spectrum of manganese; they found five triplets with wave number differences of 173 and 129 K (K=kayser = 1 cm−1) and arranged them in two spectral series, one "sharp" and one "diffuse," like those in the simpler spectra of alkaline-earth elements. (This precious information was presented in a footnote on page 104 of a paper primarily dedicated to the spectra of tin, lead, arsenic, antimony, and bismuth.)</p><p>Nearly three decades after Kayser and Runge [3] discovered the first rudimentary series in the arc spectrum of manganese, Catalán [4] extended those series and calculated the first ionization potential of manganese as 7.41 electron volts. Catalán said (p. 146) "Whilst the manganese series of ordinary type were under investigation, it was noted that there was a strong tendency for lines of similar character to appear in groups (of 9, 12, or 15 lines) and that such groups included some of the most intense lines in the spectrum". For such a group of related spectral lines, "multiplet" was suggested (p. 147). "The accuracy of the separations, some of them being identical with those of the ordinary series, together with the fact that the lines of each group are of the same character, strongly suggests that the multiplets have a real physical significance. Further evidence for their reality is afforded by the occurrence of similar multiplets in the spectra of other elements" (p. 163). In fact, Catalán's first paper on regularities in the spectrum of manganese reporting triplets and multiplets that reveal terms with five or six levels [4] also reported three multiplets in the spectrum of ionized manganese and three multiplets of the neutral atom of chromium. Even before that paper was printed, a manuscript copy of it inspired Sommerfeld [5] to write on the explanation of complex spectra (manganese, chromium, etc.) according to the method of inner quantum numbers. Almost simultaneously, Back [6] accurately measured the Zeeman effect of 49 lines in the arc- and 12 lines in the spark spectrum of manganese with the object of investigating magnetic term splitting of the complex terms found by Catalán, and Landé [7] provided an explanation of all Zeeman patterns with the aid of magnetic quantum numbers and experimental magnetic splitting factors for different types of spectral terms and multiplicities.</p><p>It was then clear that the regularities among 169 lines of Mn I presented by Catalán [4] arose from spectral terms belonging to sextet and octet systems. As a check of Landé's scheme, and an example of the usefulness of Zeeman effect in analyzing complex spectra, Back [6] found from Zeeman data the first multiplet of the quartet system. Since multiplets usually occur in triplicate (Δl = 0,±1), Catalán [8] promptly found two additional quartet multiplets associated with the one described by Back.</p><p>In 1926 McLennan and McLay [9] extended the work of Catalán and Back on Mn I by making absorption experiments, finding 14 additional multiplets, providing energy diagrams for quartets, sextets, and octets, and tables of 90 energy levels and 257 classified lines ranging in wavelength from 1874.7 Å to 17607.5 Å. Although terms of doublet multiplicity were sought, none was found at that time. This list of levels and terms, with some additions from Russell [10] and from Catalán [11], and with modernized notation, appeared in a volume of Atomic Energy States published in 1932 by Bacher and Goudsmit [12]; it presented 99 energy levels distributed in 17 quartet, 16 sextet, and 15 octet, terms then recognized for the Mn I spectrum. The following year, T. Dunham, Jr., [13] found ten new quartet and two sextet terms (but never published them), and Meggers [14] detected one quartet and two sextet terms in new observations of near infrared spectra. Aside from the above, fifteen years passed without progress in the analysis of Mn I. A strong incentive to extend this analysis came in 1946 when C. E. Moore began her critical and comprehensive compilation of Atomic Energy Levels as Derived from the Analyses of Optical Spectra.</p><p>In 1949 a major advance in the analysis of Mn I was made by Miss Olga Garcia-Riquelme [15] who was assigned the task as a thesis problem by Professor Catalán at the University of Madrid. By assembling all known energy levels of Mn I and by compiling wavelengths and intensities of manganese lines from many spectroscopic papers (by 15 different authors), Garcia-Riquelme succeeded in finding 58 new energy levels and 15 new terms for Mn I, thus producing a total of 30 even terms (85 levels), 30 odd terms (124 levels), plus 21 miscellaneous odd levels beyond the ionization limit. The permitted transitions between these levels accounted for 711 observed lines ranging in wavelength from 1876.48 A to 17607.5 Å. All the established spectral terms of Mn I were assigned to quartet, sextet, and octet systems; doublet terms were still unrecognized.</p><p>In order to complete the bibliography on description and interpretation of Mn I spectra during the period 1931 to 1949, we mention a paper by Slevogt [16] and one by Paul [17]. The former quotes earlier data on 233 lines (between 6024.67 and 8933.03 A) and remeasured the wavelengths of 113 of these; the latter reported observations on 57 Mn I lines with wavelengths between 1923.05 Å and 1085.01 Å absorbed by manganese vapor. Paul explained 10 of these as transitions from the ground state to highly excited levels, but only three of these have been confirmed in this analysis of Mn I.</p><p>In 1948 Catalán decided that an improved description of the Mn I spectrum (including reliable wavelengths, realistic relative intensities for several thousand lines, and Zeeman data for hundreds of lines) was prerequisite to a more complete and satisfactory term analysis of this spectrum.</p><!><p>To improve the data of Mn I Catalán spent most of 1948 in the United States, where he recorded the vacuum ultraviolet arc spectrum of manganese at Princeton University, and photographed the arc spectrum throughout the visible and adjacent ultraviolet at the National Bureau of Standards.</p><p>The vacuum ultraviolet arc and spark spectra of manganese were photographed with a two-m radius grating at Princeton University through the courtesy of Allen G. Shenstone who previously described [18] the apparatus and procedure. The manganese spectra were recorded in this vacuum spectrograph from 1460 Å to 2180 Å with a scale of 4.8 Å per mm, and all these spectrograms were measured and converted to wavelengths by Catalán.</p><p>In 1933, Meggers photographed and measured an ultraviolet range (2100 Å to 2800 Å) of manganese spectra with the largest quartz Littrow spectrograph constructed by Adam Hilger, Ltd., of London. Provided with a quartz lens of three-m focal length and Cornu prisms of 60-cm total base, this spectrograph produced reciprocal dispersions of 0.4 to 1.0 Å/mm at the above-mentioned wavelengths. These results were presented to Catalán in 1948 in addition to several dozen new lines between 10500 Å and 12000 Å recorded by Meggers on Eastman 1–Z photographic plates to extend his earlier observations [14] made with Eastman 1–Q plates.</p><p>In 1948 Meggers and Catalán reobserved the arc and spark spectra of manganese between 2200 Å and 7800 Å with the aid of a concave grating supplied by R. W. Wood [19]. In a Wadsworth-type mounting, this grating of 30,000 lines per inch on aluminized pyrex glass with 22 feet radius of curvature provided ghost-free spectra with reciprocal dispersions of 1.7 to 2.4 Å per mm in the first order. The second order spectra with double dispersion were photographed between 2500 Å and 4500 Å. All these spectrograms were measured relative to international standards of wavelength provided by the iron arc, and improved wavelengths and estimated intensities of more than 2000 lines characteristic of Mn I were thus obtained in 1949. Excepting several hundred wavelengths since reported in papers on the Zeeman effect, none of these new data has been published heretofore. To complete this description of the Mn I spectra, we (like others) have quoted 16 infrared wavelengths (12900 Å to 17608 Å) emitted by arcs of 60 to 80 amperes and detected with a thermopile in 1919 by Randall and Barker [20].</p><p>This improved description of the Mn I spectrum promptly led to the detection of doublet terms and extension of other multiplicities, so that by 1952 as compared with 1949, the total number of even levels had increased to 118 from 87, the number of odd levels to 217 from 124, and the number of classified lines to 1500 from 711. This improvement in the term analysis of Mn I, including g (magnetic splitting) factors for 108 levels, was summarized in 1952 by Charlotte E. Moore [21].</p><p>Although Back [6] in 1923 clearly demonstrated the great value of Zeeman effect in spectral term analyses, a quarter of a century elapsed before further determinations of inner quantum numbers and magnetic splitting factors were undertaken for Mn I.</p><p>Through the courtesy of George R. Harrison, Catalán was invited, in 1949, to photograph the Zeeman effect in manganese spectra with the Bitter magnet and large concave gratings previously described by Harrison and Bitter [22]. The electrodes were prepared with pure manganese powder mixed with pure silver powder, then pressed and sintered to form solid rods about 3 mm square. These electrodes were ignited in a d-c arc operated in a magnet producing a field of 85,000 oersteds, and the spectra were photographed between 2300 Å and 6500 Å. These spectrograms were measured and computed partly at the National Bureau of Standards in Washington and partly at the Institute of Optics in Madrid.</p><p>In those experiments, the magnetic field intensity was determined from the splitting of the resonance lines (3280.7 and 3382.9 Å) of the silver matrix, and/or from the resonance lines (3933.7 Å and 3968.5 Å) of ionized calcium, present as an impurity, assuming that the Zeeman patterns of those lines agree exactly with the Landé predictions [7]. Then all displacements of Zeeman components and magnetic splitting factors were expressed in Lorentz units (the unit displacement characterizing all singlet levels). As a result of these experiments, Zeeman data have been obtained for 440 lines of Mn I, ranging from 2461.0 Å to 6021.8 A, thus providing material for several reports extending over a decade.</p><p>The observations of normal Zeeman splitting in Mn I by Back [6] in 1923 were made in magnetic fields of 37,000 oersteds, whereas the Zeeman-effect spectrograms obtained by Catalán in 1949 recorded magnetic splitting in fields of 85,000 oersteds. Catalán and Velasco [23] observed changes in g-values of Mn I with magnetic fields above 80,000 oersteds which distort some Zeeman patterns. These distortions in the case of z6F0½° and z6F1½° levels were ascribed to repulsions between magnetic levels with equal M-values, belonging to adjacent levels of a spectral term, and it was shown that the theory of partial Paschen-Back effects provides a simple rule for obtaining correct g-values in spite of asymmetries in Zeeman patterns. This was elaborated by Catalán [24] who discussed in detail a dozen distorted patterns and concluded that the experimental g-values agreed with those predicted for LS coupling by Landé [7]. Finally, Espinosa [25, 26], in connection with his doctoral thesis, made a complete theoretical interpretation of the very complex Paschen-Back patterns of the z 6P°—e 6D and a 6S—z 6P° multiplets of Mn I.</p><p>The major contributions to g-factors for Mn I levels have been published in two papers separated by nine years. The first, by Catalán and Garcia-Riquelme [27], reported measurements of Zeeman patterns for 128 lines ranging in wavelength from 2794.817 Å to 4823.528 Å, and derived g-factors for 93 levels, the latter ranging in value from 0 to 67752.84 K. The second, by Riquelme et al. [28], presented measurements and interpretations of the Zeeman patterns of 314 lines (2573 Å to 6022 Å), including derived g-values for 105 energy levels. The data for 251 lines were totally interpreted, whereas 63 lines showing some Paschen-Back effect were partially interpreted as confirming the spectral classification. In addition to the above, Garcia-Riquelme [29] has compiled and exploited inferior Zeeman data for about 100 lines of Mn I, most of which appeared as pseudo doublets, triplets, or quartets because they were incompletely observed or resolved. In most cases, the Laudé type of Zeeman pattern could be recognized and for classified lines the observed Zeeman effect confirms the classification quantitatively. Because most of the Zeeman data for Mn I have been published elsewhere [23, 24, 25, 26, 27, 28] they will not be repeated here; only the type numbers [32, 34] will be listed in our list of classified lines, and the average derived g-values in our table of spectral terms.</p><p>Following the new observations of wavelengths and intensities of more than 2000 spectral lines belonging to Mn I, and the measurement of Zeeman patterns for nearly 500 lines, Catalán and Garcia-Riquelme undertook a complete revision of the analysis and quantum interpretation of this spectrum. A preview of this overall revision was summarized by Moore [21] in 1952 when 368 energy levels were reported to account for more than 1500 lines. This work continued practically until Catalán's death in 1957, and further progress was made by Garcia-Riquelme until 1962 when the total number of accepted levels rose to 404 and classified lines exceeded 2000. At this point, several hundred unclassified lines, mostly weak, hazy, and without Zeeman data, were sent to the Nat ional Bureau of Standards where Meggers applied an electronic computer to a final search for new energy levels by adding the wave numbers of lines to the wave numbers of levels and seeking constant sums within tolerated limits. Among hundreds of "new energy levels", only two were accepted as physically real; they constitute the term a2I with level values of 37148.66 and 37164.25 K. We conclude that the available material on Mn I does not permit further progress in the analysis of this spectrum. This conclusion and the fact that our material is more homogeneous and extensive than any existing description of Mn I justify publication of this paper.</p><!><p>Our final results for Mn I classified lines are presented in table 1, in which col. 1 contains the observed wavelength (λ) in angstroms (Å); col. 2, the intensity and character; col. 3, the wave number (σ) in kaysers (K); col. 4, the observed minus calculated wave number in kaysers (K); col. 5, the symbols for associated energy levels; and col. 6, the Zeeman type. Since the "Air" wavelengths were measured before 1950, they were converted to vacuum wave numbers with the aid of Kayser's Table [30] which was recently superseded by a new table published by Coleman, Bozman, and Meggers [31]. Because the average differences do not exceed 0.03 K, we have retained the older values.</p><p>In col. 2, some intensity numbers are accompanied by literal symbols for line character; these have the following meanings, c=complex, d=double, h=hazy, H=very hazy, l=shaded long ward, r=narrow self-reversal, R=wide self-reversal, s=shaded short ward, w=wide. Excepting self-reversals, the remaining symbols for line character frequently suggest unresolved hyperfine structure because the manganese nucleus has a spin of 5/2 (h/2π) and a magnetic moment of 3.5 nuclear magnetons.</p><p>Table 1 contains 2030 classified lines of Mn I, including 55 accepted double classifications for lack of adequate spectroscopic resolving power. The energy levels derived from these classified levels are symbolized in col. 5, and the difference between their numerical values and the vacuum wave number associated with the measured wavelength is shown in col. 4, O—C. The average O—C for all classified lines is 0.06 K.</p><p>The electron configurations, numerical values, and g-factors associated with the atomic-energy-level symbols in col. 5 of table 1, will be found in table 2. In the last column of table 1 appear Zeeman-type numbers for 390 classified lines of Mn I according to the description of basic types of Zeeman patterns by Back and Landé [32, 34.] Briefly, types 4, 5, 6 are restricted to even multiplicities (doublets, quartets, sextets, octets) whose energy levels always have half-integral inner-quantum number (J-values). In type 4, the level with larger J has the smaller g, in type 5 the level with larger J also has the larger g and in type 6, the two combining levels have equal J but unequal g values. Type 7b is a special case of types 4, 5, 6 in which the g values of both combining levels are equal so that the observed pattern is a pseudo triplet with one p component and two n components. In the case of equal J and equal g, the type is represented by 6, 7b, and the displacement of the n components in Lorentz units expresses both g values.</p><p>The letter C attached to a dozen type numbers in col. 6 indicates the lines with asymmetrical patterns which Catalán [24] analysed in detail and for which he derived proper g values. Finally, instead of type numbers, the letters P—B appear for fifty lines; these are additional lines with Zeeman patterns that exhibit partial Paschen-Back effects partially interpreted by Garcia-Riquelme et al. [28]. The Zeeman data were most helpful in this analysis of Mn I; they have confirmed our interpretation almost completely and have permitted the designation of many terms belonging to the doublet system which was the most difficult to establish. The available Zeeman data for Mn I [6, 21, 23, 24, 27, 28, 29] prove that most of the levels arising from low-energy configurations (d5s2, d5s, d5sp, d6p) exhibit a remarkably pure LS coupling of electrons, and their g-factors are usually identical with the theoretical Landé values, within the error of measurement. Some of the upper levels of both the even and the odd configurations present anomalous g-values that may be explained by intermediate coupling or by incorrect grouping of levels in designated terms. Many more Zeeman patterns could be observed if a modern light source that promotes the emission of Mn I radiation were used.</p><p>The latest information on spectral terms derived from this analysis of Mn I is presented in table 2 where electron configurations (including series limits), spectral-term designations in standard notation for LS electron coupling, J-values, relative numerical values, intervals, and observed g-factors are shown in successive columns. The totals are 42 even terms with 125 levels and 60 g values, and 94 odd terms with 266 levels plus 13 miscellaneous and 164 g values. In 1952 [21] the totals were 36 even terms with 109 levels and 26 g values, and 66 odd terms with 214 levels plus 33 miscellaneous and 81 g values, not counting 12 abandoned levels. It is seen that the present analysis of the Mn I spectrum is a considerable improvement over any preceding, but, if a comparison is made of the presently recognized terms with the table of predicted terms [21, pp. XIV, XV], any claim that it is satisfactorily completed is unjustified. In order to show the progress that has rewarded this problem during the past 40 years, we briefly summarize in table 3 the number of classified Mn I lines and derived atomic energy levels at different times during this period.</p><p>The present analysis has effectively exhausted all available data on wavelengths, intensities, and Zeeman patterns of Mn I so that no further progress can be made with that material. But now we know that an arc between manganese electrodes at atmospheric pressure, with which all observations were made, is an inferior light source. Because manganese is a relatively light atom (A-55), and the arc has a high temperature (>5000 °C), it produces broad lines which are always very hazy and unsymmetrical when they involve high energy levels. All complex Mn I terms that converge to a7S3 or a5S2 limits in Mn II have very small intervals and are mostly unresolved because of the excessive line widths. Furthermore, the traditional arc in air always produces a strong background of molecular spectra that often masks atomic lines. All of these spectroscopic objections to an arc in air can now be avoided by using highly evacuated electrodeless (quartz tube) lamps containing a trace of metal or halide compound excited with ultrahigh frequency as demonstrated by Meggers and Westfall [33] for mercury and by Corliss and Meggers [34] for hafnium. At elevated temperatures (ca. 800 °C) such lamps favor spectra of neutral atoms, especially important for faint lines and for observing first spectra in strong magnetic fields; we mention this to aid anyone who becomes ambitious to make further progress in the description and quantum interpretation of the Mn I spectrum. That such progress is desirable becomes obvious when the now known spectral terms (table 2) are compared with the predicted ones [21]. Although many electron configurations and spectral terms have been identified, rarely is a configuration represented by all its terms and in some cases the terms themselves are fragmentary or even uncertain.</p><p>In 1922, Catalán derived, from spectral series of running s electrons, the absolute energy of the ground state of Mn I, and computed the first ionization potential to be 7.41 electron volts. Thirty years later Catalán and Velasco [35] systematically studied the first three ionization potentials of elements in the iron group; for Mn I they obtained an absolute value of 59960 K for the ground state, the equivalent 7.432 eV. These values were quoted in the second volume of Atomic Energy Levels [21]. A decade later, Garcia-Riquelme [36] applied a Ritz formula to a series of 6F terms with four members (4f, 5f, 6f, 7f) and found the limit near 59979 K or 7.434 eV. We decided to adopt the average of the last two determinations resulting in a limit of 59970 K or ionization potential of 7.433 eV for normal manganese.</p>

PubMed Open Access

New potently bioactive alkaloids from Crinum erubescens

Antimalarial bioassay-guided fractionation of the swamp lily Crinum erubescens led to the isolation of four compounds with potent antiplasmodial activity. Compounds 1 and 2 were determined from their spectroscopic data to be the known pesticidal compound cripowellin A and the known pesticidal and antiproliferative compound cripowellin B. 1D and 2D-NMR techniques were used to determine the identities of 3 and 4 as the new compounds cripowellin C and D. A fifth compound was identified as the known alkaloid hippadine, which was inactive against Plasmodium falciparum. The antiplasmodial IC50 values of compounds 1 \xe2\x80\x93 4 were determined to be 30 \xc2\xb1 2, 180 \xc2\xb1 20, 26 \xc2\xb1 2, and 260 \xc2\xb1 20 nM, respectively, and their antiproliferative IC50 values against the A2780 human ovarian cancer cell line were 11.1 \xc2\xb1 0.4, 16.4 \xc2\xb1 0.1, 25 \xc2\xb1 2, and 28 \xc2\xb1 1 nM.

new_potently_bioactive_alkaloids_from_crinum_erubescens

1. Introduction<!>2.1. Isolation<!>2.2. Structure Elucidation<!>2.3. Circular Dichroism and Stereochemistry<!>2.4. Bioassay Data<!>3. Conclusions<!>4.1. General<!>4.2. Plant material<!>4.3. Extraction and Isolation<!>4.4. Antimalarial Bioassay<!>4.5. Antiproliferative Bioassay<!>4.6. Compound Information

<p>The genus Crinum contains around 110 accepted species with over 270 synonyms belonging to the family Amaryllidaceae.1 Members of the genus Crinum possess large extravagant flowers on leafless stems and are distributed in moist sites, such as forests, river edges, seasonal pools, or saltpans, and can be found throughout, the tropics of Africa, Asia, America, Southern Africa, Madagascar, and Mascarene and the Pacific Islands.2 Extracts from Crinum species have been used traditionally to treat a variety of ailments including fever, pain management, swelling, sores and wounds, cancer, and malaria.3 As a member of the Amaryllidaceae family, the Crinum genus is known to be a rich source of norbelladine type alkaloids, including lycorine, crinine, and narciclasine.4 Alkaloids isolated from various Crinum species have shown activity in a wide variety of assays including analgesic, anticancer, antibacterial, antiviral, antifungal, and antimalarial assays.3</p><p>Malaria is a tropical disease that has a disproportionate effect in poor and underdeveloped countries without access to western medicine, and with the appearance of artemisinin resistant parasites in five countries there is an urgent need for new and affordable medicines.5 Since plants have been a successful source for antimalarial medicines such as quinine and artemisinin, our group has been investigating plant extracts from the Natural Products Discovery Institute (NPDI) collection for new compounds or known compounds with new activity against Plasmodium falciparum.6 An extract of Crinum erubescens L. f. ex Aiton displayed strong antimalarial activity from the initial screening and was selected for isolation.</p><!><p>A methanol extract of C. erubescens was partitioned between aqueous methanol and hexanes, the aqueous methanol was then dried and suspended in water and extracted with ethyl acetate to afford an active ethyl acetate fraction (IC50 ⋘ 1.25 μg/mL). Due to the high likelihood of the extract containing a large amount of alkaloids, an acid/base extraction was performed on the ethyl acetate fraction. The fraction was suspended in 2% sulfuric acid and extracted with ethyl ether to produce a "neutral" fraction. The sulfuric acid solution was then adjusted to a pH of 10 with a 20% ammonium hydroxide solution and then extracted with ethyl acetate to generate a "basic" fraction. The basic fraction was found to be active (IC50 ⋘ 1.25 μg/mL) and was subjected to a C18 solid phase extraction (SPE). The SPE column was loaded and eluted with methanol, chloroform, and finally with water to remove any salts that were carried over from the acid/base extraction. Separation of the active methanol SPE fraction (IC50 ⋘ 1.25 μg/mL) by HPLC over a C18 column with methanol/water afforded five active fractions (IC50 ⋘ 1.25 μg/mL) with slight impurities. Final HPLC purifications were conducted on the five active fractions over a C18 column using acetonitrile/water, affording compounds 1-5.</p><!><p>Compound 1 (Figure 1) was isolated as a white amorphous solid with a chemical formula of C25H31NO12 as determined by HRESIMS (m/z 538.1916 [M+H]+, calcd for C25H32NO12+, 538.1919). Proton NMR data (Table 1) revealed the presence of an aromatic ring moiety, a methylenedioxy bridge, a methoxy, and a glucose moiety. A literature search using the chemical formula and the key structural features confirmed the structure of 1 as the known compound cripowellin A.7 The structure of 2 (Figure 1) was elucidated as the known compound cripowellin B from the HRESIMS data (m/z 524.2127 [M+H]+, calcd for C25H34NO11+, 524.2126) and comparison of its 1H NMR data (Table 1) to the published literature values.7 Both 1 and 2 possessed an intense fragment in their HRESIMS data at 320.1119 m/z, corresponding to the [Aglycone+H]+ fragment.</p><p>Compound 3 (Figure 1) was isolated as a white amorphous solid. It had the chemical formula C25H31NO11 as determined by HRESIMS (m/z 522.1968 [M+H]+ calcd for C25H32NO11+, 522.1970). As with the HRESIMS data for cripowellins A and B, another high intensity fragment was observed correlating to the aglycone of the molecule (320.1119 m/z [Aglycone+H]+, calcd for C16H18NO6+, 320.1129 m/z). Comparing the 1H NMR of 1 to 3 it could be observed that the aglycones of the compounds were identical in structure, with the key differences being observed in the sugar moiety. The anomeric proton observed at δH 4.44 (d, J = 7.9 Hz, 1H) along with a doublet methyl at δH 1.36 (d, J = 6.1 Hz, 3H) indicated the presence of a 6-deoxypyranosyl sugar. Two-dimensional NMR spectroscopic data, including COSY, HSQC, HMBC, and NOESY were used to determine the connectivity and configuration of the sugar moiety. HSQC was used to determine all direct proton to carbon attachments while COSY correlations were used to determine the connectivity around the pyranosyl sugar from H-1′ to H-6′. An HMBC correlation from the anomeric proton observed at δH 4.44 to δC 84.3 (C-15) confirmed the attachment of the sugar moiety to C-15. The more unusual protons associated with the sugar moiety were found in the 1,3,5-trioxepane ring system, which is formed by part of the sugar moiety (C-2′ and C-3′) and by two isolated methylene units containing four diastereotopic protons. The first methylene unit had two protons at δH 4.94 (d, J = 6.0 Hz, 1H) and δH 4.82 (d, J = 6.0 Hz, 1H) both attached to δC 91.5 (C-1″), while the second methylene unit had two protons at δH 5.01 (d, J = 5.6 Hz, 1H) and δH 4.87 (d, J = 5.6 Hz, 1H), both attached to δC 92.3 (C-2″). HMBC correlations from H-1″b to C-2′ and C-2″, and from H-2″b to C-3′ and C-1″ confirmed the attachment and location of the 1,3,5-trioxepane ring system. The methoxy group at δH 3.54 / δC 61.0 (s, 3H) had a single HMBC correlation to C-4′, confirming its attachment to the sugar moiety. NOESY correlations from H-1′ to H-3′ and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as 15-([2′,3′][1,3,5]-trioxepane-4′-methoxy-β-D-quinovose). Compound 3 was assigned the name cripowellin C.</p><p>Compound 4 (Figure 1) was isolated as a white amorphous solid. It had the chemical formula C25H33NO11 as determined by HRESIMS (m/z 508.2131 [M+H]+, calcd for C25H34NO10+, 508.2177). As in the HRESIMS data for cripowellins A, B, and C another high intensity fragment was observed correlating to the aglycone of the molecule (m/z 320.1096 [Aglycone+H]+, calcd for C16H18NO6+, 320.1129). Comparison of the 1H NMR data of 4 with that of 3 indicated that the aglycones of the two compounds were identical, that the 6-dexoypyranosyl sugar moiety was also present, there were two additional methoxy methyl groups, and that the 1,3,5-trioxepane-ring moiety was absent. Again, 2D NMR spectroscopic data, including COSY, HSQC, HMBC, and NOESY, were used to determine the connectivity and configuration of the sugar moiety. An HMBC correlation from the anomeric proton observed at δH 4.31 (d, J = 7.9 Hz, 1H) to δC 84.7 (C-15) confirmed the attachment of the sugar moiety to C-15. Once the connectivity around the ring from H-1′ to H-6′ had been established as before from HSQC and COSY correlations, HMBC was used to determine the locations of the methoxy methyl groups. The methoxy methyls at δH 3.44 / δC 60.6 (s, 3H), δH 3.58 / δC 60.8 (s, 3H), and δH 3.53 / δC 60.7 (s, 3H) each had a single HMBC correlation to their respective attachment locations at C-2′, C-3′, and C-4′ respectively. NOESY correlations from H-1′ to H-15, H-3′, and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as 15-(2′,3′,4′-methoxy-β-D-quinovose). Compound 4 was assigned the name cripowellin D.</p><p>Compound 5 (Figure 1) was isolated as an amorphous solid with the composition C16H9NO3 as determined by HRESIMS (264.0659 m/z [M+H]+, calcd for C16H10NO3+, 264.0655). The 1H NMR spectrum displayed signals for five aromatic protons, two olefinic doublets, and a methylenedioxy group. Three of the five aromatic protons were determined to be in the same spin system due to the observed coupling constants from δH 7.94 (br d, J = 7.7 Hz, 1H), δH 7.76 (dd, J = 7.6, 0.7 Hz, 1H), and δH 7.49 (t, J = 7.7 Hz, 1H). The broad doublet at δH 7.94 is most likely the result of unresolved meta coupling. The remaining two aromatic signals are both isolated singlets at δH 8.00 (s, 1H), and δH 7.68 (s, 1H). There was also a characteristic methylenedioxy resonance at δH 6.17 (s, 2H). Lastly, the two olefinic protons were both coupled to one another with resonances at δH 6.91 (d, J = 3.6 Hz, 1H) and δh 8.05 (br d, J = 3.6 Hz, 1H). Putting the observed proton resonances, molecular formula, and the thirteen degrees of unsaturation together the structure of hippadine was produced. Comparison of the 1H NMR literature data for hippadine with the observed spectroscopic data confirmed the identity of 5 as the known compound hippadine.8</p><!><p>Optical rotations of cripowellins A and B matched the published values,7 and the measured optical rotations for cripowellins C and D were very similar to those of cripowellins A and B. This observation combined with the NOESY data provided evidence to support the indicated stereochemistry for cripowellins C and D. To further support this claim ECD spectra were obtained on cripowellins A – D in methanol and it was observed that all four compounds had very similar spectra.</p><!><p>Compounds 1 – 5 were evaluated for their antiparasitic activity against the chloroquine/mefloquine-resistant Dd2 strain of Plasmodium falciparum. Compounds 1 – 4 exhibited potent antimalarial activity, with IC50 values of 30 ± 2, 180 ± 20, 26 ± 2, and 260 ± 20 nM, respectively. Comparing the IC50 values of 1 – 4 indicates that replacement of the hydroxyl at 6′ by methyl does not change the activity significantly, while removal of the 1,3,5-trioxapane-ring decreases the activity by about on order of magnitude. Compound 5 was found to be inactive at 38 μM. This loss of activity as compared with the activity of fraction containing 5 is most probably due to the small amount of 2 (cripowellin B) that was present in these fractions until the final purification of 5.</p><p>Compounds 1 – 4 were also tested for antiproliferative activity against the A2780 human ovarian cancer cell line, and were found to have IC50 values of 11.1 ± 0.4, 16.4 ± 0.1, 25 ± 2, and 28 ± 1 nM, respectively. Compound 2 has also been shown to exhibit nM antiproliferative activity against human melanoma (A375), colon (SW620), and cervical (HeLa) cell lines.10 Compound 5 was not tested in the A2780 assay since it has been previously reported to be inactive against multiple human cells lines including; lung (A549), colon (LoVo), lymphocytic leukemia (6T-CEM), and promyelocytic leukemia cells (HL-60).11</p><!><p>The genus Crinum continues to be a rich source of bioactive alkaloids, but although antiplasmodial activity has been reported for some Crinum alkaloids,3, 12-13 this work adds cripowellins A – D as a new class of antiplasmodial alkaloids characterized by a nanomolar level of activity. The two new compounds 3 and 4 were isolated along with the three known compounds, 1, 2, and 5 from C. erubescens.</p><p>The unusual 1,3,5-trioxepane ring moieties of 1 and 3 might conceivably be considered to be artefacts formed from reaction of a putative diol precursor with formaldehyde (present as a trace impurity) under the acidic conditions of partition with aqueous sulfuric acid. A comparison of the 1H NMR spectra of compounds 1 and 3 with that of the crude extract was thus conducted, and the spectrum of the crude extract was shown to contain peaks corresponding to the methylene protons of the trioxepane groups. An HPLC comparison of the crude extract with that of a fraction containing compounds 1 – 4 also indicated the presence of these compounds in the crude extract. Compounds 1 – 4 are thus all considered to be authentic natural products.</p><p>When 1 and 2 were originally isolated from Crinum powellii in 1997 they were found to possess insecticidal activity,14 and in 2006 2 was reported to possess potent antiproliferative activity against a variety of human cancer cell lines.10 Although compounds 1 – 4 have now been shown to possess potent antiplasmodial activity, their antiproliferative activities against human cancer cell lines and the complex nature of the cripowellin structure regrettably combine to make the cripowellins less than ideal candidates for development as antimalarial drugs unless their antiplasmodial and antiproliferative activities can be separated by appropriate structure modifications.</p><!><p>Optical rotations were recorded on a JASCO P-2000 polarimeter, and UV spectra were measured on a Shimadzu UV-1201 spectrophotometer. ECD analysis was performed on a JASCO J-810 spectropolarimeter with a 1 cm cell in methanol at room temperature under the following conditions: speed 100 nm/min, time constant 1 s, bandwidth 1.0 nm. 1H and 13C NMR spectra were obtained either on a Bruker Avance 600 or Bruker Avance 500 spectrometer. Mass spectra were obtained on an Agilent 6220 LC-TOF-MS spectrometer. Semipreparative HPLC was performed using Shimadzu LC-10AT pumps coupled with a Shimadzu SPD-M10A diode array detector, a SCL-10A system controller, and a Phenomenex 5 μm, 100 Å, Luna C18(2) (250 × 10 mm) column (Column A) or a Cogent 4 μm, 100 Å, Bidentate C18 (250 × 10 mm) column (Column B). Solid phase extractions were conducted with Thermo Scientific HyperSep C18 500 mg, 3 mL SPE cartridges, and all reverse phase TLC was conducted with Sorbtech C18-W silica TLC plates, w/UV254 on an aluminum backing with a thickness of 150 μm.</p><!><p>Specimens of above-ground parts of Crinum erubescens were collected by J. Francisco Morales of the Instituto Nacional de Biodiversidad, Costa Rica, along the road leading to Playa Cacao in Llano Bonito, Puntarenas. Vouchers are on deposit at INBIO under accession number FM01522.</p><!><p>Dried, powdered plant material was exhaustively extracted with MeOH to give a MeOH-soluble extract designated 13103-C4 X-2776; a total of 478 mg of this extract was made available to Virginia Tech. This extract had an IC50 value ⋘ 3 μg/mL against P. falciparum Dd2 strain. The extract of C. erubescens (478 mg) was dissolved in 200 mL of MeOH and to which water (20 mL) was added. This aqueous methanolic solution was then extracted with 8 × 200 mL of hexanes. The aqueous MeOH was then concentrated in vacuo, and suspended in 200 mL of water. The resulting aqueous suspension was then extracted with 8 × 200 mL of EtOAc to afford an active EtOAc fraction (342 mg) with an IC50 value of ⋘ 1.25 μg/mL. The dried EtOAc fraction was suspended in 200 mL of a 2% H2SO4 solution and extracted with 3 × 200 mL of Et2O to produce a "neutral" fraction, which was then dried over Na2SO4. The H2SO4 solution was then adjusted to a pH of 10 with a 20% NH4OH solution, and extracted with 3 × 200 mL of EtOAc to generate a "basic" fraction.15 Any insoluble material that formed was kept with the aqueous fraction throughout the acid/base extraction. The "basic" fraction (68 mg) was found to be active with an IC50 value of ⋘ 1.25 μg/mL and was then further separated on a C18 solid phase extraction (SPE). The sample was dissolved in MeOH and loaded onto the SPE cartridge and eluted with 100 mL of MeOH to generate the first fraction. Then the SPE cartridge was eluted with 100 mL of chloroform to form the second fraction, and finally with 50 mL of water to remove any salts that were carried over from the acid/base extraction. The MeOH fraction (59 mg) was found to have an IC50 value of ⋘ 1.25 μg/mL and was then separated on C18 HPLC. A H2O/MeOH gradient was developed using Column A, starting from 62:38 to 30:70 over 40 minutes followed by a maintained flow at 0:100 for 20 minutes. A total of thirteen fractions were collected, fractions 6 (tR 25.90 min), 8 (tR 29.12 min), 9 (tR 31.33 min), 10 (tR 32.20 min), and 12 (tR 35.10 min) all possessed IC50 values of ⋘ 1.25 μg/mL but still contained slight impurities. Fraction 6 was purified with Column A using an isocratic water/acetonitrile flow of 72:28 for 22 minutes, which yielded 1.2 mg (tR 20.20 min; IC50 30 ± 2 nM) of 1 (cripowellin A). Fraction 8 was purified with Column B using a water/acetonitrile gradient from 45:55 to 35:65 over 20 minutes, which yielded 1.4 mg (tR 11.14 min; IC50 180 ± 20 nM) of 2 (cripowellin B). Fraction 9 was purified using Column A with a water/acetonitrile gradient starting from 55:45 to 45:55 over 30 minutes, which yielded 0.4 mg (tR 25.32 min; IC50 not active) of 5 (hippadine). Fraction 10 was purified using Column A with an isocratic water/acetonitrile flow of 58:42 for 15 minutes, which yielded 1.2 mg (tR 11.94 min; IC50 26 ± 2 nM) of 3 (cripowellin C). Fraction 12 was purified using Column A with an isocratic water/acetonitrile flow of 52:48 for 12 minutes, which yielded 0.7 mg (tR 9.57 min; IC50 260 ± 20 nM) of 4 (cripowellin D).</p><!><p>Each fraction and isolated compound was tested against the chloroquine/mefloquine-resistant Dd2 strain of Plasmodium falciparum in a 72-hour treatment using the malaria SYBR green I-based fluorescence assay as described previously.16-17 Artemisinin was used as the positive control with an IC50 of 6 ± 1 nM.</p><!><p>The A2780 ovarian cancer cell line assay was performed at Virginia Polytechnic Institute and State University, as previously reported.18-19 The A2780 cell line is a drug-sensitive ovarian cancer cell line.20</p><!><p>Compound 1 (cripowellin A): [α]21D -28.5° (c 5.5×10-6, MeOH); UV (MeOH) λmax (log ε) 208 (3.19), 291 (2.32) nm; ECD (MeOH) [Δε]302 nm +2.80, [Δε]282 nm -4.40, [Δε]249 nm +0.80; HRESIMS [2M+Na]+ m/z 1097.3602 (calcd for C50H62N2NaO24+ 1097.3585), [M+Na]+ m/z 560.1744 (calcd for C25H31NNaO12+ 560.1738), [M+H]+ m/z 538.1916 (calcd for C25H32NO12+ 538.1919), [Aglycone+H]+ m/z 320.1121 (calcd for C16H18NO6+ 320.1129). 1H and 13C NMR in CDCl3 see Table 1.</p><p>Compound 2 (cripowellin B): [α]21D -51.8° (c 1.1×10-5, MeOH); UV (MeOH) λmax (log ε) 212 (3.09), 290 (2.49) nm; ECD (MeOH) [Δε]302 nm +1.35, [Δε]281 nm -2.09, [Δε]248 nm +0.48; HRESIMS [2M+Na]+ m/z 1069.4022 (calcd for C50H66N2NaO22+ 1069.3999), [M+Na]+ m/z 546.1955 (calcd for C25H33NNaO11+ 546.1946), [M+H]+ m/z 524.2127 (calcd for C25H34NO11+ 524.2126), [Aglycone+H]+ m/z 320.1129 (calcd for C16H18NO6+ 320.1129). 1H and 13C NMR in CDCl3 see Table 1.</p><p>Compound 3 (cripowellin C): [α]21D -41.1° (c 5.9×10-6, MeOH); UV (MeOH) λmax (log ε) 209 (3.26), 290 (2.44) nm; ECD (MeOH) [Δε]306 nm +2.90, [Δε]270 nm -3.14, [Δε]251 nm +1.34; HRESIMS [2M+Na]+ m/z 1065.3638 (calcd for C50H62N2NaO22+ 1065.3686), [M+Na]+ m/z 544.1784 (calcd for C25H31NNaO11+ 544.1789), [M+H]+ m/z 522.1968 (calcd for C25H32NO11+ 522.1970), [Aglycone+H]+ m/z 320.1119 (calcd for C16H18NO6+ 320.1129). 1H and 13C NMR in CDCl3 see Table 1.</p><p>Compound 4 (cripowellin D): [α]21D -87.1° (c 2.9×10-6, MeOH); UV (MeOH) λmax (log ε) 208 (3.43), 291 (2.50) nm; ECD (MeOH) [Δε]305 nm +1.77, [Δε]273 nm -2.29, [Δε]250 nm +0.84; HRESIMS [2M+Na]+ m/z 1037.4022 (calcd for C50H66N2NaO20+ 1037.4101), [M+Na]+ m/z 530.1969 (calcd for C25H33NNaO10+ 530.1997), [M+H]+ m/z 508.2131 (calcd for C25H34NO10+ 508.2177), [Aglycone+H]+ m/z 320.1096 (calcd for C16H18NO6+ 320.1129). 1H and 13C NMR in CDCl3 see Table 1.</p><p>Compound 5 (hippadine): HRESIMS [M+H]+ m/z 264.0659 (calc. for C16H10NO3+ 264.0655); 1H NMR (500 MHz, CDCl3) δH 8.05 (br d, J = 3.6 Hz, 1H), 8.00 (s, 1H), 7.94 (br d, J = 7.7 Hz, 1H), 7.76 (dd, J = 7.6, 0.7 Hz, 1H), 7.68 (s, 1H), 7.49 (t, J = 7.7 Hz, 1H), 6.91 (d, J = 3.6 Hz, 1H), 6.17 (s, 2H); 1H NMR (500 MHz, MeOD) δH 8.14 (br d, J = 7.7 Hz, 1H), 8.04 (d, J = 3.6 Hz, 1H), 7.92 (s, 1H), 7.87 (s, 1H), 7.82 (dd, J = 7.6, 0.8 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.02 (d, J = 3.6 Hz, 1H), 6.21 (s, 2H).</p>

PubMed Author Manuscript

The effect of layer thickness and immobilization chemistry on the detection of CRP in LSPR assays

The immobilization of a capture molecule represents a crucial step for effective usage of gold nanoparticles in localized surface plasmon resonance (LSPR)-based bioanalytics. Depending on the immobilization method used, the resulting capture layer is of varying thickness. Thus, the target binding event takes place at different distances to the gold surface. Using the example of a C-reactive protein immunoassay, different immobilization methods were tested and investigated with regard to their resulting target signal strength. The dependency of the target signal on the distance to the gold surface was investigated utilizing polyelectrolyte bilayers of different thickness. It could be experimentally demonstrated how much the LSPR-shift triggered by a binding event on the gold nanoparticles decreases with increasing distance to the gold surface. Thus, the sensitivity of an LSPR assay is influenced by the choice of immobilization chemistry.The C-reactive protein (CRP) is an important biomarker for inflammation and infection of the human body 1-4 . CRP is an acute phase protein of the pentraxin family formed in the liver, as a marker of general or post-operative infectious diseases primarily for bacterial infections 5 , acute myocardial infarction, and other diseases 6 . In healthy people, the concentration of CRP in serum is below 10 mg/L. Between 10-40 mg/L is typical for mild inflammation and viral infections, while active inflammation and bacterial infections result in levels of 40-200 mg/ L 3 . Thus, the CRP level correlates with the stage of the diseases and is a decisive criterion for the prescription of antibiotics for the patient 7 . Therefore, diagnostic detection is very important. Additionally, CRP detection allows for a discrimination between bacterial and viral infections 8 . The most used diagnostic methods for CRP are rapid point-of-care tests (POCT) based on lateral flow-assays with a sensitivity of 10 mg/L. Though surface plasmonic resonance sensing for CRP is becoming more common there are still very few such methods close to diagnostic use [9][10][11] . Several plasmonic nanoparticle-based methods are established in enzyme-linked immunosorbent assay (ELISA) platforms. These assays use labeled secondary antibodies in sandwich assays [12][13][14][15][16] or metal-enhanced optical signals by enzymatic deposition 15 or metal-enhanced fluorescence 17 for the signal enhancement.Direct detection of CRP with plasmonic nanoparticles is possible altogether avoiding labels and secondary antibodies. A simple detection is the main advantage of colorimetric assays. The binding of CRP on the particles either stabilizes against salt-induced aggregation, or competes with bound aptamers, leading to destabilization of the nanoparticle solution 18 . Besides colorimetric detection, which yield somewhat qualitative results, plasmonic nanoparticles can also act as transducers. This is accomplished by using the change in spectroscopic properties (resonance wavelength) upon refractive index change (binding of molecules on the surface) enabling quantitative detection. Examples include the direct binding of CRP on anti-CRP antibodies (anti-CRP-AB)-modified gold nanospheres 19 , nanorods modified with single chain variable fragment (scFv) 20 , or silver nanoprisms modified with cytidine 5´-diphosphocholine (PC) 21 . In combination with straightforward optical readout units, LSPR sensors can also provide a new field of application for on-site diagnostics.Because the capture-antibody-immobilization determines the critical analytical parameters such as sensitivity, reproducibility, and robustness, this step has to be adapted and optimized for a given assay on a given technological platform. In the case of LSPR, gold nanoparticles (AuNP) represent the sensors used, and have to be modified with the detection antibodies. A wide range of antibody immobilization approaches have been developed during the past few decades, starting with passively adsorbing the antibodies on the substrate, and subsequently establishing various functionalization and cross-linking strategies, overcoming certain shortcomings of earlier

the_effect_of_layer_thickness_and_immobilization_chemistry_on_the_detection_of_crp_in_lspr_assays

Materials and methods<!>LSPR instrument.<!>CRP-assay design.<!>Chemical immobilization of α-hCRPc via EDC/NHS.<!>Results<!>CRP detection.<!>Discussion<!>Conclusions

<p>Materials. Poly(allylamine hydrochloride) (PAH), polystyrene sulfonate (PSS) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany), N-Hydroxysuccinimid (NHS), glacial acetic acid, sodium chloride (NaCl), sodium hydroxide (NaOH), glycine, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid-hydrochlorid (EDC), ethanol, HCl, 3-triethoxysilylpropylamine (APTES), 10 × PBS Buffer and BSA were purchased from Carl Roth GmbH (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Sodium acetate was purchased from Merck KGaA (Darmstadt, Germany). Human C-reactive protein, anti-hCRP-capture-antibody biotinylated (α-hCRPcb) and non-biotinylated (α-hCRPc) and anti-hCRP-detector-antibody (α-hCRPd) was purchased from Senova GmbH (Weimar, Germany). All proteins were used as received without further purification and all used antibodies are monoclonal. Sodium acetate buffer (NaAc) was prepared by dissolving sodium acetate close to 10 mM in ultrapure water, pH was adjusted with HCl/NaOH to 4, 4.5 or 5 and filled up with ultrapure water to a final concentration of 10 mM NaAc. Glycine-HCl Buffer (Gly-HCl) was pre-pared similarly but with glycine and adjusted to pH 2.5.</p><p>AuNP-chip preparation. Schott borosilicate wafers were cut into 25 × 16 mm glass substrates. After washing with soap and water by hand, they were cleaned 10 min each under sonication in water, ethanol, acetone, rotisol, ethanol and water. Finally, they were blow-dried with nitrogen. The glass chips were activated by treatment with oxygen plasma etching for 1 h at 380 W in a 200G Plasma System (TePla GmbH, Wettenberg, Germany) and afterwards directly transferred in a preactivated (10 min stirring) 1% APTES solution with 1 mM Figure 1. Scheme of the studied immobilization approaches for anti-CRP antibodies. Left: Biotinylated anti-CRP antibodies are attached to the gold surface by thiolated streptavidin. Right: After a surface modification by a self-assembled monolayer of MUA, EDC chemistry is utilized to attach unmodified anti-CRP antibodies. The inset shows the decrease of the sensor signal with increasing surface distance for layer-by-layer (LbL) deposition with charged polyelectrolyte (PEL) bilayers. acetic acid for 10 min. A 5 min sonication in ultrapure water was the next step before the glass chips were again blow-dried with nitrogen and stored under an argon atmosphere or were used directly. On the respective APTES chips a droplet of 20 µl 10 × concentrated spherical 80 nm AuNP from BBI (British BioCell International, Cardiff, UK) were deposited and incubated for 1 h at room temperature. If coverslips were used, they were treated similar. The produced AuNP chips were then dipped two times in water for washing and carefully blow-dried with nitrogen. Stored in a closed container, the chips can be used from several month up to years.</p><!><p>All microfluidic assays used a custom build LSPR instrument (figure S3) at IPHT-Leibniz. It consists of a halogen light source HL-2000-FHSA (Ocean Optics, USA), an optical fiber connected UV/ VIS linear photodiode array spectrometer USB 2000+ (Ocean Optics, USA), a peristaltic pump (Ismatec Reglo-ICC, Cole-Parmer GmbH, Wertheim, Germany), a 2-way valve (Bio-Chem Fluidics Inc, Boonton, USA) for the waste and a custom designed 3D printed microfluidic chamber (figure S4) and sealing with two inputs and one output capillary at each channel side similar to an earlier published setup [28][29][30] . For some measurements singleuse flow cells "Basic sensor platform II" (# 10001354, microfluidic chip shop GmbH, Jena, Germany) were used with adhesive tape gasket for Fl. 1005-rhombic chamber shape # 10001361. The in-and outputs were used in the same way as for the 3D printed chamber by blocking unused channels with a plug from the same company. For the pump and valve control, a custom-built Python program was used which also records the spectral information and calculates the centroid position of the LPSR peak in nm which is then visualized in a sensogram (plot of peak wavelength against time). The evaluation of the measured sensogram was also done via a Python script to extract mean values, standard deviations of mean values and wavelength shift (Δλ) values. Dried AuNP-Chips with or without SAM were attached to 3D printed microfluidic chamber with specific 3D printed sealing or glued in the commercial chamber. If the commercial chamber was used, it is mentioned in the method. All chips were cleaned before use (in the case of SAM before the SAM deposition) 3 min under ozone (UV ozone cleaner UVC-1014 Nano-BioAnalytics, Berlin, Germany). Before and after each measurement, the whole system was flushed with water for at least 15 min. All solutions used in the microfluidic system were filtered with a 0.22 µm Syringe filter (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and afterwards degassed for at least 30 min under vacuum (Air Admiral, Cole-Parmer GmbH, Wertheim, Germany) in a desiccator (Nalgen, Thermo Fisher Scientific, Waltham, USA). At the beginning of each measurement, a lamp spectrum without an AuNP-chip was recorded for background subtraction and noise reduction. The specific refractive indices of the used buffers were measured with a portable refractometer PAL-RI B331500 (ATAGO, Tokio, Japan) and entered into the software. Afterwards, a calibration script was used to calculate the bulk sensitivity of the chip by alternately flushing two different buffers with different refractive indices and recording the associated centroid wavelength. The bulk sensitivity (S B ) is calculated according to Eq. 1:</p><p>where Δλ is the wavelength shift of the centroid position and Δn b is the refractive index change for the specific buffer solutions. Three times the standard deviation of a mean over at least 50 s of buffer injection (using as reference or blank) was used as threshold value for the limit of detection (LOD) calculation.</p><p>Layer by layer deposition. For a pure PAH/PSS assay without any CRP, both 1 mM PAH and 1 mM PSS (with respect to the monomer) were dissolved in 0.1 M NaCl, respectively. The solutions were pumped alternately over the AuNP-chip with a 100 s buffer (0.1 M NaCl) injection before and after each 150 s polyelectrolyte injection. The flowrates were set to 10 µl/min. The surface sensitivity (S S ) is calculated according to Eq. 2:</p><p>where λ is the wavelength of the centroid positions (Δλ = λ bilayer n − λ bilayer n−1 ) and n l is the refractive index of the layer (Δn l = n bilayer − n buffer ).</p><p>The decay length (l d ) of the immobilized particles and the refractive index sensitivity (m) was calculated by plotting the recorded plasmon shifts (Δλ) against the layer thickness (d) and fitting the data with Eq. 3 31,32 . The layer thickness of a PAH/PSS bilayer in aqueous environment is ~ 4 nm 33 and the refractive index of the bilayer is ~ 1.5 RIU except for the first two to three bilayer 34 . With respect to the refractive index of the 0.1 M NaCl buffer which was 1.3305 RIU, Δn = 0.1695 RIU.</p><p>Equation 3:</p><p>For the measurement of the CRP deposition on three or four PAH/PSS bilayers, coverslips and the commercial flow-chamber were used. Therefore, 2 mg/ml PAH, 2 mg/ml PSS and 31 µg/ml CRP each were dissolved in 0.5 M NaCl buffer. The PAH and PSS solutions were pumped alternately over the AuNP-chip with a buffer injection of 0.5 M NaCl before and after each polyelectrolyte injection up to 3 or 4 bilayers. Then the CRP was injected followed by another buffer step. The flowrates were set to 20 µl/min and all injection times were 150 s.</p><!><p>For all the assay measurements, the flowrates were set to 10 µl/min except for capture and target solutions which were pumped with 5 µl/min. For a clear distinction between bulk signals and binding events, the same buffer was injected before and after all reagents. In general, NaAc-Buffer (pH 4; 4.5 or 5) was used for immobilization and 1 × PBS as running buffer (Supporting Table S1). While running the first step, the channel for the second step was already pre-flushed with 3 µl/min directly to waste as well as further steps analogously. This was ensured by injecting the solutions alternately from different sides of the chamber, and opening www.nature.com/scientificreports/ the corresponding outlet valves. Due to the pre-flow and alternating flow techniques, the selected solutions ran through the channel directly after switching to the corresponding step. The software records all the injection steps accurately. Due to a time resolution of the spectrometer in the second range (1.5-3 s per measuring point), kinetic measurements are also possible. After the regeneration, the chip could be used for further target injections or a negative control. All capture reagents and BSA were dissolved in immobilization buffer. Targets and negative controls were dissolved in running buffer.</p><!><p>Carboxyl-groups, which are required for EDC/ NHS coupling, were realized on the AuNP-Chips with MUA as SAM. Therefore, the chips were incubated overnight in 1 mM MUA, 0.5 mM MUA with 5 mM MUD or 0.5 mM MUA with 5 mM 1-OT in ethanol to obtain different SAMs of MUA, MUA/MUD or MUA/1-OT on AuNP-Chips. MUA, MUD and 1-OT were purchased from Sigma (Sigma-Aldrich, Munich, Germany). Before using the chips, they were washed again shortly in ethanol and water. To identify the optimal pH for the immobilization buffer, 0.25 mg/ml α-hCRPc was dissolved in buffers with different pH and an immobilization pH scouting was carried out 35 . Therefore, the capture solutions pH 4, 4.5 and 5 were shortly pumped over the non-activated surface and electrostatically bound molecules were washed away with ethanolamine. The solution with the highest λ shift-pH 5-was chosen for the chemical immobilization. To obtain a covalent binding of the capture antibody to the AuNP-Chip, the carboxyl groups were activated by flushing 0.4 M EDC and 0.1 M NHS parallel with 5 µl/min from the same side in the chamber (finally 0.2 M EDC and 0.05 M NHS). After this activation, the capture solution was flushed over the surface for 400 s and the antibodies were bound randomly via their free amino groups (e.g., lysine). In the next step, the chip was flushed with ethanolamine for 500 s to wash away unspecific bound proteins and to react with the remaining activated carboxyl groups 36 . BSA blocking with 10 mg/ml in NaAc pH 4 for 300 s and a flow rate of 10 µl/min was still necessary.</p><p>Thiol-streptavidin mediated immobilization of α-hCRPcb. Thiol modified streptavidin (SH-SA)</p><p>was purchased from Protein Mods LLC (Waunakee, USA) and used as received without further purification or dilution. Blank AuNP-Chips can be easily functionalized with SH-SA by flushing a solution of 1 mg/ml over the surface for 200 s with 5 µl/min. In order to sufficiently block the sensor surface, various concentrations of BSA were tested in different buffers. Starting from 1 mg/ml over 10 mg/ml in PBS up to 1; 2 and 2.5 mg/ml in NaAc pH 4. Flow rates and injection times were also slightly varied between 5/10 µl/min and 200/250 s. After the blocking step a solution 0.25 mg/ml of biotinylated antibody α-hCRPcb was injected for 300 s with 5 µl/min. So, the chip was ready to use for hCRP detection without further blocking steps. For immobilization outside the chamber, one drop each of the SH-SA and the α-hCRPcb solutions were pipetted onto the AuNP chip, one after the other. Each of these were incubated for 1 h at 23 °C with 15-20% humidity, prior to washing 10 min with PBS (150 rpm horizontal shaking) and flushed with ultrapure water and dried in nitrogen stream (here called SH-SA outside).</p><!><p>Signal and sensitivity dependency on layer thickness. LSPR detection yielded the sensor response for attachment or binding events on the sensor surface. The well-established layer-by-layer deposition using PAH and PSS was utilized to demonstrate the sensing approach 34,[37][38][39] : The deposition of each additional layer on a gold nanoparticle chip (80 nm spheres) in a microfluidic system resulted in a longer wavelength (red) shift of the resonance peak. This is visible as individual steps in the sensogram, which plots the LSPR response over time (Fig. 2 inset). The first two to three bilayers were improperly formed due to a higher water content, resulting in lower refractive index and lower signal shifts than expected. After the 3rd bilayer, the refractive index of the bilayers was approximately 1.5 34 , the bilayer thickness in the wet state was approximately 4 nm 33 . The centroid of the LSPR-peak was measured over time and the shifts for each bilayer were plotted against the number of layers (Fig. 2). The evidence shows the signals for the bilayers decreased with increasing number of bilayers. This is due to a higher surface distance, where the field decay results in decreasing values. Also, the surface sensitivity was decreasing with increasing distance to the surface of the gold nanoparticles. With Eq. 3 the decay length (l d ) of this AuNP-chip was calculated to be 79.5 nm with refractive index sensitivity (m) = 141.1 nm/RIU. The before measured bulk sensitivity (S B ), calculated with Eq. 1 using the calibration, was with S B = 115.0 nm/RIU, i.e. slightly lower. For 3 bilayers, the S S was calculated to be 66.9 nm/RIU.</p><p>Although silver particles would provide better plasmonic properties, the higher chemical stability of gold results in a broader use of gold nanoparticles in LSPR sensing 40 . The spherical 80 nm gold nanoparticles therefore used in our investigations were developed over the years 28,41 as a good compromise between larger (and therefore more sensitive, but less homogenous synthesis) and smaller (less sensitive but better reproducible regarding shape and size distribution) sized nanoparticles. Anisotropic shaped particles would provide higher sensitivity, but are more complex to synthesize and usually difficult to biofunctionalize, therefore they were not utilized in this study.</p><!><p>Immobilization via thiolated streptavidin. It is well established that thiolated compounds form molecular layers on gold surfaces. One example are thiol-alkanes as well-studied model systems in the field of SAMs. On the other hand, biotin/(strept)avidin coupling is a powerful platform for nanoscale fabrication with many different applications in science, medicine, and nanotechnology. Combining these two well-established and straightforward attachment approaches, a scheme using thiolated streptavidin layers on the gold nanoparticle surfaces, and subsequent attachment of biotinylated antibodies on the SAMs, was utilized to prepare sensor substrates for CRP detection (Fig. 1 www.nature.com/scientificreports/</p><p>The preparation and subsequent performance of these sensor substrates for CRP detection is documented in Fig. 3. In the beginning (1), immobilized gold nanoparticles in a 10 mM NaAc pH 5-filled fluid cell result in a localized surface plasmon resonance (centroid) of 532.01 nm. Then, the cell was flushed with thiol-streptavidin, the resulting significant increase in centroid wavelength (to 534.388 nm) indicates strong binding on the gold nanoparticle surface (2). After buffer washing steps (3,4) a 1 mg/ml BSA passivation (5) induced only a small signal shift of 0.033 nm, which pointed to a weak BSA absorption on the gold particles in PBS buffer. Now, after a wash step (6), the biotinylated antibody (α-hCRPcb) was binding (7) with a shift of 0.521 nm. Afterwards, again the buffer (8) was injected, and a negative control (9) was conducted by applying the secondary antibody (α-hCRPd), which should bind on CRP only in case of a successful capturing. Because no CRP was present at this moment, this secondary antibody had no specific binding partner. However, the LSPR signal increased, indicating www.nature.com/scientificreports/ the presence of the secondary antibody at the surface. However, the subsequent washing step (10) appeared to remove it completely, the signal decreased to the previous level (cf. levels at 8 and 10). Next a regeneration step was introduced to check for-and remove-loosely bound molecules from the surface (11). The utilized solution seemed to have a lower refractive index as the standard buffer in 10, so the initial steep decrease in 11 can be attributed to this difference. However, afterwards the buffer from 10 was used again (12), allowed for a reproducible measurement: about 0.3 nm decrease between 10 and 12. Comparing 12 with 6 indicates that some of the attached biotinylated antibodies (7) were still on the substrate. After this preparation of the sensor surface, the actual CRP sensing step followed: 310 µg/ml CRP was flushed through the liquid cell leading to a measurable signal increase (13). This slightly decreased in a subsequent washing step (14), so that an overall CRP signal of about 0.2 nm resulted. Now, the already introduced secondary antibody was applied (15), testing on one hand the specificity of the CRP binding and, on the other hand, demonstrating possible signal amplification. There was a significant signal decrease of about 0.4 nm after a subsequent washing (16), which indicates a removal of some of the secondary antibodies. However, when CRP was flushed in again (19), this time one-tenth of the original concentration, the similar increase in signal was observed as before in step 13. This points to the fact that a saturation of the binding capacity was reached, even with the low concentration. A closer look at 13 and 19 reveals that the initial part of the curve was steeper in the case of higher concentration (13) compared to the lower one (19). When the secondary antibody was flushed in again (21), the same signal increase resulted as before in 15, indicating good reproducibility. This sensogram was selected to provide an example to highlight potential problems and challenges such as sensor regeneration, insufficient blocking or non-specific binding. Details will be discussed in the following paragraphs.</p><p>Limit of detection. Despite a significant lower response of SH-SA mediated CRP binding the measured limit of detection was 0.3 µg/ml (0.3 mg/L). With suitable regeneration solutions, the target molecules could be washed away, so that the sensor chip was available for another sample. Considering three times the standard deviation of a PBS buffer step (blank), the LOD was close to 0.3 µg/ml (Fig. 3 inset) 42 . In addition, the LOD could be improved by using signal amplification with secondary antibodies (sandwich immunoassay). Here, the secondary antibodies were used to evaluate the specificity of the assay. In an example measurement (Fig. 3), blocking with 1 mg/ml BSA in PBS was insufficient since unspecific binding of α-hCRPd in seen in step 9. But, as visible in step 10, the buffer was quickly washing away the "negative control" in the presence of CRP (steps 15/16 & 21/22). We assume that were close to, or above, the upper limit of quantification with the concentrations shown in Fig. 3 for CRP 1:10 (310 µg/ml). We support this claim with the observed shift for 310 µg/ml CRP being slightly lower than for 31 µg/ml CRP. The higher concentration still showed a higher shift during the injection (Step 13), but decreased relatively quickly in Step 14, which could indicate a saturation of the available binding sites. Another explanation for the decreased signal at higher concentrations could be the "hook effect", whereby the effectiveness of antibodies to form immune complexes is sometimes impaired when concentrations of an antibody or an antigen are very high. This can occur in sandwich immunoassays as well as in competitive format at high target concentrations and was also manifested by lower signal at higher concentrations 43 . However, such a high 31 µg/ml CRP signal as shown in Fig. 3 could not be confirmed with any other SH-SA assay. The other measurements were significantly lower, as shown in the comparison of the mean values in Fig. 5 and also in the LOD inset (Fig. 3). The regeneration with 10 mM glycine-HCl pH 2.5 also appeared to be too harsh, which was noticeable by the fact that the buffer steps showed slightly lower centroid signal after each regeneration (steps 11/12; 17 /18 & 22/23). This problem could not be solved. Still after 5 or more regenerations the signal was stable, and the target shift was comparable.</p><p>Immobilization pH scout for EDC/NHS chemistry. The efficiency of a chemical coupling of a capture molecule to a SAM via EDC/NHS strongly depends on the electrostatic interaction between the molecule and the surface. The pH value of the used immobilization buffer should be preferably adjusted between the isoelectric point (pI) of the capture molecule and the pKa of the SAM 35 . In this way, an electrostatic concentration of the protein to be immobilized will take place on the SAM surface, ensuring optimal binding. It is known from the literature that the pKa of MUA SAMs on particles is significantly higher (pKa = 6.8 for particles of 5 nm diameter and pKa ≈ 10 for flat surfaces) than for MUA in solution (pKa = 4.8) and also increases with increasing particle size 44,45 . Even the concentration and the size of surrounding ions have an impact on the pKa of MUA on nanoparticles, as well as the presence of other SAM-forming molecules such as 1-OT or MUD 44 . The pI value of the α-hCRPc used was not known and therefore immobilization scouting was performed with different pH values (4, 4.5 or 5) in 10 mM NaAc buffer (Figure S1). 1 M ethanolamine hydrochloride pH 8.5 was used to remove electrostatically bound protein from the not yet activated MUA surface. The highest plasmon shift was visible at pH 5. This pH was therefore chosen for the final immobilization.</p><p>Immobilization via MUA and subsequent EDC/NHS chemistry. The results with the thiolated streptavidinimmobilization chemistry (reported in the previous section) yielded a stable and measurable signal for the studied relevant CRP concentrations. However, as the scheme in Fig. 1 shows, the resulting construct of streptavidin and anti-CRP antibody spans a significant distant away from the sensor surface. As demonstrated in the layer-by-layer adsorption experiments in Fig. 2, an increasing distance from the surface hampered the achieved signal. In order to address this shortcoming, another immobilization approach was considered, which would allow decreasing the distance of the binding target to the sensor surface. It is based on a MUA self-assembled monolayer, binding on one side via thiol to the gold, and providing the means for an EDC/NHS-attachment of the anti-CRP antibody on the other side (Fig. 1, right). www.nature.com/scientificreports/ Blocking for EDC/NHS chemistry. A blocking step was essential to ensure that the "target" molecule under investigation was bound specifically to the immobilized captured molecule and not already adhered non-specifically to the sensor surface. In the initial SH-SA measurements with 1 mg/ml BSA in PBS as blocking reagent, nonspecific binding was clearly visible (Fig. 3). By increasing the BSA concentration to 10 mg/ml and changing the buffer to 10 mM NaAc pH 4, the blocking of the sensor surface was significantly improved. The step 9 in Fig. 4 showed a sufficient blocking with 10 mg/ml BSA, demonstrated by the secondary anti-human CRP antibody (α-hCRPd) as negative control in step 12, which showed no detectable unspecific binding. Only after CRP injection (step 16) was the α-hCRPd able to bind specifically, which resulted in a significant shift (step 18). On the other hand, the observed binding of α-hCRPd in the SH-SA assays (Fig. 3 step 9) could be due to a weak cross-reactivity to SH-SA. This would also explain why the sensogram course in Fig. 3 Steps 16 and 22 (dissociation of α-hCRPd after CRP injection) drops similarly as before CRP in Step 10. Such a dissociation course (obviously no 1:1 binding 46 ) is not observed in the sensograms of the measurements without SH-SA (SAM method, Fig. 4 Steps 12/13 and 18/19). Since this effect was not investigated further, we chose the term 'nonspecific' .</p><!><p>When considering a LSPR experiment for the detection of a target molecule one should consider the optimal immobilization method of the capture molecule. It depends on the (physico)chemical nature of the capture molecule (DNA, proteins, lipids and others) and the possible required modifications for its immobilization. Proteins, for example, are known to bind directly on bare gold surfaces. However, this can have an impact on their stability and functionality 30 and can result in weakened target binding. Different immobilization methods have been developed in the past to address this challenge, as described above. Another important factor for an efficient target binding (and therefore signal strength) is the distance of the binding site from the surface. This parameter can be modulated by the choice of the immobilization chemistry, as highlighted in this study.</p><p>The PAH/PSS bilayer deposition experiments yielded an optimal range for the distance of the target molecules from the sensor's surface. For the AuNP chips used in this study, the first 24 nm from the surface would result in a significant shift (Δλ) of at least 1.5 nm for the binding of a well packed PAH/PSS bilayer with n layer = 1.5 RIU. If the decay length (l d ) would be higher, the signal shift for each bilayer would be smaller in comparison to shifts on particles with lower l d . On the other hand, the decrease of the signal shift for each following bilayer would also be smaller because the total sensing distance is higher, but the absolute maximum shift would be the same. For higher absolute maximum shift the increase of the refractive index sensitivity (m; m = S B if d approaches to infinite 32 ) would be necessary, which can be attained with bigger particles, other materials or particles with anisotropic shapes 32,47 . The calculated m and l d with 141.1 nm/RIU and 79.5 nm for the utilized AuNP-chips differ slightly from the values expected by theory. This is reasonable because the refractive index of the layer and the layer thickness were taken from the literature. In the case of the CRP deposition, the measured plasmon shift was lower than 1.5 nm. This is confirmed in supporting Figure S2, 1.212 ± 0.032 nm/0.918 ± 0.012 nm on the 3rd/4th PAH/PSS bilayer corresponding to 12/16 nm away from the gold nanoparticle surface. Because of the water content the refractive index of a CRP layer is reported to be ≤ 1.45 RIU 48,49 which is also reflected by a lower plasmon shift. It is observed that at least one bilayer more, approximately 4 nm, reduces the signal of the target significantly (by roughly 25%). After the 3rd PAH/PSS bilayer, the total centroid shift was 10.095 nm/10.607 nm, which indicates that the chips behave similarly having comparable surface and bulk sensitivities (S S = 61.0 nm/ RIU and 64.1 nm/RIU for three PAH/PSS bilayer; S B = 97.87 nm/RIU and 115.09 nm/RIU). The methods used for CRP detection in this work (SH-SA and SAM method) are based on covalent bonding to the gold surface. Both MUA (1-OT, MUD) and SH-SA have free thiol groups (-SH) that form a covalent Au-S bond at neutral to basic pH 50 . In the SH-SA method, antibody binding occurs via a biotin-streptavidin bond, which is one of the strongest non-covalent bonds in nature. In the SAM method, the antibodies are again covalently bound to the already anchored MUA via chemical immobilization. In this process, the free carboxyl groups first react with protonated EDC to form active O-acylisourea intermediates, which is then stabilized by NHS to form a succinimidyl ester 25 . Free amine side chains of the antibodies to be immobilized (e.g., lysine) can then react with the formed ester to create an amide bond with MUA 26 . This process is a zero-length crosslinking. Nevertheless, the orientation of the antibodies is most likely similarly random in both cases since the biotinylation of the antibodies also occurs via EDC/NHS in many cases. The binding of the antigen (CRP) then takes place via non-covalent bonds such as hydrogen bonding or hydrophobic interaction, which ensures the regenerability of the sensor 51 .</p><p>Using different immobilization methods for the capture antibodies, method-dependent differences are visible (Fig. 5). For the samples 'SH-SA outside' , which was prepared outside of the fluid cell and air dried after antibody immobilization, a much smaller shift is observed, apparently air drying has a negative impact on the function of the detection antibody. However, for thiol-streptavidin (SH-SA) mediated immobilization, resulting in a greater surface distance, the plasmon shift for 31 µg/ml CRP was significantly lower than for the SAM method (independent samples T-test: p = 0.046; n ≥ 5 per group) which allows the target binding closer to the nanoparticle surface. On average, due to the thicker SH-SA layer, the target binding should be about 5 nm closer for the SAM method. On the other hand, it takes more time to produce the SAM chips (overnight), and also the EDC/NHS reaction is more time consuming during the assay. SH-SA immobilization and binding of the biotin capture antibody is straightforward and can be done completely in the microfluidic system. Blocking the surface against non-specific binding is necessary for both methods and could be sufficiently optimized for the assay. Regeneration of the sensor for antigen binding with 10 mM glycine HCl pH 2.5 was also successful. However, the LSPR signal after regeneration was always somewhat lower than before antigen binding, pointing to partial removal and/or inactivation of capture molecules, which indicates a need for optimization. The scatter of the CRP response was quite large. This could be due to less homogenous distribution of the AuNP including aggregations. This is also reflected in the bulk sensitivities (S B ) observed during calibration, as they showed a large variance (70.5-225.3 nm/RIU). However, as can be seen from the LbL data, this value cannot be equated with the calculated S B . Also, the measured surface sensitivities (S S ) of the AuNP chips show a significantly lower variance (61.0-66.9 nm/RIU after 3 bilayer of PAH/PSS). With the detection limit of 0.3 mg/L CRP for SH-SA mediated immobilization, this immobilization method is fully sufficient for clinical applications and allows the measurement of several samples in one assay. When further improvement in sensitivity is required, the short-thiol mediated approach could increase the detection limit, but requires a more cumbersome preparation.</p><!><p>The effect of the thickness of the capture probe attachment layer on the LSPR signal has been characterized. Using plain gold nanoparticle chips the straightforward SH-SA method is sufficient to determine CRP in clinically relevant concentrations. The immobilization layer is significantly thicker than with short thiols and thus also provides lower target signals. Using LbL technology, distance layers comparable to both studied immobilization chemistries could be prepared and compared regarding their LSPR signal shift upon binding of detection antibodies. The regenerability of the used sensors and the high time resolution of the utilized setup enables versatile applications such as diverse binding kinetic studies. The studied detection method is well suited for the discernment of protein biomarkers such as CRP in clinical applications allowing the measurement of several samples in one assay.</p>

Scientific Reports - Nature

Ultrasensitive SERS immunoassay based on diatom biosilica for detection of interleukins in blood plasma

An ultrasensitive surface-enhanced Raman scattering (SERS) immunoassay based on diatom biosilica with integrated gold nanoparticles (AuNPs) for the detection of interleukin 8 (IL-8) in blood plasma has been developed. The SERS sensing originates from unique features of the diatom frustules, which are capable of enhancing the localized surface-plasmon resonance of metal nanostructures. The SERS immune tags ware fabricated by functionalizing 70-nm Au nanoparticles with DTNB (i.e., 5,5′-dithiobis(2-nitrobenzoic acid)), which acted as a Raman reporter molecule, as well as the specific antibodies. These DTNB-labeled immune-AuNPs can form a sandwich structure with IL-8 antigens (infection marker) and the antibodies immobilized on the biosilica material. Our method showed an improved IL-8 detection limit in comparison to standard ELISA methods. The current detection limit for IL-8 using a conventional ELISA test is about 15.6 pg mL−1. The lower detection limit for IL-8 in blood plasma was estimated to be 6.2 pg mL−1. To the best of our knowledge, this is the first report on the recognition of IL-8 in human samples using a SERS-based method. This method clearly possesses high sensitivity to clinically relevant interleukin concentrations in body fluids. The average relative standard deviation of this method is less than 8%, which is sufficient for analytical analysis and comparable to those of classical ELISA methods. This SERS immunoassay also exhibits high biological specificity for the detection of IL-8 antigens. The established SERS immunoassay offers a valuable platform for the ultrasensitive and highly specific detection of immune biomarkers in a clinical setting for medical diagnostics. Graphical AbstractThe SERS-based immunoassay based on naturally generated photonic biosilica for the detection of interleukin 8 (IL-8) in human plasma samples Electronic supplementary materialThe online version of this article (10.1007/s00216-017-0566-5) contains supplementary material, which is available to authorized users.

ultrasensitive_sers_immunoassay_based_on_diatom_biosilica_for_detection_of_interleukins_in_blood_pla

Introduction<!>Reagents<!>Blood sample preparation<!><!>Fabrication of the immune platform<!>Synthesis of Au nanoparticles<!>Synthesis of anti-IL18/AuNPs-DTNB<!><!>Immunoassay protocol<!>Washing conditions<!>Raman and SERS measurements<!>Electron microscopy characterization<!>Characterization of the substrate and Raman-reporter-labeled immune Au nanoparticles<!><!>Characterization of the substrate and Raman-reporter-labeled immune Au nanoparticles<!><!>Characterization of the substrate and Raman-reporter-labeled immune Au nanoparticles<!><!>SERS immunoassay detection of IL-8<!><!>Quantitative analysis<!><!>Conclusions<!>

<p>Interleukins are secreted proteins that are members of the cytokine family of immune system molecules that regulate immune cell activity. Interleukins are produced by immune system cells such as lymphocytes, macrophages, and monocytes [1]. They modulate inflammation and immunity by regulating the growth, mobility, and differentiation of lymphoid and other cells [2]. The analysis and quantitation of interleukins in body fluids is important as it allows us to broaden our understanding of their immunological functions. Cytokine levels in body fluids can provide information useful for disease diagnosis and staging, prognostication, and thus the selection of an appropriate disease therapy [3]. Several analytical procedures for quantifying interleukins in body fluids and tissue culture supernatants have been developed. Enzyme-linked immunosorbent assays (ELISA) are the most popular methods of quantitating secreted cytokines due to their high specificities and sensitivities [4, 5]. Intracellular staining, the ribonuclease protection assay (RPA) [6], the polymerase chain reaction (PCR) [7], and cytometric assays [8] have also been used in recent decades. However, each of these techniques has at least one significant limitation. For instance, problems with cytokine assays, including a lack of accuracy, have been reported; a number of factors have been shown to affect the validity and quality of measurements obtained with such assays [9–12].</p><p>Therefore, there is a need to develop a more sensitive, selective, stable, and durable method for analyzing these biomarkers. Recent advances in nanotechnology and instrumentation development have permitted the development of a highly sensitive and chemically specific technique for biomolecular system recognition that uses surface-enhanced Raman spectroscopy (SERS) [13]. The phenomenon of SERS can be explained as a combination of an electromagnetic mechanism (EM) and a chemical mechanism related to charge transfer between a substrate and an adsorbed molecule [14]. Theoretically, the electromagnetic enhancement can reach factors of 103 to 1011, whilst chemical enhancement factors of up to 103 have been calculated [15, 16]. This huge enhancement in Raman scattering—even single molecules can be observed—ensures that Raman spectroscopy is very effective for ultrasensitive bioanalysis [17]. Another interesting characteristic of SERS is the linear dependence of the SERS intensity on the power of the incident light, despite the nonlinear signal enhancement that is achieved with this technique. Thus, SERS could potentially be applied for the quantitative measurement of analytes with ultrahigh sensitivity.</p><p>SERS biosensing has been used to detect various biological samples and diseases, including various cancers [18], Alzheimer's disease [19], and Parkinson's disease [20]. The most notable recent advances in SERS include the application of this technique to immunosensing. SERS-based immunoassays have attracted significant research interest due to their (i) high detection sensitivities and selectivities [21], (ii) reduced susceptibility to photobleaching [21], and (iii) narrow spectral bandwidth, allowing multiplex analysis [22].</p><p>Wang et al. [23, 24] demonstrated the detection of the interleukins IL-6 and IL-8 from buffer solution. Typically, SERS immunoassays are realized on flat glass, on a noble metal (Au, Ag, or alloys of them) surface with nanoscale roughness [25], or on photonic crystals [26]. Unfortunately, silver—which gives the best signal enhancement factors—undergoes oxidation, so when a silver surface is used, SERS must be carried out before the surface oxidizes. Photonic crystals, despite their excellent properties, are expensive to develop as techniques requiring high-tech equipment (i.e., PVD sputtering, photolithography, focused ion beam, and others) are needed.</p><p>In the work reported in the present paper, we investigated a more accessible approach based on natural photonic crystals (materials) such as diatom biosilica. Diatoms are photosynthesizing algae that possess a siliceous skeleton called a frustule [27] comprising complex hierarchical micro- to nanosized structures under natural conditions. We employed diatom silica frustules produced by Pseudostaurosira trainorii as an inexpensive and easy to prepare and modify functional material to use in a novel SERS immunoassay.</p><p>Obtaining three-dimensional (3D) structures of inorganic materials is one of the main challenges involved in the development of nanotechnology. The shape and the pattern of a frustule are unique to the particular diatomic species that produced it [28]. Thus, highly individual 3D silica structures can be obtained from single-celled diatoms without the need to use complex and expensive nanofabrication methods. Diatom frustules exhibit unpredictable optical properties due to their quasi-ordered pore patterns, such as diffraction-driven self-focusing [29] and gas-sensitive photoluminescence emission [30]. Researchers have investigated whether the unusual optical properties of frustules could be used in practical applications. Gale et al. [30] demonstrated the use of an antibody-functionalized diatom biosilica frustule as a microscale biosensor platform for the selective and label-free photoluminescence-based detection of biomolecules. Kong et al. [31] fabricated a photonic biosilica SERS substrate by integrating Ag NPs into microchannels of diatom frustules to identify explosive molecules in nanoliter solutions.</p><p>To the best of our knowledge, the present paper represents the first report of the application of a SERS immunoassay based on diatom biosilica to the detection of interleukin 8 (IL-8) in human blood plasma. IL-8 is an inflammatory cytokine that also plays an important role in breast cancer. There have been a few studies of the biological activity of this cytokine; for instance, Yokoe et al. [32] measured serum IL-8 in 12 heavily pretreated patients with recurrent breast cancer, and reported that IL-8 levels were higher in patients with refractory progressive disease but were almost unchanged in patients showing a partial response or no change after systemic therapy. In this paper we demonstrate the use of diatom biosilica as a SERS immune substrate.</p><!><p>Recombinant human interleukin 8 (CXCL8) and monoclonal anti-interleukin-8 antibody produced in mouse (clone 6217) were purchased from Sigma (St. Louis, MO, USA) and used as received. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), L-ascorbic acid, gold(III) chloride trihydrate, trisodium citrate dihydrate, hexadecyltrimethylammonium bromide (CTAB), aminopropyltriethoxylsilane (APTES), bovine blood plasma albumin (BSA), and phosphate-buffered saline (PBS) packs (10 mM, pH 7.2) were also obtained from Sigma.</p><!><p>In our experiments, we used human blood samples from 10 healthy volunteers. These samples were made available courtesy of the Regional Blood Center (Warsaw, Poland). The samples underwent morphological analyses prior to use and revealed no abnormalities. All experiments were performed in compliance with the relevant laws and institutional guidelines. The study protocol was approved by the Ethics and Bioethics Committee of Cardinal Stefan Wyszynski University in Warsaw.</p><!><p>Images of the algae cultivation setup and colonial chains of living diatoms</p><!><p>Diatom frustules are mainly composed of amorphous hydrated SiO2 and are thus amenable to simple chemical functionalization for multiple applications (e.g., in photonics and analytical chemistry). To make the diatom frustules (biosilica) useful for immune biosensing, glass slides were modified with the frustules, which was achieved as follows. The biosilica was dispersed in ethanol at a concentration of 0.5 mg mL−1. The cleaned glass slides were covered with 100, 300, 800, and 1000 μl of this diatom biosilica solution, respectively. All the samples were placed in an oven for 1 h at 450 °C. In the next step, the surface silanol groups of the biosilica were functionalized with amine groups using aminopropyltriethoxylsilane (APTES) according to the procedure described by Yang et al. [33]. The glass slides with diatom frustules were incubated in 3 mL of a 1% solution of APTES in ethanol for 1 h at 50 °C. The substrates were then removed from solution and rinsed with ethanol and DI water after 5 min. The amino-modified surfaces were functionalizedd with anti-interleukin-8 (anti-IL-8) antibodies by immersing the frustules in a mixture of 25 μL of 150 μg mL−1 anti-IL-8 in phosphate-buffered saline (PBS) and 5μL of activation solution (0.2 M EDC/0.05 M NHS; mixture in deionized water). Covalent bonds were formed between activated carboxyl groups on the anti-IL-8 antibodies and free amino groups on the diatoms. After 1 h, the substrates with immobilized antibodies were rinsed with DI water and dried with nitrogen gas. Remaining active surface groups were blocked by immersing the substrates in 15 μL of 2% BSA in PBS buffer solution (pH 7.2). Finally, the samples were rinsed twice with 5 mL of 10 mM PBS buffer solution and stored at 4 °C in PBS for future use.</p><!><p>Gold nanoparticles 70 nm in diameter (see Fig. S1 in the "Electronic supplementary material," ESM) and capped with CTAB (AuNPs@CTAB) were obtained via a two-step seeding protocol [34]. In the first step, AuNPs 15 nm in diameter were synthesized using the Turkevich approach [35]. In this approach, seeds were obtained by first adding HAuCl4.3H2O (49 mg, 0.125 mmol) to 250 mL of boiling water and then adding trisodium citrate dihydrate (125 mg, 0.425 mmol). In a second step, CTAB (2.74 g, 7.5 mmol) was dissolved in 500 mL of water heated to 35 °C. A solution of HAuCl4.3H2O in water (2.5 mL, 0.1 M) was then added and stirring was continued until the mixture became clear. Next, a solution of ascorbic acid (2 mL, 2.5 mM) was injected and, after complete discoloration of the reaction mixture, 6.5 mL of the seed solution were added quickly. The mixture was stirred gently for 1 h and then centrifuged and carefully decanted. The precipitate was dispersed in 8 mL of CTAB water solution (0.1 M) and left overnight to allow the shape-selective separation of nonspherical from spherical particles formed during the growing process. The solution of spherical AuNPs@CTAB was then carefully collected from above the sediment of unwanted nonspherical particles. The concentration of gold atoms in the final solution, as determined from the absorption spectrum, was 15.42 mM.</p><!><p>The solution of AuNPs@CTAB (1 mL) was centrifuged (6000 rpm, 10 min) in order to remove excess CTAB. The supernatant was carefully decanted and the precipitate was dissolved in water (1 mL). A solution of DTNB (18 mg, 0.045 mmol, in 5 mL of ethanol) was added during stirring and the obtained mixture was left overnight. The AuNPs@DTNB were collected by centrifugation (2000 rpm, 10 min) and purified by sixfold dissolution in acetonitrile, precipitation with methanol, and centrifugation (6000 rpm, 10 min) and then by tenfold dissolution in water and centrifugation (7500 rpm, 10 min). The purified AuNPs were dried and suspended in 2.5 mL of water. Next, the 10 μL of solution containing the DTNB-modified Au nanoparticles were mixed with the solution of anti-IL8 in PBS buffer (5 μL, 60 μg mL−1). For the synthesis of conjugate, the coupling reagents (0.2 M EDC/0.05 M NHS; in the volume ratio of 5:1, in deionized water) were added and then the resulting mixture was incubated at 4 °C for 4 hours. The Raman-reporter-labeled immune Au nanoparticles (anti-IL8/AuNPs-DTNB) were separated from the solution by centrifugation at 10,000 rpm for 5 min. The suspensions of anti-IL8/AuNPs-DTNB were then passivated with 2.5 μL of 2% BSA in PBS buffer solution. After 2 h, the mixture was centrifuged again for 10 min at 20,000 rpm and then re-suspended in 1 mL of the PBS solution. The prepared anti-IL8/AuNPs-DTNB were stored at 4 °C for future use. The maximum number of antibodies bound to a nanoparticle was estimated to be ca. 540 [36]. The average diameter of a gold nanoparticle was about 70 nm according to SEM images and a histogram of Au nanoparticle diameters (ESM Figs. S1 and S2).</p><!><p>a–d Schematic illustration of the SERS-based immunoassay: a glass slide was modified with diatom frustules (b shows an SEM image of a portion of the modified slide); c antibody capture and immobilization using the SERS immunosensor for interleukin 8 detection (d shows a close-up illustrating the bonding to the antibody)</p><!><p>The first layer of this sandwich structure is composed of immobilized antibodies against IL-8 captured on the amino-modified biosilica substrate. The second layer contains the complementary interleukins (proteins) captured by these selective antibodies. The third layer consists of Raman reporter DTNB-labeled immune Au nanoparticles. The presence of IL-8 in the analyzed samples is identified through the appearance of the SERS spectrum of the DTNB bonded with the specific antibody against the studied interleukin.</p><!><p>To limit nonspecific binding interactions, the immunoassay was washed three times with PBS buffer solution (pH 7.2).</p><!><p>SERS measurements were performed using a Renishaw (Wotton-under-Edge, UK) inVia Raman system equipped with a 632-nm He-Ne laser excitation source. The light from the laser was passed through a line filter and focused on a sample mounted on an x-y-z translation stage with a 50× microscope objective (N.A. = 0.75). The Raman-scattered light was collected by the same objective through a holographic notch filter to block out Rayleigh scattering. A grating with 1800 grooves per mm was used to provide a spectral resolution of 5 cm−1. The Raman scattering signal was recorded by a 1024 × 256 pixel RenCam CCD detector. The beam diameter was approximately 5 μm. Typically, the SERS spectra were recorded over an integration time of 30 s with a laser output power of 2.5 mW by mapping an area of size 50 μm × 50 μm.</p><!><p>The morphological and structural features of the cleaned diatom frustules were examined by high-resolution scanning electron microscopy (SEM). SEM measurements of diatoms on the glass slide were done under high vacuum using the FEI (Hillsboro, OR, USA) Nova NanoSEM 450 with an accelerating voltage of between 2 and 30 kV. The glass slide was attached to the SEM table with conductive silver paste.</p><p>The elemental composition of the diatom frustules was determined using transmission electron microscopy (FEI Tecnai F20 X-Twintool) coupled with energy-dispersive X-ray detection (EDX), with the sample being placed on a carbon-coated copper grid (lacey carbon support film, 400 mesh). In the study, samples of diatom frustules immobilized on glass slides and diatom frustules dispersed in ethanol were used.</p><!><p>According to research results obtained by Sprynskyy et al. [37], the examined biosilica can be characterized as a macroporous material that contains a network of meso- and micropores with a BET surface area of 16.9 m2/g. The pores showed size ranges of 1–1.5 nm, 10–20 nm, and 25–70 nm. The biosilica was identified as opal-A by X-ray diffraction and was termed "naturally organic functionalized 3D silica" due to the presence of residual organic functional groups related to biosilica-associated proteins as well as the functional groups characteristic of the amorphous silica framework. Also, the biosilica exhibited photoluminescence in the mid-ultraviolet region as well as in the blue-green region of the visible spectrum [37].</p><!><p>a–b SEM images of a diatom frustule assembly (a) and view of a single diatom valve from above (b)</p><!><p>Figure 3a shows an assembly of uniform and well-preserved diatom frustules. The external and internal surfaces as well as sidewall regions of the diatom valves can also be observed. It is clear that the valves possess round to slightly elliptical forms with an average diameter of 4–5 μm. The inset in Fig. 3a shows a macroscopic image of the dried diatom frustules. Figure 3b reveals the intricate design of the external surface of a single diatom valve. The valve surface is split in two halves axially by a solid strip (raphe), with each half perforated by parallel linear rows of oval pores (areolae). The rows of pores are oriented perpendicular to the central axis and each contain 4–5 pores of size 100–200 nm. The pore size decreases towards the central axis. Neighboring rows are separated by about 450 nm and neighboring pores in a row are separated by about 100 nm. TEM-EDX analysis (ESM Fig. S3) showed that the cleaned diatom frustules were 98% silica. The dominant peaks in the TEM-EDX spectrum of the frustules were from silicon and oxygen. The low O/Si atomic ratio (1.92) observed is characteristic of an amorphous hydrated silica (such as opal). Peaks attributed to Cu derive from the copper TEM grid employed. The detection of carbon indicated the presence of strongly bound residual organics in the cleaned diatom frustules. Furthermore, the initial iron concentration in the nutrient solution was very low: 0.65 mg L−1, and the concentrations of other trace elements (Zn, Cu, Co) in the nutrient solution were an order of magnitude lower still.</p><!><p>a–d SEM images of glass slides covered with a 100 μL, b 300 μL, c 800 μL, and d 1000 μL of a 0.5 mg mL−1 diatom frustule solution</p><!><p>It is clearly apparent that very dense frustule packing was obtained by drop-casting multiple coats of the 800 μL of 0.5 mg mL−1 frustule solution onto the glass substrate.</p><p>Subsequently, the SERS activities of the DTNB-labeled immune Au nanoparticles were examined. The successful binding of the DTNB and IL-8 antibodies to the Au nanoparticles was demonstrated by UV experiments (ESM Fig. S4). As shown in Fig. S4a in the ESM, the as-received gold nanoparticle solution has a strong extinction maximum at 609 nm. This wavelength is indicative that the individual nanoparticles average approximately 70 nm in diameter. The spectrum of AuNPs redshifted a little after coating them with the Raman reporter (ESM Fig. S4b), as the LSPR band of AuNPs is very sensitive to the refractive index of the surrounding medium. The redshift of the surface plasmon resonance peak [38] demonstrates the successful binding of DTNB to the gold nanoparticles. After modification with IL-8 antibodies, there was a large decrease in the strength of this band and it redshifted from 609 nm to 637 nm, which indicates that residual surface vacancies of DTNB-labeled AuNPs are occupied by anti-IL-8 antibodies (ESM Fig. S4c). Similar results have been observed and detected using the ATR/FTIR technique [39]. Additionally, according to the literature [40], the broadening and redshift may also indicate aggregation.</p><!><p>a SERS spectrum of DTNB (Raman reporter) adsorbed onto Au-NPs, and b SERS spectrum of DTNB-labeled immune-AuNPs</p><p>a–d SERS spectra of the marker band at 1326 cm−1 with different densities of diatom frustules on the surface of the glass slide: a 100 μL, b 300 μL, c 800 μL, and d 1000 μL of a 0.5 mg diatoms per mL of ethanol solution. The inset presents the relationship between the intensity of the marker band at 1326 cm−1 and the density of diatom frustules on the surface of the glass slide</p><!><p>These experimental results prove that the morphology of the capturing substrate presented in Fig. 4c is best suited to achieving the highest IL-8 detection sensitivity, and so this substrate was applied to a quantitative analysis of IL-8 in plasma blood samples.</p><!><p>(A) SERS spectra obtained for various concentrations of IL-8: (a) 0.0; (b) 0.005; (c) 0.01; (d) 0.1; (e) 0.5; (f) 1.0; (g) 2.5; (h) 5.0; (i) 30.0 ng mL–1 in blood plasma. (B) Relationship between the normalized intensity of the marker band at 1326 cm−1 and the concentration of IL-8 in the range from 0 to 30 ng mL–1 for (a) a glass slide with no diatom frustules and (b) a glass slide with diatom frustules deposited on its surface. Each SERS spectrum is the average of 20 measurements obtained at different points on the SERS substrate surface using the mapping mode. Each error bar indicates the standard deviation calculated from 20 measurements obtained at different spots on the surface for a particular IL-8 concentration</p><!><p>In a first step, a control experiment was performed to verify the specificity of the immunological recognition. The unspecific Akt blocking peptide for anti-IL-8 antibody was employed in the same immunoassay protocol as described for Fig. 2. The Akt blocking peptide specifically binds only to the Akt (pan) Rabbit mAb antibody. As can be seen in Fig. S8a of the ESM, after the Akt blocking peptide was added, there were no strong bands, only two extremely weak bands at about 840 cm−1 and 1326 cm−1 originating from the Raman reporter molecules. This may indicate that a small number of DTNB-labeled immune AuNPs were adsorbed onto the capturing substrate without any immune recognition. However, more importantly, the presence of these marker bands demonstrates that this technique can detect the low levels of IL-8 that are usually present in normal control blood plasma samples. When the anti-IL8/AuNPs-DTNB were incubated with the IL-8 in the plasma samples, strong signals originating from the Raman reporter molecules appeared in the SERS spectrum (ESM Fig. S7b–d). As can be seen in Fig. S7 of the ESM, the difference in intensity between the SERS signals from DTNB that were obtained in the specific and unspecific recognition regimes clearly demonstrates the high specificity of this immunocomplex.</p><p>In our study, we also compared the efficiency of the developed SERS immunoassay with that of a conventional SERS sensor based on flat glass. The glass slide was employed in the same immunoassay protocol as described earlier in this paper. Based on the constructed SERS response curve (b) shown in Fig. 7b, the lowest detectable concentration was 2.5 ng mL−1, which is two orders of magnitude worse than that obtained for the diatom-frustule-based SERS immune substrate. Although the reason for this enhanced sensitivity of the technique developed here requires further study as it is a rather complex phenomenon, it is very likely due to the unique properties of the diatom frustules.</p><p>Diatoms are photonic crystals that can increase the local field intensity, leading to Raman signal enhancement [44] and thus improving the detection limit of the SERS-based immunoassay sensor. The periodic two-dimensional pore arrays permit guided-mode resonances (GMRs) at visible wavelengths. Theoretical and experimental investigations indicate that the electric field amplitude of localized surface plasmons (LSPs) for nonmetallic structures can be significantly amplified through the coupling of the LSPs with the plasmonic structure of the diatom [45]. As a result, the presence of the diatom frustules lowers the detection limit for IL-8 to 6.2 pg mL−1, which is two orders of magnitude better than that achieved with a conventional glass-based SERS- immunoassay. Apart from the sensitivity, there are also other advantages of the SERS-based immunoassay sensor based on diatoms. The porous morphology of the diatom frustules leads to unique physical and chemical properties of the frustules, such as rapid mass transport inside the microchannels and outstandingly high surface area [46]. In the SERS-based assay reported here, this large surface area can magnify the number of hotspots and adsorption sites for the analyte compared with planar glass or metallic nanostructures [47], thus improving the immunoassay sensing. The abundant hydroxyl groups on their surfaces make diatom frustules very hydrophilic in comparison with a flat glass slide. Their highly hydrophilic surfaces with highly ordered nanopores of diatoms can drive the liquid flow from the glass towards the diatom frustule as a result of capillary forces [48]. In effect, the highly hydrophilic and porous diatom frustules provide a driving force that concentrates the target molecules, which may lead to a reduction in the detection limit of several orders of magnitude [49].</p><p>The current detection limit for IL-8 using a conventional ELISA test is about 15.6 pg mL−1 [50], which does not always meet the requirements of clinical diagnosis. Our method improves on that detection limit and potentially permits the quantitative detection of interleukins in complex biological fluids.</p><!><p>a Reproducibility of three separately prepared SERS immunoassays exposed to different concentrations of IL-8 in blood plasma (0.1, 0.5, and 30.0 ng mL−1). SERS spectra were recorded at 15 randomly selected spots on the substrate in each SERS assay. b Representative two-dimensional SERS spectra recorded in SERS assays of 30.0 ng mL−1 IL-8 performed at 40 different spots on the SERS surface. The spectra were collected over a distance of 1 mm in 10 μm steps (40 spectra are shown). Each point on the map was recorded using 5 mW of excitation at 632 nm and an integration time of 10 s</p><!><p>We have demonstrated, for the first time, a SERS-based immunoassay that utilizes naturally created photonic biosilica to detect the interleukin IL-8 in human plasma samples.</p><p>The most notable features of the developed SERS immunoassay include (i) the creation of an immune substrate based on anti-IL-8-functionalized diatom biosilica and (ii) the application of appropriately designed Raman-reporter-labeled AuNPs, which produce very strong SERS enhancement. The DTNB-labeled immune AuNPs can form a sandwich structure with the antigen and the antibody, as shown by the characteristic spectrum of the Raman reporter molecules (DTNB). The estimated lower detection limit and average standard deviation of the selected marker band for IL-8 at 1326 cm−1 show that the method is highly sensitive to clinically relevant concentrations of this interleukin and has excellent reproducibility, which is desirable for analytical analysis. This SERS assay also exhibits high biological specificity for the detection of IL-8 in complex fluids. Furthermore, the experimental results confirm that diatom frustules amplify the sensitivity of the immunosensor in comparison to a conventional sensor based on flat glass. The detection limits for IL-8 on diatom frustules and when using the glass-based SERS immune substrate in human blood plasma were found to be 6.2 pg mL−1 and 2.5 ng mL−1.</p><p>The SERS immunoassay presented here can be used for the sensitive and selective detection of immune markers in biological fluids and for point-of-care analysis.</p><!><p>(PDF 2233 kb)</p><p>Electronic supplementary material</p><p>The online version of this article (10.1007/s00216-017-0566-5) contains supplementary material, which is available to authorized users.</p>

PubMed Open Access

Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development.Graphical abstractElectronic supplementary materialThe online version of this article (10.1007/s00216-020-02796-1) contains supplementary material, which is available to authorized users.

exploring_sample_preparation_and_data_evaluation_strategies_for_enhanced_identification_of_host_cell

Introduction<!>Materials<!><!>Sample preparation for peptide analysis<!>Peptide analysis by HPLC-MS/MS<!>Data analysis<!>Data and code availability<!>Direct HCP identification in commercial drug products<!><!>HCP identification upon drug substance depletion<!>Improved HCP identification via probabilistic protein inference and in silico peptides<!>Comparative analysis of HCP profiles<!><!>Comparative analysis of HCP profiles<!><!>Comparative analysis of HCP profiles<!>Conclusions<!>

<p>Therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins are conventionally produced in mammalian expression systems such as Chinese hamster ovary (CHO) or human embryonic kidney cells. Naturally, these cells express not only the recombinant target protein but also a plethora of endogenous proteins essential for cellular growth and viability. Despite rigorous clean-up procedures during downstream processing, minor amounts of these host cell proteins (HCPs) may be co-purified with the therapeutic protein and remain in the final drug product (DP) [1–5]. Since these contaminating proteins may affect DP quality [6–12] or provoke immune responses when the drug is administered [13, 14], HCPs are generally considered in the context of critical quality attributes (CQAs) and product quality attributes (PQAs) [15]. Hence, sensitive and reliable analytical procedures for identifying and quantifying these impurities are indispensable for the production and release of biopharmaceuticals. Although there are no general guidelines specifying maximum acceptable HCP loads in DPs, manufacturers commonly aim at amounts below 1 to 100 ng of HCP per mg of drug substance (DS) in the final product (i.e., 1 to 100 ppm) [16]. Consequently, challenges in HCP characterization arise from low amounts of the contaminating proteins at large excess of DS, thus requiring a wide dynamic range to be covered by analytical methods.</p><p>To date, enzyme-linked immunosorbent assay (ELISA) represents the gold standard for HCP detection and quantification. In this context, polyclonal antibodies raised against the supernatant of null cells, i.e., cells that do not express the product gene, are used as primary antibodies for HCP detection [17, 18]. Due to its robustness and simplicity, ELISA can be performed in a high-throughput manner [19]. In addition, common cell lines and similar upstream conditions allow the development of platform assays that can be used for analyzing multiple DPs [20–22]. Yet, sensitivity of ELISA depends on the immunogenicity of each individual protein in the null cell supernatant, thus hindering relative quantification of different HCPs [23].</p><p>As an alternative, HCP analysis based on high-performance liquid chromatography (HPLC) combined with tandem mass spectrometry (MS/MS) has emerged [24, 25]. HPLC-MS/MS-based methods are orthogonal to ELISA in that they are independent from specific antibodies for detection. These workflows involve proteolytic digestion of HCP-containing samples and subsequent HPLC-MS/MS analysis. Fragment ion spectra of HCP peptides allow protein identification based on comparison with theoretical spectra derived from an HCP sequence database. Thus, HPLC-MS/MS-based workflows outperform ELISA in that they provide information on individual HCP identities and amounts rather than a total HCP content [26]. However, their main drawback lies in a dynamic range limited to three to four orders of magnitude, while analysis of low-abundant HCPs requires up to six orders of magnitude considering the DS to HCP ratio [27]. In addition, co-elution of DS- and HCP-derived peptides may result in ion suppression of low-abundant HCP peptides, preventing detection of the corresponding contaminant [28].</p><p>To overcome these limitations, two strategies have been described. On the one hand, multidimensional HPLC setups have been implemented to tackle sample complexity, thereby offering lower limits of detection and quantification [29–31]. However, these setups suffer from low throughput as well as limited robustness and reproducibility, which hampers their application in quality control [32, 33]. On the other hand, protein A affinity chromatography may be exploited to deplete Fc domain-containing DS, i.e., mAbs and Fc-fusion proteins, by highly specific interaction between protein A and the Fc domain before tryptic digestion and HPLC-MS/MS analysis [34, 35]. This enables identification of HCPs with abundances in the low ppm range applying a single chromatographic dimension [36, 37]. Moreover, wash solutions containing various additives may be applied to the DS captured on the protein A column to facilitate elution and subsequent analysis of HCPs that interact with the DS or the affinity resin [38–40]. Alternatively, enrichment of HCPs by applying a molecular weight cutoff filtration step has recently been described [41].</p><p>Previous studies tended to describe optimization of laboratory protocols with respect to the number of HCPs identified, while acquired MS/MS data was frequently analyzed using standard settings. Here, we describe an optimized data evaluation protocol that enhances process-independent HCP identification based on established analytical techniques, i.e., DS depletion via protein A affinity chromatography followed by reversed phase-HPLC-MS/MS. This data evaluation protocol combines (a) probabilistic protein inference based on all peptides identified from fragment ion mass spectra with (b) peptide detection on the full-scan MS level, i.e., even in the absence of MS/MS spectra. We demonstrate generic applicability of our workflow across Fc domain-containing biotherapeutics by assembling HCP profiles for a panel of structurally diverse, commercial grade DPs. Furthermore, a comparative analysis of non-depleted DPs reveals synergistic benefits of our data evaluation protocol and the depletion workflow for HCP identification. Thus, our approach represents a powerful tool that may be implemented into existing HPLC-MS/MS setups for DP characterization as well as in the context of process development.</p><!><p>Acetonitrile (≥ 99.9%) was purchased from VWR International (Vienna, Austria). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, ≥ 98.0%), iodoacetamide (≥ 99.9%), formic acid (98.0–100%), guanidine hydrochloride (≥ 99%), disodium phosphate dihydrate (HNa2PO4·2H2O, ≥ 99.0%), tetramethylammonium chloride (TMAC, ≥ 99.0%), glycine (≥ 99.0%), l-arginine monohydrochloride (≥ 98%), sodium chloride (NaCl, ≥ 99.5%), ammonium bicarbonate (LC-MS grade), ethanol (LC-MS grade), E. coli β-galactosidase, and bovine β-lactoglobulin were obtained from Sigma-Aldrich (Vienna, Austria). Tris(hydroxymethyl)aminomethane (Tris, ≥ 99%) was purchased from SERVA Electrophoresis (Heidelberg, Germany). Trypsin (Mass Spec Grade, V5111) was obtained from Promega (Madison, WI, USA). The Hi3 E. coli standard, consisting of six synthetically prepared, highly ionizing peptides derived from E. coli chaperone protein ClpB, was purchased from Waters (Milford, MA, USA). For all experiments, ultrapure water produced in-house by a Millipore Integral 3 unit (Merck/Millipore, Billerica, MA, USA) was used.</p><p>The following DPs were examined: MabThera® (batch H0139B01 expiring 01/2016, 10 mg mL−1 rituximab) and Avastin® (batch B7214HO9 expiring 03/2018, 25 mg mL−1 bevacizumab) from F. Hoffmann-La Roche Ltd. (Basel, Switzerland); Enbrel® (batches E11132 expiring 03/2011 and 1040542 expiring 03/2016, 50 mg mL−1 etanercept) from Pfizer (New York, NY, USA); and Benepali® (batch CT0056 expiring 09/2018, 50 mg mL−1 etanercept) from Samsung Bioepis UK Limited.</p><!><p>Schematic representation of the workflows used for HCP discovery in a drug product, which comprises a drug substance (i.e., the therapeutic protein) and minute amounts of HCPs. The two strategies applied involve direct analysis of HCPs in drug products or analysis of fractions obtained upon affinity depletion of the Fc domain-containing therapeutic protein. Peptide mixtures obtained upon tryptic digestion were analyzed by HPLC-MS/MS in triplicate. Data evaluation yields HCP profiles of replicates, which may be aggregated to HCP profiles of fractions and, ultimately, drug products, as indicated at the bottom of the figure</p><!><p>Flow-through and wash fractions collected during protein A affinity chromatography (2.0 mL each) were concentrated to 50 μL using Amicon Ultra 0.5 mL 10 kDa MWCO centrifugal filters (Sigma-Aldrich) at 14000×g, 25 °C. Samples were denatured and reduced by addition of 450 μL 6.0 M guanidine hydrochloride and 5.0 μL 500 mM TCEP, respectively, for 1.0 h at 37 °C while shaking at 1000 rpm. After alkylation with 15 μL 500 mM iodoacetamide for 30 min at 25 °C in the dark, the buffer was exchanged to 50 mM ammonium bicarbonate, pH 7.80, using Amicon filters. Five micrograms of trypsin was added to each sample, followed by incubation for 16 h at 37 °C, 1000 rpm. Samples were then fully dried at 45 °C for 2 h at 1000 rpm using a vacuum centrifuge, and subsequently dissolved in 10 μL 0.10% (v/v) aqueous formic acid. For direct HCP identification from DPs, 200 μg of each therapeutic protein, 20 ng β-galactosidase, and 20 ng β-lactoglobulin were digested with trypsin and dried as described above. Samples were adjusted to a concentration of 4.0 μg μL−1 with 0.10% (v/v) formic acid and stored at − 20 °C until analysis. To each sample of the depletion workflow, 250 fmol Hi3 standard were added immediately before injection.</p><!><p>HPLC-MS/MS measurements were carried out on a Thermo Scientific™ UltiMate™ 3000 HPLC system with flow splitting (1:100) coupled to a Thermo Scientific™ Q Exactive™ Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Both instruments were operated with Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) version 7.2.6. Tryptic peptides were separated on a 150 × 0.30 mm Waters ACQUITY UPLC® M-Class CSH™ C18 column with 1.7 μm particles (130 Å pore size) at a flow rate of 2.0 μL min−1 and constant column temperature of 50 °C. A linear gradient from 2.0 to 35% mobile phase B over 80 min was employed (mobile phase A: 0.10% (v/v) formic acid in ultrapure water; mobile phase B: 0.10% (v/v) formic acid in acetonitrile). At the end of each run, the column was flushed with 100% mobile phase B for 10 min followed by equilibration with 2.0% B for 20 min. The mass spectrometer was operated in positive electrospray ionization mode employing a Thermo Scientific™ Nanospray Flex™ ion source with silica emitter tips (New Objective, Woburn, MA, USA) of 30 μm inner diameter at the orifice. The applied spray voltage corresponded to + 2.50 kV. The transfer capillary was heated to 320 °C and the S-Lens RF level was adjusted to 55. Detection was performed in data-dependent mode with the following settings: mass range 400–2000 m/z; top 15 method for peptide fragment fingerprinting; one microscan per MS/MS event; dynamic exclusion for precursor selection for 20 s; resolution setting for full MS and MS/MS scans set to 70,000 and 17,500, respectively (defined at 200 m/z). Peptide fragmentation was induced by higher-energy collision-induced dissociation with a normalized collision energy of 28. Maximum injection time was set to 50 ms for full-scan MS and to 120 ms for MS/MS scans. Automatic gain control target was adjusted to 3 × 106 for full-scan MS and to 2 × 104 for MS/MS scans, respectively, with an underfill ratio of 1%. The isolation width for precursor selection was ± 0.75 m/z; only precursors with charges between 2+ and 4+ were included.</p><!><p>HCP identification was accomplished using the Protein Metrics Inc. Byos® v3.4-72-g5fd2d85e63 x64 software, which employs Byonic® v3.4.0 for peptide identification and protein inference, as well as Byologic® v3.4-72-g5fd2d85e63 x64 for quantification. Fragment spectra were searched against a CHO protein sequence database obtained from UniProt (reference proteome UP000001057, last modified on 2019-01-24, downloaded on 2019-07-01, containing 23,885 sequences), to which the following sequences were added: porcine trypsin (UniProt ID P00761), S. aureus protein A (P38507), bovine β-lactoglobulin (P02754), E. coli β-galactosidase (P00722), the Hi3 standard peptide sequences, and the respective DP sequence; thus, there was a distinct database for each DP, and each database contained 23,891 entries. Search parameters included carbamidomethylation of cysteine as a fixed modification; deamidation of asparagine and oxidation of methionine or tryptophan as common dynamic modifications; and rare dynamic modifications of N-terminal acetylation and formation of pyroglutamate from N-terminal glutamine or glutamate. Precursor and fragment ion mass tolerances were set to 7 and 10 ppm, respectively. The false discovery rate was estimated via a two-dimensional target decoy strategy [42] and adjusted to 1% (or 20 reverse count). An automatic cutoff for the Byonic® peptide score was chosen.</p><p>After initial data analysis via the default HCP workflow provided by Byos®, putative in silico peptides were added in Byologic® using the Add missing in silico peptides via existing peptides software component. In brief, this algorithm requires a collection of MS data files representing samples that were analyzed under identical chromatographic conditions, leading to sufficiently repeatable retention times (Electronic Supplementary Material (ESM) Fig. S1). For each peptide detected at least once via a fragment ion spectrum, the algorithm collects all retention times from all samples where this peptide was found on the MS/MS level. It then searches in the remaining samples on the full-scan MS level for signals at these retention times (with predefined tolerances for peak position and width), since such signals might represent peptides that did not trigger a fragmentation event. For each of these signals, the software calculates two measures to evaluate if the signal indeed originates from the respective peptide: (1) the deviation of the experimental mass from the theoretical peptide mass and (2) the so-called MS1 correlation, which is the Pearson correlation coefficient between the experimental isotope distribution and the isotope distribution of a theoretical peptide that has the same number of residues but consists entirely of averagine. (Averagine is a model for the "average" amino acid; its molecular formula is C4.9384H7.7583N1.3577O1.4773S0.0417, corresponding to an average molecular mass of 111.1254 Da [43].) The search for putative peptide signals on the full-scan MS level employed the following tolerance settings: a shift of the retention time of up to 0.5 min (parameter "FeatureCenterTolerance"), and a change in the peak width of up to 0.1 min (parameter "FeatureDurationTolerance").</p><p>Data exported from Byologic® was further processed and visualized in R [44] using packages from the tidyverse [45] and Bioconductor [46]. In particular, any provisional in silico peptide was dismissed if either (1) its mass deviation was larger than the 2.5th or 97.5th percentile of the mass deviations of all precursor ions for which a fragment ion spectrum was available, or (2) its MS1 correlation coefficient was smaller than the 5th percentile of the coefficients of all precursor ions with a fragment ion spectrum (ESM Fig. S2). Moreover, the data analysis script ensured that peptides derived from keratins or "non-HCPs" (i.e., standard peptides, trypsin, protein A, DPs) were not erroneously used to identify HCPs.</p><!><p>Raw mass spectrometry data and Byonic search results are available from Zenodo (10.5281/zenodo.3778440). All input files and data analysis scripts used in this study are available as ZIP archive (see ESM).</p><!><p>For our study, we assembled a panel consisting of five Fc domain comprising biopharmaceuticals, including two IgG1-type mAbs, that is, rituximab (MabThera®) and bevacizumab (Avastin®), as well as the Fc-fusion protein etanercept. With regard to the latter, two different Enbrel® production batches distributed in the USA and the European Union as well as the approved biosimilar Benepali® were analyzed. All of these therapeutic proteins originate from recombinant expression in CHO cells. Initially, we performed HCP identification in these commercial DPs via a direct workflow involving tryptic digestion of the respective DP, peptide analysis by RP-HPLC-MS/MS and protein identification against a CHO cell database supplemented with sequences of the respective DS, protein A, trypsin, and standard proteins (Fig. 1). With respect to the latter, we added E. coli β-galactosidase and bovine β-lactoglobulin to each DP prior to affinity chromatography, and spiked six standard peptides derived from E. coli ClpB into all samples prior to HPLC-MS measurements.</p><!><p>(a) Number of peptides and (b) number of proteins identified in the direct workflow (D) and in the combined flow-through and wash fractions of the depletion workflow (F+W) for each drug product. Each bar summarizes the results from three replicates (considering protein identification in one replicate sufficient). Colored bar sections highlight HCP-derived peptides and HCPs, respectively; moreover, they indicate credibility of detection and, in the case of peptides, their uniqueness. By contrast, light gray bars illustrate the total number of identified proteins or peptides, including putative contaminants (keratins) and "non-HCPs" (standard peptides, trypsin, protein A, drug products). "MS/MS" refers to identifications based on fragment ion spectra; "in silico" denotes identifications based solely on full-scan mass spectra. A peptide is considered "unique" if it matches a single sequence in the database; otherwise, it is classified as a "shared" peptide. Individual underlying peptide and HCP identifications are shown in ESM Fig. S3</p><p>Relative protein quantification in the direct workflow (D) and in the flow-through (F) and wash (W) fractions of the depletion workflow for each drug product. Each value was obtained by (1) extracting and integrating precursor ion currents of up to three of the most intense (unique and shared) peptides per protein, (2) averaging these areas, (3) calculating the mean over three technical replicates, and (4) scaling to the maximum value in each column (M) so that all other values in this column are given as percent of the maximum. The two topmost rows show cumulative abundances of all keratins and HCPs, respectively</p><!><p>Indeed, the depletion workflow (applying conventional data analysis) identified significantly more HCP-derived peptides and hence HCPs than the direct workflow. The increase in HCP identifications was most pronounced for Enbrel® EU, where the number of unique peptides and HCPs detected in at least one of the flow-through or wash fractions increased from 13 to 218 (Fig. 2a, blue bar segments) and 13 to 64 (Fig. 2b, violet bar segments), respectively. The substantial increase in the number of identifications upon depletion can be attributed to two factors: First, milligram amounts of the therapeutic protein loaded on the protein A column implicated comparatively high absolute amounts of HCPs. Second, DS capture prior to analysis prevented overloading of the RP-column with DS-derived peptides; thereby, it maintained separation performance, reduced overlapping elution of DS- and HCP-derived peptides, and notably lowered the dynamic range, as evident from an increase in relative HCP abundance (Fig. 3).</p><!><p>Although the depletion workflow greatly improved the number of identified peptides and HCPs compared with the direct workflow, conventional data analysis still neglected valuable information present in the acquired spectra. Hence, we aimed at optimizing MS data evaluation by (a) employing a probabilistic protein inference algorithm that includes shared peptides, and (b) peptide detection on the full-scan MS level in the absence of MS/MS spectra.</p><p>Evidently, protein inference based on unique peptides will ignore any peptide detected by MS/MS if it matches more than one protein in the sequence database. Several algorithms have been proposed that include these so-called shared peptides when determining the protein content of a sample [50, 51]. Here, we relied on the probabilistic model implemented in Byonic™, which ranks protein identifications according to their total evidence in the peptide library and assigns each shared peptide to the highest-ranking protein [52]. Moreover, this model simultaneously controls both the peptide-to-spectrum match and protein false discovery rate via a two-dimensional target decoy strategy. Thereby, it warrants a large number of protein identifications while ensuring that both false discovery rates remain reasonably low [42]. When applied to our data from the depletion workflow, the probabilistic algorithm considered between ten and 29 shared peptides (in Enbrel® US and Enbrel® EU, respectively; Fig. 2a, green bar segments). These peptides enabled identification of one to three additional HCPs (in Avastin® and the Enbrel® batches, respectively; Fig. 2b, red bar segments).</p><p>Furthermore, we expanded peptide detection to the full-scan MS level, as low-abundant peptides may fail to trigger an MS/MS event. For this purpose, we assessed samples of each DP taking into account retention times, intact peptide masses, and isotope patterns [53, 54]. Accordingly, a peptide was identified in silico (i.e., in the absence of a fragment ion spectrum), and therefore denoted as "in silico peptide", if it met the following three criteria: (1) The retention time of its intact peptide ion deviated at most 30 s from the retention time window spanned by all identical intact peptide ions identified via MS/MS in another sample of the same drug product. (2) The mass deviation of its intact peptide ion (as measured in ppm) fell within the 2.5th and 97.5th percentile of the mass deviations of all precursor ions for which a fragment ion spectrum was available (ESM Fig. S2a). (3) The experimental isotope pattern of its intact peptide ion adequately matched the theoretical averagine distribution (i.e., its Pearson correlation coefficient exceeded the 5th percentile of the coefficients of all precursor ions with a fragment ion spectrum) (ESM Fig. S2b). By re-assessing data from the depletion workflow in light of these criteria, we were able to detect up to 17 additional peptides (in Avastin®; Fig. 2a, yellow bar segments) and up to four additional HCPs (in Benepali®; Fig. 2b, yellow bar segments) that had been previously detected only in the direct workflow via MS/MS.</p><p>The different methods of identifying peptides and inferring proteins may even be used to classify detections on an ordinal scale of credibility (as indicated by the color scheme used in Fig. 2). One might consider HCP identifications supported by fragment ion spectra of several unique peptides to be more credible than those supported merely by in silico peptides, i.e., peptides identified only on full-scan MS level. Likewise, peptides identified via MS/MS are more credible than these in silico peptides.</p><!><p>In total, joint application of the depletion workflow and in-depth data analysis identified 127 distinct HCPs across all samples, based on 537 different peptides (ESM Fig. S3). Interestingly, although all investigated drugs were produced in CHO cells, only three HCPs were found in all of them, namely titin, nestin, and anionic trypsin-2 (ESM Fig. S4). These common HCPs were presumably co-purified with the therapeutic protein, probably by interacting with the IgG1-type Fc-subunit that is present in all investigated DSs, as previously suggested [37]. Moreover, several of the HCPs listed in ESM Fig. S3, e.g., clusterin, E3 ubiquitin-protein ligase, peroxiredoxin-1, glyceraldehyde-3-phosphate dehydrogenase, and 78-kDa glucose-regulated protein, have previously been identified in various DSs [26, 32, 37, 55, 56]. Thus, these HCPs may represent commonly occurring contaminants in CHO-based production systems. Several provisional HCP identifications (cationic trypsin-3, G3HUA1; anionic trypsin-2 fragment, G3HUC0; and Ig-like domain-containing protein, G3IMG2) were based solely upon peptides that could also be generated from non-HCPs via unspecific cleavage; hence, these proteins were removed from the final report. Nevertheless, other arguable HCP identifications were retained. For instance, identification of serum albumin (G3IAL6) may be attributed to carry over of bovine serum albumin that was used as standard protein in quality control runs. However, four of five peptides were unique for the hamster protein. Likewise, desmoplakin (G3HD94) and junction plakoglobin (G3HLU9) might be associated with contaminating keratins, but were nevertheless considered as HCPs, since they were identified only in a single drug product.</p><p>Although description of individual HCP identifications may provide basic insight into the HCP contents of the analyzed DPs, we aimed at an overall comparison of HCP profiles, i.e., the set of all HCPs identified in a given replicate, fraction, or DP (Fig. 1, bottom). To this end, HCPs and peptides of different samples were compared by calculating pairwise Jaccard indices, an elementary measure of set similarity: Applied to HCP profiles, a value of 1 indicates that exactly the same HCPs have been identified in two samples, while a value of 0 denotes two samples that do not share any HCPs. Computation of Jaccard indices facilitated the evaluation of (a) workflow repeatability (via comparison of HCP profiles on the replicate level), (b) co-purification tendency of HCPs during protein A chromatography (flow-through and wash fraction level), and (c) drug-to-drug versus batch-to-batch variability (DS level).</p><!><p>Repeatability of the depletion workflow depending on the minimum level of credibility required for identification of (a) HCPs or (b) HCP-derived peptides. Each point represents a comparison between two replicates of a fraction as quantified by the Jaccard index (two points have been annotated exemplarily). Boxes summarize all 30 comparisons for a given level of credibility (5 drug products × 2 fractions × 3 replicate combinations) via their quartiles, with whiskers extending to the smallest or largest value no further than 1.5 interquartile ranges away from the lower or upper quartile, respectively. Colors of x-axis tick labels correspond to the ones used in Fig. 2</p><!><p>Second, to characterize the co-purification tendency of HCPs during protein A chromatography, HCP profiles of the flow-through and wash fractions were compared for each DP (ESM Fig. S5; the HCP profile of a fraction comprised all HCPs detected in at least one replicate of this fraction). Most HCPs were identified within both fractions, corresponding to Jaccard indices ranging from 0.75 to 0.91. This high degree of similarity suggests low-affinity interactions between the shared HCPs and the DS or the protein A resin, resulting in their partial retention [11, 38–40]. Yet, some HCPs occurred exclusively in the wash or flow-through, indicating high or no affinity, respectively. Wash-exclusive detections might flag HCPs that should be particularly monitored during the production process and extensively tested for any immunogenic effects: As these HCPs bind tightly to the DS, they are most likely to be co-purified with the therapeutic protein.</p><!><p>Comparison of HCP profiles on the level of (a) DPs, (b) flow-through (F) and wash (W) fractions from the depletion workflow, and (c) replicates 1 to 3 for these fractions. Heatmap colors correspond to Jaccard indices J, whose numerical values (in percent) appear in panels (a) and (b). Dendrograms and derived row and column orders result from hierarchical clustering employing Jaccard distances 1 − J as measure of dissimilarity. Calculation of Jaccard indices involved HCPs irrespective of credibility detection. The antibody and etanercept clusters are highlighted in blue and red, respectively</p><!><p>Overall, dendrograms suggested that the HCP content of a given DP depends on the structural properties of the respective DS, with similar structures implicating similar HCP profiles: The HPC profiles of the two mAbs are about as similar as the profiles of Benepali® and the Enbrel® cluster. Within a single structural class, different manufacturing processes result in distinct HCP profiles, as exemplified by etanercept: Here, the two Enbrel® batches are more similar to each other (comparable manufacturing process) than to Benepali® (biosimilar with different manufacturing process). Despite these observed tendencies, comprehensive analyses of a larger number of production batches will be required to compensate for batch-to-batch variations as well as inherent variability of the method.</p><!><p>In this study, we explored different evaluation strategies of HPLC-MS/MS data for HCP discovery in highly pure DPs of biotherapeutics. Our data analysis protocol extended conventional approaches, i.e., HCP identification based on fragment ion mass spectra of unique peptides, by probabilistic protein inference and detection of peptides on the full-scan MS level. In conjunction with an experimental workflow involving DS depletion by protein A affinity chromatography and bottom-up proteomics via HPLC-MS/MS, this protocol significantly increased the number of identified peptides and HCPs. These detections allowed us to quantify similarities in HCP repertoires of several technical replicates and differently manufactured biotherapeutics, which may be used to evaluate workflow repeatability and drug-to-drug versus batch-to-batch variability, respectively. Taken together, these results underline the importance of utilizing the depth of information available from experimental HPLC-MS/MS data which is only partially used by standard data evaluation strategies. What is more, the demonstrated applicability of full-scan MS data for HCP identification purposes lays the basis for extended data evaluation strategies. For example, one might systematically determine accurate mass-retention time tags for all peptides from a given expression host, and then include this information in the algorithm for scoring full-scan MS and even MS/MS identifications [54].</p><p>Our findings demonstrate that the improved data analysis protocol is a flexible tool both for HCP discovery and for comparability studies of structurally diverse DPs based upon HCP profiles. If monitoring or absolute quantification of individual HCPs is required, results from our workflow may be directly used to devise targeted proteomics approaches based upon reaction monitoring techniques. Moreover, the protocol can easily be implemented into existing bottom-up setups based on HPLC-MS/MS. Notably, while we employed protein A affinity chromatography for DS depletion, our data analysis protocol is independent of this method and will readily work with novel techniques such as molecular weight cutoff enrichment [41]. The presented data evaluation workflow may thus be implemented at different stages of biopharmaceutical production, with comparative HCP profiles being especially attractive in the context of biosimilar development.</p><!><p>(PDF 4231 kb).</p><p>(7Z 13101 kb).</p><p>Chinese hamster ovary</p><p>Drug product</p><p>Drug substance</p><p>Enzyme-linked immunosorbent assay</p><p>Host cell protein</p><p>High-performance liquid chromatography</p><p>Monoclonal antibody</p><p>Mass spectrometry</p><p>Tandem mass spectrometry</p><p>Published in the topical collection featuring Female Role Models in Analytical Chemistry.</p><p>Publisher's note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p><p>Wolfgang Esser-Skala, Marius Segl and Therese Wohlschlager contributed equally to this work.</p>

PubMed Open Access

Chemoselective Synthesis of Uniform Sequence-Coded Polyurethanes and Their Use as Molecular Tags

Gunay and colleagues synthesized digitally encoded polyurethanes via a facile orthogonal iterative solid-phase approach. The polymers formed exhibited uniform molecular structure and controlled monomer sequences. Furthermore, these coded polyurethanes were very easy to read by tandem mass spectrometry sequencing. Thus, these polymers can be used as readable molecular labels and therefore open up interesting avenues in product-identification and anticounterfeiting technologies. For example, sequence-coded polyurethane tags were included in the present work in polystyrene cast films and 3D-printed polymethacrylate sculptures.

chemoselective_synthesis_of_uniform_sequence-coded_polyurethanes_and_their_use_as_molecular_tags

INTRODUCTION<!>RESULTS<!>DISCUSSION<!>EXPERIMENTAL PROCEDURES Materials and Reagents<!>Synthesis of the a-Linker for Modification of the Wang Resin<!>Resin Modification<!>General Procedure for Coupling Step 2<!>Iterative Repetition of Coupling Steps 1 and 2<!>Cleavage Procedure<!>Labeling of Materials ATRP Synthesis of Polystyrene<!>Preparation of Polystyrene Films Containing a Coded Polyurethane Label<!>Preparation of 3D-Printed Objects Containing a Coded Polyurethane Label<!>SEC<!>Thermogravimetric Analysis<!>ESI-MS<!>General Rules for MS/MS Polymer Sequencing<!>MS and MS/MS Analysis of the Polyurethane-Tagged Polystyrene Films<!>SUPPLEMENTAL INFORMATION

<p>Commodity plastics such as polyesters, polyamides, and polyurethanes (PUs) are usually prepared by step-growth polymerization, 1 which is a straightforward but poorly controlled process. For instance, PUs are generally obtained by reacting a diisocyanate with a diol, leading to polymers containing two oppositely oriented carbamates per repeat unit (Figure 1A). Although such PUs are widely applied as sealants, coatings, and adhesives, 2 they exhibit ill-defined molecular structures with a dispersity value ä of about 2. In addition, polymers formed through a step-growth mechanism exhibit, in general, simple repeating monomer sequences. 3 In contrast, it has been shown in recent years that uniform sequence-controlled synthetic polymers (ä $ 1), which are as molecularly defined as natural polymers such as nucleic acids and proteins, can be prepared using molecular machines, 4 nucleic acid templates, 5,6 or multistep-growth approaches, in which monomers are assembled one by one on a support. 3 This new generation of precision polymers is promising for a wide range of applications 7 and, in particular, for the development of informationcontaining macromolecules. 8 In such materials, information is stored at the molecular level in the form of a coded monomer sequence and can be recovered using a sequencing technique. 9,10 Some types of information-containing macromolecules have already been reported, [11][12][13][14][15][16] but their molecular structures differ markedly from those of standard commodity polymers. Here, we report that classic plastics such as PUs can also be used for information storage. Some routes for preparing sequence-defined oligocarbamates have already been reported in the literature, but they involve protecting-group-based protocols inspired by solid-phase peptide</p><p>The Bigger Picture Product identification is an important topic in modern consumption societies. Indeed, the massive production of counterfeit goods leads to major economic losses and represents a threat to health and the environment. Thus, anticounterfeiting technologies are crucial in many areas such as the pharmaceutical, cosmetics, food, and chemical industries. In particular, methods allowing product labeling at the nano or molecular scale are promising because they may not be easy to mimic. For example, synthetic polymers containing defined sequences of monomers can be used as molecular barcodes for product identification. Here, we report that digital sequences can be written at the molecular level on polyurethanes, which are cheap commodity plastics, using a very simple synthesis procedure. Moreover, the binary sequences formed can be easily deciphered by mass spectrometry. These coded polyurethanes can be used as molecular tags and blended in small amounts in other plastic materials. synthesis. [17][18][19] Synthetic chemistry can be greatly simplified by the use of protectinggroup-free strategies. 20 For instance, it has been shown that the preparation of uniform sequence-defined polymers is facilitated by the use of submonomer strategies involving chemoselective coupling steps, [21][22][23][24][25][26][27] but no route of this type has been discovered so far for the synthesis of PUs. In the present work, we demonstrate that uniform sequence-coded PUs can be prepared using a very simple chemoselective multistep-growth process. In addition, it was discovered that the polymers formed are remarkably easy to sequence by negative-mode tandem mass spectrometry (MS/MS). These advantages make this new class of coded plastics potentially interesting for applications in anti-counterfeiting technologies. Indeed, the massive production of counterfeit goods is a major problem in the global world economy. Although different types of nanoscale materials have been proposed for product identification, 28,29 the use of molecular barcodes that can be directly blended in an organic or inorganic matrix is a tempting option. In that regard, cheap sequencecoded plastics could be an interesting alternative to DNA, which is primarily investigated nowadays in such applications. 9,30 Therefore, sequence-coded PUs were tested in the present work for the labeling of different polymer materials.</p><!><p>The strategy for synthesizing uniform polyurethanes relies on the use of two successive chemoselective steps as shown in Figure 1B. Although potentially applicable on a variety of supports, this chemistry was tested in the present work on a hydroxyfunctionalized crosslinked polystyrene resin. This solid support was obtained by modifying a commercial Wang resin with a hydroxy-functional linker (leading after cleavage to a C5 acid chain-end moiety, noted as a in this article). In a first coupling step 1, the hydroxy group was reacted with N,N 0 -disuccinimidyl carbonate (DSC) to afford an unsymmetrical active carbonate. 31 It is known that the adduct formed reacts readily with primary and secondary amines, 31 as well as azides, 32 to form carbamate linkages. Interestingly, it was shown that the reaction with amines is chemoselective and can be performed in the presence of unprotected alcohols. 31 Thus, in step 2, the adduct formed was reacted with an amino alcohol to afford selectively a hydroxy-functional carbamate. The iterative repetition of coupling steps 1 and 2 allows facile protecting-group-free synthesis of uniform polyurethanes. Various monomers were tested in this study to verify this concept (Figure 1C). First, model homopolymers were synthesized using DSC and C5 0 . Different solvents, reactions times, and temperatures were screened to identify the optimal experimental conditions for the synthesis of uniform PUs. The use of acetonitrile and microwave irradiation in step 1 allowed synthesis of uniform polymers with ä $ 1 (P1 in Table S1). For instance, Figure S1 shows single-peak high-resolution electrospray mass spectra obtained for homopolymers of C5 0 (P1 in Table S1). These results imply that steps 1 and 2 are near quantitative and that no side reaction competes significantly with the iterative synthesis. Given these promising results, monomer alphabets were then developed in order to implement readable coded sequences in the uniform PUs. As shown in our previous works, 13,33,34 the use of a simple H/CH 3 molecular variation is sufficient to implement a 0/1 digital code that can be deciphered by MS/MS. Following this established principle, amino alcohols with or without methyl side groups were used to build sequence-coded PUs. In particular, two different binary monomer alphabets, C4 0 /C4 1 and C3 0 /C3 1 (Figure 1C), were tested in the present study. Both languages allowed the synthesis of uniform copolymers (P2-P16 in Table S1). As an example, Figure 2A shows the electrospray ionization (ESI)-MS and MS/MS spectra obtained for a copolymer (P9 in Table S1) synthesized with monomers C4 0 /C4 1 and containing the information sequence a-10001. The ESI-MS spectrum indicates the formation of a uniform structure. It is important to specify that this spectrum corresponds to a final product that was not purified by high-performance liquid chromatography or any other fractionation method. Results of comparable quality were obtained for all polymers (Figures S1-S13), including those synthesized with the alphabet C3 0 /C3 1 (P15 and P16 in Table S1 and Figures S14 and S15). Figures S16 and S17 also show typical nuclear magnetic resonance (NMR) spectra and size-exclusion chromatograms that were recorded for sequence-coded PUs. Sizeexclusion chromatography (SEC) measurements indicated the formation of uniform polymers with apparent ä values of about 1.01. Interestingly, the coupling step 2 seems to lead to similar yields using methylated (1-bit) or non-methylated (0-bit) amino-alcohol monomers. This is a noteworthy advantage over previously reported types of digitally encoded polymers such oligo(triazole amide)s or oligo(alkoxyamine amide)s, 11,13 in which the coupling yields can be affected by the molecular structure of the building blocks. Furthermore, the overall yields of PU synthesis were near quantitative in all cases (Table S1). The formed digitally encoded polymers were also remarkably easy to sequence by negative-mode MS/MS. In positive-ionmode MS/MS, protonated PUs undergo C-O and C-N carbamate fragmentations that lead to relatively complicated spectra (Figure S18). 18 In comparison, activation of deprotonated oligomers in the negative-ion mode leads only to C-O carbamate bond cleavage, and therefore the digital sequences written in the polymers are very easy to read (Figure 2A). It is important to specify that this analysis method permits unequivocal detection of a coded binary sequence. For instance, isobaric oligomers with different sequences can be easily distinguished by negative-mode MS/MS (Figure 2B). Although deprotonation occurs on the terminal carboxylic acid, the analysis of longer coded sequences is also possible, and the quality of sequencing is not affected by chain length (Figures 2C and S13).</p><p>Hence, sequence-coded PUs seem to be an interesting class of materials for anticounterfeiting applications. Indeed, these polymers can be easily encoded through a facile chemoselective process and decoded in a very short time. These polymers S1). (B) MS/MS discrimination of two isobaric oligomers containing different binary monomer sequences (P10 and P11 in Table S1). (C) MS/MS characterization of a sequence-coded oligomer containing a byte of binary information (P13 in Table S1).</p><p>were blended in different types of polymer matrices to demonstrate their relevance as molecular tags. Thermogravimetric analysis of these short coded polymers indicated that thermal decomposition starts to occur around 150 C (Figure S19), thus excluding their use in high-temperature processing conditions such as hot-melt extrusion. However, these molecular tags can be dispersed in solid polymer materials via many other procedures, such as film casting, mechanical blending, spray deposition, or in situ polymerization. For instance, Figure 3A shows the analysis of a polystyrene film in which a small amount of a coded PU tag was dispersed. Polystyrene and PUs are, in general, thermodynamically immiscible. 35 However, in this study, the molecular tags were used at very-low-weight fractions and could therefore be dispersed in a polystyrene matrix. The homogeneity of the PU tag dispersion was verified by studying different fractions of the polystyrene films that were cut, dissolved in deuterated tetrahydrofuran (THF-d 8 ), and analyzed by 1 H NMR. In all cases, the spectra contained specific PU signals, and compared with polystyrene peaks, they were integrated more or less the same in all fractions (Table S2). Although the formation of nanostructured PU domains cannot be excluded, this experiment suggests a homogeneous tag dispersion. After being included in the films, the PU tags can also be extracted and studied by MS. To do so, a small portion of the film was cut and immersed in methanol, which is a non-solvent of polystyrene. Even though the sequence-coded polyurethanes are also poorly soluble in methanol, they can be selectively extracted in sufficient quantity to be analyzed by ESI-MS. As shown in Figure 3A, a single-peak ESI spectrum was obtained, and S1). The oligomer barcode could be extracted from the film by selective dissolution in methanol and characterized by ESI-MS and MS/MS. (B) Labeling of a 3D-printed crosslinked poly(methacrylate) structure, in which 0.25 wt % of a polyurethane containing the binary sequence a-000101was incorporated (P12 in Table S1). The oligomer could be found by grinding the skirt of the 3D print into a thin powder, dissolving the powder in methanol, and analyzing the powder by ESI-MS and MS/MS. its MS/MS sequencing permitted easy recovery of the coded binary information. These results confirm that PUs tags can be used efficiently to label polymers films. As a second proof of concept, a sequence-coded PU tag was included in a 3D print obtained through methacrylate-based photopolymerization. As shown in Figure 3B, a sculpture representing a 3D DSC molecule was printed. In 3D printing, a significant amount of polymerized matter (i.e., draft, skirt, and brim) is unused and separated from the sculpture after printing. Small amounts of these residues can be kept attached to the sculpture as an anti-counterfeit strap that can be cut and analyzed. In the present case, some parts of the skirt were ground into a thin powder that was dissolved in methanol. MS analysis of this solution led to a mixture of peaks in which the signal of the PU tag had very weak intensity (inset of MS in Figure 3B). However, this peak can be found and efficiently sequenced by MS/MS. These results indicate that a sufficient fraction of the PU-coded tag survives the photopolymerization process and therefore enables practical 3D-sculpture labeling.</p><!><p>A practical new class of information-containing macromolecules that can be easily synthesized and sequenced was identified in the present work. The orthogonal iterative approach introduced here is probably the easiest method ever reported for the synthesis of uniform sequence-defined PUs. The two consecutive coupling steps studied in this work proceed in near-quantitative yields and are chemoselective. As a consequence, uniform macromolecules with a predetermined molar mass can be prepared in high overall yields using this process. Taking into account that the frontier between oligomers and polymers is often assumed to be 1,000 Da, 36 the present method is not restricted to the synthesis of short oligomers but can be used to prepare sequence-coded polymers, as shown in the present work with the synthesis of samples P13, P14, and P16. Moreover, the iterative process is experimentally convenient and can most probably be automatized for the synthesis of longer macromolecules. In addition, these uniform polymers can be molecularly encoded using pre-defined monomer alphabets. 9 In the present work, digitally encoded polymers were prepared using two different binary molecular alphabets in which non-methylated and methylated amino-alcohol monomers were used as 0and 1-bit motifs, respectively. It was observed that coupling step 2 led to high yields with both methylated and non-methylated building blocks. This constitutes an obvious advantage over previously reported classes of information-containing polymers that sometimes rely on the use of coded monomers of different reactivity. 13 The iterative process introduced here is not limited to the synthesis of digitally encoded polymers and could also be used for the synthesis of more complex coded macromolecules (e.g., using ternary or quaternary alphabets). 9 In addition, the coded monomers that can be used in this process do not necessarily have to be methylated or non-methylated building blocks. Many different types of coded side chains can potentially be used in this approach.</p><p>Furthermore, this new class of sequence-coded PUs, containing a carboxylic acid group at their a-chain end, is particularly easy to sequence by negative-mode MS/ MS. These macromolecules exhibit high ''readability'' because they always deprotonate on their a terminus only and hence undergo solely predictable C-O carbamate fragmentations. This is a clear advantage over positive-ion-mode MS/MS sequencing, in which randomly protonated PUs undergo C-O and C-N carbamate fragmentations that lead to complex spectra. Hence, these PUs can be used as anti-counterfeit tags to label different types of materials. The efficient molecular labeling of cast polystyrene films and photopolymerized 3D polymethacrylate prints provide proof of principle. In both cases, the PU tags were found in the materials, and their coded sequences could be efficiently assessed by negative-mode MS/MS. Here, the use of common plastics such as PUs is an interesting advantage over previously studied information-containing macromolecules. The physicochemical properties of PUs have been extensively investigated and are well documented in the literature. 2 For example, the blending behavior of PUs with other conventional plastics such as polystyrene, polypropylene, and poly(methacrylate)s is well documented. 35 Thus, the use of PU molecular barcodes is a practical and valid option for anti-counterfeit protection of industrial polymer products, as well as valuable objects such as 3D-printed art.</p><!><p>The following were used as received: citric acid (Alfa Aesar, >99%), hydrochloric acid (HCl; Sigma-Aldrich, 37%), p-toluenesulfonic acid monohydrate (PTSA; Sigma-Aldrich, 98.5%), trichloroacetic acid (TCA; Sigma-Aldrich, 99%), 4-amino-1-butanol (TCI, 98%), 3-amino-2,2-dimethyl-1-propanol (TCI, 97%), 4-amino-2-methyl-1butanol (TCI, 98%), 5-amino-1-pentanol (TCI, >95%), 3-amino-1-propanol (Alfa Aesar, 99%), 4-(dimethylamino)pyridine (DMAP; Sigma-Aldrich, 99%), N,N,N 0 ,N 0 ,N 00pentamethyldiethylene-triamine (PMDETA; Aldrich, 99%), anhydrous pyridine (Sigma-Aldrich, 99.8%), sodium hydroxide (NaOH; VWR, 99%), triethylamine (TEA; Merck, >97%), acetic anhydride (Alfa Aesar, >99%), ammonium chloride (NH 4 Cl; VWR, R99.5%), 4-benzyloxybenzyl alcohol polystyrene (Wang resin 100-200 mesh; Iris Biotech, 0.94 mmol/g resin), (1-bromoethyl)benzene (Alfa Aesar, 97%), 2-oxepanone (Sigma-Aldrich, 97%), N,N 0 -dicyclohexylcarbodiimide (DCC; Alfa Aesar, 99%), 4,4-dimethoxytrityl chloride (DMT-Cl; ChemGenes), N,N 0 -disuccinimidyl carbonate (DSC; TCI, >98.0%), sodium bicarbonate (NaHCO 3 ; SDS, 99%), anhydrous sodium sulfate (Na 2 SO 4 ; VWR, R99%), anhydrous acetonitrile (dry ACN; Sigma-Aldrich, 99.8%), cyclohexane (Carlo Erba), anhydrous dichloromethane (dry DCM; Sigma-Aldrich, R99.9%, 40-150 ppm amylene), DCM (Sigma-Aldrich, R99.9%), diethyl ether (Carlo Erba), anhydrous N,N-dimethylformamide (dry DMF; Sigma-Aldrich, 99.8%), DMF (Sigma-Aldrich, R99.0%), ethanol absolute (VWR, 99.9%), ethyl acetate (Carlo Erba), and tetrahydrofuran (THF; Aldrich, 99%, stabilized with 2,6-di-tert-butyl-4-methylphenol). Copper(I) bromide (CuBr; Alfa Aesar, 98%) was purified by stirring in acetic acid and rinsing with ethanol and diethyl ether and then dried. Styrene (Sigma-Aldrich, R99%) was distillated over CaH 2 and then degassed by bubbling argon through it. Gray photopolymer resin for Form 1+ (Formlabs, version FLGPGR02) was used for 3D printing. A Monowave 300 (Anton Paar) microwave reactor was used for step 1 of the PU synthesis. Step 2 was conducted in solid-phase extraction (SPE) tubes using a KS 130 basic (IKA) shaker.</p><!><p>This molecule was synthesized in four steps as shown in Scheme S1.</p><p>Step 1: Synthesis of 6-Hydroxyhexanoic Acid 2 NaOH (4.4 g, 110 mmol, 2 equiv) was added to a stirred mixture of 2-oxepanone (6.1 mL, 55 mmol, 1 equiv) in water (150 mL). The reaction mixture was stirred at room temperature (RT) for 16 hr before being acidified (pH 3-4) by the addition of HCl and extracted with ethyl acetate (3 3 100 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and evaporated, affording the targeted compound as a crystalline white solid. Yield: 5.9 g (82%); 1 Step 2: Synthesis of Ethyl 6-Hydroxyhexanoate 3 PTSA (107 mg, 0.56 mmol, 0.013 equiv) was added to a stirred solution of 6-hydroxyhexanoic acid (5.9 g, 45 mmol, 1 equiv) in absolute ethanol (50 mL), and the reaction was stirred at 65 C for 16 hr. The solvent was evaporated, and the resulting oil was dissolved in ethyl acetate (50 mL) and washed with saturated aqueous NaHCO 3 solution (2 3 50 mL) and with brine (1 3 50 mL). The aqueous layers were combined and extracted with ethyl acetate (3 3 50 mL). The organic layers were dried over Na 2 SO 4 , filtered, and evaporated, affording the targeted compound as a yellow oil. Yield: 6.2 g (87%). 1 H NMR (400 MHz, CDCl 3 ): d = 4.12 (q, J = 7.2 Hz, 2H, CH 2 ), 3.65 (t, J = 6.3 Hz, 2H, CH 2 ), 2.31 (t, J = 7.4 Hz, 2H, CH 2 ), 1.55-1.69 (m, 4.5H, 2 3 CH 2 ), 1.40 (m, 2.5H, CH 2 ), 1.25 (t, J = 7.2 Hz, 3H, CH 3 ); 13 C NMR (100 MHz, CDCl 3 ): d = 173.9 (C=O), 62. 8, 60.4, 34.4, 32.4, 25.4, 24.8, 14.4; HRMS (ES+) m/z: [M + H] + calculated 161.1172, found 161.1169.</p><p>Step 3: Synthesis of Ethyl 6-(Bis(4-methoxyphenyl)(phenyl)methoxy)hexanoate 4 A degassed solution of DMAP (0.8 g, 6.55 mmol, 0.5 equiv), DMT-Cl (5 g, 14 mmol, 1.1 equiv), and ethyl 6-hydroxyhexanoate (2.2 g, 13.7 mmol, 1 equiv) in anhydrous pyridine (25 mL) was stirred overnight for 16 hr at RT. The solvent was evaporated, and the resulting mixture was dissolved in ethyl acetate (100 mL) and washed with water (2 3 100 mL). The combined aqueous layers were extracted with ethyl acetate (150 mL). The organic layers were combined, dried over Na 2 SO 4 , filtered, evaporated, and purified by column chromatography (ethyl acetate/cyclohexane/TEA, 7:93:3) affording the target compound as a white oil. Yield: 6.0 g (95%); 1 H NMR (400 MHz, CDCl 3 ): d = 7.18-7.45 (m, 9H, Ar-H), 6.83 (m, 4H, Ar-H), 4.12 (q, J = 7.2 Hz, 2H, CH 2 ), 3.80 (s, 6H, 2 3 3H), 3.05 (t, J = 6.5 Hz, 2H, CH 2 ), 2.29 (t, J = 7.5 Hz, 2H, CH 2 ), 1.61 (m, 4H, 2 3 CH 2 ), 1.40 (m, 2H, CH 2 ), 1.25 (t, J = 7.2 Hz, 3H, CH 3 ); 13 C NMR (100 MHz, CDCl 3 ): d = 173.9, 158. 5, 145.5, 136.8, 130.2, 128.3, 127.8, 126.7, 113.1, 85.8, 63.3, 60.3, 55.3, 34.5, 29.9, 26.1, 25.1, 14.4; HRMS (ES+) m/z: [M + Na] + calculated 485.2298, found 485.2297.</p><p>Step 4: 6-(Bis(4-methoxyphenyl)(phenyl)methoxy)hexanoic Acid 5 An aqueous NaOH solution (6 M, 10 mL) was added to a stirred solution of ethyl 6-(bis(4methoxyphenyl)(phenyl)methoxy)hexanoate (3) (2.36 g, 5.1 mmol, 1 equiv) in absolute ethanol (5 mL). The reaction was stirred for 16 hr. Saturated aqueous NH 4 Cl was added to the reaction mixture until a white precipitate formed. The mixture was filtered and the residue was acidified with a solution of citric acid and water (1% m/m, 100 mL) and extracted with DCM (2 3 100 mL). The combined organic layers were washed with water (50 mL), dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure, affording the target compound as a white crystalline foam. Yield: 1.97 g (88%); 1 H NMR (400 MHz, CDCl 3 ): d = 7.18-7.46 (m, 9H, Ar-H), 6.8 (m, 4H, Ar-H), 3.79 (s, 6H, 2 3 3H), 3.06 (t, J = 6.6 Hz, 2H, CH 2 ), 2.34 (t, J = 7.6 Hz, 2H, CH 2 ), 1.62 (m, 4H, 2 3 CH 2 ), 1.43 (m, 2H, CH 2 ); 13</p><!><p>Wang resin (300 mg, 0.94 mmol/g, 0.28 mmol, 1 equiv) was placed into an SPE tube and swollen in DCM for 5 min. A solution of DMAP (53 mg, 0.43 mmol, 1.5 equiv), DCC (175 mg, 0.85 mmol, 3 equiv), and 6-(bis(4-methoxyphenyl)(phenyl)methoxy) hexanoic acid 5 (360 mg, 0.83 mmol, 3 equiv) in DCM was then added to the resin at RT, and the solution was shaken at RT for 36 hr. The resin was washed with DMF (103) and then with DCM (23) before proceeding to the next step. A solution of acetic anhydride in dry pyridine (1/5, 6 mL) was added to the SPE tube containing the modified resin, and the solution was shaken for 2 hr at RT. The resin was washed with DCM (53) before proceeding to the next step. A solution of TCA in DCM (3/97, w/w, 5 mL) was added to the SPE tube containing the modified resin, and the solution was shaken for 30 min at RT. The resin was washed with DCM (53). This step was repeated two times. The resin was then washed with diethyl ether (13).</p><p>General Procedure for the Iterative Synthesis of Polyurethanes General Procedure for Coupling Step 1 N,N 0 -disuccinimidyl carbonate (6 molar equiv) was first solubilized in dry acetonitrile by gentle heating. Then, triethylamine (12 molar equiv) was added to the solution. The mixture was then added to a microwave-suitable glass tube containing a hydroxy-functional solid support (1 molar equiv of hydroxy functions), and the tube was closed under an argon flow. The reaction was then stirred for 1 hr in a laboratory microwave for chemical synthesis (60 C, 8 W). After the reaction, the support was transferred into an SPE tube and washed several times with N,N-DMF before proceeding to step 2.</p><!><p>A solution of triethylamine (20 molar equiv) and an amino alcohol (10 molar equiv) in dry N,N-DMF was added to an SPE tube containing a solid support activated by N,N 0 -disuccinimidyl carbonate as described in step 1. The tube was then closed under an argon flow, and the reaction was shaken for 20 min at RT. After the reaction, the support was washed several times with DMF and diethyl ether and eventually transferred back to a microwave tube for a further step 1.</p><!><p>Steps 1 and 2 were performed consecutively and repeated a certain number of times to reach the desired polyurethane chain length. At each step 2, the molecular structure of the amino-alcohol building block could be varied to synthesize polymers with controlled sequences of information.</p><!><p>The cleavage procedure depends on the type of support and linker used for the synthesis. The following describe a solid support containing a labile Wang-type ester linkage. When a polymer with the desired chain length was obtained, the solid support was transferred into a vial. The cleavage was performed by adding a mixture of TFA and dry DCM (5:5 v/v) to the resin. The cleavage reaction was conducted for 2 hr. The support was then filtered off, and the filtrate was collected. TFA and DCM were evaporated under reduced pressure to obtain the desired polyurethane as a white solid.</p><!><p>CuBr (0.0156 g, 0.109 mmol) was added to a 25 mL flask sealed with a septum, and then the flask was degassed through argon-vacuum cycles. Styrene (5 mL, 43.5 mmol) and PMDETA (0.0227 mL, 0.109 mmol) were poured into the round flask and stirred under an argon flow for 20 min; (1-bromoethyl)benzene (0.0148 mL, 0.109 mmol) was then added, and the reaction took place at 85 C for 5 hr. After the required reaction time, the viscosity of the mixture was increased. THF (10 mL) was added to the flask, and then the mixture was precipitated in 50 mL of cold methanol, filtered, and dried. SEC in THF: M n = 31,500 g/mol, ä = 1.26.</p><!><p>Coded polystyrene films were prepared by dissolving polystyrene and the sequence-coded polyurethane P1 in THF (8% w/v) at 40 C where the weight ratio of polystyrene/PU was 99:1. The solution obtained was poured onto a glass plate, and the membranes were formed after 20 hr at RT. The films were further dried under a vacuum until complete removal of the solvent. Homogeneous and transparent membranes were obtained.</p><!><p>Printing of the 3D object including a PU tag was performed using a Form 1+ SLA 3D printer equipped with a class 1 laser and a diode of violet color of 405 nm wavelength provided by Formlabs. The PU (0.25%, w/v) was dissolved in the photosensitive liquid resin. The tank was filled with the resin mixture, and with the help of the laser, it was cured layer by layer to build the 3D model. After the building of a layer, the laser was raised before continuing with the rest of the layers. The construction of 639 layers took 1 hr 40 min. The volume of the structure obtained was about 8 mL. The final 3D model was characterized from a detailed and high-resolution structure. It was generated on a support. After the end of the building, it was immersed in isopropyl alcohol to rinse the parts and clean liquid uncured resin from the outer side of the 3D model. The 3D object was further cured under UV light of 350 nm wavelength for a final photo-crosslinking curing to completely harden the sculpture. The support was removed using tools, and the surface was sanded to give a smooth finish.</p><!><p>Two different SEC setups were used in this work. The first was equipped with four PLGel Mixed C columns (5 mm, 30 cm, diameter = 7.5 mm), a Wyatt Viscostar-II viscometer, a Wyatt TREOS light scattering detector, a Shimadzu SPD-M20A diode array UV detector, and a Wyatt Optilab T-rEX refractometer. This setup was used for polymer characterization (1,000-3,000,000 g/mol). The other setup was equipped with a Shimadzu RiD-10A refractometer, a Shimadzu SPD-10Avp UV detector, and four monoporosity PLGel columns (5 mm, 30 cm, diameter = 7.5 mm): 50, 100, 500, and 1,000 A ˚. This setup was used for characterization of oligomers and short polymers (100-20,000 g/mol). In both setups, the mobile phase was THF with a flow rate of 1 mL/min. Toluene was used as the internal reference. The calibration was based on linear PS standards from Polymer Laboratories.</p><!><p>Thermogravimetric analysis (TGA) was recorded on a Mettler Toledo TGA 2 Star System. The temperature range was from 25 C to 600 C, and the heating rate was 10 C/min in 100 mL/min N 2 .</p><!><p>HRMS and MS/MS experiments were performed using a QqTOF mass spectrometer (QStar Elite; Applied Biosystems SCIEX) equipped with an ESI source operated in negative mode (capillary voltage, À4,200 V; cone voltage, À75 V). Some experiments were conducted in positive-ion mode with a capillary voltage of +5,500 V and a cone voltage of +75 V. In MS mode, the mass of the ions was measured accurately in an orthogonal acceleration time-of-flight (oa-TOF) mass analyzer using polyethylene glycol oligomers adducted with an acetate anion as internal standards. In MS/MS mode, precursor ions were selected in a quadrupole mass analyzer before entering a collision cell filled with nitrogen, and product ions were measured in the oa-TOF. In this instrument, air was used as the nebulizing gas (10 psi), and nitrogen was used as the curtain gas (20 psi). Instrument control, data acquisition, and data processing were achieved using Analyst software (QS 2.0) provided by Applied Biosystems. Oligomers (1-2 mg) were dissolved in methanol (300 mL) in an ultrasonic bath (15 min). Samples were further diluted (1:100 to 1:1,000 v/v) in a methanol solution of ammonium acetate (3 mM) and injected in the ESI source at 10 mL/min using a syringe pump. Oligomers were ionized in negative-ion-mode ESI, and the deprotonated molecules formed were subjected to collision-induced dissociation.</p><!><p>Fragmentation of deprotonated oligocarbamates only proceeds by competitive cleavage of all C-O carbamate bonds as a result of transfer of a proton from the alkyl segment on the right-hand side of the dissociating bond. Because the negative charge remains located in the a termination, only products containing this end group are detected as ions in MS/MS spectra. As a result, the distance between two consecutive peaks in MS/MS spectra is equal to the mass of a coded unit, as shown in Scheme S2 for P6 (MS/MS data shown in Figure S6). Consequently, measuring the m/z difference between consecutive peaks starting from the precursor ion allows the oligomer sequence to be readily reconstructed from the right to the left. Product ions were named after the nomenclature established by Wesdemiotis et al. 37 for synthetic polymers. This nomenclature recommends that a-containing fragments be designated with letters from the beginning of the alphabet (a, b, c, etc., corresponding to product ions obtained after cleavage of the first, second, third, etc., bond in the monomer, respectively). However, in order to adopt a similar nomenclature for all polycarbamates regardless of the alphabet (C3, C4, and C5) used to code information in their structure, this unique ion series was named a i À (where i is the number of the entire monomeric unit contained in the product ion).</p><!><p>A small portion ($1.0 mg) of the PS film was cut and sonicated in methanol containing 3 mM ammonium acetate (200 mL) for 10 min. Because of the low solubility of carbamates in the extraction medium (chosen as a non-solvent for PS), the turbid solution obtained was further diluted (1:10 v/v) in the same solvent before it was injected in the ESI source for MS analysis and MS/MS sequencing.</p><p>MS and MS/MS Analysis of the Polyurethane-Tagged 3D-Printed Object Small amounts (5-10 mg) of the thin powder obtained by grinding residues cut from the 3D-printed sculpture were sonicated in 400 mL of methanol containing 3 mM ammonium acetate for 15 min (similar results were obtained when using THF as the extraction solvent). The extraction medium was centrifuged (130 rpm), and the supernatant was further diluted (1:10 v/v) in methanol supplemented with ammonium acetate before being injected in the ESI source for MS analysis and MS/MS sequencing.</p><!><p>Supplemental Information includes 19 figures, two tables, and two schemes and can be found with this article online at http://dx.doi.org/10.1016/j.chempr.2016.06.006.</p>

Chem Cell

Oligomeric Structure and Three-Dimensional Fold of the HIV gp41 MPER and Transmembrane Domain in Phospholipid Bilayers

The HIV-1 glycoprotein, gp41, mediates fusion of the virus lipid envelope with the target cell membrane during virus entry into cells. Despite extensive studies of this protein, inconsistent and contradictory structural information abounds in the literature about the C-terminal membrane-interacting region of gp41. This C-terminal region contains the membrane-proximal external region (MPER), which harbors the epitopes for four broadly neutralizing antibodies, and the transmembrane domain (TMD), which anchors the protein to the virus lipid envelope. Due to the difficulty of crystallizing and solubilizing the MPER-TMD, most structural studies of this functionally important domain were carried out using truncated peptides either in the absence of membrane-mimetic solvents or bound to detergents and lipid bicelles. To determine the structural architecture of the MPER-TMD in the native environment of lipid membranes, we have now carried out a solid-state NMR study of the full MPER-TMD segment bound to cholesterol-containing phospholipid bilayers. 13C chemical shifts indicate that the majority of the peptide is \xce\xb1-helical, except for the C-terminus of the TMD, which has moderate \xce\xb2-sheet character. Intermolecular 19F-19F distance measurements of singly fluorinated peptides indicate that the MPER-TMD is trimerized in the virus-envelope mimetic lipid membrane. Intramolecular 13C-19F distance measurements indicate the presence of a turn between the MPER helix and the TMD helix. This is supported by lipid-peptide and water-peptide 2D 1H-13C correlation spectra, which indicate that the MPER binds to the membrane surface whereas the TMD spans the bilayer. Together, these data indicate that full-length MPER-TMD assembles into a trimeric helix-turn-helix structure in lipid membranes. We propose that the turn between the MPER and TMD may be important for inducing membrane defects in concert with negative-curvature lipid components such as cholesterol and phosphatidylethanolamine, while the surface-bound MPER helix may interact with N-terminal segments of the protein during late stages of membrane fusion.

oligomeric_structure_and_three-dimensional_fold_of_the_hiv_gp41_mper_and_transmembrane_domain_in_pho

Introduction<!>Solid-phase peptide synthesis of site-specific labeled gp41 MPER-TMD<!>Membrane protein sample preparation for SSNMR<!>Lipid mixing assays<!>Circular dichroism experiments<!>Solid-state NMR experiments<!>Simulation of 19F CODEX and 13C-19F REDOR intensities<!>Structural modeling of the MPER-TMD trimer<!>MPER-TMD is fusogenic and is predominantly \xce\xb1-helical in lipid membranes<!>Depths of insertion of the MPER-TMD in lipid bilayers<!>MPER-TMD causes negative Gaussian curvature to PE membranes<!>MPER-TMD is immobilized in lipid membranes<!>MPER-TMD is trimerized in the lipid bilayer and has a turn between the MPER and TMD<!>Oligomeric state and three-dimensional fold of gp41 MPER-TMD in lipid membranes<!>Comparison of the oligomeric structure in lipid bilayers with previous structures in bicelles<!>Backbone conformation of the MPER-TMD protomer<!>Implication of the trimeric helix-turn-helix structural motif for HIV membrane fusion

<p>Human immunodeficiency virus (HIV) enters susceptible cells using the trimeric envelope glycoprotein, Env, which fuses the virus lipid envelope with the cell membrane 1-2. Proteolytic cleavage of the Env precursor yields two subunits, gp120 and gp41. The former binds to cell-surface receptors and dissociates from the complex, which triggers a cascade of conformational changes in the membrane-interacting subunit, gp41. This cascade starts with refolding of gp41 to expose and insert an N-terminal fusion peptide (FP) into the target cell membrane while the protein remains anchored in the virus envelope through a C-terminal transmembrane domain (TMD) 3. The extended intermediate then bends onto itself to appose two ectodomain heptad repeats, NHR and CHR (Fig. 1A), to form an antiparallel coiled-coil 4. This trimer of hairpins, characteristic of the post-fusion structure of class I viral fusion proteins 5-6, brings the two lipid membranes into proximity. The FP and TMD then disorder the cell and virus membranes by mechanisms that are still poorly understood, causing high-curvature membrane intermediates, eventually giving rise to a single merged bilayer. In addition to the FP and TMD, gp41 contains a membrane-proximal external region (MPER) N-terminal to the TMD, which contains highly conserved epitopes for several broadly neutralizing antibodies (bNAbs) 7-10. A large amount of biochemical and biophysical evidence has shown that both the MPER and TMD are important for HIV entry into cells. For example, Trp-to-Ala mutations in the MPER and deletion of the MPER abrogated virus entry and membrane fusion 11. Replacement of the gp41 TMD with the vesicular stomatitis virus-G fusion protein TMD severely impacted fusion activity 12. Truncation of the C-terminus of the TMD in the simian immunodeficiency virus fusion protein reduced fusogenicity 13-14.</p><p>Despite the importance of the MPER and TMD for HIV virus-cell fusion, most high-resolution structural studies of gp41 removed all or most of this region due to the difficulty of crystallizing gp41 constructs containing these hydrophobic domains. NMR and EPR studies of gp41 that included part of the MPER-TMD 15-19 have yielded three divergent structural topologies and oligomeric states, as summarized in Fig. 1B-G. The first structural model depicts the MPER as bound to the membrane surface while the TMD spans the bilayer (Fig. 1B). This model is based on NMR and EPR studies of gp41 MPER (residues 662-683) in DPC micelles, which found a distorted helix on the micelle surface 20. Three polar Asn (N671, N674, and N677) face the aqueous solution, four Trp residues (W666, W670, W672 and W678) are buried in the micelle, and a hinge is present at residues F673-N674 (Fig. 1C). This model is also supported by NMR and EPR studies of the Ebola virus fusion protein MPER-TMD, which found a helix-turn-helix motif, with the MPER lying at the membrane-water interface 21. However, DEER EPR data showed no intermolecular interactions, indicating that the Ebola MPER-TMD is monomeric in micelles, bicelles and nanodiscs. The second structural model depicts the MPER-TMD as a continuous helix, oriented perpendicular to the membrane plane (Fig. 1D). This model is based on solution NMR studies that included part of the MPER and TMD. For example, a study of gp41 (residues 671-693) found no helical interruption at K683 but a helix kink at G690 in the GxxxG motif of the TMD 22. Two studies of gp41 (residues 677-716) in DMPC/DHPC bicelles 19, 23 reported an uninterrupted helix from the MPER to the TMD (Fig. 1E). However, these studies differed on the oligomeric state: one study concluded a trimeric coiled coil based on intermolecular cross peaks 19, while the other study found residual dipolar couplings that are inconsistent with C3 symmetry, and paramagnetic relaxation enhancement and DEER data that indicate an absence of intermolecular contacts 23. The monomeric helical structure suggests a very tilted orientation for this long helix, to reduce the hydrophobic mismatch between the peptide and the bilayer portion of the bicelle. The third structural model postulates a trimeric MPER and monomeric TMD helices (Fig. 1F), based on studies of a designed MPER peptide (residues 656-683) that is fused with the trimerization domain of bacteriophage T4 fibritin 24. The structure shows the expected trimeric helices for the N-terminal portion, which splays apart towards the C-terminus of the sequence. This result suggests that bNAbs may interact with individual MPER helices (Fig. 1G) in competition with the membrane surface, and the TMD helices may not be tightly associated in the membrane.</p><p>These divergent structural conclusions may arise from the truncated nature of the peptide sequences in most studies, but could also reflect an inherent conformational plasticity of this region of gp41, which may be important for the protein to fold into different structures at different stages of virus-cell fusion. To distinguish these two scenarios, we have synthesized a gp41 peptide (residues 665-704) that spans the entire MPER-TMD region, and investigated the structure of this peptide in phospholipid bilayers using solid-state NMR. The use of lipid bilayers avoids the potential problem of high-curvature micelles and small bicelles to cause non-native protein structures or destabilize protein assembly 25. We examine the MPER-TMD structure in two membranes, a complex cholesterol-containing membrane that mimics the HIV lipid envelope composition and a POPE membrane that probes the effect of membrane negative spontaneous curvature on the peptide structure. We address three key aspects of the structure: the three-dimensional fold that describes the relative orientation of the MPER and the TMD, the membrane insertion depths of the MPER and TMD, and the oligomeric state of the entire peptide. We show that in both virus-mimetic membranes and negative-curvature POPE membranes, gp41 MPER-TMD is predominantly α-helical, with the TMD spanning the bilayer while the MPER residing on the membrane surface. Intramolecular distance measurements indicate a turn between the MPER and TMD, while intermolecular distance measurements unequivocally show that the peptide is trimerized. Therefore, this C-terminal domain of gp41 assembles into a trimeric helix-turn-helix in native-like lipid membranes, thus providing structural constraints to mechanistic models of HIV virus-cell fusion.</p><!><p>The peptide sequence in this work corresponds to residues 665-704 of gp41 (KWASLW NWFNITNWLW YIKLFIMIVG GLVGLRIVFA VLSI) of HIV-1 clade B HXB2 isolate 26 (UniProtKB/Swiss-Prot: P04578.2) (Table 1).</p><p>The peptide was synthesized using Fmoc solid-phase methods on a custom-designed flow peptide synthesizer 27. 0.05 mmol H-Rink amide ChemMatrix® resin (0.1 g at 0.5 mmol/g loading size) was swelled in the reaction syringe for 5 min in ~5 mL N,N-dimethylformamide (DMF) at 70°C. Ten-fold excess (0.5 mmol) of unlabeled amino acid and four-fold excess (0.2 mmol) of isotopically labeled amino acid were singly and doubly coupled, respectively, using a coupling time of 50 s and 70 s. After the final coupling step, the peptide was deprotected and cleaved from the resin by addition of TFA/Phenol/H2O/TIPS solution (88 : 5 : 5 : 2 by volume) for 3 h. The resin was filtered off, and the crude peptide was precipitated and triturated three times with cold diethyl ether and dissolved in 80% HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol) solution. Crude peptide was purified by reverse-phase HPLC using a Vydac C4 column with a linear gradient of 20-99% channel A over 120 min at a flow rate of 15 mL/min (channel A is 1 : 1 v/v acetonitrile : isopropyl alcohol and channel B is acetonitrile). MALDI-MS analysis verified the mass to be 4818.3 Da for LWIGA and 4835.1 for Da for WLIGF, in agreement with the calculated masses. The combined synthesis and purification yield was ~8%. Three peptide samples with different isotopically labeled positions were prepared (Table 2). 13C, 15N-labeled residues were placed at L669 in MPER, L684, I686, G694, and A700 in the TMD, while fluorinated residues were placed at 5F-W678, 5F-W680, and 4F-F699, where 5F corresponds to the Hζ3 position of Trp and 4F corresponds to the Hζ position of Phe.</p><!><p>The MPER-TMD peptide was reconstituted into the POPE membrane and an HIV virus-mimetic membrane (VMS), which consists of POPC, POPE, POPS, sphingomyelin (SM), and cholesterol at a molar ratio of 30 : 15 : 15 : 10 : 30. The VMS composition differs from the previous virus-mimetic membranes (VM and VM+) 28-29 by including phosphatidylserine, which is present in the HIV-1 virus envelope at non-negligible levels 30. Phospholipids were co-dissolved in chloroform and SM was dissolved in a chloroform/methanol mixture. The peptide was dissolved in 2,2,2-trifluoroethanol (TFE) and mixed with the lipid solution. The solvents were removed under nitrogen gas, then the samples were dried under vacuum overnight. The dried powder was resuspended in pH 7.5 HEPES buffer (10 mM HEPES-NaOH, 1 mM EDTA, 0.1 mM NaN3) and dialyzed against pH 7.5 HEPES buffer for a day with two buffer changes to remove salt and residual TFA and TFE. The vesicle solutions were spun at 40,000 rpm using a Beckman SW60Ti rotor at 4 °C for 4 hours to obtain wet membrane pellets, which were allowed to equilibrate in a desiccator to ~40 wt% water by mass. The samples were then spun into magic-angle-spinning (MAS) rotors through a pipette tip. Most samples in this study have a peptide : lipid (P/L) mole ratio of 1 : 15. For the 4F-F699 labeled peptide reconstituted into the POPE membrane, we prepared two samples at P/L ratios of 1 : 15 and 1 : 45, to investigate whether the oligomeric state of the MPER-TMD is sensitive to the peptide concentration in the range studied here.</p><!><p>To verify the fusion activity of the MPER-TMD peptide, we conducted lipid mixing assays on POPC/POPG (4:1) vesicles 31. Two solutions of large unilamellar vesicles (LUVs), with and without fluorescent dyes, were prepared. The unlabeled POPC/POPG LUVs were prepared in 10 mM HEPES buffer at pH 7.5 by 10-12 cycles of freeze-thaw between liquid nitrogen temperature and 35°C, followed by 15-20 cycles of extrusion through 100 nm polycarbonate membranes (Whatman). The fluorescently labeled vesicles differ by containing 2 mol% NBD-PE (1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) and 2 mol% of the quenching lipid Rh-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)). The unlabeled and labeled vesicles were mixed at a 9 : 1 mole ratio and have a total lipid concentration of 75 μM. Then the MPER-TMD peptide was added from a formic acid solution to reach a peptide: lipid mole ratio of 1 : 20. If MPER-TMD causes mixing of the labeled and unlabeled vesicles, then the distances between NBD-PE and Rh-PE will increase, thus dequenching the fluorescence intensity. A HORIBA Fluoromax-P fluorimeter was used to measure fluorescence intensities at an excitation wavelength of 465 nm and an emission wavelength of 530 nm, with a bandwidth of 4 nm. The measurement was carried out at 21°C, under continuous stirring in 2 ml LUVs, with a 1 s time increment.</p><p>We designate the initial fluorescence intensity before peptide addition as F0 and peptide addition as Ff. The maximum fluorescence intensity (Fmax) is taken as the intensity when 20 μL of 10% Triton X-100 was added to 2 ml of lipid vesicle solution. The percent lipid mixing was calculated using the equation % lipid mixing =[(Ff – F0)/(Fmax – F0)]·100. To test whether the formic acid itself causes lipid mixing, we conducted a control experiment where 5 μL of peptide-free formic acid was added to the lipid vesicle solution. We found that 5 μL formic acid caused only ~5% lipid mixing, compared to 7.2 μL of peptide-containing solution, which induced 75% lipid mixing. Thus the MPER-TMD peptide is chiefly responsible for the observed lipid mixing.</p><!><p>Circular dichroism (CD) spectra were measured on an AVIV 202 spectrometer using a 1 mm path-length quartz cuvette to evaluate the secondary structure of the MPER-TMD. The peptide was dissolved in three solutions: TFE, POPC/POPG (4 : 1) membrane, and DOPC/DOPE (2:1) membrane. The membrane samples used a P/L ratio of 1 : 20. 0.05 - 0.10 mg of peptide was dissolved in 0.5 ml vesicle solution and TFE. Control spectra of peptide-free solutions were subtracted from the spectra of peptide-containing samples.</p><!><p>Solid-state NMR spectra were measured on Bruker 400 MHz (9.4 Tesla), 600 MHz (14.1 Tesla), and 800 MHz (18.8 Tesla) spectrometers using 4 mm and 3.2 mm MAS probes. 13C chemical shifts are reported on the TMS scale using the 38.48-ppm CH2 signal of adamantane and the 14.0-ppm Met Cε signal of the tripeptide N-formyl-Met-Leu-Phe-OH (f-MLF) as indirect standards. 31P chemical shifts were referenced to the 2.73-ppm 31P signal of hydroxyapatite on the phosphoric acid scale. 19F chemical shift was referenced to the −122 ppm 19F signal of Teflon on the CFCl3 scale.</p><p>2D 13C-13C DARR correlation spectra 32-33 with a mixing time of 100 ms were measured at 263 K to assign 13C chemical shifts. To determine the depth of insertion of the peptide in lipid bilayers, we conducted a 2D 1H-13C correlation experiment in which a 1H T2 filter of 0.8 ms was applied to suppress the protein 1H magnetization while retaining water and lipid 1H magnetization 34-35. After 1H chemical shift evolution, a 1H spin diffusion mixing period of 9 – 400 ms was used to transfer 1H magnetization to the protein and detected through 13C after cross polarization 34. The experiments were conducted on the LWIGA-labeled peptide in POPE membranes at 303 K under 9 kHz MAS. Cross peak intensities are plotted as a function of the square root of the spin diffusion mixing time, since the solution to the diffusion equation in a two-phase system has an inherent √t dependence 35-36.</p><p>To probe the water accessibility of different residues in detail, we measured water-edited 2D 13C-13C correlation spectra at 263 – 273 K under MAS frequencies of 9 kHz and 10.5 kHz 35, 37-39. At this temperature, with 1H T2 filters of 0.6 ms to 0.8 ms, the lipid and protein 1H magnetization is largely suppressed, leaving water as the only 1H polarization source. This is followed by a 1H mixing period of 4 – 16 ms before 13C-13C correlation spectra were measured.</p><p>Two types of long-distance experiments were conducted to probe the three-dimensional fold and self-assembly of the MPER-TMD in the lipid membrane. Intramolecular 13C-19F distances between 5F-W678 and L684 and I686 Cα were measured using a frequency-selective REDOR experiment 40. An 800 μs Gaussian 13C π pulse was applied in the middle of the REDOR period to invert the 13C spin of interest and to suppress 13C-13C scalar couplings. The REDOR mixing time ranged from 5 ms to 25 ms, and the experiments were conducted under 5 kHz MAS at 235 K to immobilize the peptide.</p><p>Intermolecular distances were measured using the 19F CODEX experiment 41-43. Two trains of 19F 180° pulses spaced every half a rotor period were applied before and after a longitudinal mixing period. The 19F 180° pulses recouple the 19F chemical shift anisotropy (CSA) and cause the formation of a stimulated echo. 19F spin diffusion during the mixing period due to oligomerization decreases the echo intensity. To correct for T1 relaxation effects, two experiments, a control (S0) and a dephasing (S) experiment, in which the mixing time was interchanged with a z-filter after the second π-pulse train, were measured, and the intensity ratio S/S0 indicates the extent of 19F spin diffusion. The 19F CODEX experiments were conducted under 8 - 10 kHz MAS at 233 K, in order to immobilize the peptide and ensure detection of only spin diffusion and not slow motion 43.</p><p>To investigate the mobility of the MPER-TMD in the lipid membrane, we measured 13C-1H dipolar couplings using a 2D double-quantum-filtered (DQF) DIPSHIFT experiment 44-46. FSLG 47 was used for 1H homonuclear decoupling and SPC5 48 was used for 13C-13C recoupling and double-quantum filtering. The experiment was conducted under 7 kHz MAS at 303 K. The time-domain data were fitted to obtain the apparent couplings, which were divided by the FSLG scaling factor of 0.577 to obtain the true couplings. The C-H order parameter SCH was calculated as the ratio of the true coupling to a rigid-limit value of 22.7 kHz.</p><p>Static 31P spectra of lipid membranes were measured at 298 K to investigate the effects of MPER-TMD on membrane morphology 49-51. To determine the effect of the peptide on the membrane-surface hydration, we measured 2D 1H-31P heteronuclear correlation (HETCOR) spectra at 298 K under 5 kHz MAS using a 100 ms 1H mixing time 52-55.</p><!><p>The 19F CODEX S/S0 intensities were fit using a home-written MATLAB program to obtain 19F-19F distances 43. The program uses the exchange matrix formalism to treat multi-spin diffusion. For trimeric systems, the 3 × 3 exchange matrix contains rate constants that are proportional to an overlap integral and the 19F dipolar coupling of interest. Based on model compound results, the value of the overlap integral is 37 μs at 8 kHz MAS and 41 μs at 10 kHz MAS 42. To simulate F699 distances, we considered two oligomeric structures: 50% of an α-helical trimer and 50% of a β-sheet consisting of three parallel β-strands. The inter-strand distances were adjusted to 5.8 and 6.8 Å for the POPE and VMS data, respectively, in order to fit the fast initial decay, while the inter-helical distances were varied to fit the slow decay at longer mixing times. 13C-19F REDOR S/S0 intensities were fit using the SIMPSON program 56 to obtain intramolecular distances. An intensity scaling factor of 0.85 was applied to the calculated curves to represent 19F pulse imperfections in the REDOR data 57.</p><!><p>We carried out molecular dynamics (MD) simulations in explicit membranes to assess the energetic stability of the MPER-TMD trimer structures in lipid bilayers and to obtain a view of the turn structure between the MPER and TMD. We used the monomer structure of the Ebola virus envelope glycoprotein (GP2) MPER-TMD (PDB code: 5T42) 21 as the initial template, and substituted the Ebola sequence with the gp41 sequence (residues K665 – I704) by aligning P653 in the Ebola protein with one of seven residues in gp41 (L684, K683, I682, Y681, W680, L679, and W678) (Tables S1, S2). The (ϕ ψ) angles of two solution NMR structures (PDB: 5JYN and 6B3U) were then applied to the TMD residues I688-I697 in the model. Each monomer was trimerized, then inserted into a lipid bilayer containing 60 POPC, 30 POPE, 30 POPS, 20 SM, and 60 cholesterol molecules. This membrane matches the composition of the VMS membrane used in the experiments, and was created using the Charmm-Gui Membrane Builder 58. The simulation box, obtained from the CHARMM-GUI website server, consists of 7750 TIP3 waters and 21 potassium ions. MD simulations were carried out in Gromacs 59 using measured 13C-19F and 19F-19F distance constraints. In addition to these experimental constraints, we fixed N – N, Cα – Cα, and C' – C' distances between residue i and residue i+4 for residues K683–S703 to maintain the TMD backbone conformation (Tables S3). During the simulations, we found that the three intermolecular 19F-19F restraints were insufficient to stabilize the trimer; therefore, we added three interhelical restraints of 9.4 Å, 6.5 Å, and 8.0 Å at G690 Cα, V693 Cα, and R696 Cα, obtained from the solution NMR structure of trimeric TMD (PDB: 5JYN), to help stabilize the trimer 19. The energy of the system was minimized using a steepest decent strategy followed by a six-step equilibration process at 303 K for 3.75 ns. After equilibration, the production run was carried out at the same temperature for 100 ns.</p><!><p>To determine the three-dimensional fold and oligomeric structure of gp41 MPER-TMD in lipid membranes, we synthesized a 40-residue peptide corresponding to residues 665-704 of the HXB2 isolate of gp41 (Table 1). 13C, 15N-labeled residues and 19F-labeled residues were placed at strategic positions in the peptide (Table 2) to investigate the conformation and intermolecular assembly of this domain. A semi-automated continuous-flow Fmoc peptide synthesis protocol was used to allow each amino acid to be incorporated into the sequence every 3~4 minutes. CD spectra of the peptide in POPC/POPG and DOPC/DOPE vesicles showed α-helical conformations, with slightly lower helical content in the DOPC/DOPE membrane (Fig. S1B). This trend is consistent with the conformation of the PIV5 fusion protein TMD, which showed a pronounced membrane-induced conformational change from α-helix in lamellar bilayers to β-sheet in negative-curvature phosphatidylethanolamine (PE) membranes 55. To determine whether the MPER-TMD is fusogenic, we carried out fluorescence lipid mixing assays. MPER-TMD caused rapid mixing of POPC/POPG (4:1) vesicles (Fig. S1C): ~50% of the vesicles undergo mixing in less than 30 s, and by 10 minutes the extent of mixing increased to ~75%. Therefore, the MPER-TMD peptide is fusogenic in vitro.</p><p>2D 13C-13C correlation spectra provided information about the backbone conformation of the peptide in lipid membranes. We prepared a cholesterol-containing membrane (VMS) to mimic the virus envelope, and a POPE membrane to investigate the effect of membrane curvature on peptide conformation. Fig. 2 shows that L669 in the MPER and L684, I686, and G694 in the TMD exhibit exclusive α-helical chemical shifts in both membranes. In comparison, the C-terminal residue A700 shows a weak β-strand Cα-Cβ cross peak in addition to the α-helical cross peak in the VMS membrane (Fig. 2A). The POPE membrane increased the β-strand cross peak intensity to be comparable to the intensity of the α-helical peak (Fig. 2B). Thus, the C-terminus of the TMD has a moderate propensity to form β-strand structures in lipid membranes. These 2D spectra were measured at moderate low temperatures of 263 K to freeze the peptide motion and to obtain higher sensitivity. Comparison of the 1D 13C spectra at 303 K and 235 K (Fig. 2D) indicate that the peptide has the same chemical shifts between physiological temperature and low temperature, thus the structure is unchanged within this temperature range, and the structural constraints measured at the low temperatures used here also apply to the physiological temperature.</p><!><p>We next investigated the depth of insertion of MPER-TMD by correlating the lipid and water 1H chemical shifts with the protein 13C chemical shifts in a 2D experiment 34. Fig. 3A shows a representative 2D spectrum, measured using a 1H spin diffusion mixing time of 400 ms, for the LWIGA-labeled peptide bound to the POPE membrane. At this long mixing time, all 13C-labeled residues show cross peaks with both water and lipid CH2 protons. The different depths of insertion of the residues can be discerned from the relative intensities of their water versus lipid CH2 cross peaks.</p><p>Fig. 3B shows the 1H cross sections of L669 Cα and Cβ at various 1H mixing times. It can be seen that the water cross peak intensity has equilibrated by 100 ms while the lipid-chain cross peak is weak and continues to grow till the longest mixing time of 400 ms. In comparison, the I686 cross sections show significantly higher lipid cross peaks compared to L669. Fig. 3C shows the 13C cross sections at the lipid CH2 and water 1H chemical shifts from the 9 ms 2D spectrum, illustrating the different relative intensities of lipid and water cross peaks for different residues. Fig. 3D summarizes this information as polarization transfer curves from water to protein and from lipid chains to protein. The lipid-to-protein buildup curves show significant differences between residues: I686 has the fastest polarization transfer from the lipids while L669 has the slowest transfer. The difference between the water polarization transfer is less pronounced, but still shows the expected opposite trend. Together, these data indicate that I686 is the most membrane-embedded residue, followed by A700 near the C-terminus of the TMD, whereas L669 lies on the membrane surface, the furthest away from the middle of the bilayer.</p><p>Since the water-to-protein polarization transfer curves do not differ significantly between residues from the 1H-13C correlation experiment, we measured water-edited 2D 13C-13C correlation spectra to better distinguish the water accessibilities of different residues 37-38. Fig. S2 show 2D 13C-13C correlation spectra of LWIGA-labeled peptide in POPE and VMS membranes. The water-edited spectra were measured using a short 1H T2 filter of 0.6 – 0.8 ms, followed by a 1H spin diffusion mixing time of 4 – 16 ms. At these short 1H mixing times, most of the 1H polarization comes from water. For both POPE and VMS membrane samples, L669 shows the highest residual intensities in the water-edited 2D spectra, followed by A700, whereas I686 cross peaks are mostly removed. Therefore, the water-edited spectra are fully consistent with the 2D 1H-13C correlation spectra in showing that L669 is the most exposed to aqueous solution whereas I686 is the most buried in the lipid bilayer. The intensity ratios of the water-edited spectra and the control spectra (Fig. S2C) are lower for the POPE-bound peptide than for the VMS-bound peptide at the same mixing time of 9 ms, suggesting that the peptide may dehydrate the POPE membrane more than the VMS membrane.</p><!><p>To obtain information about the interaction of the MPER-TMD with lipid membranes, we measured 31P static and MAS spectra. Static 31P lineshapes of four membranes in the absence and presence of the peptide are shown in Fig. S3A. POPC/POPG and VMS membranes exhibit uniaxial powder lineshapes that are similar with or without the peptide, indicating that MPER-TMD does not perturb the lamellar structure of these membranes. For comparison, the peptide caused a small isotropic peak to the POPE membrane. The peptide-free DOPE membrane shows a powder lineshape that is characteristic of an inverted hexagonal phase (HⅡ), as expected for this lipid, while the peptide-bound DOPE membrane exhibits a dominant isotropic peak. We have shown before that this isotropic peak is indicative of negative Gaussian curvature 55, 60, which is essential for membrane remodeling during hemifusion and post-fusion stages of the virus-cell fusion. This negative Gaussian curvature has also been observed in other membrane-active peptides 49-50 and has been quantified using small-angle X-ray scattering experiments 61-62.</p><p>Since membrane dehydration is expected at some stages of viral fusion, we investigated the membrane-surface hydration of the POPE and VMS membranes using 2D 1H-31P correlation experiments. Fig. S3B shows that the lipid phosphate groups of both membranes have a strong water cross peak, indicating that both membranes remain well hydrated in the presence of the MPER-TMD. This behavior differs from the PIV5 fusion protein TMD, which significantly dehydrates PE membranes in which the peptide is predominantly β-strand. We attribute the high hydration to the α-helical conformation of the MPER-TMD, and suggest that the β-strand conformation is correlated with membrane dehydration.</p><!><p>Information about the three-dimensional fold and oligomeric state of the MPER-TMD in the membrane can be obtained from peptide dynamics, since a monomeric membrane-spanning α-helix is expected to undergo rapid uniaxial diffusion in the membrane 63-65 while a highly oligomerized peptide or a peptide that contains an extended segment on the membrane surface is expected to be immobilized 66-67. We measured the 13C-1H dipolar order parameters of LWIGA-labeled MPER-TMD in the VMS membrane at 303 K using a double-quantum-filtered (DQF) 2D DIPSHIFT experiment 46. The double-quantum filter removes the lipid natural abundance 13C signals, which partly overlap with the peptide 13C peaks (Fig. 4A). The resulting Cα-Hα dipolar dephasing curves (Fig. 4B) indicate relatively large C-H order parameters of 0.89 – 0.92 for both the MPER residue L669 and the TMD residues I686 and A700, indicating that both the MPER and TMD are immobilized in the membrane. This suggests that the MPER-TMD may be oligomerized in the membrane. To directly determine the oligomeric state of the protein, we next carried out the spin-counting 19F CODEX experiment 41.</p><!><p>We measured the oligomeric state of MPER-TMD in lipid bilayers by taking advantage of the presence of many aromatic residues, which can be readily fluorinated. The 19F CODEX experiment reports the number of 19F spins in close proximity in terms of the T1-corrected intensity (S/S0) of a stimulated echo at equilibrium 41. A trimeric complex with 19F-19F distances within about 15 Å should manifest equilibrium echo intensities of 1/3, while a monomer should have a full echo intensity of 1. To prevent slow motion from contributing to the exchange effect during the mixing time, we conducted the CODEX experiments at 233 K 43, 67. 13C chemical shifts measured at 235 K and 303 K show no differences (Fig. 2D), indicating that the MPER-TMD structure is unchanged in this temperature change. We introduced 4F-F699 in the TMD and 5F-W678 and 5F-W680 in the MPER to measure the oligomeric state and potential intermolecular distances (Fig. 5A). When the 4F-F699 labeled peptide was reconstituted into the membranes at a P/L ratio of 1 : 15, the CODEX intensities decayed to 0.32±0.07 for the POPE-bound peptide and 0.35±0.07 for the VMS membrane-bound peptide (Fig. 5B, C). Therefore, these data directly prove that the MPER-TMD is trimerized in both POPE and VMS membranes. In comparison, the W678 and W680 CODEX data show higher equilibrium values. For example, at a mixing time of 2 s, the W680 S/S0 ratio is 0.51 ± 0.07 in the POPE membrane and 0.64±0.08 in the VMS membrane. But since the F699 result dictates that the MPER-TMD forms trimers, the Trp CODEX data must be fit to a three-spin model, albeit with longer internuclear distances. Both W680 and W678 CODEX decays are single exponential. Using a symmetric trimer model, we obtained best-fit distances of 11.0 Å for POPE-bound W680 and 12.0 Å for the VMS-bound peptide (Fig. 5D). The VMS membrane-bound W678 exhibits similar decay rate as that of W680, with a best-fit distance of 12.0 Å (Fig. 5E).</p><p>Compared to the two Trp residues' CODEX decays, the 4F-F699 CODEX data in both POPE and VMS membranes show an initial fast decay and a slow decay at longer mixing times, with approximately equal weights. We attribute this bi-exponential decay to the conformational heterogeneity at A700, which exhibits β-strand and α-helical chemical shifts with similar intensities. Therefore, we fit the F699 CODEX data using a two-component model, in which a linear chain of three spins, representing parallel in-register β-strands, is combined with a symmetric trimer, representing a three-helix bundle. The inter-strand distance was adjusted to fit the initial decay while the interhelical distance was adjusted to fit the slow decay. We obtained a best-fit interhelical distance of 11.0 Å for the POPE-bound peptide and 11.5 Å for the VMS-bound peptide. The inter-strand distance that accounts for the initial fast decay is 5.8 Å for the POPE-bound peptide and 6.8 Å for the VMS-bound peptide (Fig. S4). These inter-strand distances are longer than the backbone separation of ~4.7 Å in cross-β amyloid fibrils, which may result from sidechain disorder for the Phe rings or loose association of the β-strand at the C-terminus of the peptide due to spatial constraints by the rest of the peptide. We note that this two-conformation model does not affect the trimer conclusion, since any deviation from the trimer model in either the helix or the strand population of the peptide would change the CODEX equilibrium value from the measured value of ~0.33.</p><p>To investigate whether the oligomeric state of MPER-TMD is sensitive to the peptide concentration in the membrane, we prepared another POPE-bound sample with a three-fold lower P/L ratio of 1 : 45, and measured the CODEX data. Fig. 5C shows that this diluted peptide sample has similar CODEX intensities to the 1 : 15 sample, thus indicating that the MPER-TMD trimer is insensitive to the peptide concentration within the concentration range studied here.</p><p>To investigate whether a turn is present between the MPER and TMD, we measured 13C-19F distances between 5F-W678 and 13C-labeled I686 and L684 in the WLIGF-labeled peptide, and between 5F-W680 and 13C-labeled I686 in LWIGA-labeled peptide. If a turn exists between the MPER and TMD, then the intramolecular distances are expected to be shorter than those of a continuous helix. Various structures of truncated constructs of gp41 containing part of the MPER or part of the TMD indicate distances longer than 13 Å between the W678 sidechain and the L684 and I686 backbone, which are too long to be detectable by 13C-19F dipolar couplings. In the W680-fluorinated LWIGA sample, I686 Cα exhibits negligible dephasing by 5F-W680 (data not shown), indicating that the distance is longer than 13 Å. In contrast, I686 and L684 Cα are clearly dephased by 5F-W678. Fig. 6 shows the frequency-selective 13C-19F REDOR data of WLIGF-labeled MPER-TMD bound to the VMS membrane. Representative S0 and S spectra of the Cα region (Fig. 6A) show significant intensity differences without and with 19F pulses, indicating detectable dipolar couplings. The normalized REDOR intensities (S/S0) for the two Cα carbons (Fig. 6B) decay to ~0.50 for I686 Cα and ~0.75 for L684 Cα by 25.6 ms. To fit the experimental dephasing curves, we took into account the 19F CSA of 47.5 ppm for 5F-W678 and 19F 180° pulse imperfections 57, 68-69. Best-fit simulations gave a distance of 9.2 Å for 5F-W678 to I686Cα and 10.0 Å for 5F-W678 to L684Cα.</p><p>Based on these 13C-19F and 19F-19F distances and the depths of insertion of different residues, we built a trimer model of MPER-TMD to assess the energetic stability of the trimer in lipid membranes and to obtain a low-resolution view of its structure. Since the 1H-13C correlation spectra place the MPER L669 on the membrane surface and the TMD I686 in the membrane interior, we used the helix-turn-helix structure of the Ebola GP2 MPER-TMD (PDB code: 5T42) as the initial structure template 21. To define the turn position between the MPER and TMD, we aligned the gp41 sequence with the Ebola sequence in seven ways, matching each residue between L684 and W678 in MPER with the Ebola residue P653 (Table S1). Using the two dominant rotamers of t90 (χ1 = ±180°, χ2 = +90°) and t-105 (χ1 = ±180°, χ2 = −105°) for the W678 sidechain 70, we found that template E, matching W680 with P653, gave good agreement with the measured 13C-19F distances between 5F-W678 and L684 and I686 Cα, while other alignments produced distances or secondary structures that deviated significantly from experimental data (Table S2). For example, matching L684 of gp41 with P653 (template A) resulted in long distances of 14-16 Å from L684Cα and I686Cα to 5F-W678. Matching P653 with K683 (template B), which is traditionally considered the boundary between the MPER and TMD, caused either long distances or non-helical structures of I684 and L686. In this way, we ruled out four of the seven templates. The three remaining alignments were subjected to MD simulations under the constraints of the measured 13C-19F and 19F-19F distances. Based on the W680-P653 alignment (template E), we generated two monomer models by using the backbone (ϕ ψ) torsion angles from the recently reported solution NMR structures of gp41 TMD (5JYN 19 and 6B3U 23) for residues I688–I697. The resulting helix-turn-helix monomers were each trimerized, inserted into a lipid bilayer with the composition of the VMS membrane, and subjected to 100 ns MD simulations 59 in Gromacs under the constraints of the measured 13C-19F and 19F-19F distances. Specifically, the intramolecular 5F-W678 to L684Cα distance and 5F-W678 to I686Cα distance are 10.0 Å and 9.2 Å, respectively, while the intermolecular 19F-19F distances at residues W678, W680, and F699 are 12.0 Å, 12.0 Å, and 11.5 Å in the VMS membrane, respectively (Table 3). In addition, we fixed backbone Cα – Cα, N - N, and C' – C' distances between residues i and i + 4 of the TMD (from K683 to S703) to stabilize the monomer conformation during simulations (Table S3). We also introduced three additional intermolecular distance restraints at G690 Cα, V693 Cα, and R696 Cα, obtained from the PDB structure 5JYN, to help stabilize the trimer assembly.</p><p>The results of these simulations based on the template-E alignment are shown in Fig. 7 and Fig. S6. Both structural models point W678 towards the helix-helix interface and W680 towards solution. The two Trp residues predominantly adopt t90 and t-105 rotamers (Table S4) 70. Residues W678 to K683 show non-helical torsion angles (Fig. S5), but most MPER residues retain the α-helical (ϕ ψ) angles from the input values, without changing to non-helical structures during the 100 ns simulations. The L669 sidechain faces the membrane, consistent with the observed lipid cross peaks of this residue. Moreover, the 5F-W680 to I686Cα distance is longer than 16 Å in both models, in good agreement with the negligible dipolar dephasing between 5F-W680 and I686 in the REDOR experiments.</p><p>While these simulations based on the W680-P653 alignment gave structures that agree with the experimental data, simulations using the Y681-P653 alignment (template D) and the W678-P653 alignment (template G) did not (Fig. 8). The template-D structure shows a short distance of less than 7 Å between 5F-W680 and I686Cα, which contradicts the experimental data. The template-G alignment produced a trimer structure in which L669 points to aqueous solution while W680 is embedded in the lipid bilayer, with a distance of less than 7 Å to I686 Cα. Both features disagree with the experimental data. Therefore, residues Y681 and W678 do not correspond to the turn positions and these alternative structural models can be ruled out.</p><!><p>The 19F CODEX data of 4F-F699, showing equilibrium intensities of ~0.33 (Fig. 5B, C), unambiguously indicate that the MPER-TMD self-associates into trimers in lipid membranes at peptide concentration of 2–7 mol%. This trimerization is observed in both lamellar virus-mimetic membranes containing 30% cholesterol and in negative-curvature POPE membranes. Trimer formation in the absence of the water-soluble ectodomain suggests that this C-terminal region of gp41 may be the trimerization core of the protein, and may stabilize the trimeric state of the ectodomain 71-72. Trimerization is also consistent with the high Cα-Hα dipolar order parameters of two TMD residues and one MPER residue (Fig. 4). These high and similar order parameters indicate that only small-amplitude local motions are present, while fast uniaxial rotational diffusion of the entire peptide is absent. The latter is true because the TMD and MPER residues have very different Cα-Hα bond orientations relative to the bilayer normal, thus they would give rise to very different order parameters if whole-body uniaxial diffusion were present 64, 73.</p><p>Intramolecular 13C-19F distance measurements provided important constraints about the relative orientation of the TMD and MPER. If the two segments form a continuous α-helix, as reported in a number of structural studies (Fig. 1), then distances of 11-19 Å would be expected between the W678 sidechain and L684 and I686 backbone (Fig. 6D). Instead, we measured 13C-19F distances of 10.0 Å and 9.2 Å, which constrain W678 to be part of a turn that deposits the MPER onto the membrane surface. The helix-turn-helix fold is supported by the fact that L669 is not only well exposed to water but also shows cross peaks with lipid acyl chains at 1H spin diffusion mixing times of 100 ms (Fig. 3). If the MPER segment were to extend away from the membrane surface into the aqueous solution, then L669 would not be in spin diffusion contact with the lipid chains 34, 74-75. Finally, the helix-loop-helix architecture is also consistent with the observed immobilization of the MPER-TMD. Taken together, the distance data and the depth of insertion data define the three-dimensional fold and oligomeric state of the gp41 MPER-TMD as a trimeric helix-turn-helix structure (Fig. 7). The umbrella-like structural topology is held together by the trimeric membrane-spanning TMD stalk, while the MPER fans out on the membrane surface.</p><p>The surface location of MPER is fully consistent with the Trp-rich (W666, W670, W672, W678, and W680) nature of MPER, since Trp residues are known to favor the membrane-water interface due to the nonpolar aromatic ring and the ability of the indole NH to hydrogen-bond with lipid carbonyls and phosphate oxygens 76-77. Indeed, the solution NMR structure of DPC-bound MPER peptide shows that most Trp residues face the membrane interior while the polar residues in this segment face the aqueous solution (Fig. 1C). Thus, the helix-turn-helix structure of the MPER-TMD is favorable for stabilizing both the Trp-rich amphipathic MPER helix and the hydrophobic TMD helix. The topology of the gp41 MPER-TMD resembles the pinwheel structure of the pentameric phospholamban (PLN), which regulates calcium homeostasis in cardiac muscles. There, orientational NMR measurements, paramagnetic relaxation enhancement data, and DEER distance data, indicate that the N-terminal cytoplasmic domain of PLN lies on the membrane surface while a C-terminal TMD spans the bilayer 78-79.</p><p>While the present data clearly define the tertiary and quaternary structures of the MPER-TMD, they do not uniquely constrain the turn structure between W678 and K683 at the atomic level. For example, slightly different TMD backbone conformations cause different rotamers of W678 and W680, which all agree with the measured 13C-19F distances and 19F-19F distances (Fig. 7 and Fig. S6). Further experiments measuring the 13C and 15N chemical shifts and many interresidue contacts will be necessary to fully determine the atomic-resolution structure of this turn between the MPER and TMD.</p><!><p>The trimeric helix-turn-helix structural model obtained from the current study differs from two solution NMR structures of gp41 (residues 677-716), but in different ways 19, 23. The previous construct, denoted TMD-CT below, includes six MPER residues, the TMD, and ten residues of the cytoplasmic domain. Both structures were solved in small bicelles with DMPC to DHPC molar ratios of 1 : 2 to 1 : 2.5 (q = 0.4-0.5). One study, conducted at a P/L molar ratio of 1 : 225, concluded a trimeric structure based on intermolecular 15N-1H NOE's between 13C, 1H-labeled protein and 15N, 2H-labeled protein 19; however assignment of the methyl 1H chemical shifts is ambiguous. In contrast, the second study, conducted at a P/L molar ratio of 1 : 300, found that residual dipolar couplings of the protein in different alignment media disagree with C3 symmetry; paramagnetic relaxation enhancement data show no intermolecular association, and DEER EPR data at P/L ratios of 1 : 4000 to 1 : 300 also indicate an absence of intermolecular dipolar couplings 23. Therefore these data indicate a monomeric helix in these small bicelles 23. We speculate that these divergent findings may result from heterogeneity in the protein oligomeric states, differences in the bicelle stability and size, and the different P/L ratios used. At the higher P/L ratio of 1 : 225, an oligomeric population may coexist with a monomeric population to give rise to the intermolecular NOE's, while the lower P/L ratios may shift the protein conformational equilibrium to the monomeric state.</p><p>The experimental conditions in the current study promote more stable membrane environments as well as more stable protein structures compared to the conditions of these solution NMR studies. First, the current gp41 construct includes the entire MPER region in addition to the entire TMD. The Trp-rich MPER is expected to have significant propensity for binding to the membrane surface 20, which should tether the protein to the membrane to increase the probability for oligomerization. Second, the phospholipids in the current study present essentially an infinite membrane plane with weak curvature, thus placing no spatial restrictions on potential trimer formation. In comparison, the q = 0.4-0.5 DMPC/DHPC bicelles in the solution NMR studies have an average width of only ~45 Å for the flat portion of the bicelle, which is capped by the round DHPC edges 19. The thickness of the bilayer portion is only ~30 Å, dictated by the DMPC chain length. This restricted volume may shift the protein structural equilibrium towards the monomeric state. Third, the current solid-state NMR experiments use relatively high P/L molar ratios of 1 : 15 to 1 : 45 to obtain sufficient sensitivity, which should shift the equilibrium towards trimers. Future experiments are required to address the question of whether the MPER-TMD remains trimeric at much lower P/L ratios in lipid bilayers. It is informative to review existing biochemical and biophysical evidence for the environmental dependence of the oligomerization of viral fusion protein TMDs. Analytical ultracentrifugation data of the PIV5 fusion protein TMD 80 indicate weak homo-oligomerization tendency in DPC micelles. In comparison, oxidative crosslinking of Cys mutants of full-length PIV5 F protein in HeLa cell membranes show cross-linking even at very low protein concentrations 81. Solution NMR studies of full-length gp41 at low protein : detergent molar ratios of 1 : 500 detected only signals of the N-terminal ~110 residues, indicating that the C-terminal MPER and TMD are immobilized, which suggest oligomerization 15. Analytical ultracentrifugation and liposome release assay of a series of gp41 constructs in DPC micelles found that the oligomeric state depended on pH, construct length, and P/L ratios in a complex manner 18. Trimer formation is favored by high pH and by inclusion of the water-soluble ectodomain heptad repeats. At a very low protein : DPC molar ratio of ~1 : 3000, a construct that includes both the ectodomain heptad repeats and the MPER-TMD was found to be trimeric at high pH while monomeric at low pH.</p><!><p>Since most structural studies of MPER and TMD peptides found α-helical conformations, we sparsely labeled our peptide with 13C, 15N-labeled residues, with the goal of assessing the influence of the membrane on the peptide backbone conformation. Out of the labeled residues, only A700 at the C-terminus of the TMD exhibits partial β-strand conformation. The β-strand peaks account for ~50% of the total intensities of this residue in POPE membranes, while in the VMS membrane the β-strand peak intensities were initially low and gradually equilibrated to ~50%. Similarly, PE membranes induced β-strand conformation of the PIV5 fusion protein TMD 55, but the β-strand chemical shifts are observed for more residues in the peptide. We attribute the partial β-strand character of the gp41 TMD to a combination of the amino acid sequence and the spontaneous negative curvature of PE membranes. It has been shown that membrane peptides rich in β-branched Ile and Val residues have a significant propensity for the β-sheet conformation 82-84. Both PIV5 and HIV fusion protein TMDs contain ~40% Ile and Val residues, thus isolated TMD peptides in lipid membranes with the appropriate curvature may have a significant tendency to change to the β-strand conformation. The presence of the MPER in the current study may have shifted the conformational equilibrium towards the α-helix, thus restricting the β-strand segment to the C-terminal end of the TMD. In analogy, a FP-TMD chimera of the PIV5 fusion protein also exhibited membrane-independent α-helical conformation for the entire protein 85. Additional studies are necessary to understand the significance of the C-terminal β-strand conformation for virus-cell fusion. It is noteworthy that the N-terminal fusion peptide of gp41 has been shown to adopt β-strand conformation in complex cholesterol-containing membranes 86-87. Thus, we speculate that the β-sheet structures of both domains may promote close association of the FP and TMD in the lipid membrane, to cause the necessary saddle-splay curvature 55, 61 in late stages of membrane fusion.</p><!><p>The trimeric helix-turn-helix structure of the gp41 MPER-TMD in lipid bilayers has two implications for the mechanism of HIV virus-cell membrane fusion. First, the segment from W678 to K683 may be a site of conformational plasticity, and the turn conformation may be important for the protein to interact with lipid components such as PE and cholesterol to induce membrane curvature. The 31P lineshapes (Fig. S3) confirm membrane-curvature induction by the MPER-TMD. In support of this notion, recent lipid mixing assays showed that only a peptide that spans the junction between the MPER and TMD (residues 671-693) has fusogenic activity, while peptides corresponding only to the MPER (residues 656-683) or only to the TMD (residues 684-705) are not fusogenic 88. The fusion activity of the MPER-TMD junction not only increases with the peptide concentration but also increases with the cholesterol concentration. MD simulations suggest that this cholesterol dependence may arise from the stabilizing effect of cholesterol to focal points of negative curvature created by the aromatic-rich residues between the MPER and TMD, which cause phospholipid protrusion and acyl-chain splay to promote membrane fusion 88. Among anti-MPER antibodies, the two most broadly reactive antibodies, 4E10 and 10E8, both bind to residues spanning the MPER-TMD junction. For example, the epitope residues recognized by 10E8 include W672, F673, W676 as well as K/R683 in the turn 7. The precise interactions among 10E8, the turn between the MPER and TMD, and the lipids, are currently unknown. Crystal structures of 10E8 in complex with an MPER peptide and short-chain lipids have recently been reported 89, suggesting that the 10E8 epitope may consist of both the MPER and lipids.</p><p>Second, the surface orientation of the MPER helix, detected from the 2D 1H-13C correlation spectra and water-edited 2D 13C-13C correlation spectra, may be important for recognition of the N-terminal fusion peptide proximal region (FPPR) during late stages of virus-cell fusion. Fluorescence resonance energy transfer and lipid mixing data indicate that the TMD and FP of gp41 interact with each other in lipid membranes 90. This implies that their respective neighboring segments of the MPER and FPPR may also interact with each other after the formation of the ectodomain six-helix bundle. For the Ebola virus fusion protein, titration of the FP caused chemical shift perturbation of the surface-bound MPER residues 21, also supporting an interaction between the MPER and N-terminal regions of the fusion protein. Future studies determining the atomic-resolution structure of the MPER-TMD in lipid membranes will be required to understand the precise mechanistic roles of this domain for HIV entry into cells.</p>

PubMed Author Manuscript

Investigation on the Interactions of NiCR and NiCR-2H with DNA

We report here a biophysical and biochemical approach to determine the differences in interactions of NiCR and NiCR-2H with DNA. Our goal is to determine whether such interactions are responsible for the recently observed differences in their cytotoxicity toward MCF-7 cancer cells. Viscosity measurement and fluorescence displacement titration indicated that both NiCR and NiCR-2H bind weakly to duplex DNA in the grooves. The coordination of NiCR-2H with the N-7 of 2′-deoxyguanosine 5′-monophosphate (5′-dGMP) is stronger than that of NiCR as determined by 1H NMR. NiCR-2H, like NiCR, can selectively oxidize guanines present in distinctive DNA structures (e.g., bulges), and notably, NiCR-2H oxidizes guanines more efficiently than NiCR. In addition, UV and 1H NMR studies revealed that NiCR is oxidized into NiCR-2H in the presence of KHSO5 at low molar ratios with respect to NiCR (≤4).

investigation_on_the_interactions_of_nicr_and_nicr-2h_with_dna

1. Introduction<!>2.1. Materials and General Methods<!>2.2. Viscosity Experiments<!>2.3. Oxidation of NiCR by K H S O 5<!>2.4. Fluorescence Displacement Titration<!>2.5. 1 H NMR Analysis of NiCR with 5′-dGMP<!>2.6. UV Analysis of Oxidation of NiCR<!>2.7. Analysis of Reactions of NiCR and NiCR-2H with DNA Containing a Bulge by Denaturing PAGE<!>2.8. The MTS Assay<!>2.9. Statistical Analysis<!>3.1. NiCR-2H Is More Cytotoxic to MCF-7 Cells than NiCR<!>3.2. Both NiCR and NiCR-2H Bind Weakly in the DNA Grooves<!>3.3. NiCR-2H Coordinates More Strongly to the N-7 in 5′-dGMP than NiCR<!>3.4. NiCR-2H Oxidizes Guanines More Efficiently than NiCR<!>3.5. NiCR Is Oxidized into NiCR-2H by KHSO5 at Low Ligand-Oxidant Ratios<!>4. Conclusions<!>

<p>Natural and synthetic nickel [especially Ni (II)] complexes (Figure 1) can oxidatively damage nucleic acids via redox reactions, resulting in direct strand breaks and modified bases (lesions) [1–5]. If not repaired properly, DNA lesions can be mutagenic and have been implicated in aging and diseases such as cancer [6, 7]. Therefore, nickel-containing complexes that oxidize DNA are of biological importance. A classic example is Ni(II)∙Gly∙Gly∙His, a naturally occurring metallopeptide, found in the N-terminal Cu (II) or Ni (II) chelating domain of the serum albumins [8], human sperm protamine P2a [9], and the histatins [3]. Its mechanism of action involves redox reactions of Ni (II) in the presence of exogenous chemical oxidants to produce a ligand- or metallopeptide-based radical, which subsequently abstracts hydrogen(s) from proximate DNA backbones to induce strand breaks [10, 11]. Over the years, synthetic nickel (II) complexes mimicking their natural counterparts have been developed and investigated for their oxidation of DNA. Bailly and coworkers and others showed that Ni(salen) coordinated complexes can form adducts with guanines in RNA or DNA via a phenolic radical [12, 13]. Burrows and coworkers studied NiCR that was formed by coordination of Ni (II) with 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]-heptadeca-1,2,11,13,15-pentaene (CR) as a ligand [2, 14]. NiCR preferentially oxidizes guanine(s) in single-stranded nucleic acids, at the end of DNA duplexes, and in the DNA duplex regions where guanine residues do not adopt standard Watson-Crick base pairing. Exogenous oxidants such as oxone are required for such oxidation, and the oxidation is believed to involve an unstable Ni (III) complex intermediate [15, 16]. In addition, oxidation of DNA by NiCR cannot directly produce DNA strand breaks unless DNA is further treated with hot alkaline conditions (e.g., piperidine). The same investigators have also successfully utilized NiCR as a molecular probe for detecting unique DNA structures containing guanine(s) such as bulges, loops, and hairpins [14, 17, 18].</p><p>Although NiCR and other Ni (II) complexes as DNA damaging agents have been rigorously characterized, new biochemical properties keep emerging. A recent study of NiCR and its close structural analogue NiCR-2H revealed that NiCR-2H was cytotoxic (IC50: ~70–80 μM) toward MCF-7 cancer cells in the absence of any exogenous oxidant while NiCR had no effect on cell growth [19]. In the same study, strand breaks in calf thymus (CT) and plasmid DNA by millimolar concentrations of NiCR were observed in the absence of any exogenous oxidant. The explanation for the observed differences in cytotoxicity remains unclear. In this paper, we focus on analyzing the binding modes of NiCR and NiCR-2H with duplex DNA, their coordination with the N7 of 5′-dGMP, the oxidation of NiCR by oxone, and the DNA cleavage efficacy of NiCR and NiCR-2H. Our goal is to determine if the differences in molecular interactions of NiCR and NiCR-2H with DNA are responsible for the observed differences in cytotoxicity in cultured cells.</p><!><p>Oligonucleotides were purchased from Fisheroligos (Pittsburgh, PA). NiCR and NiCR-2H were synthesized based on the previously published procedures [20, 21]. Unless otherwise specified, chemicals for synthesis were purchased from Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) and used without further purification. Calf Thymus DNA (Code No.: MB-102-0100, Lot No.: 20471) was purchased from Rockland (Gilbertsville, PA). Viscosity data were collected using an Ostwald-type viscometer. 1H NMR spectra were collected on a JEOL ECA 600 MHz FT-NMR spectrometer (Redding, CA). UV spectra were collected on a Varian Cary 100 Bio UV-Vis spectrophotometer (Walnut Creek, CA). Fluorescence spectra were collected on a Perkin-Elmer LS 55 fluorescence spectrophotometer (Waltham, MA). Circular dichroism spectra were recorded on a JASCO J-810 spectropolarimeter (Easton, MD) using a quartz cuvette with a 1 cm optical path length. T4 polynucleotide kinase was obtained from New England Biolabs (Ipswich, MA). [γ-32P]-ATP was purchased from MP Biochemicals (Solon, OH). Quantification of 5′  32P-labeled oligonucleotides was carried out using a Storm 860 phosphorimager and ImageQuant 5.1 software (Molecular Dynamics, Sunnyvale, CA). Cell medium and supplements (fetal bovine serum, L-glutamine, penicillin, streptomycin, amphotericin B) were acquired from Invitrogen (Carlsbad, CA). Cell Titer 96 AQueous One Solution (MTS) was purchased from Promega (San Luis Obispo, CA). Analysis of cell viability (the MTS assay) was carried out on a Thermo Scientific Multiskan Ex plate reader (Waltham, MA) and the number of cells (dye exclusion assay) was counted using a hemocytometer under a microscope. DNA labeling was performed by incubating [γ-32P]-ATP (30 μCi) and T4 polynucleotide kinase (20 units) in the presence of an oligonucleotide (10 pmol) at 37°C for 30 min. Unreacted [γ-32P]-ATP was removed using a MicroSpin G-25 column (IBI Scientific, Peosta, IA). Cells were maintained in advanced DMEM/F12 medium supplemented with 5% fetal bovine serum, L-glutamine and penicillin (50 IU/mL), streptomycin (50 μg/mL) and amphotericin B at 37°C in a humid atmosphere containing 5%  CO2 and air.</p><!><p>Calf thymus (CT) DNA was dissolved in a mixture of sodium phosphate buffer (10 mM, pH 7.0) and NaCl (100 mM). The concentration of the DNA was determined by UV spectroscopy, using a molar extinction coefficient at 260 nm (12,800 M−1 cm−1 bp−1). Small aliquots of a concentrated stock solution of ethidium bromide (EB), NiCR, or NiCR-2H were added into a 2 mL of DNA solution (1 mM) in an Ostwald-type viscometer that was immersed in a thermostated water bath at 25°C to obtain the desired ligand/DNA ratios. After each addition, the solution was mixed by bubbling with N2. The time for the level of the liquid to pass between two marks on the viscometer was recorded using a stopwatch. The relative viscosities were calculated based on the published equations [22].</p><!><p>KHSO5 (3 equivalent, 0.058 mmol or 10 equiv., 0.193 mmol) was added to an aqueous solution (400 μL) of NiCR (10 mg) and incubated for 5 min. The reaction mixture was then evaporated to dryness and the residue was suspended in acetonitrile (500 μL). After filtration, the acetonitrile solution was concentrated under vacuum. The residue was dissolved in CF3COOD and subjected to 1H NMR measurements. The spectra of NiCR and NiCR-2H in CF3COOD were also recorded.</p><!><p>CT DNA (5 μM) and ethidium bromide (5 μM) were premixed in a 2 mL of Tris-HCl buffer (10 mM) and NaCl (100 mM) and allowed to stand for 30 min at 25°C. Small aliquots of a stock solution of the ligand (NiCR, NiCR-2H, or 9-aminoacridine) were added into the DNA-EB complex solution until the 1 : 1 ligand/DNA ratio was reached. After each addition, the mixture was incubated for 15 min at 25°C prior to the fluorescence analysis (Ex: 546 nm and Em: 605 nm). The fluorescence spectra of the ligands in the absence of CT DNA and EB were measured and used as blanks.</p><!><p>The 5′-dGMP stock solution (100 mM) was prepared by dissolving 5′-dGMP in D2O followed by lyophilization to dryness twice and then redissolving in D2O. A NiCR-2H stock solution (36.37 mM) was also prepared in D2O. Each individual sample (600 μL) was prepared by mixing the 5′-dGMP stock solution with the NiCR-2H stock to make a final concentration of 50 mM 5′-dGMP and a desired ligand/5′-dGMP ratio ranging from 0 to 0.33. The samples were then subjected to 1H NMR measurements.</p><!><p>The oxone solution was freshly prepared in water prior to the reactions. Based on the molecular formula of oxone (2KHSO5∙KHSO4∙K2SO4), the concentration of KHSO5 was two fold higher than that of oxone. NiCR (1 mM) in water was mixed with a KHSO5 stock to obtain a desired ligand/KHSO5 ratio ranging from 0 to 10. The absorbance of each individual mixture was recorded from 350–800 nm.</p><!><p>All experiments were carried out in triplicate. A 15-mer oligodeoxynucleotide duplex (1, 10 μM) mixed with a small amount of 5′  32P-labeled 1 in phosphate buffer (10 mM, pH 7.0) and NaCl (100 mM) was prepared by heating at 90°C for 5 min and then slowly cooling down to 25°C, and incubated at 4°C overnight. 5′  32P-labeled 1 was added such that the radiation of DNA solution was approximately 20,000 cpm/μL. The DNA (1 μM) was incubated in a mixture (10 μL) of phosphate buffer (10 mM, pH 7.0), NaCl (100 mM), NiCR or NiCR-2H (30 μM), and KHSO5 (ranging from 0–1 mM) at 25°C for 30 min. After quenching the reaction by addition of NaHSO3 (1 μL of 200 mM stock), the DNA products were precipitated from NaOAc (0.3 M) and EtOH at −80°C. After centrifugation, removal of the supernatant, and drying, the residue was treated with 10 μL of piperidine (1 M) at 90°C for 20 min, concentrated, and resuspended in formamide loading buffer (5 μL). Analytical oligonucleotide separations were carried out using 20% polyacrylamide denaturing gel (5% crosslink, 45% urea (w/w)).</p><!><p>All the experiments were carried out in triplicate. The nickel complex (NiCR or NiCR-2H) was predissolved in the medium and filtered using 0.2 micron sterile filter. The medium solution (50 μL) containing ~3 × 103 cells was added into a 96-well microtiter plate and incubated in 5% CO2 incubator for 24 h. This would allow cells to reach ~90% confluence. The nickel complex solution (50 μL) was then added into the microtiter plate to reach a desired concentration and incubated for 72 h at 37°C. After incubation, a Cell Titer 96 AQueous One Solution (20 μL) was added to each individual well. Quantification of viable cells was done by measuring the absorbance at 492 nm using a plate reader. Untreated cells and media with no cells were used as controls.</p><p>To calculate the viability (%), the following equation was used. (1)Viability  (%)=(Atreated)−(Amedia)(Auntreated)−(Amedia)×100%.</p><p>A treated: The absorbance of the solution containing treated cells, A media: The absorbance of the media, A untreated: The absorbance of the solution containing untreated cells.</p><!><p>Minitab 15.0 software was used to determine the statistical significance. Two-sample Student's t -test was performed to show statistically significant (P < .05) and insignificant (P > .05) data.</p><!><p>Our initial attempts were to find a general trend of cytotoxicity of NiCR-2H toward different cancer cells. Three cancer cell lines, HeLa (human cervical cancer), A549 (human lung cancer), and MCF-7 (human breast adenocarcinoma) were chosen for study. Dye exclusion staining and the MTS assay were used to determine the cytotoxicity. In a dye exclusion test, dead cells are blue because they cannot exclude the dye molecule (trypan blue) in the media. In a MTS assay, the absorbance of a reduction product (formazan) from a tetrazolium salt (MTS) is determined spectroscopically. Only live cells are able to release active reductases that catalyze the reduction reaction; therefore, the absorbance of formazan is proportional to the number of live cells in culture. The results from the MTS assay are shown in Figure 2. NiCR-2H is more cytotoxic (IC50: 20 μM) toward MCF-7 cells than NiCR in a statistically significant manner, and NiCR barely has any effect on inhibition of the growth of MCF-7 cells. These observations are consistent with the previous report [19]. It is noteworthy that our observed IC50 value for NiCR-2H is less than previously reported. The IC50 values of NiCR and NiCR-2H for HeLa and A549 cells could not be determined within the concentration range used for the two nickel complexes (Figure 2). Both NiCR and NiCR-2H at high concentrations became slightly cytotoxic to HeLa and A549 cells. The reductions in cell viability for both HeLa and A549 with NiCR (200 μM) and NiCR-2H (200 μM) were 25% and 35%, respectively. Surprisingly, dye exclusion staining resulted in 90–100% cell viability for all cells at all NiCR and NiCR-2H concentrations(See Table S-1 of the Supplementary Material available online on doi: 10.115512010/619436). The disagreement between cytotoxicity of known drugs and dye exclusion results has been previously reported in [23–25]. Dye exclusion has been used as an indicator of cell membrane integrity [26]. Dead cells (e.g., the reproductively dead) that do not have major membrane damage are known to exclude the dye molecule such as trypan blue. On the other hand, the MTS assay relies on active reductases released by live cells in the media and is probably more suitable for our cytotoxicity studies. However, dye exclusion staining was useful to explain the absorbance readings representing over 100% cell viability in the MTS assay for NiCR with MCF-7 cells (Figure 2). The overmeasured absorbance must result from the cell proliferation by NiCR as determined by dye exclusion staining. Nevertheless, the results from the MTS assay have undoubtedly confirmed that NiCR-2H is more cytotoxic to MCF-7 cells than NiCR and has little effect on HeLa and A549. In order to understand the differences in cytotoxicity, we in the present paper have compared the differences in molecular interactions of NiCR and NiCR-2H with duplex DNA. The results obtained using biophysical and biochemical methods are described below.</p><!><p>The pioneering work by Burrows and coworkers revealed a minimum binding of NiCR to duplex DNA [18]. Later, studies by Hellmann-Blumberg's laboratory suggested the binding of NiCR and NiCR-2H to duplex DNA is either intercalation or groove binding, which was not clearly distinguished [19]. Both NiCR and NiCR-2H were found to significantly displace ethidium bromide out of duplex DNA [19], suggesting relatively strong binding of NiCR and NiCR-2H to duplex DNA.</p><p>Because molecular interactions of NiCR and NiCR-2H with duplex DNA had not been fully investigated until the present work, we have been able to characterize these interactions using simple and reliable procedures of viscosity measurement [27, 28]. Noncovalent binding of small molecules to duplex DNA occurs mainly via either intercalation or groove binding mode [29]. The viscosity of a duplex DNA solution varies proportionally with the concentration of an intercalator due to the elongation of DNA length by intercalation. Groove binders have no effect on DNA length; therefore, the viscosity of a DNA solution is unaffected by the groove binding. In our experiments, a calf thymus DNA solution (1 mM in base pairs) was titrated with the molecule of interest varied over the range of 0–1.1 mM. The viscosity of the CT DNA solution in the presence of NiCR or NiCR-2H remained unchanged even when the ligand/DNA ratio was raised up to 1.1 (Figure 3). In contrast, the viscosity of the DNA solution varied linearly with the concentration of ethidium bromide (a known intercalator) until the EB/DNA ratio was above 0.5, at which a clear plateau was observed (Figure 3). The formation of the plateau indicates that all the possible binding sites in DNA are saturated by EB at this EB/DNA ratio, which can be explained with the neighbor exclusion principle [30]. The use of EB here was to provide a benchmark for this study. Our experiments lead us to conclude that DNA intercalation is not the major mode on non-covalent interaction of NiCR or NiCR-2H with duplex DNA.</p><p>The interactions between small molecules and DNA can also be determined spectroscopically [1, 31]. When a preformed EB-DNA complex in solution (5 μM) was titrated with 9-aminoacridine (a known competitive DNA intercalator), a decrease in fluorescence (Ex: 546 nm and Em: 605 nm) of the solution was clearly observed. A dose-dependent reduction in fluorescence was found with up to 97% reduction at 100 μM 9-aminoacridine as compared to the control (Figure 4). In contrast to 9-aminoacridine, titrating NiCR or NiCR-2H into a preformed EB-DNA complex solution (5 μM) only gave rise to a subtle decrease in fluorescence (Figure 4). An approximate 16% fluorescence reduction was observed in the presence of NiCR (100 μM) or NiCR-2H (100 μM) after subtracting the background intensity (Figure 4), suggesting that both nickel complexes weakly displace ethidium bromide out of DNA. The rank order for binding given by the C50 values (drug concentrations required to affect a 50% reduction of the initial bound EB fluorescence) is 9-aminoacridine (~19 μM) > NiCR-2H (~278 μM) > NiCR (~327 μM). The C50 values for NiCR-2H and NiCR were obtained by extending the titration curves to reach the theoretical 50% reduction. A quantitative analysis [32] of these C50 values in conjunction with the previously published binding constant of EB (107 M−1) and the EB concentration (5 μM) gives the apparent binding constant of 2.6 × 106 M−1, 1.8 × 105 M−1, and 1.5 × 105 M−1 for 9-aminoacridine, NiCR-2H, and NiCR, respectively. The binding constant of 9-aminoacridine to CT DNA derived from the fluorescence titration experiments (Figure 4) is compatible with a previously reported value [33]. However, the binding constants of NiCR-2H and NiCR could be overestimated because the binding site sizes of NiCR-2H and NiCR should not be the same as EB (groove binding versus intercalation) [34]. Our results revealed that the ability to displace EB out of duplex DNA by NiCR or NiCR-2H is much weaker than previously reported in [19], and our data are actually in line with Burrows' conclusion. The disagreement between our fluorescence titration results and Hellmann-Blumberg's is probably due to the use of different salt concentrations in the experiments. The salt concentration used in our experiments was 100 mM, which is 10-fold more than that used by the other group and is commonly used for in vitro studies. It is known that cations (e.g., Na+, K+) can prevent positively charged species (NiCR and NiCR-2H in our case) from binding to DNA due to the electrostatic repulsion. Hence, the binding of NiCR or NiCR-2H with DNA at 100 mM NaCl is expected to be weaker than that in 10 mM NaCl. Based on the results of viscosity and fluorescence titration, we conclude that NiCR and NiCR-2H bind weakly to duplex DNA in the grooves under physiological conditions.</p><p>Further evidence for the weak binding comes from the UV denaturation experiments. The melting temperatures of a 22-mer (AT tracts) or a 16-mer (mixed base) DNA oligonucleotide duplex are independent of the concentration of NiCR or NiCR-2H (See Figure S-1–Figure S-3 of the Supplementary Material), suggesting that both complexes cannot stabilize duplex DNA probably due to the minimal binding. Collectively, the little quantitative differences in the binding of NiCR and NiCR-2H with DNA lead us to conclude that the binding of the two with DNA should not be responsible for the differences in cytotoxicity.</p><!><p>Metal complexes are known to coordinate with guanine because the N-7 position of guanine is the most nucleophilic site [35]. The coordination of NiCR or NiCR-2H with ligands (such as H2O and guanine) changes its geometry from square planar to octahedral (Scheme 1). The coordinated complexes become paramagnetic, perturbing the chemical shift and the relaxation of the proximate protons of guanine. In our experiments, 5′-dGMP (Scheme 1) was used as a model compound to coordinate with NiCR-2H. Each solution in D2O containing 5′-dGMP (50 mM) and NiCR-2H varied over the range of 0–16.5 mM was individually prepared to guarantee an accurate 5′-dGMP/NiCR-2H molar ratio, and the 1H NMR spectra of these solutions were recorded. The resulting spectra between 3–10 ppm are shown in Figure 5. The proton signals of the coordinated NiCR-2H were not observable because it is paramagnetic. The relaxations of several proton signals of 5′-dGMP as a function of the concentration of NiCR-2H were observed, and the relaxations were distance dependent. The H-8 at 8.2 ppm had the strongest relaxation response to the concentration of NiCR-2H (Figure 5). The relatively weak relaxations of the H-1′ at 6.3 ppm and the H-5′at 3.9 ppm were also observed (Figure 5). Because the H-8 is the most proximate proton to the coordinated paramagnetic NiCR-2H as compared to the H-1′ and the H-5′ (Scheme 1), its signal has the most influence from NiCR-2H. Interestingly, a previous result from Burrows' group showed that the relaxations of protons were minimal when incubating NiCR with 5′-dGMP [17]. Together with their result, we conclude that NiCR-2H coordinates more strongly with the N-7 of 5′-dGMP than NiCR. The difference in coordination strength of NiCR and NiCR-2H with 5′-dGMP may result from their structural properties. According to the electronic spectra, NiCR-2H has more charge transfer in nature than NiCR [20]. The charge transfer from metal to the isolated imine in NiCR-2H could make its metal center more positive, enhancing the coordination with ligands. It is also well known that the coordination of metal complexes with guanine can promote the oxidation of the complexed guanine. Because NiCR-2H coordinates with guanine more strongly than NiCR, we predict that NiCR-2H should more readily oxidize guanine. The oxidation of guanine by NiCR-2H and NiCR will be discussed in more detail in the next section.</p><!><p>NiCR can selectively oxidize guanines present in distinctive DNA structures such as bulges and loops in the presence of an oxidizing agent [18]. Information on DNA damage by NiCR-2H to our knowledge is very limited. Hence, a side-by-side comparison of DNA damage by these two nickel complexes can be a useful addition to this field and may provide evidence to address our inquiry about the previously observed differences in cytotoxicity. We chose a 15-mer 5′  32P-labeled DNA oligonucleotide duplex containing guanines in a bulge region (1, Figure 6) for the DNA cleavage studies because its reactions with NiCR in the presence of KHSO5 have already been characterized [36]. In our experiments, no noticeable DNA damage was detected when incubating NiCR or NiCR-2H with 1 in the absence of KHSO5 at room temperature for 30 min (See Figure S-4 of the Supplementary Material). KHSO5 is proved to be a necessity to produce detectable amounts of DNA damage products under the same conditions. Like NiCR, NiCR-2H in the presence of KHSO5 could not directly produce strand breaks in DNA. However, it undoubtedly damaged DNA because strand breaks (faster moving DNA cleavage products) were detected by gel electrophoresis after treatment of reacted 1 with hot piperidine. The overall cleavage patterns of 1 produced by NiCR-2H are similar to those by NiCR (Figure 6). The Maxam-Gilbert [37] lane (lane 2, Figure 6) shows that these observed migrating bands represent the DNA scission at the guanine residues of 1. In the presence of KHSO5 varied from 100 to 500 μM, the most abundant DNA fragments produced by both NiCR and NiCR-2H were at G2 and G3 in the bulge region (lane 3–6 for NiCR and lane 9–12 for NiCR-2H, Figure 6). Interestingly, a substantially greater amount of cleavage product at G2 was detected compared with that at G3 in both cases (Figure 6). G2 prefers to remain in the bulge in the equilibrium of two bulge conformers (Figure 6) [36]; therefore, it is more prone to oxidation. The amounts of cleavage products of 1 produced by NiCR and NiCR-2H are listed as a bar graph in Figure S-5 of the Supplementary Material. For instance, in the presence of KHSO5 (200 μM), the amounts of cleavage products at G2 and G3 by NiCR were (22.3 ± 1.3)% and (11.4 ± 0.9)% and the amounts of cleavage products at G2 and G3 by NiCR-2H were (29.4 ± 0.9)% and (16.5 ± 1.2)%, respectively. NiCR-2H in general provides 5%–9% more of damaged guanine products than NiCR (Figure 6), suggesting that NiCR-2H more readily oxidizes guanine than NiCR. When the concentration of KHSO5 was raised above 500 μM, the cleavage products at G1 and A1 became dominant. For instance, in the presence of KHSO5 (1 mM), the sums of the cleavage products at G1 and A1 for NiCR and NiCR-2H were (57.4 ± 4.8)% and (68.4 ± 1.6)%, respectively. We believe the changes in damage sites result from the destabilization of 1 by the high concentration of KHSO5. This destabilization effect was confirmed by circular dichroism (See Figure S-6 of the Supplementary Material). The destabilization of 1 was only observed when NiCR and KHSO5 were both present in the solutions, and KHSO5 alone had no effect on the stability of 1 (Figure S-7 of the Supplementary Material). When 1 dissociates into random coils, the bulge region no longer exists. In the random coils, G1 and A1 located at the end of the DNA are less well protected than G2 and G3 in the middle of the DNA; therefore, the nickel complexes mainly hit on the less-protected nucleobases. The oxidation of adenine (A1) observed in our experiments has not previously been reported; however, A1 could simply be overoxidized by the large excess of KHSO5. Our results for the first time directly compare the efficiency of NiCR and NiCR-2H to oxidize DNA. Both complexes mainly oxidize guanines present in the bulge of 1 in the presence of KHSO5, and NiCR-2H more readily oxidizes guanines than NiCR. The oxidation potentials of NiCR and NiCR-2H should not be responsible for their difference in guanine oxidation because both complexes have similar oxidation potential values (1.03 V versus Ag/Ag+ for NiCR and 1.05 V versus Ag/Ag+ for NiCR-2H, in CH3CN) as previously determined in [20]. In fact, the better guanine oxidation by NiCR-2H might be attributable to its stronger coordination with guanines as described in 1H NMR.</p><p>Because the cytotoxicity was determined by incubating NiCR or NiCR-2H with cultured cells in the absence of any exogenous oxidant, we then investigated the DNA damage by NiCR or NiCR-2H in the absence of KHSO5 at the physiological temperature with a prolonged incubation time. DNA 1 was incubated with either NiCR or NiCR-2H at various concentrations (30, 300, and 600 μM) in the absence of KHSO5 at 37°C for 18 h followed by hot piperidine treatment. The DNA cleavage products obtained under this reaction condition was only 1%–5%, which is much less as compared to those obtained in the presence of KHSO5. The Maxam-Gilbert method confirmed that both complexes still mainly oxidized guanines in 1 but with no preference to G2 and G3 in the bulge region. (See Figure S-8 of the Supplementary Material). NiCR-2H produced ~2–3.5% more cleavage products (the sum of all product bands) than NiCR. The minimal DNA oxidation by NiCR and NiCR-2H in the absence of KHSO5 seems not to be responsible for the observed differences in cytotoxicity to MCF-7 cells. However, this conclusion is drawn without taking endogenous oxidants into consideration. Endogenous oxidants such as reactive oxygen species (ROSs) are known to promote the DNA damage induced by metal complexes [38, 39]. NiCR and NiCR-2H in cultured cells in principle could efficiently oxidize guanines in the presence of endogenous oxidant(s), leading to the differences in cytotoxicity.</p><!><p>Previous studies on oxidation of DNA by NiCR always adopted high-ligand oxidant ratios [18]. A Ni (III) complex was also proposed as an important intermediate for oxidation of guanine [15]. Because the cytotoxicity of NiCR-2H was observed without any exogenous oxidant, it is necessary to study oxidation of the nickel complexes at low-oxidative stress conditions. Oxidation of NiCR without an exogenous oxidant is very slow and therefore is not suitable for study. The oxidation of NiCR by KHSO5 with different molar ratios to NiCR was first investigated using UV absorption spectroscopy. The spectrum of NiCR showed a maximum absorption at 399 nm and a weak absorption at 720 nm (Figure 7). Because of its low extinction coefficient, 1 mM NiCR was used for this study. The maximum absorption (λ max ) increased dramatically as a function of the concentration of KHSO5 ranging from 0 to 4 mM (KHSO5/NiCR ≤ 4). A blue shift of λ max from 399 nm to 394 nm was observed (Figure 7). The weak absorption at 720 nm in the spectrum of NiCR also decreased accordingly (Figure 7, insert). The changes in the UV spectra indicate the oxidation of NiCR. Interestingly, the UV spectrum of this newly formed oxidation product is very similar to that of NiCR-2H, which also has a λ max at 394 nm and no absorption at 720 nm as well. When the concentration of KHSO5 was over 4 mM (KHSO5/NiCR > 4), the absorption at 394 nm decreased accompanying a red shift of 14 nm to λ max at 408 nm, suggesting that a secondary oxidation occurred (Figure 7).</p><p>Additional support for the oxidation of NiCR into NiCR-2H at low KHSO5/NiCR ratios (≤4) was gleaned from 1H NMR. The diamagnetic NMR spectra of NiCR and NiCR-2H were obtained using CF3COOD as a solvent, suggesting that CF3COOD is a weak ligand that cannot form a paramagnetic complex with NiCR or NiCR-2H. The reactions of NiCR with 3 or 10 equivalents of KHSO5 were carried out, and the resulting products were measured by 1H NMR. The spectra of NiCR, NiCR-2H, and the oxidation products are shown in Figure 8. The spectrum (Figure 8(c)) of the product obtained from the reaction of NiCR with 3 equivalents of KHSO5 is very similar to that of NiCR-2H (Figure 8(b)). The signal at 8.2 ppm clearly indicates the formation of the imine group. In addition, the methyl protons of the product appear as two 1 : 1 singlet peaks at 2.60 and 2.62 ppm, suggesting an asymmetrical structure. In contrast, the methyl protons of NiCR appear as a singlet at 2.5 ppm (Figure 8(a)) because of its symmetrical structure. Oxidation of NiCR with 10 equivalents of KHSO5 gave a 1H NMR spectrum containing no signals between 7.5 and 8.5 ppm, which is completely different from that of NiCR or NiCR-2H (Figure 8(d)). The results presented here are direct evidence for oxidation of NiCR into NiCR-2H by KHSO5 under physiological conditions, which have not been previously reported. Because such oxidation occurs at low oxidant/ligand molar ratios, we believe that it is possible to oxidize NiCR into NiCR-2H in vivo in the absence of an exogenous oxidant. NiCR-2H is relatively stable under physiological conditions and may survive a long period of time in cultured cells. The role of NiCR-2H in oxidation of guanine by NiCR may be underestimated in previous studies, and NiCR-2H could be an important precursor for the proposed Ni (III) intermediate.</p><!><p>Amongst three cancer cell lines, NiCR-2H is only cytotoxic to MCF-7 cells (IC50: 20 μM) and both NiCR and NiCR-2H have neglectable effect on HeLa and A549. In order to understand the differences in cytotoxicity, we in this paper have investigated the interactions of NiCR and NiCR-2H with DNA. We conclude that the differences in cytotoxicity should not result from the differences in the binding of NiCR and NiCR-2H with DNA because both complexes bind weakly in the grooves of DNA with no quantitative differences. Both NiCR and NiCR-2H damage DNA with a similar sequence preference, and NiCR-2H more readily oxidizes guanine than NiCR in the presence of KHSO5 probably due to its stronger coordination with guanine. The differences in oxidation of guanine between NiCR and NiCR-2H could be a key to the differences in cytotoxicity. However, this is not conclusive because the role of exogenous oxidants is unknown. We have also obtained the direct evidences for oxidation of NiCR into NiCR-2H at low molar ratios of KHSO5/NiCR, suggesting NiCR-2H could act as an important precursor for the previously proposed Ni (III) intermediate. The investigation of molecular interactions of NiCR and NiCR-2H with DNA is the first step toward understanding the differences in cytotoxicity. The ultimate explanation on this matter must be more complicated and requires understanding of the biological responses of NiCR and NiCR-2H in vivo such as cellular uptake and cellular metabolism.</p><!><p>Details of experimental procedures and spectra for UV denaturation of DNA oligonucleotide duplexes with NiCR and NiCR-2H, circular dichroism titration of 1 with oxone, and DNA cleavage studies under various conditions, and dye exclusion studies. This material is available online on doi: 10.1155/2010/619436.</p><p>Click here for additional data file.</p>

PubMed Open Access

Auranofin disrupts selenium metabolism in Clostridium difficile by forming a stable Au-Se adduct

Clostridium difficile is a nosocomial pathogen whose incidence and importance are on the rise. Previous work in our laboratory characterized the central role of selenoenzyme dependent Stickland reactions in C. difficile metabolism. In this work we have identified, using mass spectrometry, a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. X-ray absorption spectroscopy supports the structure that we proposed based on mass spectrometric data. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. This study demonstrates that targeting selenium metabolism provides a new avenue for antimicrobial development against C. difficile and other selenium-dependent pathogens.

auranofin_disrupts_selenium_metabolism_in_clostridium_difficile_by_forming_a_stable_au-se_adduct

Introduction<!>Reaction of Hydrogen Selenide with Auranofin<!>X-Ray Absorption Spectroscopy<!>Growth of Escherichia coli<!>Growth of Clostridium difficile, C. perfringens and C. tetani<!>75Se Incorporation into selenoproteins<!>75Se Uptake studies<!>HPLC analysis of 75Se in C. difficile growth media<!>Auranofin reacts with HSe\xe2\x88\x92 to form an adduct<!>Characterization of Au-Se adduct using XAS<!>Auranofin impacts the growth of anaerobically grown E. coli<!>Auranofin inhibits growth of C. difficile<!>Auranofin inhibits selenoprotein synthesis in E. coli model and C. difficile<!>Auranofin prevents the uptake of selenium by E. coli and C. difficile<!>Auranofin forms a complex with selenium in C. difficile growth media<!>Selenium supplementation prevents auranofin dependent growth inhibition<!>Discussion

<p>Auranofin [2,3,4,6-tetra-o-acetyl-1-thio-β-D-glucopyranosato-S-(triethyl-phosphine) gold] is a Au(I) complex containing a Au-S bond stabilized by a triethyl phosphine group [1]. It is used clinically to treat rheumatoid arthritis [2]. It is a potent inhibitor of the mammalian selenoenzyme thioredoxin reductase (TrxR) and it is proposed that the mechanism of action in arthritis treatment is, in part, due to its activity against this and other selenoenzymes [3, 4]. Recently, auranofin has been shown to inhibit growth of the parasite Trypanosoma brucei [5]. It has also exhibited activity against Schistosoma mansoni in a mammalian host [6]. In both reports the proposed mechanism of action was the inhibition of selenoenzymes critical for survival. It is hypothesized that auranofin inhibits selenoenzymes through interactions with the reduced selenocysteine residues at the active sites [4].</p><p>Recently we found that auranofin blocks the incorporation of selenium into selenoproteins in mammalian cells in culture [7], however the mechanism of this inhibition has not yet been determined. Selenoprotein synthesis is well defined in both prokaryotic and eukaryotic systems. It should be noted, however, that transport and metabolism of selenium upstream of the specific selenoprotein synthesis machinery is not well understood. In the first step of selenoprotein synthesis, the highly reactive, reduced form of selenium, hydrogen selenide (HSe−), serves as the substrate for selenophosphate synthetase (SPS) [8-12]. SPS produces selenophosphate in an ATP dependent manner. Selenocysteine synthase subsequently catalyzes the reaction of selenophosphate with a serine charged tRNA to form selenocysteine [13, 14]. Specialized translation factors and a stem-loop structure within the mRNA (selenocysteine insertion sequence or SECIS) then direct the insertion of selenocysteine into the polypeptide chain [15]. Given its reactivity with active site selenols, the possibility exists that auranofin could interact with reactive selenium metabolites upstream of SPS, such as HSe−, thus blocking selenoprotein synthesis entirely.</p><p>The role of selenium and selenoproteins in human health has been studied extensively. At least 25 human selenoproteins, have been identified [16]. In recent years studies have focused on the role of human selenoproteins as catalytic antioxidants and the impact of selenium supplementation on cancer incidence. In addition to humans, several pathogens, including, but not limited to, Clostridium difficile, Treponema denticola and Plasmodium falciparum also produce selenoproteins [17-19]. The importance of these selenoproteins and how they impact pathogenesis has yet to be fully elucidated. These unique enzymes and their specialized assembly machinery present an intriguing target for antimicrobial development.</p><p>Clostridium difficile is a gram positive, anaerobic, spore forming bacillus that has emerged as a significant nosocomial pathogen. Pathogenesis is mediated by two large clostridial cytotoxins, toxins A and B, and symptoms typically range from mild to severe diarrhea. In more severe infections, patients develop pseudomembranous colitis [20]. C. difficile associated disease (CDAD) contributes an estimated $1 billion in excess health care costs annually [21]. Recently the emergence of an epidemic strain (NAP1/O27) that exhibits increased virulence, and increasing mortality rates in the United States has been of particular concern [22, 23]. In addition, isolates of this strain have exhibited a wide array of antibiotic resistance [22, 24]. Analysis of data collected before and after the emergence of NAP1/O27 indicated a reduction in the effectiveness of vancomycin over metronidazole in treating C. difficile infection [25]. A recent update of the literature regarding this epidemic has suggested that C. difficile now rivals methicillin resistant Staphylococcus aureus (MRSA) as a significant clinical pathogen [26]. Given the increased incidence in the clinic and emerging resistant strains, new approaches to target this pathogen are certainly justified.</p><p>In this report we demonstrate that auranofin reacts with HSe− to form a stable complex. Subsequently, we show that auranofin blocks selenium utilization by both Escherichia coli, a model organism for prokaryotic selenoprotein synthesis, and C. difficile, a significant human pathogen. In addition, auranofin exhibits antimicrobial activity against C. difficile. We propose that the molecular mechanism of this growth inhibition is the formation of the complex with HSe− which prevents uptake and nutritional utilization of selenium by C. difficile.</p><!><p>Hydrogen selenide (HSe−) was synthesized by reaction of elemental selenium with sodium borohydride as previously described [27]. For experiments examining the oxidation of hydrogen selenide to elemental selenium, equal volumes (50μL) of varying concentrations of auranofin (in DMSO) and hydrogen selenide were added to the wells of a 96 well plate, mixed thoroughly by pipetting and incubated for 30 minutes under anaerobic conditions. The reactions were then exposed to ambient atmospheric conditions for 30 minutes followed by visual examination. Under these conditions hydrogen selenide oxidizes to Se0 and forms a red precipitate. Similarly, equal volumes of varying concentrations of auranofin and hydrogen selenide were reacted anaerobically before analysis by mass spectrometry or high performance liquid chromatography (HPLC).</p><p>HPLC-MS data were collected on an Agilent 1100 HPLC and Agilent 1969A time of flight mass spectrometer. The capillary voltage was 5,000 V, and the fragmentor voltage was 100 V. The nitrogen gas temperature was 300° C. The range scanned was from 145 to 2000 amu at 10,000 transients/scan in positive ion mode. The HPLC column was a Vydac C18 (218TP5105) with flow at 20 μL/min and temperature held at 30° C. Solvent A was 0.05% trifluoroacetic acid and solvent B was 0.05% trifluoroacetic acid in acetonitrile. The gradient ran from 0 to 95% B at 2%/min. The 0.05% TFA is added to the HPLC solvents to improve chromatographic separation and to increase the solubility of eluted compounds in acetonitrile [28]. However, TFA can severely suppress ionization of proteins, so 20 μL/min neat acetic acid is mixed in a tee placed between the HPLC UV detector and the electrospray needle. The resulting 50% acetic acid displaces TFA from the protein [29]. Some spectra were obtained by direct injection of the sample into the mass spectrometer, without HPLC separation, as shown in Figure 2. The mass of the Auranofin-selenide adduct was the same whether identified by direct injection or after separation by LC.</p><p>HPLC analysis (UV-visible detection) was performed using a Hewlett Packard 1050 system (diode array detection). 20 μL samples were loaded onto a C18 column at a flow rate of 0.5 mL per minute. The starting solvent (used for injection) was 0.05% trifluoroacetic (TFA) acid in H2O. A linear gradient (50 minutes) was developed to 100% acetonitrile, 0.05% TFA. Eluting compounds were monitored spectrophotometrically at 254 nm.</p><!><p>We used Se K edge and Au L3 edge XAS to characterize the major product of reaction of hydrogen selenide with auranofin. The samples were prepared as described above, resulting in solutions containing either 3.0 mM auranofin + 2.0 mM hydrogen selenide in 60% DMSO (used for Se XAS) or 2.0 mM auranofin + 3.0 mM hydrogen selenide in 40% DMSO (used for Au XAS). As controls, we also measured Au XAS of a solution of 2.5 mM auranofin in 50% DMSO and Se XAS of a solution of 2.5 mM hydrogen selenide in 50% DMSO. Each sample was loaded into one 8 μL well of a polycarbonate cuvet with an X-ray transparent Kapton window on one side and quickly frozen in LN2 for shipment on dry ice. XAS data collection was performed on beam line 9-3 of the Stanford Synchrotron Radiation Laboratory with the SPEAR3 ring operating at 3.0 GeV and 85-100 mA. Beam line mirrors were configured for a nominal 15 keV cutoff for harmonic rejection and the focused beam passed through the LN2-cooled double crystal monochromator using Si[220] crystals at full tuning. The monochromatic x-ray beam was apertured to 1 × 1 mm to illuminate the sample in each well of a 5-well cuvet suspended in an Oxford continuous-flow LHe cryostat; all data were collected with the sample maintained at 10 K. Fluorescence excitation data were collected using a Canberra 30-element intrinsic Ge solid state detector, using Soller slits and fluorescence filters consisting of 6 absorption lengths of As (for Se K edge XAS) or of Ga (for Au L3 edge XAS). Three 22 min spectra were collected for each sample and the averaged data were used for analysis. Residual x-ray intensity that penetrated the sample was used to collect transmission data on an elemental standard (either Au foil or powdered elemental Se) positioned between two ionization chamber detectors downstream of the sample cryostat. We also collected Au L3 XAS data by transmission on three structurally characterized ("model") complexes presented as finely ground powders diluted with BN in the same LHe cryostat: KAuBr4 [30, 31], KAuCl4 [32] and Na3Au(S2O3)2 [33, 34]. These were used to learn about scattering characteristics of Au-Br (similar to Au-Se), Au-Cl, and Au-S (the latter also similar to Au-P). Data reduction was accomplished by standard techniques using the EXAFSPAK program suite (http://ssrl.slac.stanford.edu/exafspak.html). Energy calibration using the internal standard spectra relied on assuming the first inflection point of the L3 edge of Au foil and the K edge of elemental Se occur at 11920.0 and 12658.0 eV, respectively. Pre-edge subtraction used a Gaussian model and fit the data through 11885 eV for Au L3 and through 12620 eV for Se K edge data. Extended X-ray absorption fine structure (EXAFS) data were extracted using a third-order three-region spline function with spline points of 11930, 12190, 12450, and 12710 eV for Au L3 and 12665, 12928, 13191, and 13454 eV for Se K edge data. E0 (k = 0) values were assumed to be 11930 and 12665 eV, respectively. EXAFS data of model compounds were analyzed by constructing a molecular model of the known structures, using the software package feff version 7 (feff v7, [35]) to calculate multiple scattering paths, then importing these into the OPT program of EXAFSPAK. OPT was used to optimize ΔE0 as well as fine tune the values of other parameters including first-shell distances (Ras) and Debye-Waller factors (σas2). The amplitude reduction factor (S02) was fixed at 0.9 in all fits. In all cases, optimized distances were within ± 0.02 Å of crystallographic distances, if they were available. Debye-Waller factors from the fits of Au model compounds were used to judge the validity of coordination numbers resulting from fits of Au L3 EXAFS of auranofin-containing samples, which were carried out in a similar fashion, starting with a hypothetical structure. Se K EXAFS were analyzed using the same molecular models, again using feff v7 to generate multiple-scattering paths for Se as absorber.</p><!><p>Wild type E. coli (MC4100) and a selenoprotein deficient strain (WL400, ΔselD) were cultured in modified Luria broth (10 gm/L tryptone, 5 gm/L torula yeast extract, 5 gm/L NaCl, 1% dextrose) at 37°C in a Coy anaerobic chamber. A 1% inoculum of an overnight culture was used in each experiment. Optical density measurements were taken 24 hours after inoculation and hydrogen production was assessed by Durham tubes and/or bubbling of cultures upon vigorous shaking. For experiments utilizing E. coli, auranofin was dissolved in ethanol rather than DMSO because the latter inhibits gas production in this organism.</p><!><p>Four strains of C. difficile were used in this study, ATCC 9689, VPI 10463, NAP1/O27 and strain 630. C. difficile 630 was kindly provided by Peter Mullany (Eastman Dental Institute, London, United Kingdom), and NAPI/027 was provided by Dr. Michel Warny (Acambis, Inc., Cambridge, MA). Two pathogenic clostridia that do not produce selenoproteins, C. perfringens (ATCC 19406) and C. tetani (ATCC 10543), were used as experimental controls. Cultures were grown in brain heart infusion (BHI; Oxoid) supplemented with 0.5 g/L L-cysteine to pre-reduce the culture media. All cultures were grown at 37°C in a Coy Laboratories anaerobic chamber under an atmosphere of 98% nitrogen, 2% hydrogen. A 1% inoculum of an overnight culture was used in each experiment. Auranofin (Alexis Biochemicals, San Diego, CA) was diluted in DMSO before addition to the culture media. Equal volumes of the resulting auranofin dilutions, or DMSO as a vehicle control, were added to each culture. To examine the role of selenium metabolism in the toxicity of auranofin the growth medium was supplemented with selenium (sodium selenite or L-selenocysteine) as indicated. Optical density measurements of cultures at 600 nm were determined using a Spectramax multiwell plate reader (Molecular Devices, Sunnyvale, CA) from 200 μL of culture after 24 hours of growth in each experiment.</p><!><p>One mL cultures of E. coli (MC4100) were prepared in modified Luria both as described above in the presence of increasing concentrations of auranofin (5, 10, and 50 μM). For the identification of selenoproteins, 6 μCi of 75Se (University of Missouri, Columbia), in the form of sodium selenite (100 nM), was added to each culture. To examine total protein synthesis, 20 μCi of 35S (methionine/cysteine mixture) was added to replicate cultures. After 24 hours of growth under anaerobic conditions, cells were harvested by centrifugation for 5 min at 5,000 × g and resuspended in lysis buffer (25 mM Tris, pH 8.8, 1 mM DTT, 0.5 mM EDTA, 0.1 mM benzamidine). Cells were lysed by sonication using a sonic dismembranator, model 100 (Fisher Scientific), for 10 seconds at a power output of 12 W, and the resultant crude cell extracts were clarified by centrifugation at 13,500 × g for 10 minutes. Protein concentration was determined by the Bradford assay using bovine serum albumin (Pierce) as a standard [36]. Selenoproteins and total protein synthesis were analyzed by separating 25 μg of cell extracts using a 12% SDS-polyacrylamide gel, and radioisotope-labeled proteins were detected by PhosphorImager analysis (Molecular Dynamics).</p><p>The impact of auranofin on selenoprotein synthesis in C. difficile (NAP1/O27) was determined in 1 mL mid-logarithmic phase cultures. A 20% inoculum of an overnight culture was used in each experiment. C. difficile was cultivated in BHI + cysteine for 4 hours before addition of auranofin and 6 μCi of 75Se in the form of sodium selenite (100 nM) or 20 μCi of 35S (methionine/cysteine mixture). The cultures were incubated for an additional 4 hours before harvesting. Cell extracts were prepared and analyzed as described for E. coli.</p><!><p>The impact of auranofin on selenite uptake in E. coli and C. difficile (NAP1/O27) was determined in 1 mL mid-logarithmic phase cultures. A 20% inoculum of an overnight culture was used in each experiment. Both organisms were cultivated in BHI + cysteine for 2 hours before the addition of chloramphenicol (30 μg/mL) to inhibit protein synthesis during uptake analysis in response to drug treatment. Various concentrations of auranofin (0 to 10 μM) were added to the cultures immediately followed by 4 μCi of 75Se in the form of sodium selenite (2 nM selenium). Cultures were incubated for 20 minutes at 37°C. Cells were harvested by centrifugation (1 minute at 16200 × g). Culture media supernatants were placed on ice for further analysis by HPLC. Cells were washed once in 500 μL PBS and harvested by centrifugation (1 minute at 16,200 × g). The supernatant was discarded and the cells were resuspended in 500 μL PBS before measuring the total uptake of 75Se using a Model 1470 Gamma counter (Perkin–Elmer, Wellesley, ME).</p><!><p>20 μL samples of reserved growth medium from the 75Se uptake study were separated by HPLC as described above for the mixtures of auranofin and selenide (above). Fractions were collected every minute and the distribution of 75Se (counts per minute) was measured using a gamma counter (Perkin-Elmer).</p><!><p>HSe− is highly reactive and exquisitely sensitive to oxidation under aerobic conditions. When it is exposed to oxygen it is rapidly oxidized to elemental selenium forming a red precipitate. We took advantage of this simple oxidation reaction to determine if HSe− interacts directly with auranofin. Reactions of HSe− and auranofin were prepared anaerobically and subsequently exposed to ambient conditions. HSe− alone formed the expected red precipitate; however, reaction with auranofin protected it from oxidation to elemental selenium, suggesting formation of an oxygen-resistant complex (data not shown).</p><p>To isolate and identify this apparently stable complex, mixtures of auranofin and selenide were separated by HPLC using reverse phase chromatography. Auranofin alone elutes as a single peak (denoted as peak 1 in Figure 1). When HSe− is added to auranofin, this results in the disappearance of peak 1 and coincides with the appearance of an earlier selenium-dependent peak (Peak 2, Figure 1). Similar reactions containing sodium selenite or selenocysteine did not alter the elution profile of auranofin, indicating that these forms of selenium are not reactive (data not shown). Reaction of auranofin with 75Se labeled selenide (formed by reaction of 75Se labeled selenite with DTT) confirmed that the earlier peak (peak 2) contained selenium (data not shown). Mass spectrometry revealed that the major product of an equal molar reaction of auranofin and HSe− exhibits a selenium isotope signature and has a mass of 1025.10 atomic mass units (amu) (Figure 2). Based upon the mass obtained, we hypothesize that hydrogen selenide displaces the sulfur atoms in auranofin (Figure 3a) to form a stable complex (Figure 3b).</p><!><p>We used both the Au L3 edge and Au L3 and Se K EXAFS of the product of this reaction to evaluate the hypothesized molecular structure of this compound, referred to as the auranofin-selenide adduct (Figure 3b). We prepared and analyzed two reaction mixtures with either HSe− or auranofin in substoichiometric amounts to assure that all the Se or Au, respectively, was in a single species (the auranofin-selenide adduct). Figure 4 compares the Au L3 edge spectra of this adduct with that of the auranofin reactant and three structurally characterized compounds, two containing Au(III) (KAuBr4, KAuCl4) and one containing Au(I) (Na3Au(S2O3)2). The electronic environment of Au dominates these edges and the auranofin-selenide adduct has a similar electronic environment to the parent auranofin, containing predominantly Au(I). Au L3 EXAFS provides information about the local structural environment around the Au including precise metric details and Table 1 summarizes the detailed curve-fitting optimization results for the auranofin-selenide adduct, compared to structural models and to the parent auranofin (Figure 3a). We were also able to investigate local structural details around the Se in the adduct using Se K EXAFS; these results are also summarized in Table 1. Graphical depiction of the observed EXAFS and FT data compared with the best-fit simulations is provided in Figures 5 (auranofin-selenide adduct) and Supplementary Figures S1 and S2 (model compounds, auranofin).</p><p>Au L3 EXAFS analysis of the structure of the auranofin-selenide adduct depended on scattering parameter values obtained from the parent auranofin and model compound data. Fit 4 of Table 1 describes a multiple scattering feff fit to a model of the Au environment of auranofin that assumes two identical P ligands (P and S have very similar EXAFS scattering characteristics and display nearly identical Au-(P,S) distances of 2.28 Å in many structurally characterized compounds). This model provides an excellent fit (Figure S2, bottom) even reproducing the minor FT peak at 4.3 Å (associated with the P-Au-(P,S) angle of 180°) and defines the auranofin Au-P interaction that we assumed was present in the Au EXAFS of the auranofin-selenide adduct (Fit 5, Table 1) as hypothesized in the structure of Figure 3b. The Au EXAFS data for the adduct requires the presence of another first-shell ligand and an excellent fit results (Fit 5 of Table 1, Figure 5) by assuming this is a single Se bound 180° from the P at a Au-Se distance of 2.40 Å. The Au-Se coordination number is validated by the value of σas2, which is nearly identical to that for the Au-Br scattering in KAuBr4 (Fit 1, Table 1); Au-Se and Au-Br scattering are expected to be nearly indistinguishable.</p><p>Se EXAFS of the auranofin-selenide adduct was used to provide an alternate view of local structure, this time around the Se atom. Fit 6 of Table 1 (and Figure 5) shows that the hypothesized Se environment of the adduct, containing three Au-PEt3 auranofin fragments as required by the MS-determined molecular weight, provides a very good fit to the Se EXAFS, although the higher values for the σas2 parameters may suggest some disorder in Se-Au distances and more disorder in the outer-shell phosphine environments. The resulting Se-Au distance is within 0.02 Å of the Au-Se distance determined from Au EXAFS. Generally speaking, the results from the Au L3 and Se K EXAFS analyses show that these data are fully consistent with the hypothesized structure of the auranofin-selenide adduct shown in Figure 3b and provide metric details for the structure: Se-Au distances of 2.41 ± 0.02 Å, Au-P distances of 2.28 ± 0.02 Å, Se-Au-P angles of ca. 180°.</p><!><p>Given that auranofin reacts with HSe− in vitro, we next examined whether formation of this adduct impacts microbial growth in culture. Although E. coli does not require selenium under normal laboratory conditions, selenoproteins play an important role in mixed acid fermentation during anaerobic growth. The most predominant selenoprotein in anaerobically grown E. coli is formate dehydrogenase (FDHH). FDHH production is required for activity of the formate hydrogen lyase complex (FHL) and the production of hydrogen gas [37]. Thus, E. coli can be used to study dynamically the impact of auranofin on prokaryotic selenoprotein synthesis.</p><p>We tested the effect of auranofin on growth and gas production in wild type E. coli (MC4100). Auranofin reduced the growth yield of MC4100 at 24 hours when present at concentrations of 25 and 50 μM (Supplementary figure S3). The growth inhibition observed under these conditions was similar to that seen with the deletion of selD and may be attributed to build-up of formic acid in the growth medium. In addition, growth of the isogenic selD deletion mutant (WL400) was not affected by auranofin. Further, gas production was significantly reduced in MC4100 cultures containing 10 and 20 μM auranofin (as assessed by Durham tubes) and was completely absent in those grown with 50 μM auranofin (data not shown). The effect of auranofin on MC4100 could be due interruption of selenoprotein synthesis, or possibly due to direct inhibition of FDHH.</p><!><p>Recent work in our laboratory demonstrated the central role of Stickland reactions in the growth of C. difficile [17]. Stickland reactions are described as the coupled fermentation of amino acids in which one, the Stickland donor, is oxidized and the other, the Stickland acceptor, is reduced [38, 39]. Glycine reduction results in production of acetyl phosphate, and thus ATP synthesis via substrate-level phosphorylation [40, 41]. Reduction of proline has been tied to membrane gradients [42]. The enzymes that catalyze these reactions in C. difficile are glycine reductase and D-proline reductase respectively. Both are selenoproteins [17, 43].</p><p>Based upon the knowledge that C. difficile utilizes Stickland reactions for energy metabolism and the enzymes that catalyze these reactions are selenoproteins, we decided to determine the impact of auranofin on the growth of the organism. To determine the antimicrobial activity of auranofin, it was tested against four strains of C. difficile. As with E. coli, variable concentrations of auranofin (0.25 to 2 μM) were added to rich culture medium (BHI) before inoculation. The turbidity of C. difficile cultures was measured spectrophotometrically (at 600 nm) following 24 hours of anaerobic growth. Growth of C. difficile is potently inhibited by auranofin and this growth inhibition is consistent among all four strains (Figure 6). At 2 μM auranofin, no appreciable growth was observed. A sharp decrease in growth occurred between 750 nM and 1 μM auranofin in all strains tested. The estimated IC50 values are as follows: NAP1/O27, 775 nM; VPI 10463, 1000 nM; 630, 800 nM; ATCC 9689, 750 nM. Thus all strains were significantly inhibited by concentrations of auranofin in the high nanomolar range. In order to insure that vegetative growth rather than spore germination was examined, a 1% inoculum of mid-exponential phase cells was used in these experiments. Moreover we followed the inhibition of growth of one strain for the entire batch growth period and found concentration dependent inhibition of growth at each time point (Supplementary figure S4).</p><p>C. tetani and C. perfringens are human pathogens that are classified with C. difficile in a group of organisms known as the toxigenic clostridia. We therefore tested the growth of these organisms in the presence of auranofin to determine the relative specificity within this class. No significant inhibition of growth was observed at concentrations of auranofin up to 10 μM for C. tetani or C. perfringens (Supplementary figure S5). A similar pattern of sensitivity of C. difficile relative to other toxigenic species was observed in cultures grown in reinforced clostridial medium (data not shown). Based on genomic DNA sequence, neither C. tetani nor C. perfringens carry genes encoding glycine or D-proline reductase. There is also no evidence presented in the literature that these strains can catalyze Stickland reactions. In addition, neither strain carries genes encoding the needed components for selenoprotein synthesis (selA, selB, selC or selD) [44]. We experimentally confirmed that no specific selenoproteins were expressed in C. perfringens using 75Se radiolabeling (data not shown). The inhibitory action of auranofin in C. difficile can be attributed to the organism's reliance upon selenium and selenoproteins for growth.</p><!><p>Previous work in our laboratory has demonstrated that auranofin prevents the incorporation of selenium into selenoproteins in mammalian cells. To determine the impact of auranofin on overall selenoprotein synthesis in E. coli, we cultivated MC4100 in the presence of several concentrations of auranofin with the addition of radiolabeled selenium (75Se) in the form of selenite. Cell extracts were prepared after 24 hours of anaerobic growth. The results clearly demonstrate a concentration dependent decrease of selenium incorporation into FDHH(Figure 7a). Total protein synthesis was not impaired by auranofin as indicated by 35S labeling (Figure 7b).</p><p>Because auranofin potently inhibits the growth of C. difficile we treated exponentially growing cells with several concentrations of auranofin plus 75Se and prepared cell extracts after four hours of anaerobic growth. We have previously used these same techniques to describe the presence of and expression of both glycine and D-proline reductase in C. difficile [17]. This allowed us to examine if auranofin also interferes with the incorporation of selenium into selenoproteins in this organism. As was the case with E. coli, a clear decrease in selenoprotein synthesis was observed (Figure 7c), but there was no impact on total protein synthesis (Figure 7c). A slight increase in the selenoprotein component of D-proline reductase (PrdB, Figure 7c) is also observed. The cause for this increase is not yet known. Nonetheless, these results clearly demonstrate that auranofin prevents selenoprotein synthesis in both organisms. The mechanism of C. difficile growth inhibition is not likely due to direct inhibition of glycine or D-proline reductase. Rather, auranofin does not allow the production of these critical selenoenzymes.</p><!><p>The mechanisms of selenium uptake in both prokaryotes and eukaryotes are undefined. Thus far we have shown that auranofin forms a complex with HSe− in vitro and auranofin inhibits selenoprotein synthesis in both E. coli and C. difficile. We examined whether the formation of the auranofin-selenide adduct prevents the uptake of selenium from the growth medium. Uptake of radiolabeled selenite (75Se) occurs rapidly in mid-logarithmic cultures of both organisms in rich media (BHI) and is easily quantified using a gamma counter. For this study, actively growing cultures were treated with varying concentrations of auranofin immediately followed by the addition of 75Se (2 μCi). The uptake of selenium was followed kinetically for a period of 60 minutes and was found to be essentially linear over this period (data not shown). Using a fixed time point (20 minutes) we observed a clear inhibition in the amount of radiolabel transported into the cells (Figure 8) when auranofin was added. This inhibition was also concentration dependent. For C. difficile, 500 nM auranofin reduced the uptake of 75Se by approximately 50%. The slight variation in the effect of auranofin on 75Se uptake between E. coli and C. difficile may be attributed to differences in the cell membranes of these organisms (E. coli is gram negative whereas C. difficile is gram positive).</p><!><p>Media supernatants from the uptake study with C. difficile were fractionated by HPLC as described for the analysis of the mixtures of auranofin and selenide above. In media treated with DMSO alone (vehicle control) a small peak of radioactivity was observed at the beginning of the trace with no other distinguishable peaks (Figure 9a). In contrast, media treated with 10 μM auranofin exhibited a clearly defined peak of 75Se at approximately 22 minutes (Figure 9b). These data indicate that the earlier identified auranofin-selenium complex is formed in the growth media preventing uptake and nutritional utilization of selenium by C. difficile.</p><!><p>C. difficile expresses at least three major selenoproteins that could be directly inhibited by auranofin (glycine reductase, D-proline reductase, and selenophosphate synthetase). If the formation of the auranofin-selenide adduct is indeed blocking the metabolic use of selenium in bacterial cultures, then supplementation would alleviate this inhibition. Conversely, if auranofin is directly inhibiting one or more selenoproteins, then supplementation would be unlikely to affect the action of the drug. Thus, we evaluated the impact of selenium supplementation on growth of the NAP1/O27 strain in the presence of inhibitory concentrations of auranofin.</p><p>The addition of 5 μM sodium selenite to the growth medium significantly reduces the impact of auranofin on C. difficile, with lower concentrations of selenite also exhibiting a protective effect (Figure 10a). Selenocysteine was not as potent, but the addition of 5 μM selenocysteine to the growth medium was also protective (Figure 10b). This disparity may be due to differences in the ability of the organism to utilize selenium from selenite versus selenocysteine. It should be noted that supplementation of BHI with selenite or selenocysteine did not significantly increase growth yield alone.</p><!><p>Recently, targeting selenoproteins has become an interesting avenue for the development of anticancer therapies [45, 46]. These strategies provide a new angle to combating an immensely complex human health problem. In addition to their role in mammalian cells, selenoproteins are necessary for the growth of several significant human pathogens. It is becoming clear that the potential of selenoenzyme inhibition and interruption of selenoprotein synthesis as a means for antimicrobial development cannot be overlooked. The unique enzymatic characteristics of selenoproteins and their complex assembly machinery provide several prospective antimicrobial targets.</p><p>C. difficile continues to be a major cause of hospital acquired infection that warrants further attention. With the mortality rates from C. difficile increasing by 35% per year from 1999 to 2004 [47], and an increasingly poor response to metronidazole, the preferred treatment for CDAD, [48] new tactics for combating this disease must be developed. In this study we capitalized on C. difficile's unique reliance on selenoenzymes for energy metabolism. We demonstrated that auranofin diminishes the growth of C. difficile at nanomolar concentrations.</p><p>Based upon the data that we have gathered so far, we cannot eliminate the possibility that auranofin may directly inhibit one or more of the selenoenzymes in C. difficile. Preliminary experiments did not indicate that D-proline reductase was inhibited by auranofin (data not shown), but C. difficile expresses at least two other selenoproteins, glycine reductase and selenophosphate synthetase. The observed effect of selenium supplementation on auranofin toxicity may be due to relief of enzyme inhibition through competitive binding to free hydrogen selenide. Figures 9 and 10, however, clearly show a reduction in the incorporation of radiolabeled selenium into selenoproteins with the addition of auranofin to the growth medium. In this context it appears that potential direct inhibition of glycine reductase, or D-proline reductase, is irrelevant.</p><p>Previous studies suggest that Au(I) containing compounds, such as auranofin, inhibit selenoenzymes by binding to the reduced selenocysteine at the active site. Although there is substantial literature examining the chemical interactions between Au and Se, little research has focused on the biological implications. The alteration of mammalian selenium metabolism by Au(I) containing drugs has been demonstrated suggesting that covalent reactions between Au(I) and the nucleophilic metabolites of selenium could limit the nutritional availability of selenium for the production of selenoproteins [49]. In this report we have clearly shown that auranofin reacts with HSe− in vitro to form a stable complex. This is consistent with other chemical studies utilizing organic selenolate compounds to demonstrate similar Au-Se interactions [50]. If auranofin reacts with hydrogen selenide in vivo then the pool of available selenium for selenoprotein synthesis would be reduced. We have shown that this complex occurs in bacterial culture to prevent the uptake and nutritional utilization of selenium by both E. coli and C. difficile. The implications of these results for mammalian systems must be further studied.</p><p>Recently there have been several studies that have demonstrated the activity of auranofin against significant eukaryotic human pathogens [5, 6]. They examined inhibition of selenoenzymes in these organisms, but did not consider the possibility that auranofin could inhibit overall selenoprotein synthesis. In light of our results, this warrants further examination.</p><p>The mechanisms of selenium transport and reduction to hydrogen selenide remain enigmatic. Our results provide some insight into the metabolism of selenium upstream of selenophosphate synthetase, suggesting that the reduction of selenite to HSe− occurs before it is taken up by the bacterial cell. In addition to its potential implications for human health, auranofin may be used as a tool to study selenium metabolism. We can take advantage of the ability of auranofin to form a complex with hydrogen selenide to further elucidate prokaryotic selenium metabolism upstream of selenophosphate synthetase.</p><p>Finally, given that auranofin appears to block the metabolic use of an essential nutrient, rather than acting upon a single enzyme, the development of resistant strains is improbable. Studies are underway to determine whether strains can be isolated that are resistant to auranofin. Given its importance in energy metabolism, and the fact that multiple enzymes require selenium, resistance is unlikely to occur by point mutation.</p>

PubMed Author Manuscript

Rapid Enrichment and Sensitive Detection of Multiple Metal Ions Enabled by Macroporous Graphene Foam

Nanomaterials have shown great promises in advancing biomedical and environmental analysis because of the unique properties originated from their ultrafine dimensions. In general, nanomaterials are separately applied to either enhance detection by producing strong signals upon target recognition; or to specifically extract analytes taking advantage of their high specific surface area. Herein, we report a dual-functional nanomaterial-based platform that can simultaneously enrich and enable sensitive detection of multiple metal ions. The macroporous graphene foam (GF) we prepared display abundant phosphate groups on the surface and can extract divalent metal ions via metal-phosphate coordination. The enriched metal ions then activate the metal-responsive DNAzymes and produce the fluorescently labeled single-stranded DNAs that are adsorbed and quenched by the GF. The resultant fluorescence reduction can be used for metal quantitation. The present work demonstrated duplexed detection of Pb2+ and Cu2+ using the Pb and Cu-responsive DNAzymes, achieving a low detection limit of 50 pM and 0.6 nM, respectively. Successful quantification of Pb2+ and Cu2+ in human serum and river water were achieved with high metal recovery. Since the phosphate-decorated GF can enrich diverse types of divalent metal cations, this dual-functional GF-DNAzyme platform can serve as a simple and cost-effective tool for rapid and accurate metal quantification in determination of human metal exposure and inspection of environmental contamination.

rapid_enrichment_and_sensitive_detection_of_multiple_metal_ions_enabled_by_macroporous_graphene_foam

<!>Chemicals<!>Synthesis and characterization of GF<!>Preparation of DNAzymes<!>Detection of metal ions based on GF<!>ICP-AES analysis<!>Fluorescence measurements<!>Characterization of GF<!>GF for metal ion enrichment<!>Construction of the GF sensor for the detection of metal ions<!>Performance of the GF sensor in the detection of metal ions<!>Application of the GF sensor<!>CONCLUSIONS

<p>Heavy metals such as copper and lead are continuously released to our environment through industrial and human activities like gasoline processing, electronic waste disposal, fertilizer usage, etc.1–3 They are difficult to be degraded and make their ways to plants and living organisms, imposing persistent risk to our ecosystems. Metal pollution in the environment also represents a great threat to human beings, because they could cause severe health issues like memory loss, blindness and deafness, kidney damage, cancers, etc.4–6 In particular, childhood exposure to lead can damage learning and recognition capabilities for the entire life-time; and copper can induce the pathogenesis of hepatic disorder, neurodegenerative changes and other disease conditions.7–9 Thus, it is of paramount importance to constantly survey heavy metal contents in environmental samples as well as in clinical specimen for pollution reduction and human exposure prevention.</p><p>With the acute toxicity of single heavy metals well documented, safety guidelines and regulations are established for individual metals in water, sediment or other environmental subjects. However, little progress has been made to evaluate the impact of metal mixture in the environment.10–12 Metals in the mixture would compete or share binding sites to biological receptors, leading to different toxicity and uptake behaviors than single metals.13,14 The high complexity of the metal mixtures found in the environment and its potentially enhanced danger to the ecosystem and human health call for simple survey techniques that can detect multiple metals selectively and sensitively in a fast and high-throughput manner.</p><p>Detection of metal mixtures in complex biological or environmental samples demands higher sensitivity and selectivity compared to single metal detection. Electrochemical sensors have been developed for measurement of heavy metals, but with poor discrimination capability and low sensitivity.15–18 Mass spectrometric and optical spectroscopic methods are still the main approaches for assessment of metal mixtures in the environment, which include flame atomic absorption spectroscopy (FAAS), electrothermal atomic absorption spectroscopy (ETAAS), inductively coupled plasma optical emission spectroscopy (ICP-OES), and ICP-mass spectrometry (MS). While such instrumental analysis permit very sensitive and simultaneous detection of a large numbers of metals, they are expensive, take up a lot of space, and require well-trained scientists to operate, making it difficult for on-site and real-time detection.19,20 New methods for detection of metal mixtures are desired for field-survey of environmental contamination and point-of-care applications.</p><p>Our approach to overcome the aforementioned problems is to combine the high selectivity of the metal-responsive DNAzymes and the strong absorptivity of nanomaterials in designing sensors for ultrasensitive and multiplexed metal detection. Metal-responsive DNAzymes have been discovered by systematic evolution of ligands by exponential enrichment (SELEX), showing good catalytic ability and binding activity towards many specific metal ions.21–23 Nanomaterials, with judicious design, can provide large specific surface areas and tunable functional groups to facilitate metal ion absorption. They could also possess superior optical property or quenching capability to enable sensitivity and simple fluorescent or colorimetric detection. Herein, we constructed our sensor by combing the macroporous graphene foam (GF) with the Cu- and Pb-specific DNAzymes for simultaneous enrichment and detection of Cu2+ and Pb2+ from aqueous solutions. The GF acts as both an extractor for metal ions and a quencher for the fluorophores that label the DNAzymes (Scheme 1). The dual functionality comes from the phosphate groups on the GF surface that can coordinate with the metal cations for their extraction; and the graphene backbone that can bind to single-stranded DNA (ssDNA) strongly and quench the fluorophores attached to the ssDNAs. Once Cu2+ and Pb2+ are enriched on the surface of GF, they can activate the corresponding DNAzymes and release the ssDNA products that are linked to two different fluorophores. The turn-off fluorescence from the released fluorophores then allows quantitative measurement of the contents of these two metals simultaneously. Sensitive detection of Pb2+ and Cu2+ in serum and environmental water samples were attained in the present work.</p><!><p>The Pb2+-specific DNAzyme (Pb-Sub: 5′-/5Cy3/ACT CAC TAT rAGG AAG AGA TG -3′ and Pb-Enz: 5′-CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG AGT-3′) and the Cu2+-specific DNAzyme (Cu-Sub: 5′-TTT TTT TTT TAG CTT CTT TCT AAT ACrG GCT TAC C/36-FAM/-3′ and Cu-Enz: 5′-GGT AAG CCT GGG CCT CTT TCT TTT TAA GAA AGA AC-3′) were synthesized and purified by Integrated DNA Technologies, Inc. (IDT) (Coralville, IA). Graphene oxide (GO), phytic acid (PA), Tris base and ascorbic acid (AA) were obtained from Sigma-Aldrich (St. Louis, MO). Cupric nitrate (Cu(NO3)2, 99%), lead acetate (Pb(CH3COOH)2, ≥98.0%), manganese nitrate (Mn(NO3)2, 99%), magnesium nitrate (Mg(NO3)2, 99%), cadmium nitrate (Cd(NO3)2, 99%), nickel nitrate (Ni(NO3)2, 99%), cobalt nitrate (Co(NO3)2, 99%), ferric nitrate (Fe(NO3)3, > 98.0%), zinc nitrate (Zn(NO3)2, 99%), magnesium chloride (MgCl2, 99%), potassium chloride (KCl, 99%), nitric acid (HNO3,) were purchased from Fisher Scientific (Waltham, MA). All the reagents were used as received without further purification. All experiments and measurements were carried out at room temperature unless otherwise stated. Deionized water (18.4 MΩ) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA).</p><!><p>Graphene foam (GF) was prepared by using phytic acid as the gelator and dopant and Graphene oxide (GO) was employed as the precursor, as reported by Chen et al.24 Briefly, 0.5 mL of PA was added into 15 mL of GO (2 mg/mL, aqueous solution) and sonicated for 40 min at room temperature. Then, the mixture was sealed in a 25-mL Teflon-lined autoclave tube and maintained at 180 °C for 12 h. Subsequently, the solid precipitate formed from the reaction was collected by tweezer after the autoclave tube was naturally cooled to room temperature. The product was washed by ethanol and water, and then freeze-dried for 24 h to obtain the desired final product, GF.</p><p>Transmission electron microscopy (TEM) images were directly taken with a JEOL 2011 microscope operated at 200 kV (JEOL, Tokyo, Japan). Samples were suspended in ethanol and spotted on a carbon-coated copper grid. The infrared spectra were obtained by using a FTIR 360 manufactured by Ni-colet (Thermofisher, USA). X-ray photoelectron spectroscopy (XPS) data were collected by an X-ray photoelectron spectrometer (PerkinElmer PHI 5000C ESCA System) equipped with Mg Kα radiation. Raman spectra were taken by a Labram-1B Raman spectrometer from Yobin Yvon with a laser (2 mW) excitation wavelength of 632.8 nm.</p><!><p>Equimolar of the enzyme strand and the Cy3 or AFM labelled substrate strand were added into the reaction buffer (50 mM MgCl2 and 5 mM Tris, pH ~ 8.0) and denatured at 98 °C for 2 min in a water bath. The obtained DNAzymes were stored at 4 °C after cooled to room temperature.</p><!><p>The procedure for extraction and detection of metal ions based on GF is shown in Scheme 1. For metal ion extraction, 20 μg of GF was added into 1 mL of the metal solution at different metal cation concentrations in 0.5 M of KCl-HCl (pH 1.5). The solution was stirred at room temperature for 30 min to reach maximum adsorption, and then the solution was centrifuged. After removal of the supernatant, the GF was re-suspended in MgCl2-Tris buffer (50 mM MgCl2 and 50 mM Tris, pH ~ 8.0, 90 μL), followed by addition of both DNAzymes reaching a final concentration of 5 nM. Then, 5 L of AA (5 mM) was supplied to reduce Cu2+ to Cu+ and incubated for 10 minutes at room temperature. Subsequently, solution fluorescence was measured as described in Fluorescence Measurement. Detection of metal contents in serum and environmental water samples were carried in the same manner.</p><!><p>The Optima 200DV Inductively Coupled Plasma - Atomic Emission Spectrometer (ICP-AES) (Perkin Elmer, Norwalk, CT) was employed to verify the quantities of all metals recovered from the standard metal solutions and the unknown samples. The samples were acidified with 10% HNO3 before analysis. The instrument was rinsed thoroughly with 10% HNO3 before injection to prevent memory effects. The argon source (> 99%) was set at 90 psi. The data was acquired using the ICP Expert II software. A blank was run at the beginning of each measurement to establish the baseline level. Then, standard solutions and unknown samples were measured in triplicate. A standard curve was generated to determine the concentration of the unknown samples.</p><!><p>Fluorescence measurements were conducted on a QM400 fluorometer (HORIBA, Japan). For detection of Pb2+ with the Cy3 labeled Pb-specific DNAzyme, the excitation and emission wavelengths (λEx and λEm) were set at 535 and 540–600 nm, respectively. Copper detection was performed with the λEx/λEm at 496 nm/500–600 nm that detected the FAM label on the Cu-specific DNAzyme. The slit width for both excitation and emission was set at 5 nm. One hundred μL sample was added to the cuvette and the fluorescence spectra were scanned. The cuvette was washed with pure water for three times and dried under N2 after each sample.</p><!><p>GF was prepared from GO by a thermal annealing approach using phytic acid (PA) as the gelator and dopant.24 PA not only reduced the GO sheets and assembled them into the compact, highly porous foam with good structural stability, but also introduced many phosphate groups onto the GO surface. As shown in the SEM image in Figure 1a, the pore size of GF was ~10 μm in diameter. The finer structure revealed by TEM (Figure 1b) illustrates the wrinkled and even folded layers of GF. Thus, GF is like a sponge containing rough surface and pores that provide high absorptivity for target compounds and rapid diffusion for the absorbed molecules. The elemental composition of GF was probed by XPS. All of the binding energies in XPS spectra were calibrated using the carbonaceous C1s line (284.6 eV) as the reference. Figure 1c reveals a graphitic C1s peak at around 284.8 eV, a strong O1s peak at around 532.7 eV, and the characteristic P2s and P2p peaks at 191 and 134.5 eV. These peaks confirm the presence of the hydrophilic oxygen-containing groups, such as the hydroxyl/epoxyl groups on GF surface, as well as prove the successful integration of PA. XPS elemental analysis also supports the rich content of C, O, and P in the material. (Figure 1d).</p><p>Chemical modification with PA on the GF was further validated by FT-IR and Raman spectroscopy. The spectra of the GF were compared with that of the GO to illustrate the key differences between these two materials. As shown in Figure 2a, both GO and GF exhibit the C=C bond stretch at 1615 and 3415 cm−1 when examined by FT-IR. The spectrum for GF also contains the distinct transmittance peaks at 1161, 1057, 1003, and 886 cm−1 which can be ascribed to the stretching vibrations of P=O, P-O-C (phosphate ester group), P-O, and P-O-H, respectively. The peak at 510 cm−1 can be assigned to the de-formation vibration of PO4. The Raman spectra reveal the typical G band at about 1580 cm−1, and the D band at about 1340 cm−1 for both GO and GF (Figure 2b). The ratio of the intensities of the D and G bands (ID/IG) can be utilized to judge the degree of structural disorder and defects. The relatively large amounts of phosphate groups originating from PA reduce the relative number of the six-membered aromatic rings, and thus increase the degree of structural disorder: the ratio of ID/IG was enlarged by 10% compared to that of GO.</p><!><p>The above examination results prove that macroporous GF was successfully synthesized. The prepared GF not only holds the basic structure of GO but also contained abundant phosphate groups on the surface. The phosphate groups can form strong coordination with transition metals, and the conjugated carbon structure on GF surface can establish the cation-π interaction with the metal ions, both making GF an excellent sorbent for metals. To test this, Pb2+ and Cu2+, the contents of which should be monitored closely in the environment and exposure patients, were chosen as the model cations for optimization of the adsorption conditions by GF. After 30 min incubation of Pb2+ and Cu2+ at different pH values (from 0.5 – 2.5) and salt conditions (0.05 – 2.0 M KCl-HCl, as well as 0.05 M of PA-HCl and glycine-HCl), the adsorption reached the maximum values in 0.5 M KCl-HCl buffer (pH~1.5) for both Pb2+ and Cu2+ (Figure S1, Supporting Information). The low pH in this buffer can ensure all metals are soluble, and the high salt content is needed for the latter steps involved DNAzymes. Moreover, we tested adsorption of diverse transition metal cations, Cd2+, Zn2+, Mn2+, Co2+, Mg2+, Ni2+, Pb2+ and Cu2+ on the GF in the optimal buffer. As shown in Figure 3a, more than 90% of the added metal ions were enriched by GF within 30 min. In particular, Pb2+ and Cu2+ exhibited the fastest adsorption rates, reaching the adsorption maximum of 92% and 96% within 120 min. The adsorption capacity was also examined at the extended incubation period of 120 min. Most of the metals, including Cu2+, can reach the maximum capacity, qmax, of 15–50 mg of metal per gram of GF (46.3±0.9, 35.7±1.8, 24.1±3.3, 21.5±0.5, 13.1±2.5, 29.1±4.3, and 27.8±2.6 mg/g GF for Cd2+, Zn2+, Mn2+, Pb2+, Co2+, Mg2+, Ni2+, and Cu2+, respectively), but the qmax of Pb2+ was 97.4±0.6 mg/g GF, more than 3 times higher than others. For both Pb2+ and Cu2+, we tested the recoveries at various metal concentrations. We found that even with lower than 1 mg/L of the metal ion, at which concentration the adsorption efficiency would be limited by the concentration-driven diffusion to the surface of GF, the recovery was more than 80% (Figure S2, Supporting Information). All of the above results confirm that GF can rapidly capture and concentrate trace metal ions. The enrichment should benefit sensitive detection of trace metals in samples.</p><!><p>Besides its large specific area functionalized with groups beneficial for metal enrichment, GF contains the graphene structure that can help with in situ detection of the enriched ions. It has been well studied that single-stranded DNA (ssDNA) can bind strongly to graphene via π-π stacking between the bases on nucleotides and the sp2-hybridized carbon atoms in the extended π-conjugation on graphene. In particular, guanine (G) shows enhanced binding via the NH-π interaction, supported by both computational simulation and experimental measurement.25,26 As shown in Figure S3 (Supporting Information), DNA can be adsorbed by GF within 10 min, with the maximum capacity reaching 11.6 mg DNA (g GF)−1. In addition, the planar carbon π system on graphitic domain can establish long range resonance energy transfer with the adsorbed dye molecules, quenching a wide range of fluorophores with high efficiency.27,28 Based on these features, we designed our metal sensor by coupling the porous GF with the fluorescently labeled, metal-responsive DNAzymes: the enriched metals on the GF surface can specifically cleave the substrate of the DNAzyme, and the cleaved product would be subsequently adsorbed and quenched by the GF.</p><p>Diverse DNAzymes have been reported for metal sensing.29–31 Thus, the two DNAzymes specific for Pb2+ and Cu2+ were chosen, and the substrate strands were labeled with Cy3 and FAM, respectively. The fluorophores were not quenched by GF when the DNAzymes were intact, owing to the double-stranded regions formed between the substrate and enzyme strands. Once the DNAzymes were mixed with the Pb2+ or Cu2+ enriched by GF, the substrate strand was cleaved and the released ssDNA was adsorbed and the fluorophore was quenched by GF. As shown in Figure S4 (Supporting Information), the 100 nM Cy3-labeled ssDNA product resulted from the Pb-induced substrate cleavage could be quenched completely by 20 μg/mL of GF, while the fluorescence of the intact DNAzyme was not affected by the presence of GF. TEM was used to examine the GF before and after metal enrichment and DNAzyme cleavage, and revealed no difference on the GF (Figure S5, Supporting Information). The presence of GF did not affect the cleavage efficiency, as proved by using gel electrophoresis to monitor product generation with or without GF (Figure S5c).</p><p>Since the salt content, concentration, and pH value of the reaction buffer could influence the structure stability of the DNAzymes and interaction between DNA and GF, we compared the quenching efficiency of GF in three kinds of common buffers (50 mM NaCl in 50 mM phosphate buffer at pH 7.4; 50 mM MgCl2 in 50 mM Tris buffer at pH 8.0; and 50 mM NaNO3 in 50 mM Tris-Acetate at pH 7.8). The quenching efficiency was defined as (F0 – F)/F0, where F and F0 are the fluorescence intensities of the DNA solutions with and without the presence of the nanomaterial, respectively. As shown in Figure S6 (Supporting Information), these buffers showed similar quenching efficiency with GF. We chose the MgCl2-Tris buffer because it exhibited better quenching stability in repeated measurements. We also compared the quenching capability of GF with other common graphene materials: graphene (G), graphene oxide (GO), and the hydrophobic macroporous graphene foam (MGF), in this buffer (Figure S7, Supporting Information). The signals were measured at 30 min after the metal ion, nanomaterials, and the DNAzyme were mixed. While fixing the concentrations of the DNAzyme and the metal ions in the mixture, the quenching efficiency increased linearly with the increase of GF concentration until reaching a plateau (larger than 90%) at around 40 μg/mL. On contrary, the other materials showed similar trends but with much slower rates of increase; and no plateau was attained even with 200 μg/mL of the material used. The higher quenching efficiency exhibited by GF compared to the other graphene-based materials could be attributed: (I) the inherent aromatic structure and amphiphilic property of GF; and (II) the increased structural disorder and defects of GF. The former feature facilitates highly efficient adsorption of ssDNA; and the latter benefits long-range energy transfer and results in enhanced quench of fluorescence.32–34 Moreover, the adsorption event occurred very rapidly: within 5 min, the quenching efficiency of GF for ssDNA reached the maximum value of 98% (Figure S8, Supporting Information). The high quenching efficiency can help improve the signal-to-noise ratio of our sensing method; and prompt adsorption of the cleaved product can ensure fast detection upon metal enrichment, allowing us to perform sensitive and quick survey of these two toxic metals in samples of interest.</p><!><p>The performance of our GF-based metal sensor was examined. Figure 4a&c shows the fluorescence spectra of the sensing system upon enriching Pb2+ and Cu2+ from the 1-mL solution at various concentrations using the GF, followed with detection in the 100-μL DNAzyme solution. The fluorescence intensity decreased dramatically as the concentrations of Pb2+ increased. The limit of detection (LOD) was calculated to be 50 pM and 0.6 nM for Pb2+ and Cu2+, respectively, using the 3σ method. These LODs are much lower than most of the previously reported approaches for Pb2+ and Cu2+ detection, as shown in Table 1 that compares the LODs of various techniques for Pb2+ and Cu2+ detection. The high sensitivity of our sensing system can be attributed to both the excellent metal enrichment capability of GF and its high quenching efficiency over the fluorescently labeled ssDNA. Furthermore, we evaluated the impact from Cu2+ to detection of Pb2+, and vice versa. The fluorescence intensity change of the Pb-specific DNAzyme caused by incubation with the GF enriching 50 nM Pb2+ did not vary with the presence of Cu2+ ranging from 1×10−12 M to 1×10−4 M. Similarly, the coexistence of 1×10−12 M to 1×10−4 M Pb2+ did not affect the signal from 0.6 nM Cu2+ (Figure S9, Supporting Information).</p><p>We further tested whether other divalent metals could affect selective detection of Pb2+ and Cu2+. The fluorescence response was monitored when the sensing system was challenged by the presence of other metal ions, including Mn2+, Mg2+, Cd2+, Ni2+, Co2+, Fe2+, Zn2+. As shown in Figure 5, the GF sensor yielded much more quenching with 10 nM Pb2+ or Cu2+, compared to that obtained with other metal ions at 100-fold higher concentrations. The excellent selectivity is originated from the high specificity of each DNAzyme to its target cation, as well as from the good capability of GF in differentiating ss- and ds-DNA.</p><!><p>The above results indicate the potential of our GF-DNAzyme sensing platform in extraction and detection of Pb2+ and Cu2+ present in complex biological and environmental systems. Most of metal ions exposure can be measured by testing ions concentration in serum.44,45 To demonstrate this, we spiked Pb2+ and Cu2+ to the human serum obtained from Sigma, and detected their contents using our sensor. Each metal was spiked at two concentrations: 0.1 and 1.0 nM for Pb2+; and 1.0 and 10.0 nM for Cu2+. The metal concentration obtained with our sensing method was divided by the actual spiked concentration to achieve the recovery, which was presented in Table 2. The recoveries for both metal ions at the two concentrations tested were more than 95 ± 3%. Switching serum with plasma, low concentrations of Pb2+ and Cu2+ were still determined successfully with excellent recoveries using the developed approach (Table S2, Supporting Information).</p><p>Environmental water is another type of samples that could be subject to survey of heavy metal contamination (Table S1, Supporting Information). We collected some water samples from the Santa Ana River at ~100 meter downstream from the waste water treatment plant for City of Riverside. The river sample did not contain detectible level of Pb2+ or Cu2+, indicating no heavy metal contamination in the discharged water from the treatment plant. If spiked with these two metal cations, the detected quantity agreed well with the true content. In human serum, plasma, and environmental water, the metal recovery found with our sensor is similarly high, which indicates that sample viscosity has no obvious influence on our sensor. All the results support our GF-DNAzyme sensing system can be used to monitor Pb2+ and Cu2+ simultaneously with desirable sensitivity and accuracy in complex samples.</p><!><p>In summary, we present a simple method for quick, sensitive, and selective detection of metal ions using the macroporous GF. Taking advantage of the abundant phosphate groups on its surface, its high specific surface area, and the unique DNA adsorption and fluorophore quenching properties, the macroporous GF enables both metal ion extraction and fluorescence-based detection. The enrichment and detection can be completed within 1 hr; and multiple metals can be enriched simultaneously, with detection limits for specific metals such as Pb2+ and Cu2+ reaching the low nM or even pM range. The sensor is also tolerant to complex sample matrices, as demonstrated by metal quantification in serum, plasma, and environmental water, eliminating the need of sample pretreatment. With the availability of numerous DNAzymes specifically targeting different metals and fluorophores detectible at various wavelengths, our method can be expanded for multiplexed detection of several metal ions for quick and easy assessment of metal contents in environmental samples and medical specimens. It will be valuable for on-site survey of heavy metal contamination and for diagnosis of metal exposure in patients.</p>

PubMed Author Manuscript

Synthesis and Biological Evaluation of Isomeric Methoxy Substitutions on Anti-Cancer Indolyl-Pyridinyl-Propenones: Effects on Potency and Mode of Activity

Certain indolyl-pyridinyl-propenone analogues kill glioblastoma cells that have become resistant to conventional therapeutic drugs. Some of these analogues induce a novel form of non-apoptotic cell death called methuosis, while others primarily cause microtubule disruption. Ready access to 5-indole substitution has allowed characterization of this position to be important for both types of mechanisms when a simple methoxy group is present. We now report the syntheses and biological effects of isomeric methoxy substitutions on the indole ring. Additionally, analogues containing a trimethoxyphenyl group in place of the pyridinyl moiety were evaluated for anticancer activity. The results demonstrate that the location of the methoxy group can alter both the potency and the mechanism of cell death. Remarkably, changing the methoxy from the 5-position to the 6-position switched the biological activity from induction of methuosis to disruption of microtubules. The latter may therefore represent a prototype for a new class of mitotic inhibitors with potential therapeutic utility.

synthesis_and_biological_evaluation_of_isomeric_methoxy_substitutions_on_anti-cancer_indolyl-pyridin

1. Introduction<!>2.1. Chemistry<!>2.2. Biological activity<!>3. Conclusions<!>4.1. Chemistry - General description<!>4.2.1. 1-Benzenesulfonyl-4-methoxyindole (5a)<!>4.2.2. 1-Benzenesulfonyl-6-methoxyindole (5b)<!>4.2.3. 1-Benzenesulfonyl-7-methoxyindole (5c)<!>4.2.4. 1-Benzenesulfonyl-5,6-dimethoxyindole (5d)<!>4.2.5. 1-Benzenesulfonyl-6-methoxy-2-methylindole (6b)<!>4.2.6. 1-Benzenesulfonyl-5,6-dimethoxy-2-methylindole (6d)<!>4.2.7. 4-Methoxy-2-methylindole (7a)<!>4.2.8. 6-Methoxy-2-methylindole (7b, Scheme 2)<!>4.2.9. 7-Methoxy-2-methylindole (7c)<!>4.2.10. 5,6-Dimethoxy-2-methylindole (7d)<!>4.2.11. 4-Methoxy-2-methylindole-3-carboxaldehyde (8a)<!>4.2.12. 6-Methoxy-2-methylindole-3-carboxaldehyde (8b)<!>4.2.13. 6-Methoxy-2-methylindole-3-carboxaldehyde (8b, Scheme 2)<!>4.2.14. 7-Methoxy-2-methylindole-3-carboxaldehyde (8c)<!>4.2.15. 5,6-Dimethoxy-2-methylindole-3-carboxaldehyde (8d)<!>4.2.16. trans-3-(4-Methoxy-2-methyl-1H-indole-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9a)<!>4.2.17. trans-3-(6-Methoxy-2-methyl-1H-indole-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9b)<!>4.2.18. trans-3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9b,Scheme 2)<!>4.2.19. trans-3-(7-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9c)<!>4.2.20. trans-3-(5,6-Dimethoxy-2-methylindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (9d)<!>4.2.21. N-Boc-5-methoxy-2-methylaniline (11)<!>4.2.22. 6-Methoxy-2-trifluoromethylindole (13)<!>4.2.23. 6-Methoxy-2-trifluoromethylindole-3-carboxaldehyde (14)<!>4.2.24. trans-3-(6-Methoxy-2-trifluoromethyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (15)<!>4.2.25. trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(2,4,6-trimethoxyphenyl)-2-propen-1-one (17)<!>4.2.26. trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(3,4,5-trimethoxyphenyl)-2-propen-1-one (18)<!>4.2.27. trans-3-(5-Methoxy-1,2-dimethyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (19)<!>4.3.1. Cell culture<!>4.3.2. SRB assays<!>4.3.3. Imaging of microtubules<!>4.3.4. Cell cycle analysis

<p>Glioblastoma multiforme (GM) remains a lethal cancer due to rapid progression and limited treatment options, namely surgical removal of the tumor followed by combined radiotherapy and chemotherapy with temozolomide [1,2]. Recurrence of disease is usually untreatable as a result of acquired drug-resistance and invasive dissemination of the tumor. Temozolomide relies on triggering programmed cell death via activation of apoptosis [3]. However, GM cells harbor specific mutations in genes that are required to promote an efficient apoptotic response [4,5]. Stimulation of nonconventional cell death pathways offers a possible solution for treating drug-resistant cancers that are able to circumvent apoptosis [6,7]. Methuosis is a recently identified caspase-independent form of cell death that displays characteristics distinct from other types of non-apoptotic cell death, such as necroptosis or autophagy [8,9]. In cultured glioblastoma cells, methuosis begins with defective macropinocytotic trafficking, causing the formation of large fluid-filled vacuoles. Accumulation of vacuoles ultimately displaces the cytoplasm and the cell membrane loses integrity and ruptures. While dysfunctional vesicular trafficking and accumulation of vacuoles appear to contribute to cell death, there is evidence that additional metabolic or cellular insults are required for execution of the methuosis cell death program [8,10,11].</p><p>The methuosis phenotype was initially observed by the ectopic expression of activated Ras and Rac GTPases in GM cells [12,13]. More recent studies have focused on the pursuit of small molecules with the potential to induce this form of cell death in a therapeutic context. An initial search for compounds reported to induce cellular vacuolization led us to an indolyl-pyridinyl-propenone (IPP, also referred to as indole-derived chalcone) as a potential lead [13]. Associated structure-activity relationship (SAR) studies revealed that the optimized scaffold for induction of methuosis consists of a 2,5-disubstituted indole and a pyridine in the para-configuration, bridged by an α,β-unsaturated ketone [11,14,15]. Our previously reported IPP compounds, and their modes of biological activity, are summarized in Fig. 1.</p><p>To date, compounds 1a–1e are the most potent inducers of methuosis, possessing activity between 2–3 μM when assayed against the human glioblastoma cell line, U251. Among these compounds 1a has been studied most thoroughly. Comparisons of structurally similar IPP's have revealed intriguing and unexpected results suggesting that the morphological appearance of vacuoles in the treated cells is not always associated with cell death. For instance, analogues with larger aliphatic substitutions (2e–2g) on the 2-indolyl position caused vacuolization but had surprisingly less cytotoxicity than the vacuole-inducing compounds with Me (1a) or Et (1b) at this position (Fig. 1) [11]. Similarly, certain 5-substituted analogues (2a–2c), as well as the 2-des-methyl derivative 2d, also induced vacuole formation but were not cytotoxic [15]. While 5-methoxy (1a) and 5-propoxyindole (1e) analogues triggered cell death by methuosis, their structurally similar counterparts, namely 5-ethoxy (2a) and 5-isopropoxyindole (2b) caused cytoplasmic vacuolization without cell death. Studies are currently underway with this series of analogues to explore the mechanistic basis for their differential cytotoxicity.</p><p>Another novel insight into the biological effects of the IPP compounds was gathered from derivatives containing electron-withdrawing functionalities at the 2-indolyl position [15]. Derivatives containing trifluoromethyl (3a) or alkyl carboxylate (3b–3d) substitutions caused minimal to no vacuolization but remained highly cytotoxic. Morphologically, cells treated with the latter series of compounds did not resemble cells undergoing methuosis. Instead, the cells displayed features consistent with disruption of tubulin polymerization and microtubule architecture. Cell cycle analysis demonstrated an accumulation of cells in the G2/M phase, with eventual death by mitotic catastrophe. In this respect, 3a–3d were quite distinct from the methuosis-inducing compounds, which did not disrupt microtubules or cause mitotic arrest at the same concentrations. The redirection of cytotoxicity from methuosis to microtubule disruption for derivatives 3a–3d was associated with a significant increase in growth inhibitory potency.</p><p>While our previous synthetic work focused on substitution at either the 2- or 5-indole positions, a lack of information exists for substitutions at the 4-, 6-, or 7- positions (Scheme 1). We noted that the importance of a 5-methoxy group for either methuosis or microtubule disruption is further dependent upon the electron withdrawing properties of the 2-substituent. In the present study we have synthesized and evaluated methoxy isomers of 1a to sequentially survey the 4-, 6- and 7-positions while initially holding the 2-position constant. Upon finding significant anti-mitotic activity for the 6-position isomer 9b, we immediately prepared its 2-trifluoromethyl version (15) by analogy to 3a where this type of functionality also led to microtubule disruption [15]. Finally, drawing from several reports describing N-methyl-indole-based trimethoxyphenyl chalcones as compounds affecting tubulin polymerization, we examined replacing the para-pyridine in our structural template with a trimethoxyphenyl group and, likewise, separately examined the effect of adding a methyl group to the indole nitrogen. The results reveal that the position of the methoxy group on the indole ring and the para-pyridine are critical determinants of the biological activities of the IPP compounds.</p><!><p>Scheme 1 illustrates the synthesis of isomers of 1a at the 4-, 6- or 7- indole position (9a-9c). A disubstituted 5,6-dimethoxy derivative (9d) was also synthesized. From commercially available 4a–4d, the indole nitrogen was protected with benzenesulfonyl chloride (5a–5d). The benzenesulfonyl group ensured regioselective methylation at the 2-indolyl position (6a–6d), which was accomplished under conditions of tert-butyllithium and iodomethane [16]. Removal of benzenesulfonyl in a mixed solvent system of EtOH/aqueous NaOH provided 7a–7d. Formylation reactions utilizing Vilsmeier conditions (8a–8d) followed by Claissen-Schmidt condensation reactions produced target compounds 9a–9d. This approach generally provided compounds in reasonable yields; however, intermediate 7b was not stable under these conditions and resulted in low yields for 9b. When 9b was synthesized by the alternative method shown in Scheme 2, intermediates were stable and produced high yields. Compound 15 was also prepared according to Scheme 2. Targets 9b and 15 were synthesized from aniline derivative 10, which was protected with BOC. Regioselective acylation of 11 with Weinreb amide [17] 12a or 12b controlled by sec-butyllithium provided a ketone intermediate, which subsequently cyclized to indole. The BOC protecting group was cleaved with TFA to produce 7b or 13 [18]. Typical conditions of formylation (8b or 14) and condensation afforded final targets 9b and 15.</p><p>The compounds synthesized in Scheme 3 probe the effects of a trimethoxyphenyl functionality in place of para-pyridine. Aldehyde 16 was condensed with either 2,4,6-trimethoxyacetophenone or 3,4,5-trimethoxyacetophenone to yield 17 or 18, respectively. Piperidine is typically employed as the base to form the enolate from aryl acetates. This was appropriate during the synthesis of 18, as well as target compounds 9a–9d and 15, but yielded no reaction for 17. Presumably, the steric hindrance of methoxy groups present at the adjacent 2- and 6- positions of acetophenone prevented the formation of enolate by piperidine. Compound 17 was synthesized using 50% aqueous KOH/MeOH, albeit in relatively low yields as compared to other analogues in this series. Additionally, derivative 19 was synthesized from 1a in one step using sodium hydride as the base and methyl iodide as the alkylating reagent (Scheme 4).</p><!><p>Previous studies with U251 glioblastoma cells have established that the sulforhodamine B (SRB) colorimetric assay is useful for evaluating the loss of viable cells and for ranking the relative potency [11,15]. This assay is sensitive to both methuosis and microtubule disruption. Growth inhibitory activities for all compounds at a 48 h end-point are listed in Table 1 as the dose able to achieve 50% growth inhibition (GI50) compared to growth within control cultures treated with only vehicle (DMSO).</p><p>In the early stages of methuosis, cells become filled with a large number of phase-lucent macropinosome-derived vacuoles, which can be readily observed by phase contrast microscopy. As the cells begin to die (usually between 24–48 h), they detach from the culture surface and lose membrane integrity. Phase-contrast microscopy pictures of cells treated with the compounds at 2.5 μM for 4 h or 48 h are compiled in Fig. 2. Consistent with our previous studies, the 5-methoxy compound 1a induced methuosis with a GI50 of 2.30 μM. By 48 h, tumor cells treated with the compound at concentrations ≥ 2.5 μM began to detach from the dish and lose membrane integrity. By comparison, the 4-methoxy compound 9a elicited much fewer vacuoles and possessed weak growth inhibitory activity. The 7-methoxy compound 9c produced no vacuoles, but was moderately growth inhibitory. A strikingly different pattern of activity was observed for the 6-methoxy compound 9b. Growth inhibitory activity increased more than 25 fold, with a GI50 of 0.09 μM. At the same concentration that 1a induced methuosis (2.5 μM), 9b caused early formation of extensive membrane blebs on the cell surface and general rounding of the cell body, with only a few vacuoles detected (Fig. 2). By 24 h the majority of the cells had rounded up and detached from the dish, and by 48 h most of the cells had disintegrated. The few remaining attached cells were either rounded or enlarged with multiple micronuclei. The higher potency and distinct morphological characteristics observed with 9b were similar to the effects we previously observed when testing compounds 3a–3d in Fig. 1 [15]. Examination of cells treated with 9b by immunofluorescence microscopy with an antibody against tubulin (Fig. 3) confirmed disruption of microtubules. Cells treated with 9b at 100 nM exhibited a dense network of microtubules, similar to the DMSO control. However, at higher concentrations, 9b caused a complete loss of normal microtubule architecture in the few cells that remained attached to the culture dish. In contrast, the microtubule network in cells treated with the methuosis inducer, 1a, was generally intact, except for distortions created by the presence of the large cytoplasmic vacuoles.</p><p>DNA histograms obtained by flow cytometry of cells treated with 9b for 24 h (attached and detached cells combined) were indicative of mitotic arrest at concentrations > 500 nM, with a large increase in the G2/M phase population (Fig. 4). In contrast, cells treated with 1a were predominantly in the G1/G0 phase of the cell cycle (Fig. 4). It is interesting to note that cells treated with 100 nM 9b exhibited an intact microtubule network (Fig. 3) and a near-normal cell cycle distribution (Fig. 4), despite the growth inhibition observed at this concentration in the SRB assay (Table 1). This raises the possibility that at low concentrations, near the GI50, 9b may inhibit cell proliferation via an unidentified mechanism separate from microtubule disruption. Based on the distinct phenotypes elicited by 1a (methuosis) and 9b (microtubule disruption), we were curious to determine what type of activity might occur with a 5,6-dimethoxyindole derivative, 9d. Surprisingly, the dimethoxy compound lost all activity in the growth assay and neither mode of action was observed in the morphology assays (Table 1, Fig. 2).</p><p>We previously reported that the methuosis-inducing activity of 1a could be switched to microtubule-disrupting activity when trifluoromethyl was substituted for methyl at the 2-position on the indole ring (3a, Fig. 1) [15]. Therefore, we postulated that a 2-trifluoromethyl substitution might increase the activity of 9b. Interestingly, the resulting compound 15 lost all detectable growth inhibitory activity (Table 1, Fig. 2).</p><p>The trimethoxyphenyl moiety and the indole scaffold are common motifs in many compounds that possess anticancer activity mediated by microtubule disruption, including several indole-based chalcones [19–23]. Features that distinguish the latter compounds from our IPP series include methylation of the indolyl nitrogen and presence of either a 3,4,5-trimethoxyphenyl [24,25] or 2,4,6-trimethoxyphenyl [26,27] group in place of the pyridine ring. Based on these observations, we evaluated whether substitution of the pyridinyl moiety in 1a with a trimethoxyphenyl moiety would switch activity from methuosis to microtubule disruption. The results indicate that neither the 2,4,6-trimethoxyphenyl (17) nor the 3,4,5-trimethoxyphenyl (18) derivative had any morphological effects on U251 cells that would be consistent with methuosis or microtubule disruption (Fig. 2). Compound 18 had no detectable effect on cell proliferation, whereas 17 was moderately growth inhibitory (Table 1). Similarly, we asked what effect methylation of the indole nitrogen might have on the activity of potent methuosis inducer 1a. The resulting N-methyl derivative 19 (Scheme 4) showed substantially diminished growth inhibition (GI50 >10 μM) and only transient vacuole-inducing activity at 2.5 μM (Fig. 2).</p><!><p>The present SAR studies demonstrate that the position of methoxy substitutions on the indoly-pyridinyl-propenone scaffold have a significant influence on anti-cancer activity. The 5-methoxy substitution is optimal for the induction of methuosis (1a). Changing the methoxy from the 5-position (1a) to the 4-position (9a) or the 7-position (9c) of the indole ring attenuates or eliminates methuosis activity. Unexpectedly, moving the methoxy group to the 6-position (9b) provided a striking enhancement of growth inhibitory potency by conferring a different type of anti-cancer activity to the compound; i.e., disruption of microtubules leading to mitotic arrest and cell death. Rather than enhancing either methuosis or microtubule-disrupting activity, combining the 5- and 6-methoxy indole modifications so as to produce dimethoxy compound 9d essentially abolished both activities. Thus, two mutually distinct substitution patterns emerge from our parent template, the 5-methoxy (1a) which induces methuosis and the 6-methoxy (9b) which can disrupt microtubules without additionally having an electron withdrawing substituent at the 2-position. Both types of arrangements are promising leads for the development of new therapeutic agents that can remain effective when cancer cells become resistant to apoptotic cell death induced by traditional anti-cancer drugs.</p><p>In addition to providing new insights regarding isomeric methoxy substitutions on the indole, the present studies also reinforce the importance of the pyridinyl moiety for the continued development of IPPs as potential anti-cancer therapeutics. Our previous studies demonstrated that changing the configuration of the pyridinyl nitrogen (e.g., from para to meta) in the context of various IPP's with either methuosis or microtubule-disrupting activity eliminated or markedly reduced activity [11,14,15]. Here we show that replacing the pyridine ring with trimethoxyphenyl substituents (17 and 18) markedly reduced or eliminated the morphological effects and growth inhibitory activity. These trimethoxyphenyl substitutions previously were shown to impart potent microtubule-disrupting activity on various indole-based chalcones containing N-methyl indole [24,25]. It thus appears that the trimethoxyphenyl substituents are not compatible with the 5-methoxy-2-methylindole moiety for generating either microtubule-targeted compounds or methuosis-inducing compounds.</p><p>Compounds that alter microtubule stability are widely used in cancer therapy [28–30]. However, these agents are not without drawbacks, such as dose-limiting toxicity, development of drug resistance, and restricted penetrance of the blood-brain barrier [31,32]. Because of these issues, there continues to be significant interest in discovery of new microtubule-targeted compounds with distinct properties. In this regard, our findings suggest that further exploration of the anti-neoplastic potential and pharmacological properties of the 6-methoxy derivative, 9b, could be productive.</p><!><p>All reactions were performed in oven-dried 2-neck round-bottom flasks under an atmosphere of either Ar or N2 and stirred with teflon-coated magnetic bars. TLC (silica gel F254 plates, Baker-flex) was used to monitor progress of all reactions with visualization performed under 254 nm UV light. Reagent grade and anhydrous solvents were purchased from Sigma-Aldrich and used without further purification unless otherwise noted. Compounds 4a–4d were purchased from Alfa-Aesar, while compound 10 was purchased from Sigma-Aldrich. Compounds 12a, 12b and 16 were reported previously [11,14,15]. Chromatography was conducted on normal phase silica gel sorbent (Fisher Scientific, 230–400 mesh) by flash column methods as described previously [15,33] utilizing a gradient of increasing polar eluent specifically indicated for each compound. Isocratic separations are denoted on an individual basis. Samples to be purified were prepared by adsorption onto silica gel before performing chromatography (previously described as "dry loading") [15]. TLC was used to monitor product elution during flash column chromatography. Appropriate fractions were combined, solvents distilled in vacuo (rotary evaporator under water aspirator vacuum) and then further dried by a vacuum pump (0.5 mm Hg) for 24 h unless described otherwise. Samples that were heated in a vacuum desiccator were equipped to a vacuum pump (0.5 mm Hg) and dried for a specified time and temperature denoted in the individual procedure. Solvent solutions dried with Na2SO4 were stored in a sealed flask and allowed to sit for at least 4 h. Upon completion, the drying agent was removed by filtration and filtrate was evaporated in vacuo and then further dried by a vacuum pump (0.5 mm Hg) for 24 h. Melting points were performed in triplicate on an Electrothermal digital melting point apparatus and are uncorrected. Proton (1H) and carbon (13C) NMR experiments were recorded on either a 600 MHz Bruker Avance, Inova 600 MHz or an Inova 400 MHz instrument. Samples were referenced to TMS when present, or the solvent residual peak for 1H and 13C, respectively: (CDCl3; 7.27, 77.13; d6-DMSO; 2.50, 39.51; d6-acetone; 2.05, 29.92). 1H NMR chemical shifts were given in ppm and coupling constants (J values) were expressed in hertz (Hz) using the following designations: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), m (multiplet). The 13C chemical shifts were reported in ppm for each compound in the experimental section and in all cases confirm structure. In a few cases 13C shifts were found to double-up in their peak locations. Fluorine (19F) NMR was recorded on an Inova 400 MHz instrument at 376 MHz. Samples were referenced externally to CFCl3. Purity for tested compounds was determined by combustion analysis (Atlantic Microlabs, Norcross GA) and HPLC. All tested compounds possess ≥95% as determined by both purity methods. Observed values for combustion analysis were considered acceptable within ±0.4% of calculated values. Synthetic derivatives reported as solvates are denoted in the text. HPLC was performed on an Alliance® instrument (#2659) equipped with a quaternary pump, an inline membrane degasser, autosampler and Photodiode Array (PDA) Detector (#2996) from Waters Corporation (Milford, MA). The column was a Nova-Pak®C18 column, 4 μm particle size (150 mm × 3.9 mm). Samples were dissolved in 60% eluent A (H2O) and 40% eluent B (CH3CN) for injection. The following procedure, termed "Gradient 1", was employed for final targets (9a–9d, 15, 17–19) and intermediate 8c: Time 0.01–2.00 min (isocratic, 20% eluent B); Time 2.01–15.00 min (linear gradient, 45% eluent B to 80% eluent B); Time 15.01–20.00 min (isocratic, 20% eluent B). Details for HPLC analysis are denoted in the individual procedures. Chromatograms are illustrated in Figure S1 (supplementary data) and were recorded at the UVmax for each compound.</p><!><p>A suspension of 4-methoxyindole (500 mg, 3.4 mmol), TBAB (10 mol%, 109 mg, 0.34 mmol) and 50% sodium hydroxide (5 mL) in THF (10 mL) and H2O (3 mL) was stirred vigorously for 20 min at rt. Benzenesulfonyl chloride (2 eq, 6.8 mmol, 1.2 g) in THF (15 mL) was added dropwise to the reaction mixture. The reaction mixture was stirred overnight then extracted with EtOAc (20 mL X 3). The combined organic layers were dried with anhydrous Na2SO4 and then concentrated in vacuo to yield a yellow oil. The product was purified using column chromatography (DCM) to yield white solid (910 mg, 92%): mp 86–88 °C. TLC Rf 0.71 (DCM). 1H NMR (600 MHz, CDCl3) δ 7.87-7.86 (d, 2H, J = 7.68 Hz), 7.61-7.59 (d, 1H, J = 8.34 Hz), 7.5155 (t, 1H, J = 7.38 Hz), 7.47 (d, 1H, J= 3.66 Hz), 7.43-7.41 (t, 2H, J = 7.8 Hz), 7.25-7.22 (t, 1H, J = 8.16 Hz), 6.78-6.77 (d, 1H, J = 3.66 Hz), 6.65-6.64 (d, 1H, J = 7.98 Hz), 3.88 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 153.3, 138.4, 136.3, 134.0, 129.4, 127.0, 125.89, 124.97, 121.3, 106.68, 106.49, 103.7, 55.6. Elemental analysis calcd for C15H13NO3S: C, 62.70; H, 4.56; N, 4.87. Found: C, 62.71; H, 4.60; N, 4.97.</p><!><p>A suspension of 6-methoxyindole (1.0 g, 6.79 mmol), TBAB (218 mg, 0.679 mmol) and 50% sodium hydroxide (10 mL) in THF (10 mL) and H2O (3 mL) was stirred vigorously for 20 min at rt. Benzenesulfonyl chloride (13.6 mmol, 2.4 g) in THF (8 mL) was added dropwise to the reaction mixture. The reaction mixture was stirred overnight then extracted with EtOAc (50 mL X 3). The combined organic layer was dried over Na2SO4 then concentrated in vacuo. The product was purified using column chromatography (5% to 25% EtOAc/hexanes) to yield white solid (1.80 g, 92%): mp 144–146 °C (140–142 °C [34]). TLC Rf 0.38 (20% EtOAc/hexanes). 1H NMR (600 MHz, CDCl3) δ 7.87-7.85 (m, 2H), 7.55-7.52 (m, 2H), 7.45-7.43 (m, 3H), 7.39-7.38 (d, 1H, J = 8.58 Hz), 6.87-6.85 (dd, 1H, J1 = 8.58 Hz, J2 = 2.34 Hz), 6.59-6.58 (dd, 1H, J1 = 3.66 Hz, J2 = 0.72 Hz), 3.88 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 157.9, 138.2, 135.9, 133.8, 129.2, 126.7, 125.06, 124.45, 121.8, 112.6, 109.2, 97.9, 55.8.</p><!><p>A suspension of 7-methoxyindole (1 g, 6.8 mmol), TBAB (219 mg, 0.68 mmol) and 50% sodium hydroxide (10 mL) in THF (20 mL) and water (6 mL) was reacted and purified in a manner similar to that for 5a to yield a white solid (1.5 g, 73%): mp 88–90 °C (89–90 °C [35]). TLC Rf 0.75 (DCM). 1H NMR (600 MHz, CDCl3) δ 7.85-7.84 (m, 3H), 7.57-7.54 (m, 1H), 7.49-7.46 (m, 2H), 7.17-7.16 (m, 1H), 7.13–7.11 (m, 1H), 6.68-6.66 (d, 1H, J = 7.86 Hz), 6.66-6.65 (d, 1H, J = 3.66 Hz), 3.64 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 147.3, 140.4, 133.67, 133.11, 128.72, 128.59, 127.1, 124.55, 124.11, 114.0, 107.12, 106.76, 55.3.</p><!><p>A suspension of 6-methoxyindole (1.01 g, 5.70 mmol), TBAB (184 mg, 0.570 mmol) and 50% sodium hydroxide (5 mL) in THF (10 mL) and H2O (10 mL) was reacted and purified in a similar manner to that for 5b to yield a white solid (1.71 g, 92%): mp 140–143 °C (130–133 °C [36]). TLC Rf 0.18 (20% EtOAc/hexanes). 1H NMR (600 MHz, CDCl3) δ 7.87-7.85 (m, 2H), 7.55-7.52 (m, 2H), 7.45-7.43 (m, 3H), 7.39-7.38 (d, 1H, J = 8.58 Hz), 6.87-6.85 (dd, 1H, J1 = 8.58 Hz, J2 = 2.34 Hz), 6.59-6.58 (dd, 1H, J1 = 3.66 Hz, J2 = 0.72 Hz), 3.88 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 157.9, 138.2, 135.9, 133.8, 129.2, 126.7, 125.06, 124.45, 121.8, 112.6, 109.2, 97.9, 55.8. 1H NMR (600 MHz, d6-acetone) δ 7.99-7.97 (m, 2H), 7.67-7.65 (m, 1H), 7.59-7.56 (m, 3H), 7.52 (d, 1H, J = 3.66 Hz), 7.08 (s, 1H), 6.67-6.66 (dd, 1H, J1 = 3.60 Hz, J2 = 0.66 Hz), 3.90 (s, 3H), 3.79 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 149.61, 148.71, 138.9, 135.1, 130.47, 130.06, 127.7, 126.0, 124.8, 110.7, 104.3, 98.4, 56.53, 56.30. Elemental analysis calcd for C16H15NO4S: C, 60.55; H, 4.76; N, 4.41. Found: C, 60.40; H, 4.84; N, 4.52.</p><!><p>Compound 5b (1.50 g, 5.22 mmol) was dissolved in THF (25 mL). The solution was cooled to −30 °C and treated with tert-butyllithium (1.7 M in pentane, 6.79 mmol, 4.0 mL) slowly to maintain an internal temperature of −30 °C. After completion, the reaction mixture stirred for an additional 30 min at −30 °C, then allowed to warm to 0 °C and stirred for another 20 min. The reaction mixture was cooled to −30 °C and iodomethane (15.7 mmol, 0.97 mL) was added drop-wise. The reaction mixture was warmed to rt and stirred for 16 h. The reaction mixture was concentrated in vacuo and redissolved in DCM (100 mL) then washed with saturated NaHCO3 (75 mL X 3). The organic layer was dried over Na2SO4, the filtrate collected and concentrated in vacuo to yield crude material which was purified by chromatography (0% to 20% EtOAc/hexanes) to yield a brown solid (984 mg, 63%): mp 68–71 °C. TLC Rf 0.29 (10% EtOAc/hexanes). 1H NMR (600 MHz, CDCl3) δ 7.76-7.74 (m, 3H), 7.54-7.51 (m, 1H), 7.43-7.41 (m, 2H), 7.27-7.26 (d, 1H, J = 8.58 Hz), 6.85-6.84 (dd, 1H, J1 = 8.52 Hz, J2 = 2.34 Hz), 6.26 (t, 1H, J = 0.84 Hz), 3.87 (s, 3H), 2.55 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 157.3, 139.2, 138.0, 136.0, 133.6, 129.3, 126.2, 123.4, 120.3, 112.3, 109.4, 99.4, 55.8, 15.8. Elemental analysis calcd for C16H15NO3S: C, 63.77; H, 5.02; N, 4.65. Found: C, 63.88; H, 5.17; N, 4.57.</p><!><p>Compound 5d (1.70 g, 5.36 mmol) was reacted and purified in a manner similar to that for 6b to yield a cream-colored solid (742 mg, 42%): mp 129–131 °C. TLC Rf 0.23 (20% EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 7.88-7.86 (m, 2H), 7.73 (s, 1H), 7.68-7.65 (m, 1H), 7.58-7.55 (m, 2H), 6.96 (s, 1H), 6.38-6.37 (t, 1H, J = 0.84 Hz), 3.88 (s, 3H), 3.79 (s, 3H), 2.56 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 148.7, 139.8, 136.6, 135.0, 131.8, 130.5, 127.2, 123.9, 111.1, 103.4, 100.1, 56.60, 56.27, 15.9. Elemental analysis calcd for C17H17NO4S: C, 61.62; H, 5.17; N, 4.23. Found: C, 61.34; H, 5.17; N, 4.19.</p><!><p>Compound 5a (700 mg, 2.44 mmol) was dissolved in THF (25 mL) under an Ar (g) atmosphere. The solution was cooled to −30 °C and then treated with tert-butyllithium (1.7 M in pentane, 3.16 mmol, 1.86 mL) slowly to maintain an internal temperature of −30 °C. After completion, the reaction mixture was allowed to stir for an additional 30 min at −30 °C then was allowed to warm up to 0 °C and stirred for another 20 min. The reaction mixture was cooled to −30 °C and iodomethane (0.46 mL, 7.3 mmol) was added dropwise to the stirring mixture. After the addition was complete, the reaction mixture was stirred and warmed to rt overnight. The mixture was concentrated in vacuo and redissolved in DCM (75 mL) and washed with saturated NaHCO3 (50 mL x 3). The organic layer was separated and then dried over anhydrous sodium sulfate. The filtrate was collected and concentrated in vacuo to yield black oil which was immediately taken to the next step. Crude 6a (1.1 g) was dissolved in a solution of 3 N NaOH/EtOH (50 mL, 1:1) and heated at 90 °C for 24 h. The reaction mixture was concentrated in vacuo and extracted with DCM (50 mL X 3). The combined organic layers were dried over Na2SO4. The filtrate was collected and concentrated in vacuo to yield a brown oil. The product was purified using column chromatography (0% to 10% EtOAc/hexanes) to yield a white solid (250 mg, 64%): mp 88 °C. TLC Rf 0.34 (20% EtOAc/hexanes). 1H NMR (600 MHz, CDCl3) δ 7.85 (s, 1H), 7.05-7.02 (t, 1H, J = 8.10 Hz), 6.94-6.93 (d, 1H, J = 7.68 Hz), 6.51-6.50 (d, 1H, J = 7.56 Hz), 6.31 (s, 1H), 3.94 (s, 3H), 2.44 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 152.5, 137.3, 133.4, 121.6, 119.3, 103.8, 99.7, 97.5, 55.3, 14.1. Elemental analysis calcd for C10H11NO • 0.15 EtOH: C, 73.64; H, 7.14; N, 8.34. Found: C, 73.83; H, 7.23; N, 7.97.</p><!><p>Compound 11 (1.20 g, 5.06 mmol) was dissolved in THF (15 mL) under an atmosphere of Argon. The solution was cooled to −40 °C over 10 min and sec-butyllithium (1.4 M, 7.93 mL) was added slowly as to maintain an internal temperature of < −25 °C. After reaching 1 equivalent of sec-butyllithium (~3.96 mL) the reaction mixture turned a bright yellow signifying de-protonation of the amide nitrogen. The reaction mixture was then cooled to −50 °C and a solution of N-methoxy-N-methylacetamide 12a (575 mg, 5.57 mmol) in THF (3 mL) was added over 5 min. The reaction mixture was warmed to −10 °C over 30 min. The mixture was partitioned between Et2O (75 mL) and 0.5 N HCl (75 mL). The aqueous layer was separated and extracted an additional two times with Et2O (50 mL). The Et2O phases were combined and washed with brine (75 mL) and then dried over Na2SO4 to yield a dark oil. The crude intermediate was dissolved in DCM (20 mL). TFA (3 mL) was added to the mixture which was then stirred at rt for 24 h. Upon completion, the reaction mixture was added to a separatory funnel and washed with saturated NaHCO3 (50mL) followed by brine (50 mL). The organic layer was separated, dried with Na2SO4 and concentrated in vacuo to provide a crude oil which was purified by chromatography (0% to 20% EtOAc/hexanes) to yield a white solid (146 mg, 18%): mp 106–108 °C. TLC Rf 0.31 (20% EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 9.77 (s, 1H), 7.27-7.26 (d, 1H, J = 8.46 Hz), 6.84 (d, 1H, J = 2.22 Hz), 6.62-6.61 (dd, 1H, J1 = 8.52 Hz, J2 = 2.28 Hz), 6.04 (m, 1H) 3.77 (s, 3H), 2.36 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 156.5, 138.2, 134.8, 124.4, 120.4, 109.4, 100.2, 95.1, 55.7, 13.6. Elemental analysis calcd for C10H11NO: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.67; H, 6.90; N, 8.66.</p><!><p>Compound 5c (890 mg, 3.10 mmol) was reacted and purified in a manner similar to that for 7a to yield a white solid (120 mg, 24%): mp 83 °C (83–83.5 °C [37]). TLC Rf 0.74 (DCM). 1H NMR (600 MHz, CDCl3) δ 8.09 (s, 1H), 7.13-7.12 (d, 1H, J = 7.92 Hz), 6.99-6.96 (t, 1H, J = 7.86 Hz), 6.58-6.57 (d, 1H, J = 7.44 Hz), 6.18 (s, 1H) 3.93 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 145.4, 134.6, 130.3, 126.2, 119.9, 112.5, 101.11, 100.65, 55.3, 13.6.</p><!><p>Compound 6d (0.732 g, 2.21 mmol) was dissolved in a solution of 3 N NaOH/EtOH (75 mL, 1:1) and refluxed at 90 °C for 60 h. The reaction mixture was then concentrated in vacuo and extracted using DCM (50 mL X 3). The combined organic layers were dried over Na2SO4, collected and then concentrated to provide a brown oil. The product was purified using column chromatography (0% to 20% EtOAc/hexanes) to yield a yellow solid (352 mg, 83%): mp 94–95 °C (90–91 °C [38]). TLC Rf 0.15 (20% EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 9.66 (s, 1H), 6.95 (s, 1H), 6.88 (s, 1H), 6.00-5.99 (m, 1H), 3.77 (s, 6H), 2.35 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 147.4, 146.1, 134.5, 132.0, 123.2, 103.8, 100.3, 96.3, 56.85, 56.63, 13.7.</p><!><p>DMF (2 mL) was cooled to 0 °C. POCl3 (0.5 mL) was added and the reaction mixture was stirred for 10 min at 0 °C. A solution of 7a (220 mg, 1.37 mmol) in DMF (2 mL) was added to the reaction mixture drop-wise over 10 min. The solution was stirred for an additional 40 min while warming to rt, then slowly poured into ice-cold 1 N NaOH (50 mL) and stirred for 10 min. The precipitate was collected, washed with ice-cold H2O and dried at 40 °C for 24 h in a vacuum desiccator yielding a tan solid (177 mg, 68%): mp 190–192 °C. TLC Rf 0.71 (80% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 12.08 (s, 1H), 10.44 (s, 1H), 7.10-7.08 (t, 1H, J = 7.92 Hz), 7.02-7.01 (d, 1H, J = 7.98 Hz), 6.73-6.72 (d, 1H, J = 7.8 Hz), 3.92 (s, 3H), 2.65 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 187.3, 153.2, 142.7, 136.0, 122.7, 116.4, 113.6, 104.8, 102.2, 55.1, 13.8. Elemental analysis calcd for C11H11NO2: C, 69.83; H, 5.86; N, 7.40. Found: C, 69.59; H, 6.00; N, 7.27.</p><!><p>Compound 6b (0.718 g, 2.38 mmol) was dissolved in a solution of 3 N NaOH/EtOH (75 mL, 1:1) and refluxed at 90 °C for 36 h. The reaction mixture was then concentrated in vacuo and extracted using DCM (50 mL X 3). The combined organic layers were dried over Na2SO4, collected and concentrated in vacuo to provide brown oil 7b (205 mg, 53%). TLC and NMR of 7b suggested the sample was rapidly decomposing. The product was immediately taken to the next step. DMF (2 mL) was cooled to 0 °C. POCl3 (0.4 mL) was added and the reaction mixture was stirred for 10 min. A solution of crude 7b (205 mg, 1.27 mmol) in DMF (1 mL) was added to the reaction mixture dropwise over 10 min. The solution was stirred for an additional 2 h. The reaction mixture was added to ice-cold 1 N NaOH (40 mL) and stirred for 10 min. The precipitate was collected, washed with ice-cold H2O and dried overnight in a vacuum desiccator set at 40 °C yielding a tan solid (50 mg, 21%): mp 219–222 °C. TLC Rf 0.47 (75% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.79 (s, 1H), 9.99 (s, 1H), 7.89-7.88 (d, 1H, J = 8.58 Hz), 6.87 (d, 1H, J = 2.22 Hz), 6.79-6.78 (dd, 1H, J1 = 8.52 Hz, J2 = 2.28 Hz), 3.77 (s, 3H), 2.64 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 184.0, 156.2, 147.8, 136.3, 120.7, 119.4, 113.7, 110.9, 95.1, 55.2, 11.4. Elemental analysis calcd for C11H11NO2: C, 69.83; H, 5.86; N, 7.40. Found: C, 69.74; H, 5.98; N, 7.23.</p><!><p>This compound was prepared from 7b-Scheme 2 (280 mg, 1.74 mmol) in a similar manner to that for 8a except the reaction was stirred for 2 h while warming to rt to yield a tan solid (297 mg, 90%): mp 223–225 °C. TLC Rf 0.57 (EtOAc). 1H NMR (600 MHz, d6-DMSO) δ 11.79 (s, 1H), 9.99 (s, 1H), 7.89-7.88 (d, 1H, J = 8.58 Hz), 6.87 (d, 1H, J = 2.22 Hz), 6.79-6.78 (dd, 1H, J1 = 8.58 Hz, J2 = 2.28 Hz), 3.77 (s, 3H), 2.64 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 184.0, 156.2, 147.8, 136.3, 120.7, 119.4, 113.7, 110.9, 95.1, 55.2, 11.4. Elemental analysis calcd for C11H11NO2 • 0.15 H2O: C, 68.84; H, 5.93; N, 7.30. Found: C, 68.48; H, 5.90; N, 7.24.</p><!><p>This compound was prepared from 7c (100 mg, 0.62 mmol) in a manner similar to that for 8a to yield a light brown solid (97 mg, 83%): mp 209 °C. TLC Rf 0.53 (80% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 12.10 (s, 1H), 10.03 (s, 1H), 7.62-7.61 (d, 1H, J = 7.80 Hz), 7.09-7.06 (t, 1H, J = 7.86 Hz), 6.78-6.77 (d, 1H, J = 7.74 Hz), 3.93 (s, 3H), 2.65 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 184.2, 147.8, 145.5, 126.9, 124.9, 122.6, 114.0, 112.5, 103.6, 55.1, 11.2. HPLC analysis: retention time = 3.338 min; peak area, 99.45%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 215 nm; injection of 10 μL of 20 μM 8c.</p><!><p>DMF (2 mL) was cooled to 0 °C. POCl3 (0.6 mL) was added and the reaction mixture was stirred for 10 min at 0 °C. A solution of 7d (344 mg, 1.80 mmol) in DMF (2 mL) was added to the reaction mixture drop-wise over 10 min. The solution was stirred for an additional 3 h at rt, then slowly poured into ice-cold 1 N NaOH (50 mL) and stirred for 10 min. The solution was transferred to a separatory funnel and extracted with EtOAc (50 mL X 4). The combined organic layers were washed with brine (100 mL) and then dried over Na2SO4. The filtrate was collected and concentrated in vacuo. The material was purified by chromatography (20% to 100% EtOAc/hexanes) to yield a beige solid (368 mg, 93%): mp 204–208 °C. TLC Rf 0.25 (75% EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 10.64 (s, 1H), 10.10 (s, 1H), 7.69 (s, 1H), 6.98 (s, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 2.69 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 184.4, 148.64, 148.00, 146.6, 130.8, 120.0, 115.6, 104.2, 96.3, 56.51, 56.44, 11.6. Elemental analysis calcd for C12H13NO3: C, 65.74; H, 5.98; N, 6.39. Found: C, 65.47; H, 5.95; N, 6.28.</p><!><p>Compound 8a (100 mg, 0.53 mmol) was dissolved in anhydrous methanol (15 mL). 4-Acetylpyridine (96 mg, 0.79 mmol) and piperidine (67 mg, 0.79 mmol) were added and the solution was heated to reflux for 24 h. A precipitate slowly formed which was collected, washed with ice-cold MeOH (50 mL) and dried at 40 °C in a vacuum desiccator for 24 h to yield a bright orange powder (143 mg, 92%): mp 234 °C. TLC Rf 0.27 (80% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.98, (s, 1H), 8.82-8.81 (m, 2H), 8.46-8.44 (d, 1H, J = 15.54 Hz), 7.84-7.83 (m, 2H), 7.44-7.41 (d, 1H, J = 15.54 Hz), 7.10-7.08 (t, 1H, J = 7.92 Hz), 7.00-6.99 (d, 1H, J = 7.98 Hz), 6.70-6.68 (d, 1H, J = 7.74 Hz), 3.93 (s, 3H), 2.64 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 188.1, 153.2, 150.5, 145.3, 141.68, 141.46, 136.9, 123.1, 121.1, 116.13, 115.46, 109.3, 104.8, 102.1, 55.2, 14.0. Elemental analysis calcd for C18H16N2O2 • 0.25 MeOH: C, 72.98; H, 5.70; N, 9.33. Found: C, 72.62; H, 5.51; N, 9.14. HPLC analysis: retention time = 5.020 min; peak area, 98.99%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 224 nm; injection of 10 μL of 20 μM 9a.</p><!><p>This compound was prepared from 8b (50 mg, 0.26 mmol) in a manner similar to that for 9a to yield a bright orange powder (33 mg, 43%): mp 250–252 °C. TLC Rf 0.39 (80% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.83, (s, 1H), 8.81-8.80 (m, 2H), 8.07-8.04 (d, 1H, J = 15.18 Hz), 7.96-7.94 (m, 3H), 7.43-7.40 (d, 1H, J = 15.18 Hz), 6.91-6.90 (d, 1H, J = 2.28 Hz), 6.84-6.82 (dd, 1H, J1 = 8.64 Hz, J2 = 2.34 Hz), 3.80 (s, 3H), 2.56 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 187.9, 156.0, 150.6, 144.96, 144.92, 139.4, 137.3, 121.43, 121.10, 119.6, 112.6, 110.44, 109.55, 95.3, 55.2, 11.8. Elemental analysis calcd for C18H16N2O2 • 0.7 H2O: C, 70.90; H, 5.75; N, 9.19. Found: C, 70.51; H, 5.32; N, 8.93. HPLC analysis: retention time = 5.779 min; peak area, 98.44%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 230 nm; injection of 10 μL of 20 μM 9b.</p><!><p>This compound was prepared from 8b-Scheme 2 (252 mg, 1.33 mmol) in a manner similar to that for 9a to yield an orange solid (338 mg, 87%): mp 248–250 °C. TLC Rf 0.32 (80% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.83, (s, 1H), 8.81-8.80 (m, 2H), 8.07-8.04 (d, 1H, J = 15.18 Hz), 7.96-7.94 (m, 3H), 7.43-7.40 (d, 1H, J = 15.18 Hz), 6.91-6.90 (d, 1H, J = 2.28 Hz), 6.84-6.82 (dd, 1H, J1 = 8.64 Hz, J2 = 2.34 Hz), 3.80 (s, 3H), 2.56 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 187.9, 156.0, 150.6, 144.96, 144.92, 139.4, 137.3, 121.43, 121.10, 119.6, 112.6, 110.44, 109.55, 95.3, 55.3, 11.8. Elemental analysis calcd for C18H16N2O2 • 0.025 H2O: C, 73.84; H, 5.53; N, 9.57. Found: C, 73.45; H, 5.52; N, 9.32. HPLC analysis: retention time = 5.932 min; peak area, 97.97%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 229 nm; injection of 10 μL of 20 μM 9b.</p><!><p>This compound was prepared from 8c (76 mg, 0.40 mmol) in a manner similar to that for 9a to yield a bright orange powder (104 mg, 89%): mp 265–266 °C. TLC Rf 0.43 (80% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 12.10 (s, 1H), 8.81-8.80 (m, 2H), 8.09-8.06 (d, 1H, J = 15.24 Hz), 7.95-7.94 (m, 2H), 7.63-7.62 (d, 1H, J = 7.92 Hz), 7.44-7.41 (d, 1H, J = 15.18 Hz), 7.16-7.13 (t, 1H, J = 7.92 Hz), 6.82-6.81 (d, 1H, J = 7.74 Hz), 3.95 (s, 3H), 2.57 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 188.0, 150.6, 145.80, 144.98, 144.91, 139.6, 127.2, 125.9, 122.26, 121.41, 113.09, 112.94, 109.9, 103.6, 55.3, 11.8. Elemental analysis calcd for C18H16N2O2 • 0.875 H2O: C, 70.17; H, 5.81; N, 9.09. Found: C, 69.78; H, 5.88; N, 8.79. HPLC analysis: retention time = 5.342 min; peak area, 98.91%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 225 nm; injection of 10 μL of 20 μM 9c.</p><!><p>This compound was prepared from 8d (355 mg, 1.62 mmol) in a manner similar to that for 9a to yield a dark orange gum (438 mg, 83%): mp 222–223 °C. TLC Rf 0.14 (80% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.75 (s, 1H), 8.81-8.80 (m, 2H), 8.07-8.05 (d, 1H, J = 15.24 Hz), 7.94-7.93 (m, 2H), 7.43 (s, 1H), 7.36-7.33 (d, 1H, J = 15.24 Hz), 6.94 (s, 1H), 3.88 (s, 3H), 3.80 (s, 3H), 2.55 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 188.1, 150.6, 146.8, 145.76, 145.17, 143.8, 139.7, 130.5, 121.4, 118.4, 112.5, 109.6, 103.7, 95.6, 56.40, 55.71, 12.0. Elemental analysis calcd for C19H18N2O3 • 0.075 H2O: C, 70.50; H, 5.65; N, 8.65. Found: C, 70.07; H, 5.64; N, 8.39. HPLC analysis: retention time = 5.311 min; peak area, 96.50%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 222 nm; injection of 10 μL of 20 μM 9d.</p><!><p>Compound 10 (2.0 g, 14.6 mmol) and di-tert-butyl-dicarbonate (3.51 g, 16.1 mmol) in THF (50 mL) were heated to reflux for 20 h. The reaction mixture was concentrated in vacuo and redissolved in DCM (75 mL). This mixture was washed with saturated NaHCO3 (75 mL), and then brine (50 mL). The organic layer was separated, dried over Na2SO4 and concentrated in vacuo to produce an oil which was purified by chromatography (0% to 20% EtOAc/hexanes) to yield a white solid (2.80 g, 81%): mp 79–80 °C (76–80 °C [39]). TLC Rf 0.55 (20% EtOAc/hexanes). 1H NMR (600 MHz, CDCl3) δ 7.56 (s, 1H), 7.03-7.02 (d, 1H, J = 8.34 Hz), 6.56-6.54 (dd, 1H, J1 = 8.34 Hz, J2 = 2.64 Hz), 6.28 (s, 1H), 3.80 (s, 3H), 2.18 (s, 3H), 1.54 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 158.8, 153.0, 137.4, 130.9, 118.3, 109.6, 105.6, 80.7, 55.6, 28.6, 17.1.</p><!><p>This compound was prepared from 11 (1.18 g, 4.97 mmol) in a manner similar to that for compound 7b-Scheme 2 except Weinreb amide 12b (859 mg, 5.47 mmol) was employed to obtain a white solid (446 mg, 42%): mp 91–95 ° C. TLC Rf 0.32 (15% EtOAc/hexanes). 1H NMR (600 MHz, CDCl3) δ 8.29 (s, 1H), 7.56-7.55 (d, 1H, J = 8.46 Hz), 6.89-6.87 (m, 3H), 3.86 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 158.4, 137.3, 124.6 (q, 2JFC = 39 Hz), 123.0, 121.52 (q, 1JFC = 266 Hz), 120.97, 112.1, 104.6 (q, 3JFC = 3 Hz), 94.4, 55.8. 19F NMR (376 MHz, CDCl3) δ −60.6 (s, 3F). Elemental analysis calcd for C10H8F3NO • 0.11 CH2Cl2: C, 54.09; H, 3.69; N, 6.24. Found: C, 54.54; H, 3.34; N, 5.78.</p><!><p>DMF (2 mL) was cooled to 0 °C. POCl3 (0.6 mL) was added and the reaction mixture was stirred for 10 min at 0 °C. A solution of 13 (286 mg, 1.33 mmol) in DMF (2 mL) was added to the reaction mixture drop-wise over 10 min. The solution was stirred for 30 min while warming to rt and then heated to 80 °C for 3 h. The mixture was slowly poured into ice-cold 1 N NaOH (50 mL) and stirred for 10 min. The solution was transferred to a separatory funnel and extracted with EtOAc (50 mL X 4). The combined organic layers were washed with brine (100 mL) and then dried over Na2SO4. The filtrate was collected and concentrated in vacuo. The material was purified by chromatography (10% to 50% EtOAc/hexanes) to yield a white solid (105 mg, 32%): mp 243–245 ° C. TLC Rf 0.19 (20% EtOAc/hexanes). 1H NMR (600 MHz, d6-acetone) δ 11.91 (s, 1H), 10.29 (s, 1H), 8.21-8.20 (d, 1H, J = 8.82 Hz), 7.07 (d, 1H, J = 2.22 Hz), 7.03-7.01 (dd, 1H, J1 = 8.82 Hz, J2 = 2.28 Hz), 3.85 (s, 3H). 13C NMR (150 MHz, d6-acetone) δ 184.7, 160.1, 137.7, 130.9 (q, 2JFC = 39 Hz), 124.3, 122.1 (q, 1JFC = 268 Hz), 119.6, 117.6, 115.5, 95.6, 55.9. 19F NMR (376 MHz, d6-acetone) δ −51.9 (s, 3F). Elemental analysis calcd for C11H8F3NO2: C, 54.33; H, 3.32; N, 5.76. Found: C, 54.49; H, 3.32; N, 5.71.</p><!><p>Compound 14 (53 mg, 0.22 mmol) was dissolved in anhydrous MeOH (10 mL). 4-Acetylpyridine (40 mg, 0.33 mmol) and piperdine (28 mg, 0.33 mmol) were added and the mixture heated to reflux for 20 h. Upon completion, volatiles were evaporated in vacuo and the sample was purified by column chromatography (30% to 70% EtOAc/hexanes). The resulting solid was recrystallized from MeOH and dried at 40 °C in a vacuum desiccator for 36 h to yield a yellow solid (40 mg, 53%): mp 305–308 °C. TLC Rf 0.37 (75% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 12.35, (s, 1H), 8.85-8.84 (m, 2H), 8.38 (s, 1H), 8.21-8.18 (d, 1H, J = 15.72 Hz), 7.98-7.97 (m, 2H), 7.87-7.85 (d, 1H, J = 15.72 Hz), 7.01-6.98 (m, 2H), 3.95 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 189.0, 156.8, 150.8, 144.1, 141.7, 139.7, 125.0 (q, 2JFC = 38 Hz), 123.2, 121.55, 121.36 (q, 1JFC = 266 Hz), 120.19, 119.46, 118.40, 104.2 (q, 3JFC = 3 Hz), 93.8, 55.9. 19F NMR (376 MHz, d6-DMSO) δ −54.5 (s, 3F). Elemental analysis calcd for C18H13F3N2O2: C, 62.43; H, 3.78; N, 8.09. Found: C, 62.22; H, 3.84; N, 8.00. HPLC analysis: retention time = 8.239 min; peak area, 98.78%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 236 nm; injection of 10 μL of 20 μM 15.</p><!><p>Compound 16 (189 mg, 1 mmol) and 2′,4′,6′-trimethoxyphenylacetophenone (210 mg, 1 mmol) were dissolved in MeOH (10 mL). KOH (50%, 10 mL) was added and the solution was heated to reflux for 7 d. Upon completion, MeOH was distilled in vacuo and the aqueous layer was extracted with DCM (50 mL X 3). The combined organic layers were washed with brine (75 mL) and dried over Na2SO4. The filtrate was collected and evaporated in vacuo. The resulting material was purified by chromatography (50% to 90% EtOAc/hexanes) to yield a yellow solid (65 mg, 17%): mp 197–200 °C. TLC Rf 0.34 (75% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.66 (s, 1H), 7.44-7.42 (d, 1H, J = 15.90 Hz), 7.27-7.25 (d, 1H, J = 8.70 Hz), 7.14 (d, 1H, J = 2.28 Hz), 6.79-6.77 (dd, 1H, J1 = 8.70 Hz, J2 = 2.34 Hz), 6.65-6.63 (d, 1H, J = 15.90 Hz), 6.31 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.72 (s, 6H), 2.36 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 192.5, 161.5, 157.9, 154.8, 142.9, 137.6, 130.9, 126.3, 122.0, 112.19, 111.82, 110.98, 108.1, 102.1, 91.0, 55.72, 55.41, 55.39, 11.8. Elemental analysis calcd for C22H23NO5 • 0.075 H2O: C, 69.03; H, 6.10; N, 3.66. Found: C, 68.63; H, 6.17; N, 3.59. HPLC analysis: retention time = 5.345 min; peak area, 96.94%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 204 nm; injection of 10 μL of 20 μM 17.</p><!><p>This compound was prepared from 16 (100 mg, 0.53 mmol) in a manner similar to that for 9a to yield a yellow solid (131 mg, 65%): mp 250–253 °C. TLC Rf 0.45 (75% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 11.75 (s, 1H), 8.03-8.00 (d, 1H, J = 15.24 Hz), 7.50-7.48 (d, 1H, J = 15.24 Hz), 7.44 (d, 1H, J = 2.34 Hz), 7.37 (s, 2H), 7.29-7.28 (d, 1H, J = 8.70 Hz), 6.81-6.79 (dd, 1H, J1 = 8.70 Hz, J2 = 2.34 Hz), 3.90 (s, 6H), 3.86 (s, 3H), 3.75 (s, 3H), 2.57 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 187.5, 154.9, 152.8, 144.2, 141.0, 137.6, 134.3, 130.8, 126.5, 113.8, 112.23, 111.45, 109.2, 105.4, 102.4, 60.2, 55.89, 55.10, 12.1. Elemental analysis calcd for C22H23NO5 • 0.2 MeOH: C, 68.75; H, 6.19; N, 3.61. Found: C, 68.35; H, 5.94; N, 3.66. HPLC analysis: retention time = 6.131 min; peak area, 97.89%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 204 nm; injection of 10 μL of 20 μM 18.</p><!><p>Compound 1a 'MOMIPP' (100 mg, 0.34 mmol) was dissolved in DMF (2.5 mL). NaH (16.4 mg, 0.68 mmol, 60% dispersion in mineral oil, unwashed) was added and stirred for 10 min. Methyl iodide (32 μL, 0.51 mmol) was then added and the reaction mixture was stirred at rt for 5 h. Upon completion, saturated NH4Cl solution (30 mL) was added to the reaction mixture and transferred to a separatory funnel. The aqueous later was extracted with EtOAc (25 mL X 3). The combined organic layer was dried over Na2SO4, the filtrate was collected and volatiles were distilled. The resulting material was purified by chromatography (2% to 5% MeOH/DCM). The resulting solid was recrystallized from MeOH to yield a yellow-orange solid (13 mg, 12%): mp 160–161 °C. TLC Rf 0.33 (75% EtOAc/hexanes). 1H NMR (600 MHz, d6-DMSO) δ 8.81-8.80 (m, 2H), 8.13-10 (d, 1H, J = 15.18 Hz), 7.95-7.94 (m, 2H), 7.50-7.48 (d, 1H, J = 8.88 Hz), 7.48 (d, 1H, J = 2.40 Hz), 7.41-7.38 (d, 1H, J = 15.18 Hz), 6.93-6.91 (dd, 1H, J1 = 8.82 Hz, J2 = 2.40 Hz), 3.88 (s, 3H), 3.74 (s, 3H), 2.58 (s, 3H). 13C NMR (150 MHz, d6-DMSO) δ 188.0, 155.5, 150.6, 146.7, 145.1, 139.4, 132.7, 125.7, 121.4, 112.8, 111.16, 110.82, 108.9, 103.7, 55.6, 30.3, 10.7. Elemental analysis calcd for C19H18N2O2 • 0.055 MeOH: C, 74.28; H, 5.96; N, 9.09. Found: C, 73.84; H, 5.90; N, 9.06. HPLC analysis: retention time = 5.788 min; peak area, 96.78%; eluent A, H2O; eluent B, CH3CN; Gradient 1 over 20 min with a flow rate of 1 mL min−1 and detection at 429 nm; injection of 10 μL of 20 μM 19.</p><!><p>U251 human glioblastoma cells were obtained from the DCT Tumor Repository (National Cancer Institute) and were maintained in Dulbecco's modified Eagle medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) (JR Scientific, Woodland, CA) at 37°C with 5% CO2/95% air. For live cell imaging, the cells were plated in 35mm dishes at 100,000 cells per dish. On the following day compounds, dissolved in DMSO, were added at a final concentration of 2.5 μM. Controls received an equivalent volume of DMSO. Phase-contrast images were captured at 4 h and 48 h after addition of the compounds using an Olympus IX70 inverted microscope equipped with a DP-80 digital camera and Cellsense imaging software.</p><!><p>The effects of compounds on cell growth were assessed using the sulphorhodamine B (SRB) colorimetric assay, as described previously [11,15]. The concentration of each compound producing 50% growth inhibition (GI50) relative to the control without drug was calculated as described in the NCI-60 human cell line screening protocol (http://dtp.nci.nih.gov/branches/btb/ivclsp.html).</p><!><p>U251 cells were seeded on glass coverslips in 60 mm dishes at 350,000 cells dish. One day after plating, fresh medium was added with compounds at the indicated concentration. Cells were fixed and stained with a monoclonal antibody against α-tubulin, followed by Alexa Fluor 568-labeled goat anti-mouse IgG as described previously [15]. Nuclear DNA was stained with 4′,6-diamidino-2-phenyl-indole (DAPI).</p><!><p>U251 cells were seeded at 3.5 × 105 cells in 60 mm dishes. On the next day the cells were treated with 9b or 1a at the indicated concentrations. After 24 h the cells were harvested by trypsinization, fixed in ice-cold 70% ethanol, washed twice by centrifugation/resuspension in PBS, and then suspended in 900 μl PBS containing 6.25 mM MgSO4 and 1 mM CaCl2. After incubation at rt for 15 min, 20 μl of a 10 mg/ml solution of RNAse A was added and cells were incubated at 37° C for 15 min. Finally, 100 μl of a 500 mg/ml aqueous solution of propidium iodide was added and the cells were analyzed with a Becton-Dickinson FACS-Calibur flow cytometer. DNA histograms were generated with CellQuest Pro software.</p>

PubMed Author Manuscript

Oxygen storage in transition metal-doped bixbyite vanadium sesquioxide nanocrystals

Bixbyite vanadium sesquioxide (V2O3) is a metastable polymorph of vanadium oxide that has been shown to have a significant oxygen storage capacity with very low temperature oxidation onset.In this work, bixbyite V2O3 nanocrystals were synthesized with titanium and manganese dopants. Doped materials with varied dopant concentration were synthesized, and all were incorporated as aliovalent metal ions. The oxygen storage capacity of these nanocrystal materials was evaluated over ten oxidation and reduction cycles. It was found that over these ten cycles, the oxygen storage capacity of all the materials fell drastically. In situ X-ray diffraction evidence shows that manganese-doped materials degrade into an amorphous manganese-containing vanadate, while titanium-doped materials form crystalline degradation products. In all cases, this degradation causes an increase in the minimum mass achieved during oxygen release, indicating irreversible oxidation.

oxygen_storage_in_transition_metal-doped_bixbyite_vanadium_sesquioxide_nanocrystals

Introduction<!>Results and Discussion<!>Conclusions<!>Material Synthesis<!>Thermogravimetric Analysis<!>Inductively Couples Plasma Atomic Emission Spectroscopy<!>Transmission Electron Microscopy<!>X-ray Photoemission Spectroscopy<!>X-ray Diffraction

<p>The bixbyite phase of vanadium sesquioxide (V2O3), a metastable phase of the material, was first reported in 2011 by Weber et. al. 1 In 2015, a method to synthesize the pure bixbyite phase V2O3 as colloidal nanocrystals was described by Bergerud et al.. 2 Shortly after, it was discovered that this material exhibits excellent reversible oxygen storage capacity, with a remarkably low oxidation onset temperature, owing to its intrinsic oxygen vacancies. 3 This property has motivated investigation into ways to maximize the oxygen storage capacity and thermal and environmental stability of this material, to facilitate its potential application as an oxygen storage material (OSM) in automotive catalysts, where it could address problems with cold start.</p><p>Automotive catalysis has been discussing the cold start problem for quite some time. Catalytic converters were introduced in the United States by General Motors in 1975, consisting of alumina support with small amounts of platinum and palladium catalyst material. 4 Their use was made nearly ubiquitous by emissions standards introduced by the US Environmental Protection Agency in that year, and practically required by updated standards as an amendment to this legislation in 1990. 5 Modern catalytic converters have added cerium oxide, or ceria, to the catalyst bed to promote high conversion and efficiency at low air-to-fuel ratios in the exhaust stream. 6 Ceria, while it has enjoyed decades of use as the OSM of choice in the automotive industry, is not without shortcomings. Below about 300 o C, the ceria surface is not reactive with oxygen. An inactive OSM is one reason that automotive catalysts are virtually inactive until the converter reaches an operation temperature of at least 300 o C. Thus high levels of emission are observed during the first few minutes of operation of an automotive engine, and this is referred to by the catalysis community as the "cold start" problem. 7,8 By contrast, V2O3 nanocrystals have been shown to absorb oxygen from an atmosphere at temperatures as low as 100 o C and have a very similar oxygen storage capacity (OSC) to this industry standard material. It struggles, however, with poor stability upon cycling, owing to the wide variety of structures available to the large number of vanadium oxides that can be formed, reflected in the complexity of its phase diagram. 9,10 Doping is a commonly employed strategy in materials design for applications where stability is desired. In fact, OSMs for automotive catalysts have long used dopants to optimize performance. 11,12 Since at least the 1990s, the ceria OSMs in most auto catalytic converters have included these deliberate impurities, which increase the material's useful lifetime and its OSC.</p><p>Through experimental and computational methods, it has been shown that zirconium impurities, as Zr 4+ , introduce strain to the ceria lattice that prevents sintering, which causes a decrease in capacity over the material's lifetime, while increasing OSC by causing partial reduction of cerium ions. 13,14 Studies on divalent dopants in ceria show that the interplay between charge imbalance and lattice strain has strong impacts on oxygen storage capacity. 15,16,17 In this work, doped V2O3 nanocrystals are synthesized to investigate the impact of dopants on the OSC and cycling stability of this novel OSM. Titanium and manganese dopants were chosen, as transition metals on either side of vanadium on the periodic table. Incidentally, titanium dopants have been studied before in vanadium oxides, where titanium was shown to stabilize the corundum paramagnetic phase. 18 It has also been shown to increase the monoclinic-to-rutile transition temperature in VO2. 19,20 Likewise, manganese is one of many dopants that have been studied as incorporated into the layered material V2O5 for use in battery and energy storage applications. [21][22][23][24][25][26][27]</p><!><p>V2O3 particles were prepared with varied concentrations of titanium and manganese dopants.</p><p>Changes in the oxidation state of the metal ions caused by doping were evaluated by X-ray photoemission spectroscopy (XPS). The OSC for each sample was measured by thermogravimetric analysis (TGA). The crystallinity and crystal phase for each material was evaluated by ex-situ X-ray diffraction (XRD) before and after processing in TGA. Further, the crystal structure of the materials during oxidation and reduction were followed by in situ XRD, which revealed different degradation mechanisms for materials with different compositions. Titanium doping resulted in particles of size and shape very similar to undoped particles. Both types of nanocrystals have flower morphology and diameter of 20-40 nm, accompanied by some smaller side products, possibly the result of secondary nucleation during synthesis. Molybdenum doping resulted in highly faceted particles with a wide distribution of sizes, ranging from 3 to 20 nm across the longest dimension. Very small, round particles observed here may be from secondary nucleation as well, which can create small crystals that do not have the opportunity to grow. The larger particles are bipyramidal in shape, with rhombohedral projections between 5 and 20 nm across the dark middle, and between 10 and 25 nm across from tip to tip. XPS of the doped vanadium oxides (Figure 2) show the 2p transitions for vanadium in all the samples, along with the 2p transitions for the dopant elements. Survey spectra are shown in Figure S2. Ti-and Mn-doped samples all show a slight shift of the V 2p transitions to higher binding energy. The oxidation state of the dopant atoms can be determined by analysis of their 2p transitions. The Mn 2p spectrum shows a shake-up feature at 647 eV, diagnostic of the Mn 2+ state. 28 To further support this finding, the Mn 3s spectrum was collected (high resolution spectra and fits shown in Figure S4). The 3s spectrum has two peaks, due to correlation with the unfilled 3d 5 shell. The binding energy difference between the 3s 7 S and 5 S multiplet component peaks was found to be 6.5 eV for both the 5% and 10% doped samples, consistent with previous literature observing Mn in the 2+ oxidation state. 29 The titanium 2p peaks have a spin-orbital splitting of 5.7 eV each, which is consistent with reports for TiO2, where Ti is in the 4+ oxidation state. 30 Both of these ions are larger than the native V 3+ ion, at 0.74 and 0.83 nm for Ti 4+ and Mn 2+ with 6-fold coordination, respectively, compared to 0.64 nm for V 3+ , so they are expected to have similar effect on lattice strain. 31 While both dopants are larger than the host ion, they introduce charge imbalance in opposite directions. All the doped samples had higher OSC than undoped nanocrystals for their first six cycles of oxidation and reduction. However, for all samples, the OSC dropped dramatically during ten cycles. The Ti-doped samples both maintained higher OSC during all ten cycles, ending with an OSC 20-30% higher than that of undoped V2O3. Vanadium oxide doped by Mn had lower OSC than undoped nanocrystals after 10 cycles. The OSC is determined by the difference between the maximum weight and the minimum weight during each oxidation and reduction cycle (Figure 4 ab), and tracking those maximum and minimum values provides more information about the decrease in OSC with cycle number (Figure 4 c-f). It is immediately evident that the minimum mass fraction for all samples rose sharply in the first four cycles, indicating irreversible oxidation and suggesting a kinetic barrier to oxygen release during the reduction half-cycle. There is a correlated drop in maximum mass fraction after oxidation for Mn-doped nanocrystals. However, for the Ti-doped samples, the maximum and minimum fractional masses both drop nearly linearly with cycle number after cycle 5, suggesting that the Ti-doped samples are degraded by some separate or additional mechanism.</p><p>The as-synthesized nanocrystals and the products after the ten oxidation and reduction treatment as described above were analyzed by powder X-ray diffraction. The results are shown in Figure 5.</p><p>Reference XRD patterns are shown for bixbyite V2O3 in black, the thermodynamically preferred corundum phase of V2O3 in red, rutile VO2 in green, and rutile TiO2 in turquoise, all at the bottom.</p><p>The samples show good crystallinity before TGA cycling and exist in the bixbyite crystal structure, apart from highly Mn-doped V2O3, which shows only very broad diffraction signal. Two peaks in the pattern for 10% Ti:V2O3, at 2θ of 28.6 o and 47.5 o (marked with a star in Figure 5), were not indexed to a known phase of titanium or vanadium oxide. For samples doped up to 5%, only bixbyite V2O3 peaks are observed. After ten cycles of oxidation and reduction, the crystallinity in both the Mn-doped samples has all but disappeared, indicating degradation to form some noncrystalline product. The undoped and Ti-doped samples lose intensity from the original bixbyite phase and develop new peaks due to crystalline impurities. The major impurities are from the thermodynamically preferred corundum structure of V2O3 and rutile VO2. While diffraction peaks of rutile TiO2 could not be differentiated from those of VO2, the amount of titanium incorporated into the material is quite small, so we expect that these peaks appear due to the formation of rutile VO2. To investigate the different mechanisms of oxidation and reduction for doped V2O3, in situ Xray diffraction was conducted. In Figure 6, the oxidation half-cycle is shown in reciprocal space for q between 3 and 4.5 A -1 . The strongest peak around 3.7 A -1 is the bixbyite (440) reflection, which appears for the ex situ diffraction at 2θ of about 55 o . At time 0, the sample is exposed to synthetic air at 150 o C. Shortly, the bixbyite reflections are seen to move slowly to lower q, indicating lattice expansion that is nearly isotropic. Peaks shown that do not shift are reflections from the sample holder stage. Some weakening in the intensity of those peaks is observed, which may indicate very slight movement of the sample due to thermal expansion of the sample holder during oxidation. We also observe that reflection peaks for the Mn-doped samples are much broader, consistent with ex situ observations showing Mn-doped samples have poorer crystallinity than their Ti-doped counterparts. Notably, the Ti-doped sample in panel b shows a splitting of the (440) peak after about 25 minutes of oxidation. This splitting is attributed to the phase transformation of bixbyite V2O3 to form rutile VO2, as discussed above. By contrast, the peak shifts to lower q for the Mn-doped case are not accompanied by formation of any new crystalline impurity phases. Rather, this transition is partially reversible upon heating to 300 o C under nitrogen.</p><p>Unabridged in situ diffraction for the oxidation and reduction of the Mn:V2O3 nanocrystals is shown in Figure S6, and for the full q range collected of oxidation of the Ti:V2O3 nanocrystals in Figure S7. All the V2O3 samples show a lattice expansion upon oxidation, as evidenced by bixbyite peaks moving to lower q during oxidation. Scherrer analysis of the ( 222), (440), and (622) reflections was conducted before, during, and after oxidation to assess the extent of lattice expansion. In the fully oxidized state, Ti-doped nanocrystals showed a 1.3% expansion, while Mndoped nanocrystals showed 1.6% expansion, compared to 0.6% expansion for undoped V2O3 seen in a previous study. 3 The main difference in these two doped materials appears to be their degradation mechanism during repeated cycling. These observations support the conclusion that doped V2O3 nanocrystals follow two different degradation mechanisms depending on the dopant.</p><p>For Mn-doped materials, oxidation causes disordering of the crystalline structure to form amorphous vanadates and manganates, which have much less storage capacity than the original vanadium oxide lattice. In contrast, Ti-doped materials experience an irreversible phase transition to monoclinic VO2 during oxidation that causes loss in OSC. This difference may be due to the Ti 4+ dopant's positive relative charge, which may stabilize oxygen interstitials, facilitating a transformation to the higher oxidation state vanadium oxide.</p><!><p>Vanadium sesquioxide nanocrystals were synthesized with titanium and manganese dopants.</p><p>These are incorporated as aliovalent dopants Ti 4+ and Mn 2+ . Doped materials were compared to the undoped by thermogravimetric analysis. Cycling these materials between oxidation and reduction ten times revealed that cycling these oxygen storage materials results in degradation of the oxygen storage capacity. This degradation is driven primarily by the irreversible oxidation of the materials during the low temperature oxidation step. Furthermore, we have found that Mndoped and Ti-doped V2O3 nanocrystals degrade by different mechanisms. For undoped and Mndoped materials, the nanocrystals tend to degrade by amorphizing the initially crystalline bixbyite phase material to form an impurity phase of inactive amorphous material. For Ti-doped V2O3, there is an additional degradation method in which the nanocrystals undergo irreversible phase transition to form rutile VO2 during oxidation, perhaps promoted by Ti 4+ , which may promote filling of oxygen vacancies because of its charge imbalance and the fact that Ti itself forms a rutile oxide. This degradation progresses over all ten cycles, resulting in an OSC that declines approximately linearly with cycle number. Aliovalent dopants may drive degradation products to a structure that the oxide of the dopant shares with a vanadate or to amorphous materials in case no favorable crystalline polymorph exists between the dopant and vanadium oxide. Future work might therefore seek a dopant that shares a structure with a vanadate which is a stable oxygen deficient structure. Cerium and indium may be prime candidates for this, since cerium oxide is a known oxygen ion conductor and indium shares the bixbyite structure in common with vanadium sesquioxide.</p><!><p>Doped bixbyite-phase vanadium sesquioxide materials were synthesized by modifying the reported vanadium sesquioxide nanocrystal synthesis, replacing some amount of vanadyl acetylacetonate with an equimolar amount of a metal dopant precursor. 2 For the titanium-doped samples, the metal dopant precursor was Ti (IV) oxyacetylacetonate. For the manganese-doped samples, the precursor was Mn (II) acetylacetonate. Nominal dopant percentages indicate the percent of vanadyl acetylacetonate that was replaced with the dopant metal precursor. Synthesis was done with 1 mmol of metal precursor, 4 mmol oleic acid, 4 mmol oleylamine, and 8 ml squalene. Chemicals were loaded into a 50 ml round-bottom 3-neck flask and degassed under dynamic vacuum at 110 o C for an hour before switching to a nitrogen atmosphere and increasing the temperature to the synthesis temperature of 370 o C. This temperature was held for 1 hour before cooling to room temperature. Nanocrystals were washed several times with isopropanol and hexane before use or characterization. Solutions were washed and stored air-free, powder or dry samples stored in nitrogen or in a vacuum dessicator.</p><!><p>TGA was collected with a Mettler Toledo TGA 2. Samples were dropcast from solution into 100 ul aluminum crucibles and allowed to dry. Multiple depositions were used until the total weight of sample in the crucible was between 5 and 10 mg.</p><!><p>Doped and undoped samples were digested with 70% nitric acid and diluted to contain 2% nitric acid. ICP AES was collected using a Varian 720-ES ICP AES.</p><!><p>TEM images were obtained using a JEOL 2010F electron microscope equipped with a Schottky field emission gun and a CCD camera, operated at 200 kV.</p><!><p>As-synthesized colloidal particles were dropcast onto p-type doped silicon substrates, and X-ray photoemission was collected with a Kratos Axis Ultra DLD spectrometer with a monochromatic Al Kα source (1486.6 eV). Resulting spectra were analyzed with CasaXPS equipped with the Kratos library of elemental relative sensitivity factors. Spectra were treated with a simple constant correction to calibrate the main carbon 1s peak to an energy of 284.8 eV.</p><!><p>In situ X-ray diffraction of doped vanadium sesquioxide materials was performed at the Stanford Synchrotron Radiation Lightsource in Stanford, California. Using the Anton Paar in situ heating cell, powder samples of vanadium sesquioxide materials deposited on silicon substrates were irradiated with an X-ray beam with an energy of 14 eV. The diffraction image was collected with a Pilatus 300k detector in landscape orientation and analyzed using the Nika and Irena X-ray analysis package with Igor. 32,33,34,35 The sample was reduced in situ under constantly flowing nitrogen gas at 300 o C for one hour before the experiment began, to remove any adventitious carbon or water on the sample's surface. The sample was then cooled to 50 o C in nitrogen, at which point the flowing gas was changed to a synthetic air mixture. In the air atmosphere, the sample was heated slowly from 50 o C to 150 o C and held at 150 o C for one hour. Then the gas was switched to nitrogen and the sample was heated to 300 o C and held at 300 o C for one hour.</p><p>Ex situ X-ray diffraction measurements were obtained using a Rigaku R-Axis Spider diffractometer with a Cu sealed tube source and a large, image plate detector.</p>

ChemRxiv

Carrier Dynamics Engineering for High-Performance Electron-Transport-Layer-free Perovskite Photovoltaics

The performance of electron-transport-layer-free (ETL-free) perovskite photovoltaics is far more sensitive to the carrier lifetime of perovskite films than analogous ETL-containing devices. A long carrier lifetime can counterbalance the inferior interface in the absence of a distinct ETL, enabling efficient carrier collection in ETL-free perovskite PV. By using perovskite films with microsecond carrier lifetime, Hu and co-workers achieved ETL-free solar cells with >19.5% power conversion efficiency, which is comparable to the analogous value of ETLcontaining devices.

carrier_dynamics_engineering_for_high-performance_electron-transport-layer-free_perovskite_photovolt

INTRODUCTION<!>ETL-free and ETL-Containing PV<!>Impact of Carrier Lifetimes on PV Performance<!>Perovskite Film with Microsecond Carrier Lifetimes<!>High-Performance ETL-free Devices<!>Conclusion<!>EXPERIMENTAL PROCEDURES Perovskite Film Deposition<!>Characterizations<!>SUPPLEMENTAL INFORMATION

<p>Perovskite photovoltaics (PV) has attracted tremendous attention because of recent advances in power conversion efficiency (PCE) and diverse processing options. [1][2][3][4][5][6][7][8] Other than high performance, factors including good stability, simpler device configuration, and low processing cost should be considered in the next-generation perovskite PV. Currently, high-performance perovskite solar cells exclusively employ p-i-n device architectures, [5][6][7] wherein distinct electron transport (n-type) and hole transport (p-type) layers are generally viewed as critical components for reliable photogenerated carrier extraction. 9 However, the deposition processes to construct a p-i-n architecture, especially for inorganic electron-transport layers (ETLs), typically require high-temperature conditions (e.g., $500 C for TiO 2 , $500 C for BaSnO 3 , and $250 C for ZnO). 7,[10][11][12] Such high-temperature steps compromise the low-cost advantage of solution-based approaches for perovskite film deposition (e.g., generally performed at 25 C-100 C), leading, for example, to increased projected payback time for perovskite PV and other optoelectronics. 13 Additionally, in building more complex optoelectronic devices, such as all-perovskite tandem solar cells, the high temperatures to process ETLs in top cells can damage the perovskite and/or other temperature-sensitive films in bottom cells. The high temperatures can also melt prospective flexible substrates and/or cause The Bigger Picture Interface properties essentially determine the performance of perovskite photovoltaics (PV). Typical electron-transport-layerfree (ETL-free) perovskite PV suffers from significant loss of performance as a result of inferior carrier dynamics at the interface. Here, we determine that the low efficiency of ETL-free perovskite PV is attributed to insufficient photoexcited carrier collection, which originates from the inhibited carrier injection at the interface without a distinct ETL. We found that improving the carrier lifetimes of perovskite films can effectively counterbalance the low-injection-rate interface (e.g., ETL-free interface), enabling efficient carrier collection in ETLfree perovskite PV. By using perovskite films with microsecond carrier lifetimes, we achieved ETLfree PV with >19% efficiency, which makes ETL-free devices comparable to p-i-n-structured perovskite devices. These results indicate a general approach to improving the performance of PV devices with inferior interfaces. ion migration from substrates, limiting the applications of perovskite technology in versatile optoelectronics. More importantly, the existence of some ETLs themselves can detrimentally affect the perovskite devices. [14][15][16][17] For example, ZnO ETLs can cause decomposition of perovskite layers during annealing 15 and TiO 2 ETLs can induce degradation of perovskite films under illumination. 16 Regardless of these performance issues, the inclusion of numerous layers in devices is not desired for effective commercialization. In short, these ETL-induced issues lead to prospects of lower processing yields, increased payback times, faster degradation, and difficulties in constructing more versatile and complex perovskite optoelectronics. To address these issues, building solar cells without distinct ETLs in the architectures is a promising direction for next-generation perovskite PV. In fact, studies have demonstrated that perovskite films possess superior properties for charge-carrier dissociation 18 and transport, 19,20 and that the transparent electrodes (e.g., fluorine-doped tin oxide glass [FTO] and tin-doped indium oxide glass [ITO]) themselves are typically n-type semiconductors. Therefore, distinct ETLs should not theoretically be necessary for high-performance perovskite PV. Unfortunately, all current-generation ETL-free perovskite solar cells suffer from low PCE, with relatively large hysteresis and inferior stability. [21][22][23][24][25][26][27][28][29] In this regard, creating deeper understanding and developing effective approaches to improving the performance are keys for the success of ETL-free perovskite PV.</p><p>In this work, we reveal that the carrier injection process is significantly inhibited at the interface in the absence of an ETL, which leads to insufficient carrier collection and severe interfacial carrier recombination. The recombination decreases the external quantum efficiency (EQE) in both short-and long-wavelength ranges and thereby compromises the performance for ETL-free perovskite PV. Moreover, we demonstrate that improving the intrinsic carrier lifetimes in perovskite films can counterbalance the inferior device interfaces and carrier recombination at the ETL-free interface. Through such carrier dynamics engineering, the carrier collection efficiency in the ETL-free perovskite PV can be remarkably tailored to approach that in ETL-containing devices. Benefiting from this discovery, we exploit perovskite films with microsecond carrier lifetimes to successfully realize ETL-free perovskite PV with a best PCE of 19.52% (18.48% on average), nearly eliminated hysteresis, and good stability. Such a high PCE is comparable to the value (20.7%) achieved for ETL-containing solar cells prepared with analogous perovskite films. Our research provides insights into ETL-free solar cells and points to a promising direction for perovskite PV and analogous optoelectronic devices, offering simultaneously high performance, simplified processing, and better prospects for ultra-low-cost device fabrication.</p><!><p>To identify the difference between typical ETL-free and ETL-containing PV, we used pristine CH 3 NH 3 PbI 3 (MAPbI 3 ) perovskite films as absorbers. We deposited the pristine MAPbI 3 films by a one-step method employing a precursor solution containing a 1:1 molar ratio of MAI and PbI 2 . ITO-coated glass was used as the transparent electrode substrate. In the ETL-containing PV devices, SnO 2 was chosen as the distinct ETL because of the wider bandgap of SnO 2 (e.g., compared with TiO 2 ), such that the distinct ETL would minimally affect the light absorption within the perovskite films during device operation (as discussed later). 30 The full details of film deposition are described in the Experimental Procedures. Top-view scanning electron microscopy (SEM) images in Figures 1A and 1B present the morphologies of typical pristine MAPbI 3 films on glass/ITO and glass/ITO/SnO 2 substrates, respectively, from which one can deduce that the MAPbI 3 films share similar film morphologies and compactness on both substrates. From X-ray diffraction (XRD) patterns (Figure S1), the MAPbI 3 films exhibit similar structural properties on both substrates. The optical absorption spectra (Figure S2) illustrate that the MAPbI 3 films also have similar optical features (E g = $1.60 eV). These results indicate that glass/ITO and glass/ITO/SnO 2 substrates do not affect the basic structural/optical properties of MAPbI 3 films. In many respects this observation is not surprising, given that both SnO 2 and ITO present a tin-oxide-based surface.</p><p>To examine the PV performance for such MAPbI 3 films in devices, we fabricated ETL-free and ETL-containing perovskite PV devices with the device architectures of glass/ITO/MAPbI 3 /2,2 0 ,7,7 0 -tetrakis[N,N-di(4-methoxyphenyl)amino]-9,9 0 -spirobifluorene (Spiro-OMeTAD)/Au and glass/ITO/SnO 2 /MAPbI 3 /Spiro-OMeTAD/Au, respectively. Figure 1C presents the statistical distributions of the PCE values, from which the ETL-free solar cells yield PCE from 7.70% to 11.40% (8.68% on average) for 24 devices, while ETL-containing solar cells yield PCE from 15.78% to 17.99% (16.97% on average) for 24 devices. The statistical results indicate that the PCE of a typical ETL-free PV device is significantly lower than that of a typical ETLcontaining device when using the pristine MAPbI 3 films, which is consistent with the earlier reports. 21,22 To ascertain the reasons for the compromised PCE, we compared the EQE for ETL-free and ETL-containing perovskite PV. From the EQE spectra (Figure 1D), the EQE response of the ETL-free device falls significantly lower than that for the analogous ETL-containing device, especially in the short (300-400 nm) and long (500-750 nm) wavelength ranges. Such results indicate that the carrier collection is less effective in ETL-free devices (e.g., owing to enhanced recombination in the ETL-free device structures). Since the glass/ITO and glass/ITO/SnO 2 substrates have nearly the same optical absorption features (Figure S3) and the perovskite films have similar grain structures, the reduced carrier collection in ETL-free devices likely derives from the different interfaces. Previous study attributed this device performance loss to the lack of effective hole blocking. 22 From this opinion, because of the absence of a distinct ETL, photogenerated holes can easily reach the ITO/MAPbI 3 interface and ITO contains a high density of electrons that would facilitate the recombination of photogenerated holes at this interface, eventually leading to the loss of photogenerated carriers and device performance. However, one study showed that the work function of ITO (À4.7 eV) is higher than the Fermi level of MAPbI 3 . 31 Therefore, The band-bending direction of MAPbI 3 films at the ITO/MAPbI 3 interface is downward and the built-in electric field formed at the interface points from ITO to MAPbI 3 film, which attracts electrons and repels holes photogenerated in MAPbI 3 films (shown in Figure S4). In addition, ITO is a typical n-type semiconductor whose valence band maximum edge is lower than that of perovskite materials. Such situations make the as-formed ITO/MAPbI 3 interface preferable for electron extracting and hole blocking. Based on this analysis, the performance loss in ETL-free perovskite PV may not be due to the lack of hole blocking.</p><p>To understand the impact of the ITO/MAPbI 3 interface on photogenerated carriers, we performed photoluminescence (PL) quenching measurements. Figure 1E presents the steady-state PL spectra for the MAPbI 3 films on quartz, glass/ITO, and glass/ITO/SnO 2 , respectively, from which we can observe that the PL signal quenches less on the ETL-free substrates. The quenching results evidently illustrate that the interfacial carrier injection process is substantially inhibited without the assistance of a distinct ETL, which suggests that the compromised performance of ETL-free perovskite PV originates from the inhibited carrier injection on the ITO substrate. One study indicated that the work function/Fermi level of SnO 2 is around À4.36 eV, which is higher than the work function of ITO. 30 Without the SnO 2 ETL in the devices, the interfacial built-in electric field may become weaker because of the relatively lower work function of ITO. The inhibited injection could be derived from this weaker built-in electric field.</p><p>At or near the interface, if the carriers photogenerated in perovskite films cannot be rapidly collected, these carriers will undergo recombination (e.g., radiative and/or nonradiative recombination) in the perovskite films. From the time-resolved photoluminescence (TRPL) result (Figure 1F), the pristine MAPbI 3 films used in typical ETL-free PV exhibit short average carrier lifetimes (t avg = 1.17 G 0.02 ns). We refer to such lifetimes as ''intrinsic'' lifetimes since they are measured for films on quartz substrates rather than for films within device structures, wherein built-in electric fields will affect the values measured. The photogenerated carriers with such short intrinsic lifetimes will more readily recombine at or near the interface if the built-in electric field cannot cause them to drift to the interface and rapidly across the interface to the contact.</p><p>For reducing the carrier recombination near the low-injection-rate interface, the typical approach involves adding an additional layer at this poor interface to provide a strong built-in electric field to quickly drift the carriers across the interface for extraction (ultimately, before recombination). However, this approach adds extra layers to the ETL-free architectures and creates an architecture that is essentially identical to that for the ETL-containing devices. Logically, if we are not able to improve the interfacial band alignment to enhance carrier drifting, the next best thing would be to improve the carrier lifetimes within the perovskite films so that the photogenerated carriers can survive long enough to diffuse to the interface and make it across the less effective interface. Based on this analysis and expectation, increasing the lifetimes for the carrier diffusion could be an effective method to compensate the weaker ''drifting ability'' (e.g., built-in electric field), which is highly desired for ETL-free PV (even more so than in ETL-containing PV). In the following section, we explore the effect of intrinsic carrier lifetimes in perovskite films on the performance of ETL-free PV devices.</p><!><p>The effect of the intrinsic carrier lifetimes on the performance of ETL-free perovskite PV was explored by introducing extra PbI 2 into the MAPbI 3 films to tune the film carrier lifetimes. 10,13 Top-view SEM images (Figure S5) show that the MAPbI 3 films with 2.5%, 5.0%, and 7.5% extra PbI 2 on glass/ITO substrates have grain size similar to that in the pristine MAPbI 3 film as shown in Figure 1A. From the atomic force microscopy (AFM) images in Figure S6, the MAPbI 3 films share similar surface roughness, within the range of 6-8 nm, illustrating that adding extra PbI 2 to the MAPbI 3 films does not significantly affect film morphology. The optical absorption spectra (Figure S7) indicate that adding 0%-7.5% extra PbI 2 in MAPbI 3 films does not remarkably affect the absorption characteristics of the films.</p><p>We performed TRPL measurements for the MAPbI 3 films on quartz substrates to evaluate the optoelectronic properties of the MAPbI 3 films with extra PbI 2 . By comparing the TRPL kinetic traces in Figures 2A and 1F, it can be observed that adding extra PbI 2 significantly increases the intrinsic carrier lifetimes of the MAPbI 3 films, from several nanoseconds to several hundred nanoseconds. To further pursue this point, we also performed steady-state PL measurements on the MAPbI 3 films (Figure 2B), showing significant intensity enhancement with extra PbI 2 addition, which is consistent with the TRPL results. In addition to the intrinsic carrier lifetimes, we also measured the steady-state PL for the MAPbI 3 films with extra PbI 2 on glass/ITO substrates to examine whether extra PbI 2 in MAPbI 3 films may change the carrier injection rate at the ITO/MAPbI 3 interface (as shown in Figure S8). Since the PL intensity of the MAPbI 3 films significantly varies with different PbI 2 levels, we define a PL quenching rate metric (QR PL )-i.e., QR PL = (I 1 À I 2 )/I 1 , where I 1 is the PL intensity of the MAPbI 3 film on quartz while I 2 is the analogous value on ITO-to better evaluate the carrier injection at the ITO/perovskite interface. By comparing the QR PL values (Figure S9), it is seen that the extra PbI 2 itself in MAPbI 3 films does not enhance carrier injection yield at the ITO/perovskite interface, as underscored by the extent of PL quenching. This result is reasonable since the relative conduction band position of PbI 2 is higher than that of perovskite materials so that prospective extra PbI 2 accumulation at the ITO/MAPbI 3 interface would not improve the band alignment for electron injection. 32 Taken together, the TRPL and PL results indicate that addition of extra PbI 2 significantly improves the carrier lifetimes in the MAPbI 3 films but does not facilitate the electron-injection rate at the ITO/MAPbI 3 interface. 10,13 To show the correlation between the intrinsic carrier lifetimes and performance for ETL-free perovskite PV, we fabricated solar cells by using extra-PbI 2 -added MAPbI 3 films with the same ETL-free architecture (glass/ITO/MAPbI 3 /Spiro-OMeTAD/Au) described above. For each PbI 2 addition level, 24 devices were used for acquiring the statistical results shown in Figures 2C and S10. It can be observed that the trend of all the PV parameters is similar to the trend of carrier lifetimes as the PbI 2 addition level changes, illustrating close correlation between the device performances and the intrinsic carrier lifetimes, and that long carrier lifetimes substantially benefit the ETL-free device performance. For comparison, ETL-containing PV devices were also fabricated. From the PCE distributions shown in Figure S11, the intrinsic carrier lifetimes are also seen to affect the performance of ETL-containing PV. To explore the degree to which the carrier lifetimes can impact on the ETL-free and ETL-containing perovskite solar cells, respectively, we calculated the correlations between extra PbI 2 addition levels and average PCE values. From Figures 2D and S12, it is found that the performance of ETL-free devices is substantially more sensitive to the intrinsic carrier lifetimes than ETL-containing PV. We also measured the EQE for the ETL-containing and ETL-free PV devices (Figures 2E and 2F) to better understand the impact of intrinsic carrier lifetimes on photogenerated carrier collection. We observed that as carrier lifetimes increased, both the short-and long-wavelength spectral responses substantially improved for ETL-free PV, whereas only the long-wavelength range increased for ETL-containing PV. For ETL-containing PV (Figure 2E), the EQE increase in the long-wavelength range can be attributed to the reduction of bulk-defect-induced recombination, enabling more long-wavelength-excited carriers to travel through the perovskite films for extraction. For ETL-free devices (Figure 2F), the additional enhancement of short-wavelength EQE could be due to reduced carrier recombination at the ETL-free interface, leading to increasing numbers of short-wavelength-excited carriers being effectively collected at the ITO/perovskite interface. The EQE spectra explain the result that the performance of ETL-free PV is more sensitive to carrier lifetimes. Moreover, comparing the EQE spectra shown in Fig- ure 2G, we see that the EQE values for the ETL-free PV progressively converge to that for ETL-containing PV for long carrier lifetimes, totally different from the situation for short carrier lifetimes (Figure 1D). Long carrier lifetimes in perovskite films therefore appear to be important for obtaining high-performance ETL-free PV. In addition, these results also suggest that use of distinct ETLs is beneficial for the device performance if the intrinsic carrier lifetimes in the associated perovskite absorbers are relatively short.</p><!><p>For further boosting the PCE of ETL-free PV, mixed-cation lead mixed-halide perovskite films (Cs 0.05 FA 0.8 MA 0.15 PbI 2.55 Br 0.45 , referred to as CsFAMA, where FA = formamidinium) with microsecond carrier lifetimes were used as the light absorbers in devices (see the Experimental Procedures for deposition details). 33 The top-view SEM image of a CsFAMA film on glass/ITO substrate (Figure 3A) reveals that the grain size is $500 nm, with good compactness and coverage over a large area (Figure S13). According to the AFM image (Figure S14), the CsFAMA films exhibit flat surfaces with roughness on the order of 20 nm. The relatively flat surface reduces the contact area at the CsFAMA/Spiro-OMeTAD interface, leading to reduced interfacial recombination and therefore benefiting the device performance.</p><p>The XRD pattern (Figure 3B) for a CsFAMA film on glass/ITO substrate demonstrates that the CsFAMA film contains only the photoactive perovskite a phase (black phase), and no nonperovskite d phase (yellow phase) exists to negatively affect the optoelectronic properties. To evaluate the optical properties of the CsFAMA film, we performed optical absorption and steady-state PL measurements (Figure 3C), which demonstrate that the optical absorption onset and PL peak are consistent (E g = $1.59 eV shown in Figure S15). To understand the carrier dynamics, we acquired TRPL data for a CsFAMA film on quartz substrate. The intrinsic carrier lifetimes extracted from the PL dynamics (Figure 3D) are t 1 = 1,231.9 G 27.7 ns and t 2 = 301.9 G 28.3 ns; these values have respective amplitudes A 1 = 71.4% and A 2 = 28.6% (t avg is 966.4 G 27.9 ns). Such microsecond carrier lifetimes, approximately three times the value achieved for the previously shown MAPbI 3 film with 5% extra PbI 2 , are expected to substantially boost the performance of ETL-free perovskite PV, as discussed above.</p><!><p>Given the above results, ETL-free perovskite solar cells were fabricated with the device architecture ITO/CsFAMA/Spiro-OMeTAD/Au. From the SEM image of the device cross-section (Figure 4A), the thicknesses of the CsFAMA, Spiro-OMeTAD, and Au layers are seen to be $650, $150, and $80 nm, respectively. The statistical distributions of PCE values (reverse scanning direction) for 24 ETL-free devices (Figure 4B) vary from 17.85% to 19.52% (18.48% on average). For comparison, analogous ETL-containing perovskite solar cells with an ITO/ SnO 2 /CsFAMA/Spiro-OMeTAD/Au structure yielded similar performance levels (Figure 4B), such that PCE values vary from 19.60% to 20.72% (20.03% on average). To evaluate the hysteresis behavior, we measured the best-performing devices by using both forward and reverse voltage-scanning directions. From the reverse (forward) scan, the best-performing ETL-free device (Figure 4C) yielded a PCE of 19.52% (18.84%), open-circuit voltage (V oc ) of 1.061 (1.069) V, shortcircuit current density (J sc ) of 23.61 (23.39) mA cm À2 , and fill factor (FF) of 77.79% (75.37%). The best-performing ETL-containing device (Figure S16) yielded a PCE of 20.72% (20.51%), V oc of 1.100 (1.100) V, J sc of 23.75 (23.51) mA cm À2 , and FF of 79.28% (79.23%). We can see that the ETL-free device hysteresis approaches that of the ETL-containing device.</p><p>The EQE spectrum of the best-performing ETL-free device (Figure 4D) illustrates high quantum efficiency for energies above the band gap, leading to an integrated J sc of 23.39 mA cm À2 , which is consistent with the results from the current densityvoltage (J-V) characteristics. The steady-state output profile (Figure 4E) shows that the best-performing ETL-free device has a steady-state output current density (J) of $21.37 mA cm À2 under 0.89 V applied bias, corresponding to a stabilized PCE value of $19.02%-i.e., showing good agreement with the J-V measurement. Such a performance level for the ETL-free devices approaches record metrics for any type of perovskite devices and represents the best PCE performance level among all currently reported ETL-free perovskite solar cells to date.</p><p>The correlations between intrinsic carrier lifetimes and device performance were examined for ETL-free/ETL-containing PV (Figure 4F). The results indicate that the PCE discrepancy between ETL-free and ETL-containing perovskite solar cells is significantly reduced as the carrier lifetime increases, and further suggest that extending the carrier lifetimes of perovskite films (e.g., >>1 ms) may boost the PCE of associated ETL-free perovskite solar cells to the same level as ETL-containing perovskite solar cells. Moreover, the PCE of the best-performing ETL-free device remains $19.1% (from J-V measurement) after 1,000 hr of storage (temperature of $25 C and relative humidity of $25% in the dark), illustrating that the ETL-free devices have good environmental stability (Figure S17). The photostability of the best-performing ETL-free perovskite solar cells was also measured using continuous light soaking (one sun) under ambient conditions (temperature of $25 C and relative humidity of $25%) without encapsulation (Figure S18). Such stability tests suggest that ETL-free PV with CsFAMA perovskite films having good stability can be made.</p><!><p>In conclusion, we use EQE to reveal that typical ETL-free perovskite solar cells with relatively low carrier lifetimes in perovskite films exhibit more substantial photogenerated carrier loss compared with ETL-containing devices. PL quenching experiments show that the injection of carriers from the perovskite to the transparent conducting oxide contact (ITO) is less effective for ETL-free devices. To address this interface issue without changing the ETL-free device design, we tailor the carrier lifetimes of the perovskite films and demonstrate that improved carrier lifetimes can enhance the carrier collection efficiency at the low-injection-rate interface, making the carrier dynamics in ETL-free devices essentially as good as those in ETL-containing devices. On the basis of such an understanding, the use of perovskite films with microsecond carrier lifetimes enables ETL-free perovskite solar cells to realize a best PCE of 19.52% with nearly eliminated hysteresis and good stability. Such high-performance ETL-free perovskite solar cells are comparable to the analogous ETLcontaining devices (PCE: 20.72%). These results offer opportunities for versatile perovskite PV with simple processing, low cost, and high performance. Our results also provide a general approach to improving the performance of PV with low-injection-rate interfaces not only limited to the perovskite PV family.</p><!><p>For deposition of the pristine MAPbI 3 films, precursor solutions were prepared with 1.2 M PbI 2 and 1.2 M MAI in the DMF/DMSO co-solvent (V DMF /V DMSO = 9:1). Extra PbI 2 with levels of 0%, 2.5%, 5%, and 7.5% (mol %, relative to stoichiometric MAPbI 3 ) was added into the MAPbI 3 precursor solutions, respectively, to tune the carrier lifetimes in the MAPbI 3 films. For each system, the precursor solutions were stirred at $25 C for 24 hr and filtered with a 0.45-mm PTFE syringe filter before further use. The MAPbI 3 films were then deposited by spin-coating the precursor solution on substrates at 5,000 rpm for 30 s. Chlorobenzene (1.5 mL) was poured on the surface of the MAPbI 3 film $5 s after commencing spin-coating. The as-deposited MAPbI 3 films were annealed at 100 C for 10 min to form the resultant films. For the Cs 0.05 FA 0.80 MA 0.15 PbI 2.55 Br 0.45 (CsFAMA) films, a 1.2 M precursor solution was prepared with 0.06 M CsI, 0.96 M FAI, 0.18 M MABr, 1.02 M PbI 2 , and 0.18 M PbBr 2 dissolved in DMF/DMSO co-solvent (V DMF /V DMSO = 4:1) and 10% extra PbI 2 (mol %, relative to CsFAMA) was added to the CsFAMA precursor solution. 7,34 The precursor solution was stirred at $25 C for 24 hr and filtered with a 0.45-mm PTFE syringe filter before further use. To obtain the CsFAMA film, we spin-coated the CsFAMA precursor solution on the substrates at 2,000 rpm for 10 s and 4,000 rpm for 20 s, respectively. During the second step, 1.5 mL of chlorobenzene was poured on the top surface of the CsFAMA film $5 s before the end of the spin cycle. The as-deposited films were then annealed on a hotplate at 100 C for 10 min to form the resultant CsFAMA films. All the preparation and deposition steps were performed in a nitrogen-filled glovebox.</p><p>Device Fabrications ITO-coated glass substrates (10 U/sq) were cleaned in soapy water, deionized water, acetone, and isopropanol with sonication. The ITO-coated glass substrates were then subjected to ultraviolet-ozone (UVO) treatment for 10 min. For the ETL-free perovskite solar cells, the perovskite films were directly deposited on the ITO substrates according to the procedure mentioned above. Li-doped Spiro-OMeTAD was then spin-coated as the hole-transporting layers on the perovskite films. A solution consisting of 72.5 mg of Spiro-OMeTAD, 28.8 mL of 4-tert-butylpyridine, 17.6 mL of Li-bis(trifluoromethanesulfonyl)imide (Li-TFSI) solution (520 mg Li-TFSI in 1 mL of acetonitrile), and 1 mL of chlorobenzene was employed with a spin speed of 3,000 rpm for 30 s. To complete the device, Au was thermally evaporated on the hole-transporting layers to serve as the electrode. For the devices with SnO 2 ETLs, the SnO 2 ETLs were deposited by spin-coating a SnO 2 suspension (15 wt % in H 2 O) in air on the UVO-treated ITO substrates and then annealing on a hotplate at 150 C for 20 min to form $20-nm-thick ETLs in air. 30 The glass/ITO/SnO 2 substrates were then treated with UVO for 10 min. Finally, the perovskite, Li-doped Spiro-OMeTAD and Au layers were sequentially deposited to complete the ETLcontaining perovskite solar cells by following the procedures described above for the ETL-free devices.</p><!><p>Morphologies of the CsFAMA and MAPbI 3 films were imaged with a scanning electron microscope (FEI XL-30 SEM-FEG). AFM images were characterized using a scanning probe microscope (Digital Instruments Dimension 3100). XRD measurements were carried out on a PANalytical Empyrean Powder X-ray diffractometer using Cu Ka radiation. The charge-carrier lifetimes were characterized via TRPL experiments using an Edinburgh FLS980 fluorescence spectrometer with excitation wavelength of 510 nm. The steady-state PL was also measured with the Edinburgh FLS980 fluorescence spectrometer with excitation wavelength of 510 nm. Optical absorption measurements were performed on a Shimadzu UV-3600 spectrophotometer. The EQE was taken using a QE-R instrument from Enlitech without bias voltage. The J-V characteristics and steady-state output were measured using a Keithley 2420 source meter. The illumination source was a Newport Oriel 92192 solar simulator with an AM 1.5G filter, operating at 100 mW cm À2 . All devices were masked with area aperture of 0.09 cm 2 to define the active areas. All the J-V characteristics of perovskite solar cells were evaluated with voltage-scanning speed of 1.0 V/s. A standard silicon solar cell from Newport Corp was used as a reference for J-V and EQE measurements. All measurements were performed under ambient conditions with relative humidity level below 30%.</p><!><p>Supplemental Information includes 18 figures and can be found with this article online at https://doi.org/10.1016/j.chempr.2018.08.004.</p>

Chem Cell

Bilateral common carotid artery ligation transiently changes brain lipid metabolism in rats

Brain lipid metabolism was studied in rats following permanent bilateral common carotid artery ligation (BCCL), a model for chronic cerebral hypoperfusion. Unesterified (free) fatty acids (uFA) and acyl-CoA concentrations were measured 6 h, 24 h, and 7 days after BCCL or sham surgery, in high energy-microwaved brain. In BCCL compared to sham rats, cPLA2 immunoreactivity in piriform cortex, and concentrations of total uFA and arachidonoyl-CoA, an intermediate for arachidonic acid reincorporation into phospholipids, were increased only at 6 h. At 24 h, immunoreactivity for secretory phospholipase A2 (sPLA2), which may regulate blood flow, was increased near cortical and hippocampal blood vessels. BCCL did not affect difference brain IB4+ microglia, glial fibrillary acidic protein (GFAP)+ astrocytes, cyclooxygenase-2 (COX-2) immunoreactivity at any time, but increased cytosolic cPLA2 immunoreactivity in one region at 6 h. Thus, BCCL affected brain lipid metabolism transiently, likely because of compensatory sPLA2-mediated vasodilation, without producing evidence of neuroinflammation.

bilateral_common_carotid_artery_ligation_transiently_changes_brain_lipid_metabolism_in_rats

Introduction<!>Chemicals<!>Animals<!>Brain lipid extraction and chromatography<!>Analysis of Acyl-CoA<!>Histology<!>Statistics<!>Unesterified fatty acids (uFAs)<!>Acyl-CoAs<!>Immunochemistry<!>Discussion

<p>The brain has a high demand for oxidation of glucose for the synthesis of adenosine triphosphate (ATP), and pathological changes occur when oxygen delivery is chronically impaired [1–3]. Reduction of cerebral blood flow (CBF) limits oxygen delivery and causes cerebral ischemia. Chronic CBF reduction is major cause of vascular dementia [4–6], which accounts for approximately 20% of age related dementias [4]. Low CBF also is considered an aggravating factor for Alzheimer disease [7], and for dementia associated with sleep apnea [8]. In Alzheimer disease and vascular dementia, the severity and persistence of cerebral hypoperfusion correlate with the severity of cognitive impairment [9–11].</p><p>Complete interference of the carotid or vertebro-basilar circulation in rodents has identified vulnerability of specific brain regions, including the hippocampus and frontal cortex [12, 13]. While the severity of brain damage following acute CBF reduction is well established, less is known about effects of chronic CBF insufficiency. Permanent occlusion of both common carotid arteries in the rat (bilateral common carotid artery ligation (BCCL)) has been used as an experimental model for chronic cerebral hypoperfusion [14]. After BCCL, CBF immediately declines to 30–60% of its control value, but recovers to approximately ~63% and ~90% of control at four- and eight-weeks, respectively [15, 16].</p><p>BCCL in the rat produced neuronal loss in the hippocampus and cerebral cortex [17–20], and altered brain carbohydrate metabolism, high-energy phosphates, neurotransmitters and behavior [21–26]. These effects may result from decreased oxidative metabolism and decreased synthesis of ATP following reduced oxygen delivery to the brain [18, 27]. About 25% of net brain ATP consumption is accounted for by lipid metabolism [28]. Disruption of brain lipid metabolism secondary to reduced blood and oxygen flow, as well as reduced ATP production, also can have far reaching consequences.</p><p>In gerbils, which lack posterior communicating arteries between the carotid and vertebro-basilar circulations, BCCL produces complete forebrain ischemia, resulting in massive release of unesterified fatty acids (uFA) within 5 min and an increase in the concentration of arachidonyl (ARA)-CoA [29–31]. BCCL in rats may not produce as severe changes, however, since the rat has posterior communicating arteries.</p><p>One early study reported that brain concentrations of palmitic, stearic, oleic, ARA and docosahexaenoic acids were increased after decapitation at 6 h after BCCL [32]. However, it now is recognized that decapitation itself markedly distorts brain lipid concentrations through activation of lipases that release fatty acids from phospholipids, and that high energy microwaving can reduce this distortion [31, 33]. Because of the relevance of chronic partially reduced CBF to human brain disease (see above), in this paper we thought it important to measure concentrations, in high-energy microwaved brain of the rat, of long-chain unesterified fatty acids (uFAs) and acyl-CoAs, intermediates for reincorporation of released uFAs into phospholipids [34, 35], at different times following BCCL, or after sham operation. Since phospholipases A2 (PLA2) and cyclooxygenase (COX)-2 are involved in the metabolism of arachidonic acid (ARA) and docosahexaenoic acid [36–38], which are released during ischemia [39], we also used immunochemistry to examine regional brain COX-2 and PLA2 proteins.</p><!><p>HPLC grade isopropanol was purchased from EM Science (Gibbstown, NJ, USA). Ammonium sulfate and acetic acid glacial were purchased from Mallinckrodt (Paris, KY, USA). Reagent grade methanol was from Mallinckrodt (Phillipsburg, NJ, USA). HPLC grade acetonitrile was obtained from Fisher Scientific (Fairlawn, NJ, USA). Potassium monobasic phosphate and acyl-CoA standards were from Sigma-Aldrich (St. Louis, MO, USA). Sodium pentobarbital was purchased from Richmond Veterinary Supply Co. (Richmond, VA, USA).</p><!><p>Male Wistar-Kyoto rats, aged 2–3 months and weighing 180–281 g (Charles River Laboratories, Wilmington, MA, USA), were maintained on a 12-h/12-h light/dark cycle in a temperature- and humidity-controlled facility with food and water available ad libitum. Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p) and both common carotid arteries were exposed through a ventral cervical U-shaped incision. The arteries were separated from their sheaths and vagal nerves, and permanently ligated with 4-0 silk sutures. Sham animals underwent the same surgical procedure but without the actual ligation. All procedures were conducted under a protocol (#05-023) approved by the NICHD Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23).</p><p>To examine the time course of responses to BCCL, brains were collected at 6 h, 24 h and 7 days after BCCL or sham surgery. Rats (n = 6–10 per group at each time point) were anesthetized with sodium pentobarbital and immediately killed by focused high energy beam irradiation (5.4 kW, 3.8 sec, Cober Electronics, Stamford, CT, U.S.A.) [31]. The brain was excised and regions posterior to the posterior communicating arteries were discarded. The forebrain was bisected in the midsagittal plane and stored at −80 °C. One hemisphere was used for uFA analysis and the contralateral hemisphere for long chain acyl-CoA analysis. For histology, rats (n = 3–5 for each group at each time point) were deeply anesthetized with pentobarbital (50 mg/kg body wt), and transcardially perfused with 100 ml cold saline followed by 4% paraformaldehyde in PBS.</p><!><p>Total lipids were extracted by the method of Folch [40]. The extracts were separated by thin layer chromatography on silica gel 60 plates (Whatman, Clifton, NJ, USA). uFAs were separated using a mixture of heptane (Fisher Scientific, Fair Lawn, NJ, USA): diethyl ether : glacial acetic acid (60:40:2 by volume) [41]. uFAs and standard bands were visualized with 6-p-toluidine-2-naphthalene-sulfonic acid (Acros, Fairlawn, NJ USA) under ultraviolet light. uFA bands were scraped and heptadecanoic acid (17:0) was added as an internal standard prior to extraction and methylation. Fatty acid methyl esters were prepared by heating the removed bands in 1% H2SO4 in methanol at 70°C for 3 hours [42]. The methyl esters were separated on a 30 m × 0.25 mm i.d. capillary column (SP-2330, Supelco; Bellefonte, PA, USA) using gas chromatography with a flame ionization detector (Model 6890N, Agilent Technologies; Palo Alto, CA, USA). Runs were initiated at 80 °C with a temperature gradient to 160° C (10° C/min) and 230° C (3° C/min) in 31 min and held at 230 °C for 10 min. Peaks were identified by retention times of fatty acid methyl ester standards (Nu-Chek-Prep, Elysian, MN, USA). Fatty acid concentrations (nmol/g brain) were calculated by proportional comparison of gas chromatography peak areas to that of the 17:0 internal standards.</p><!><p>Samples (0.3–0.4 g) plus 25 μl of 17:0 and 14:0- CoA as internal standards were placed on ice in a scintillation vial or a 15-ml conical vial prior to sonication. Then 25 mM KH2PO4 (2 ml) was added and sonicated for one min (output control 3 units). Isopropanol (2 ml) was added to the vial and the homogenate was sonicated again for 20 sec. Saturated ammonium sulphate (0.25 ml) was added and the sample was lightly shaken by hand. Acetonitrile (4 ml) was added and the sample was vortexed for 2 min prior to centrifugation. The upper phase was separated and 12 ml of 25 mM potassium phosphate was added. Each sample was run through an activated oligonucleotide purification cartridge (Applied Biosystems, Foster City, CA USA), washed with 1 ml of 25 mM potassium hydrogen phosphate (KH2PO4), and eluted with 200 μl isopropanol: 30 mM glacial acetic acid (75:25 by volume). The first 3 drops were discharged and the remaining 150 μl were collected in an auto sampler vial. Acyl-CoA species were separated using HPLC (Beckman-Coulter, Fullerton, CA, USA) with a Symmetry C-18, 5-μm column (250 × 4.6 mm; Waters-Millipore, Mildford, MA, USA) and UV absorbance was measured at 260 and 280 nm with a System Gold, 168 dual wavelength detector. Conditions were set to a 1 ml/min gradient system composed of (A) 75 mM KH2PO4 and (B) 100% acetonitrile. The gradient started with 44% B, then increased to 49% B over 25 min, and then to 68% B over 10 min. It remained at 68% B for an additional 5 min, then returned to 44% over 2 min and held for 8 min. Concentrations of acyl-CoA species (nmol/g wet weight) were identified according to the retention times of authentic standards and were measured using peak area analysis (32 Karat, version 5.0, Beckman Coulter) from HPLC chromatograms. A representative HPLC pattern of acyl-CoA concentrations in both sham (A) and BCCL (B) rat brains is shown in Figure 1. The peaks at 18 and 37 min represent internal standards of tetradecanoyl-CoA and heptadecanoyl-CoA (20 μl of each), respectively.</p><!><p>Following cardiac perfusion, brains were excised then post-fixed in situ for 6 h, bisected in the mid-sagittal plane, rinsed with PBS, dehydrated in ethanol, and embedded in paraffin. Serial 8-μm sections of the entire sagittal plane containing the hippocampus were collected, and from this pool of sections for each animal were randomly selected representing multiple planes of cut. Deparaffinized, rehydrated sections were treated to quench endogenous peroxidase and subjected to heat-induced epitope retrieval using a decloaking chamber (Biocare Medical, Walnut Creek, CA, USA). Microglia were detected by binding of Griffonia simplicifolia isolectin B4 (IB4; Sigma) at a 1:75 dilution in 1X Automation Buffer (AB; Biomedia Corp, Foster City, CA, USA) containing 0.1 M CaCl2, MgCl2, MnCl2, and 0.1% Triton X-100 overnight at 4 °C. For immunohistochemistry, sections were blocked with avidin-biotin followed by 10% normal goat serum/ 1% BSA/ 1X AB. Astrocytes were identified with a rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:1000 for 30 min; Dako, Carpinteria, CA, USA) detected with an HRP labeled streptavidin–biotin kit. Sections were incubated with anti-secretory sPLA2 or anti-cytosolic cPLA2 (1:100; overnight at 4 °C; Santa Cruz) or anti-COX-2 polyclonal antibodies (1:1500; 1 h RT; Cayman Chemicals, Ann Arbor, MI, USA) detected by avidin biotin technique. Antigen retrieval was not used for anti-cPLA2. Reaction products were visualized by 3,3′-diaminobenzidine (DAB) substrate. Sections were counterstained with modified Harris hematoxylin. Digital images of the entire brain section were acquired with a Leica DMRBE microscope (Wetzlar, Germany) or using the Aperio Scanscope T2 Scanner (Aperio Technologies, Vista, CA USA) and viewed using Aperio Imagescope v. 6.25.0.1117.</p><!><p>Data are presented as mean ± standard deviation. Statistical significance was determined by a Student's t-test at each individual time point, with significance set at p < 0.05 as indicated by *, BCCL vs. sham.</p><!><p>Table 1 presents measured concentrations of brain uFAs at different times following BCCL or sham operation. At 6 h, there was a statistically significant increase in the concentration of DHA and of total uFAs compared to sham control (p < 0.05). At 24 h and 7 days, there was no significant difference in the concentration of total or any individual uFA between groups.</p><!><p>As illustrated in Table 2, the brain concentration of ARA-CoA at 6 h after BCCL was increased 2-fold, compared to its concentration in the sham rats. There was no group difference in any acyl-CoA concentration at 24 h or 7 days after BCCL.</p><!><p>A general histopathological evaluation of brain sections indicated no overt cell death in any region within the sagittal plane of cut as a result of BCCL. When scanning the entire plane of cut, specific staining by each antibody was detected in some brain regions. Immunoreactive product for COX-2 was detected in neurons within the somatosensory cortex (Figs. 1a,b), the piriform cortex (Figs. 1c,d), and CA3 pyramidal neurons of the hippocampus (Figs. 1e,f). Staining was similar in the BCCL rats and sham controls across all time points.</p><p>At 6 h, the general staining pattern involving cPLA2 within the cortex was similar across the BCCL rats and sham controls (Fig. 2). In both groups, cytosolic cPLA2 was detected in cell bodies and in processes of cerebellar Purkinje neurons, dentate granule cells, and cortical neurons. When matched for location and plane of cut, immunoreactive processes in the piriform cortex at 6 h after ligation displayed a more distinct staining pattern, suggesting upregulation, in BCCL compared with sham rats (Figs. 2c,d). This appeared to be a transient change, as no difference in staining pattern was seen at the 24-h or 7-day time points (data not shown).</p><p>In sham controls, secretory sPLA2 was not detected and only rarely was a positive area detected in sections from the BCCL rats. This occurred only at the 24 h time point, within cells displaying a glia-type morphology suggestive of astrocytes, in close proximity to blood vessels in the cortex (data not shown). Also affected were the area between the dentate gyrus and the stratum lucidem (Fig. 3a), and the lacunosum molecular layer of the hippocampus (Fig. 3b). No immunopositive staining for sPLA2 was evident in the brain parenchyma at the later 7-day time point.</p><p>The morphology and distribution of IB4+ microglia and GFAP+ astrocytes in the hippocampus and cortex were similar between BCCL rats and sham controls. The overall staining for GFAP+ astrocytes in the hippocampus did not differ between BCCL and sham controls at any time point examined. IB4 staining for microglia indicated no significant difference between BCCL and sham controls over time (data not shown).</p><!><p>In the current study, an early indication of cerebral hypoperfusion was seen at 6 h after BCCL, as a statistically significant increase in the brain concentrations of total uFA, docosahexaenoic acid and ARA-CoA, the intermediate for reincorporation of unesterified ARA into brain phospholipids [29, 30, 34, 43]. cPLA2 immunoreactivity appeared elevated in neuronal processes of the piriform cortex. These changes were absent at later times. At 24 h however, immunochemistry showed that sPLA2 clearly was overexpressed close to blood vessels in the BCCL rats. The transient nature of the responses suggests that the uFAs released during BCCL were cleared within 24 h [29, 44].</p><p>Our mean sham-surgery uFA concentrations were comparable to reported values in microwaved rat or uFA [30, 31], but they had much larger variances than reported, as did concentrations following BCCL (Table 1). Possible causes for their increased variance are incomplete microwaving and modification by the sham operation itself, since our analytical methods were the same as used in the prior publications. The high variance limits information gathered from the uFA data, but might be overcome in future studies by using more animals [30, 31].</p><p>Our sham acyl-CoA concentrations also were comparable to published values in microwaved brain [30, 31]. The net acyl-CoA concentration was not changed significantly by BCCL, which also was found following complete ischemia [30, 31]. This constancy may be due to binding of intracellular acyl-CoA to acyl-CoA binding proteins or acyl-CoA synthetases, whose availability is not changed by ischemia [45, 46]. As after 6 h of BCCL (Table 2), ARA-CoA was the only acyl-CoA elevated by complete ischemia or decapitation in other studies using microwaved brain [30, 31]. This elevation likely mediates increased energy-dependent reincorporation of unesterified ARA into phospholipid [29].</p><p>The increased total brain uFA concentration at 6 h after BCCL is consistent with a report elevated concentrations at 2, 4 and 6 h after BCCL, when the brain was removed after decapitation without microwaving [32]. However, the magnitude of the increase in our study at 6 h (1.55 fold) is much lower than in the prior study (11.3 fold) [32], likely because we used focused beam high energy microwaving of the brain before removing it, to denature brain fatty acid releasing enzymes [31, 33].</p><p>In gerbils, which lack posterior communicating arteries, increased brain concentrations of palmitic, stearic, oleic, linoleic, ARA and docosahexaenoic acids were reported at 5 min after BCCL, when the brain was subjected to high energy microwaving [30]. The total uFA concentration was increased by 4.4 fold at 5 min, much higher than the 1.55 fold increase in rats at 6 h after BCCL. The lack of posterior communication arteries results in complete forebrain ischemia in gerbils following BCCL [30].</p><p>This study revealed a significant increase in ARA-CoA, but no significant change in other acyl-CoA species at 6 h after BCCL, and no change in any acyl-CoA including ARA-CoA at 24 h (Table 2). These results suggest upregulated ARA reincorporation into brain at 6 h after BCCL. In complete ischemia in the gerbil, a high brain ARA-CoA concentration was associated with a higher ARA concentration. A high concentration of unesterified ARA can be neurotoxic, by interfering with cell signaling, gene transcription, mitochondrial oxidative phosphorylation, cell growth and excitability, and inducing apoptosis [47–49].</p><p>The transient nature of the brain response to BCCL was evidenced by the focal expression of sPLA2 in close proximity to blood vessels at 24 h but not later, and evidence for increased cPLA2 immunoreactivity. The perivascular location of sPLA2 suggests a role in vasodilatation in the initial stages of BCCL, associated with formation of the potent ARA-derived vasodilator, prostaglandin E2 (PGE2) [50]. The transient nature of the response suggests that selective changes were induced on cerebral microvessels, which were not maintained over time. While there was no observed morphological response in microglia in the initial 24 h after BCCL, activated microglia as well as other pathological changes in gray and white matter have been noted 13 weeks after BCCL in rats [14, 51].</p><p>COX-2 is constitutively expressed in the brain in discrete neuronal populations. In the current study, COX-2 expression in neurons was not obviously different between BCCL and sham rats after 24 h (Fig. 2). With complete ischemia [50], lipopolysaccharide-induced neuroinflammation [52, 53], COX-2 was rapidly upregulated and promoted formation of pro-inflammatory eicosanoids like prostaglandin E2 (PGE2) [54–56].</p><p>Acute BCCL in rats leads to a transient cerebral hypoxia, which increases intracellular calcium [57] to activate ARA-selective cPLA2 and release ARA and increase ARA recycling in phospholipid [58]. Activation of sPLA2 by calcium [59] also may release ARA and ultimately to produce PGE2 [50] to mediate compensatory vasodilatation. Nitric oxide, free radicals and neurogenic factors during ischemia also can produce vasodilatation [60, 61]. CBF recovers to ~63% and ~90% of its control value at 4 and 8 weeks after BCCL [14–16]. It is likely that some recovery occurred in the present study even at 24 h, associated with the sPLA2 activation (Fig. 3), helping to normalize the significant disturbances in uFA and ARA-CoA concentrations evident at 6 h (Tables 1 and 2). However, autoregulation remains abnormal and the brain is more vulnerable to additional insults such as hypotension, hypoxia and further ischemia following BCCL[62].</p><p>In conclusion, BCCL in rats caused an acute statistically significant increases in the net brain uFA concentration and the ARA-CoA concentration at 6 h, immunohistochemical evidence increased cPLA2 expression in neuronal processes of the piriform cortex at 6 h and of sPLA2 expression surrounding blood vessels at 24 h, suggesting vasodilation. These results demonstrate the transient nature of the brain metabolic response, likely because of the intervention of compensatory vasodilation over time. The absence of increased immunoreactivity for COX-2, IB4+ microglia or GFAP+ astrocytes in the hippocampus and cortex in BCCL compared with sham control rats indicates that BCCL did not initiate significant neuroinflammation. Thus, BCCL exerts a low-threshold reversible ischemic brain insult, which transiently increases brain ARA metabolism and enzymes, but may increase vulnerability to further stress.</p>

PubMed Author Manuscript

Effects of Macromolecular Crowding on the Conformational Ensembles of Disordered Proteins

Due to their conformational malleability, intrinsically disordered proteins (IDPs) are particularly susceptible to influences of crowded cellular environments. Here we report a computational study of the effects of macromolecular crowding on the conformational ensemble of a coarse-grained IDP model, by using two approaches. In one, the IDP is simulated along with the crowders; in the other, crowder-free simulations are postprocessed to predict the conformational ensembles under crowding. We found significant decreases in the radius of gyration of the IDP under crowding, and suggest repulsive interactions with crowders as a common cause for chain compaction in a number of experimental studies. The postprocessing approach accurately reproduced the conformational ensembles of the IDP in the direct simulations here, and holds enormous potential for realistic modeling of IDPs under crowding, by permitting thorough conformation sampling for the proteins even when they and the crowders are both represented at the all-atom level.

effects_of_macromolecular_crowding_on_the_conformational_ensembles_of_disordered_proteins

<p>It is now accepted that a considerable proportion of proteins are disordered and play essential roles in signaling and regulation.1-12 The complex, crowded environments inside cells may significantly influence the conformational, thermodynamic, and kinetic properties of proteins.13 These influences are expected to be particularly strong for intrinsically disordered proteins (IDPs), due to their conformational malleability and propensity to oligomerize and aggregate and to bind with different cellular targets.14 While some studies15-18 have shown that IDPs preserve their disordered state under crowded conditions, these and other studies19-22 have found that the conformations of IDPs (and unfolded proteins) become more compact. Compression of the polymer PEG by Ficoll as a crowding agent23 and of unfolded RNA by PEG24 has also been observed, suggesting a common cause for the compaction in size. Here we report a computational study of the effects of macromolecular crowding on the conformational ensemble of an IDP.</p><p>Two different computational approaches have been used to study proteins under crowding. In the direct simulation approach, a test protein is simulated along with crowders;25-32 to calculate thermodynamic or kinetic properties, one has to obtain the full free energy surface that covers two end states (such as the folded and the unfolded states of a protein). To overcome the enormous computational cost of such calculations, the protein has generally been represented at a coarse-grained level, and the crowders are often treated with an even simpler representation, such as a sphere or a few linked beads. Simulations with an all-atom representation of test and crowder proteins have recently been carried out, although the relatively short simulation times limited the conformation sampling largely to near the folded state.32</p><p>We have introduced an alternative approach known as postprocessing,33-34 whereby the conformational ensemble of a protein sampled in the absence of crowders is transformed to that under crowding, through weighting by the Boltzmann factor of the excess chemical potential, Δμ, arising from protein-crowder interactions. Similar ideas have been exploited by others.21, 35 To implement the postprocessing approach, we have developed methods to accelerate the calculation of the excess chemical potential.33-34, 36 In essence, the postprocessing approach uses crowder-free simulations to predict crowding-induced changes in free energy differences and rate constants between end states,33-34, 36-42 as well as free energy surfaces of protein folding and conformational transition under crowding.39, 43</p><p>The postprocessing approach may be especially suited for studying IDPs under crowding. These proteins may not have well-defined basins of attraction and can access enormous conformational space. Sampling this conformational space is already challenging for simulations in dilute solution.44-49 The addition of crowders in these simulations may necessitate reduced representations for the IDP and the crowders. The postprocessing approach allows the more realistic representations to be preserved in modeling the effects of crowding.</p><p>The accuracy of the postprocessing approach depends on adequate sampling in the crowder-free simulations of the test protein, so as to cover all the conformational regions that would be important when crowders are present. Under-sampling of new low-energy regions that emerge in the presence of crowders is always a concern.32, 43 Previously we have validated the postprocessing approach against direct simulations for the effects of crowding on the flap open-to-closed population ratio of the HIV-1 protease dimer38 and for the folding free energy surfaces of three small proteins under crowding.43</p><p>Below we compare the conformational ensembles of an IDP under crowding, either predicted by the postprocessing approach or obtained from direct simulations with crowders present. To accommodate the direct-simulation approach, the IDP has a coarse-grained representation, with one bead for each residue.26, 50 The residues in the protein chain are connected by springs with an equilibrium length of 3.8 Å and a spring constant of 70 kcal/mol/Å2, and interact via a Morse potential</p><p>where rij is the distance between two residues, r0 = 9.5 Å, α = 0.707 Å−1, D0 = 0.207 kcal/mol, and ξ is a constant introduced to scale the strength of the intra-protein interactions. A higher ξ is expected to strengthen the attraction between the residues and thus result in more compact conformations. In the direct simulations, the crowders are soft spheres with a nominal radius of rc = 30 Å, with the interaction center located on the spherical surface with a radius of 23 Å; the crowders interact with a repulsive inverse r12 potential:26</p><p>where rαβ is the center-center distance between two crowders, and ε = 0.6 kcal/mol. When two crowders are at contact (i.e., rαβ = 60 Å), the interaction energy is ε, or roughly the thermal energy kBT at T = 300 K. Each protein residue and each crowder also interact with an inverse r12 potential:</p><p>We carried out Brownian dynamics simulations of a 99-residue IDP alone and along with the crowders, using a modified UHBD program as done previously.26, 38 Periodic boundary condition was imposed, with a cubic unit cell of 510 Å side length. The cutoff distances were 24.5 Å for the Morse potential and 75 Å for the inverse r12 potentials. For the simulations with crowders, 71 to 575 crowders were included to produce crowder volume fractions (ø) ranging from 0.06 to 0.49 (Table 1), the same as the crowding conditions in our previous study of the flap open-to-closed transition of the HIV-1 protease dimer.38 Five values of the scaling constant ξ for the intra-protein Morse potential were studied: 1, 0.8, 0.7, 0.6, and 0.5. For each combination of ø and ξ, conformation sampling consisted of 36 repeat trajectories that started from different random number seeds and lasted 30 μs each. The timestep was 50 fs; snapshots in the last 20 μs were saved at 10 ns intervals for analysis. For implementing the postprocessing approach, we also carried out crowder-only simulations, with the unit cell now enlarged to a side length of 1000 Å and the numbers of crowders increased in proportion to produce ø values matching those in the simulations of the protein-crowder mixtures.</p><p>Both without and with the crowders, the IDP sampled a wide variety of conformations, with the radius of gyration (Rg) spanning a broad range (Fig. 1). To demonstrate that the conformation sampling was adequate, in Fig. 2 we compare the histograms of Rg at ξ = 0.5 obtained from simulations starting from two very different initial conformations, one compact with Rg = 13 Å and the other extended with Rg= 93 Å.51 For both the crowder-free simulations (ø = 0) and the simulations at ø = 0.31, the histograms of Rg from the different initial conformations agree very well, indicating convergence of the simulation results. Additional results demonstrating convergence for ξ = 0.7 and ø = 0 are shown in Fig. S1. All subsequent results are from simulations with the compact initial conformation for the protein.</p><p>Comparing the histograms in Fig. 2 for ø = 0 and ø = 0.31, one sees that the peak position shifts toward a lower Rg value, meaning that the IDP becomes more compact under crowding. The corresponding results at ξ = 0.7, displayed in Fig. 3, further illustrate that the shift in peak position toward lower Rg values can be accompanied by a significant narrowing of the distribution of Rg. Despite the overall compaction under crowding, the histograms in Figs. 2 and 3 show that the same range of Rg values are spanned with and without crowders, suggesting that the accessible conformational spaces may largely overlap under crowder-free and crowding conditions.</p><p>From the direct simulations, the root-mean-square Rg (Rg;rms) values, along with their standard deviations among 36 repeat trajectories,52 for the various combinations of ø and ξ are obtained and listed in Table 1. These results show that, at a given ø, the protein chain becomes more and more extended as the intra-protein Morse interactions are weakened. Conversely, at a given ξ, the protein chain becomes more and more compact as the level of crowding is increased. Interestingly, the extent of the crowding-induced compaction is maximal at an intermediate ξ. At ξ = 0.7, Rg;rms is reduced by nearly 50% when the crowders are present at a volume fraction of 0.49. In comparison, under this crowding condition, the reduction in Rg is only 6.5% at ξ = 1 and 34.6% at ξ = 0.5. In addition, at intermediate ξ values (0.7 and 0.8), the standard deviations of Rg are lower under crowding than in the crowder-free condition, corresponding to the narrowing of the distribution of Rg shown in Fig. 3. Explanations for these intriguing observations at intermediate ξ will be presented below.</p><p>Based on studies of proteins unfolded by denaturants and/or low pH, a polypeptide chain with 99 residues is expected to have an Rg;rms ∼35 Å in dilute solution.53 An IDP probably has a comparable size. This expected size is produced by our model with intermediate ξ values (0.7 and 0.8) at ø = 0. Coincidentally, as just noted, it is at the intermediate ξ values that crowding exerts maximal compaction as well as narrowing of the distribution of Rg.</p><p>The magnitudes of crowding-induced compaction obtained in our simulations are similar to those observed in some recent experimental studies. In FRET measurements of Mikaelsson et al.,22 200 g/l Dextran 20 produced ∼10% decrease in donor-acceptor distance in urea-denatured ribosomal protein S16. Similarly, in SAXS measurements of Kilburn et al.,24 20% (w/v) PEG1000 produced a 16% decrease in the Rg of unfolded RNA. In comparison, the compaction for our IDP with ξ = 0.8 is 12% at ø = 0.18. Interestingly, there seems to be experimental evidence for crowding-induced narrowing of the distribution of Rg in the study of Mikaelsson et al.22 These authors fitted their FRET data with a Gaussian distribution for the donor-acceptor distance, and found that the width of the distribution decreased in the presence of 200 g/l Dextran 20.</p><p>We now use the above results from the direct simulations to benchmark the accuracy of the postprocessing approach, which uses the crowder-free simulations to predict the conformational ensembles of the IDP under the various levels of crowding. This entails weighting each crowder-free conformation by a factor exp(−Δμ/kBT).38 When the interactions between the test protein and crowders are hard-core repulsion only, the excess chemical potential can be calculated as Δμ = –kBTlnp, where p is the fraction of attempts to place the test protein into the crowded solution that do not result in protein-crowder clash. For spherical crowders, we developed an efficient method for calculating p, by mapping the covolume of a crowder and the protein onto a grid centered on the crowder.33 In these calculations, averages are taken over both conformations of the IDP and configurations of the crowders. Following our previous study,38 we first approximated the repulsive inverse r12 potentials as hard-core repulsion and applied the covolume-based method to calculate Δμ (using crowder configurations generated in previous hard-sphere simulations). Up to ø = 0.25, the postprocessing approach with the hard-sphere treatment accurately predicts the Rg;rms values from the direct simulations (Fig. 4). At ø = 0.31, it yields small but discernible underestimation of the crowding-induced compaction. We also used the crowder configurations generated by the crowder-only simulations here (with crowder-crowder interactions governed by the potential of eq (2)) but then treated the crowders as hard spheres in calculating Δμ. The predicted Rg;rms values were unchanged.</p><p>To understand the slight overestimation of Rg;rms at high ø, we note that, in the hard-sphere treatment, the interaction between a residue and a crowder is turned off at their nominal contact distance, even though the interaction energy there still roughly equals kBT. Therefore the crowders and the residues can approach each other to their contact distance more easily than they should, leading to slight underestimation of Δμ. This underestimation is worse for the more extended conformations (Fig. 3) and for the more crowded conditions. The former is because the more extended conformations have more residues exposed to the crowders; the latter is because, with more crowders around, the chance for them to approach the IDP increases.</p><p>We have just developed a new method for calculating Δμ that is based on fast Fourier transform (FFT) and can handle any form of protein-crowder interactions.36 The basic idea is to express the protein-crowder interaction energy as a correlation function and then evaluate this correlation function via FFT. Using the crowder configurations generated by the crowder-only simulations here and applying the FFT-based method to treat the inverse r12 form of protein-crowder interactions, the predictions for the Rg;rms values at ø = 0.31 are improved, and are now in good agreement with those obtained from the direct simulations (Fig. 4). The predicted histogram of Rg also agrees well with that from the direct simulations (Fig. 3), suggesting that the conformational ensemble under crowding is predicted well by the postprocessing approach when the protein-crowder interactions are properly treated when calculating Δμ. However, at an even higher level of crowding (ø = 0.49), the crowder-free simulations here under-sampled the most compact conformations (Fig. S2), resulting in a small overestimate of Rg;rms (35.7 Å predicted versus 34.2 ± 5.2 Å from the direct simulations, both at ξ = 0.5). More extensive sampling in the crowder-free simulations can be obtained by specialized techniques such as umbrella sampling or replica exchange.</p><p>The postprocessing approach provides a conceptual framework via which the crowding-induced maximal compaction and narrowing of the distribution of Rg at intermediate ξ can be easily understood. Recall that this approach yields the conformational ensemble under crowding through weighting each crowder-free conformation by a factor exp(–Δμ/kBT). Consider two different scenarios for the crowder-free conformational ensemble, one consists of similar conformations, resulting in a narrow distribution of Rg, while the other consists of vastly different conformations that span broad distribution of Rg. In the first scenario, reweighting by exp(–Δμ/kBT) will not produce a significant compaction, since all the crowder-free conformations would have similar weighting factors. In the second scenario, the more extended crowd-free conformations would have smaller weighting factors whereas the more compact crowd-free conformations would have larger weighting factors; the net result is significant compaction. The crowder-free conformations of our IDP have the broadest distribution of Rg at ξ = 0.7 (Fig. S3), thus explaining why crowding results in maximal compaction at this intermediate ξ.</p><p>That the broadest distribution of crowder-free Rg occurs at ξ = 0.7 requires some explanation. At ξ = 1, residue-residue attraction arising from the Morse potential keeps the IDP compact. As ξ is reduced, the protein chain can access more open conformations, leading to broadening of the distribution of Rg. However, as ξ is reduced further and further, the chain mostly samples highly extended conformations. With Rg bounded from above (at ∼100 Å for the fully extended conformation), the distribution of Rg again starts to narrow (compare the histograms of Rg at ξ = 0.6 and 0.5 in Fig. S3). Note that the standard deviations of crowder-free Rg is the largest at ξ = 0.7 (Table 1), consistent with the broadest distribution at this ξ.</p><p>Compaction under crowding can manifest either as a shift in the histogram of Rg toward lower Rg values (Fig. 2) or as a shift accompanied by a narrowing of the distribution of Rg (Fig. 3). A lower bound of Rg also exists, when the protein chain becomes extremely compact. As the histogram of Rg shifts toward lower Rg under crowding, the left envelope of the histogram may reach the lower bound of Rg. Then only the right envelope can shift leftward, resulting in a narrowed distribution.</p><p>We have demonstrated here that, from crowder-free simulations, the postprocessing approach can faithfully predict the conformational ensembles of IDPs under crowding. Note that, by varying ξ, we produced protein chains spanning a wide range of crowder-free Rg;rms values, both below and above the crowder radius; the postprocessing approach works equally well in both cases. While the demonstration carried out here uses simplified representations for the IDP and for the crowders, the postprocessing approach is now ready to model proteins and crowders both represented at the all-atom level.36 Using direct simulations with crowders included, presently achieving exhaustive conformation sampling for IDPs at such a level of realism seems impractical. The postprocessing approach may provide a viable solution. What is perhaps particularly attractive about this approach is that all the previously published simulations of IDPs in dilute solution44-49 can be postprocessed to predict results in crowded solution.</p><p>Additional technical advantages of the postprocessing approach are also worth noting. First, in implementing this approach, all locations in the crowded solution are probed for possible protein-crowder interactions.33, 36 This is in contrast to the situation with direct simulations, where the test protein can be trapped in interactions with one or two particular crowder molecules, especially when attractive components are included in the interactions. Second, the same crowder-free simulations can be used to predict conformational ensembles for many different crowding conditions (as illustrated here) and for different types of protein-crowder interactions. In particular, our FFT-based method can treat both repulsive and attractive protein-crowder interactions.36</p><p>Minton54 proposed both an equivalent hard sphere model and a Gaussian cloud model for treating unfolded protein chains. Applying these models to our IDP, we found that they significantly overestimated the magnitudes of crowding-induced compaction (Fig. S4). While simplified theoretical models can be very useful for predicting qualitative trends, predictions that have the potential to reach quantitative agreement with experimental studies of crowding effects will likely require an atomistic representation for the proteins and crowders.</p><p>Similar to what is found here for an IDP, decreases in Rg have been reported in previous computational studies for unfolded proteins under crowding.25, 29 The present study, especially with the help of the postprocessing approach, reinforces the notion that repulsive interactions with crowder molecules may be a common cause for the compaction of proteins and other polymer chains observed in experimental studies.15-16, 19-24 Crowders could also stabilize partially structured intermediates for IDPs.18 Based on thorough conformation sampling in dilute solution, the postprocessing approach holds enormous potential in realistically modeling this and other rich effects of crowding on IDPs.</p>

PubMed Author Manuscript

Versatile Online\xe2\x80\x94Offline Engine for Automated Acquisition of High-Resolution Tandem Mass Spectra

For automated production of tandem mass spectrometric data for proteins and peptides >3 kDa at >50 000 resolution, a dual online\xe2\x80\x94offline approach is presented here that improves upon standard liquid chromatography\xe2\x80\x94tandem mass spectrometry (LC\xe2\x80\x94MS/MS) strategies. An integrated hardware and software infrastructure analyzes online LC\xe2\x80\x94MS data and intelligently determines which targets to interrogate offline using a posteriori knowledge such as prior observation, identification, and degree of characterization. This platform represents a way to implement accurate mass inclusion and exclusion lists in the context of a proteome project, automating collection of high-resolution MS/MS data that cannot currently be acquired on a chromatographic time scale at equivalent spectral quality. For intact proteins from an acid extract of human nuclei fractionated by reversed-phase liquid chromatography (RPLC), the automated offline system generated 57 successful identifications of protein forms arising from 30 distinct genes, a substantial improvement over online LC\xe2\x80\x94MS/MS using the same 12 T LTQ FT Ultra instrument. Analysis of human nuclei subjected to a shotgun Lys-C digest using the same RPLC/automated offline sampling identified 147 unique peptides containing 29 co- and post-translational modifications. Expectation values ranged from 10\xe2\x88\x925 to 10\xe2\x88\x9299, allowing routine multiplexed identifications.

versatile_online\xe2\x80\x94offline_engine_for_automated_acquisition_of_high-resolution_tandem_mass_

<!>Cell Processing<!>Sample Preparation<!>Liquid Chromatography<!>Mass Spectrometry<!>Software<!>Database Searching<!>RESULTS<!>Top-Down Proteomics<!>Middle-Down Proteomics<!>Data Acquisition Times for High-Resolution MS/MS Spectra<!>DISCUSSION<!>Advanced Data Acquisition<!>Making \xe2\x80\x9cOne-Hit Wonders\xe2\x80\x9d Routine<!>Offline Advantages<!>Future Development<!>Extensibility

<p>Historically, there has been a trade-off in mass spectrometry between resolution and sensitivity. In contemporary MS-based proteomics,1 there is long-standing interest in increasing either spectral resolution, the size of peptides/proteins analyzed, or both. Such improvements will allow more protein complexity to be measured with greater certainty.2 Driven in part by new ion fragmentation approaches3,4 and improving instrumentation,5-12 the steadily advancing capabilities of MS are challenged by targeting polypeptides >3 kDa, such as intact proteins, non-tryptic peptides, and/or large endogenous peptides.13 Although proteomics has traditionally been a field ripe for automation,14 data acquisition solutions for MS/MS of proteins or peptides at >50 000 resolving power remain relatively underdeveloped.</p><p>In a typical bottom-up LC—MS/MS experiment using the new breed of ion trap-Fourier transform (FT) hybrid instruments, intact peptide data are now routinely acquired at FTMS resolution,15,16 substantially clarifying protein identifications by database retrieval. However, parameters that lead to increased MS/MS data quality (e.g., long ion accumulation times, detection by FTMS, and spectral averaging) are sacrificed to increase the speed of MS/MS sampling in order to maximize proteome coverage. This increase in sampling rate is not compatible with acquisition of high-resolution MS/MS spectra by Fourier transform mass analyzers, which are inherently slower than electron multiplier detection-based ion traps and time-of-flight (TOF) instruments more commonly employed for automated MS/MS on a chromatographic time scale.17-21</p><p>In addition to the challenges of performing proteomics with high-resolution MS/MS data, the data acquisition approaches common on modern mass spectrometers are less effective for masses >3 kDa due to charge state distributions that mask less abundant species.22 Straightforward implementation of data-dependent experimental methods with larger parent ions produced by electrospray typically fragments several charge states of the same precursor, thus making it unlikely to acquire data on a species not even an order of magnitude less abundant on a chromatographic time scale. For small peptides this is not a major concern, since 1—3 kDa peptides will usually produce only one or two charge states within the m/z range of analysis. Although online top-down proteomics is now a reality, as recently demonstrated for yeast proteins <40 kDa,23 these factors continue to argue for an offline data acquisition strategy.</p><p>Much of the current research into increasing the biological dynamic range accessible by MS/MS involves parallel24 or data-independent25 methods, where multiple precursors are selected for simultaneous fragmentation. This multiplexing methodology is very effective at improving offline throughput,26 which is particularly critical for the acquisition of high-resolution tandem mass spectra, and is one of three distinct data acquisition strategies published by Patrie et al. in 2004.27 These past solutions focused on automating more intelligent data acquisition for top-down proteomics following fractionation by RPLC and were based on the use of advanced data analysis algorithms for determination of highly accurate precursor masses. Initially, the ZSCORE implementation28 of the charge state deconvolution algorithm29 was used.22 Later, a modified version of the thorough high-resolution analysis of spectra by Horn (THRASH) algorithm30 was incorporated to improve sensitivity and accuracy.31</p><p>Although most research on this front involves innovative mass spectrometers, there are alternatives rooted in the inlet and chromatography configuration. For targeted work performed online, "peak parking" has shown promise.32-34 For proteomic work, however, a split-flow setup is necessary to extend analysis time for species eluting throughout the entire chromatographic gradient. Split-flow mass spectrometry has thus far been successfully applied to the detection of low-abundance phosphopeptides35,36 and bacterial signaling molecules,37 but has not yet been extended to whole proteome studies.</p><p>The work presented here is a natural extension of these past platforms and combines the Advion TriVersa NanoMate with a Thermo Scientific 12 T LTQ FT Ultra into an integrated system for collection of online LC—MS data with simultaneous fraction collection for intelligent acquisition of offline fragmentation data. Targets are sought online, re-infused, and fragmented offline in a highly automated fashion. This new data production engine is coupled with a streamlined version of the ProSight software suite,38-41 advancing technology for high-resolution proteomics that allows for automated acquisition of high-quality MS/MS for bottom-up, middle-down, or top-down proteome projects.</p><!><p>Washed human HeLa cell pellets (∼2 × 107 cells) were suspended in nuclear isolation buffer (NIB-250): 15 mM tris-hydrochloric acid (pH 7.5), 60 mM potassium chloride, 15 mM sodium chloride, 5 mM magnesium chloride, 1 mM calcium chloride, 250 mM sucrose, 1 mM dithiothreitol, 10 mM sodium butyrate, protease inhibitor cocktail set III (Calbiochem; San Diego, CA) at a 100:1 v:v ratio, and phosphatase inhibitor cocktail set II (Calbiochem) at a 100:1 v:v ratio plus 0.3% NP-40 at a 10:1 v:v ratio. Cells were lysed by gentle mixing and incubation on ice for 5 min. Nuclei were pelleted at 600 × g for 5 min at 4 °C and then washed twice with NIB-250 without detergent.</p><!><p>For top-down, 0.4 N sulfuric acid was added to HeLa nuclei to give a 3:1 ratio. The acid-extracted nuclei were maintained at 4 °C for 30 min and centrifuged at 2000 × g. The supernatant was transferred to a 1.5 mL microcentrifuge tube and centrifuged again at 14 000 rpm for 20 min. This supernatant (200 μL) was mixed with 150 μL of buffer A—water + 0.2% formic acid and 0.01% trifluoroacetic acid (TFA)—prior to injection.</p><p>For middle-down, isolated nuclei were suspended directly in lysis buffer containing 50 mM ammonium bicarbonate, 1 mM dithiothreitol, 10 mM sodium butyrate, 2 M urea, and 10 nM microcystin. Nuclei were lysed with pulsed sonication six times for 30 s each, and to the unclarified lysate, 20 ng of endoproteinase Lys-C (Wako Chemicals; Richmond, VA) was added to give roughly a 250:1 substrate-to-enzyme ratio. The nuclear lysate was digested overnight at 37 °C. Prior to injection, the digest was clarified at 14 000 rpm for 20 min. Buffer A was added to the supernatant to double the volume, and the sample was reclarified to remove any precipitate.</p><!><p>Top-down or middle-down samples were injected with a Gilson 235P autosampler (Middleton, WI) into an Agilent 1200 binary HPLC system with degasser (Santa Clara, CA). A flow rate of 100 μL/min was used with PLRP-S 1000 Å, 5 μm, 150 mm × 1.0 mm polymer columns (Higgins Analytical; Mountain View, CA). The gradient lasted 116 min; samples were injected with 95% solvent A (water with 0.2% formic acid and 0.01% TFA) and 5% B (90:10 acetonitrile:isopropyl alcohol with 0.2% formic acid and 0.01% TFA) as starting conditions for 5 min. The linear gradient ramped to 30% B at 10 min and to 50% B at 106 min. A majority of the proteins/peptides eluted between 30 and 50% B. At 111 min, the gradient reached 95% B and was maintained until 116 min.</p><p>The TriVersa NanoMate (Advion BioSciences; Ithaca, NY) was used in LC—MS fraction collection mode with a split such that 300 nL/min was infused into the mass spectrometer via the chip-based nanoelectrospray ionization source and the remaining 99.7 μL/min was collected for subsequent offline analyses. The first 15 min of the gradient were directed to waste. Electrospray started at 16 min, when both data acquisition and fraction collection began, and ended after fraction 96 at 111 min. The electrospray voltage was typically +2.0 kV.</p><!><p>The mass spectrometer used was a Thermo Scientific 12 T LTQ FT Ultra running LTQ Tune Plus 2.2 and Xcalibur 2.0.5 (San Jose, CA/Bremen, Germany). For top-down experiments, the instrument method consisted of nine steps of "zoom mapping", or data-independent ion trap isolation windows, detected by FT and with no subsequent fragmentation. The center of the isolation windows progressed from m/z 700 to 1100, with an isolation width of 60 m/z and a step size of 50 m/z to ensure overlap at the edges of the isolation windows. The detection range for all FT events was m/z 600–1200. This was done to ensure that all scans have sufficient data past the region of interest for the data analysis software to function optimally. After the fifth ion trap window centered at m/z 900, a full ion trap scan from m/z 600–1600 was included to enable optional assessment of data quality, but it was not analyzed by the software. Automatic gain control (AGC) targets were increased from the default of 2 × 105 to 1 × 106 for MSn FTMS, while the full ion trap was left at the default of 3 × 104. The number of microscans was 1 except where noted.</p><p>For middle-down experiments, the instrument method consisted of full FT scans (5 microscans) from m/z 500–1500, since zoom mapping fails to cover enough m/z space in which peptide precursors occur to be effective. AGC targets were increased from 5 × 105 to 2 × 106 for full FTMS. For both top-down and middle-down experiments, maximum injection times were increased from the default of 500 to 4000 ms for full FTMS, 1000 to 8000 ms for MSn FTMS, and 10 to 80 ms for full ITMS. FT resolving power was always ∼171 500 (nominally 100 000 in the software, based on a 7 T ion cyclotron resonance (ICR) cell) at m/z 400. Source-induced dissociation voltages of +10–20 V were applied to all scan events to reduce adducts.</p><p>For offline experiments, the TriVersa NanoMate was switched to direct infusion mode. An electrospray voltage of +1.8 kV and a backing gas pressure of 0.6 psi was used. The isolation width was typically 5 m/z for middle-down and 8 m/z for top-down. Collision-induced dissociation (CID) parameters were normalized collision energy (NCE) of 0.41, activation Q of 0.5, and activation time of 50 ms.</p><!><p>All non-ProSight software was written in C# using the Microsoft .NET 2.0 Framework, with the exception of THRASH which was written in ANSI C and compiled into a dynamic link library (DLL). Development was done primarily with Microsoft Visual Studio 2005. The AUTOMATION WAREHOUSE database is implemented in MySQL 5.0. For data acquisition, Component Object Model (COM) libraries were used for control of both the Thermo Scientific LTQ (LTQInstControl.dll, March 2007 release) and the Advion TriVersa NanoMate (CSVirDevice.tlb from Chip-Soft 8.1.0.901). Reading of Thermo Scientific .raw data files was performed with the XRawfile COM library (XRawfile2.dll installed with Xcalibur). Extensive .NET wrapper libraries were written to encapsulate the functionality and simplify the interface of all three COM libraries.</p><p>Online data in the Thermo Scientific .raw file format was analyzed with an application called ONLINE AUTOMATION cRAWler, which converts isotopically resolved peaks in every FT scan into neutral masses using a modified version of the THRASH algorithm.30 These peaks were then filtered on m/z, charge, mass, and mass shift relative to previously observed species and other peaks in the same data set. The filtered species were then "binned" with a 10 ppm mass tolerance and inserted into the AUTOMATION WAREHOUSE database.</p><p>Targets for offline analysis were selected from the AUTOMATION WAREHOUSE database via an application called TARGET EXTRACTOR, saved to an extensible markup language (XML) file, and loaded into the MSn APPLICATION, which was responsible for all automated data collection, controlling both the Advion TriVersa NanoMate and the Thermo Scientific LTQ FT Ultra. The MSn APPLICATION iterated through every user-enabled target in the list and collected a user-specified number of scans of various types: FT broadband (optional), IT broadband (optional), isolation, and fragmentation. Before the main acquisition on each target occurred, the software first determined whether or not it had sufficient signal abundance in a preview isolation scan, with a cutoff of 1000 typically used. If the minimum signal threshold was met, this abundance was used to determine the number of isolation and fragmentation scans to average, otherwise the target was skipped.</p><p>For each target precursor with enough signal abundance to compel MS/MS, a separate Thermo Scientific .raw file was produced; the collection of which was then batch processed by an application called OFFLINE AUTOMATION cRAWler. This software determined the mass of the precursor(s) and fragments with a modified version of the THRASH algorithm.30 This information was passed into a XML-based .puf file for searching by ProSightHT, a module within ProSightPC 2.0 (Thermo Fisher Scientific).</p><!><p>ProSight .puf files were iteratively searched against the appropriate top-down (69 435 basic sequences, 1 565 945 protein forms, 978 MB) or middle-down (3 378 894 basic sequences, 6 051 898 peptide forms, 2.5 GB) ProSight database, both shotgun annotated.42 For top-down experiments, two absolute mass searches were performed, followed by a biomarker search, both against a heavily annotated human database previously described.43 The first absolute mass search used a 10 Da precursor mass tolerance, while the second used a 300 Da tolerance. The biomarker search was performed with a 1.1 Da precursor mass tolerance. Fragment tolerance for all three searches was 10 ppm, and the expectation value (probability score44 × database size) threshold to define a positive identification was conservatively set at ≤10−5. Final results were exported to a Microsoft Excel.xls file by ProSightPC. Due to extensive modifications in the database and experimental data, the top-down results were also manually curated to ensure that only a single protein form that shows the maximum support in the fragmentation data is reported per precursor.</p><p>For middle-down experiments, two absolute mass searches were performed against an in silico digested human database that contained all Lys-C peptides from 1–50 kDa with up to 4 missed cleavages. The first absolute mass search used a 5 Da precursor mass tolerance and a 10 ppm fragment tolerance. MS/MS experiments that did not yield an expectation value within the strict confidence threshold of ≤10−5 were automatically researched with a 200 Da intact mass window. Final results were exported to a Microsoft Excel .xls file by ProSightPC.</p><!><p>The workflow described here is shown in Figure 1, with the online portion shown in panel a (top) and the offline portion shown in panel b (bottom). The AUTOMATION WAREHOUSE database links the online and offline segments of the workflow, acting as a data repository for the entire proteome project. After converting all isotopic clusters into neutral masses, filtering and binning the results of the online analysis led to several orders of magnitude reduction in the number of species. This step minimized the number of precursor targets for offline interrogation by condensing masses observed at multiple charge states and masses eluting over multiple scans into a single target that was selected at its time of maximum elution. For complete data accountability, every peak found by THRASH was stored in the AUTOMATION WAREHOUSE database.</p><p>A typical experimental result of the workflow for top-down is shown in Figure 2. First, an intact-only LC—MS run with no fragmentation is performed (Figure 2a), during which multiple species are isolated for detection (Figure 2b). Any given target is reisolated with a narrower m/z window in offline mode (Figure 2c) and subsequently fragmented (Figure 2d). ProSight analysis shows excellent fragmentation of several modified forms of intact human histone H4, the most abundant of which is N-terminally acetylated and dimethylated at lysine 20 (Figure 2e).</p><!><p>For intact proteins obtained from acid-extracted HeLa nuclei, LC—MS (2 microscans) resulted in the system recognizing 535 targets above 25 signal-to-noise ratio (S/N) in the online, intact-only data. For the offline mode, the system set up an accurate mass list for these targets in 73 of the 99.7 μL fractions collected in the whole 96-well plate. Of the 535 species targeted, MS/MS experiments were actually performed on 382 by the instrument, yielding 305 top-down identifications with ProSight expectation scores below 10−5. Identified proteins ranged from 4–16 kDa (see Supporting Information for Microsoft Excel "offline top-down.xls" file from ProSightHT).</p><p>These 305 identifications from human HeLa cells collapse to 57 forms from 30 unique genes, including several for all core histones (H2A, H2B, H3, H4), high mobility group proteins (HMGA, HMGN2, HMGN1, HMGA1), ribosomal protein 40S, and small ribonucleoproteins. By comparison, an online zoom mapping with fragmentation run of the same sample yielded 16 identifications from 16 genes with expectation values ranging from 10−13 to 10−102 (see Supporting Information for Microsoft Excel "online top-down.xls" file from ProSightHT). Of these genes, 10 were unique to the online run.</p><!><p>Online RPLC was run directly on a Lys-C digest of HeLa nuclear lysate, with the column eluent automatically split and the TriVersa NanoMate collecting 99.7 μL fractions (a total of 96 fractions). Of these, 80 fractions were analyzed by automated MS/MS with an accurate mass target list obtained from peptides observed in the online run. A typical fraction is displayed in Figure 3, with a single scan shown in the center and six typical isolation windows shown as insets. Of the seven peptides identified from the six MS/MS spectra, expectation values ranged from 10−6 to 10−14, with one example of multiplexed identifications (bottom right).</p><p>Data from all 1233 MS/MS experiments performed over the 80 fractions were iteratively searched with multiplexed searching enabled to intelligently manage multiple hits per spectrum. This resulted in identification of 256 peptides ranging from 1–13 kDa, of which 147 were unique, with expectation values from 10−5 to 10-84, (see Supporting Information for Microsoft Excel "offline middle-down.xls" file from ProSightHT). By comparison, an online data-dependent LC—MS/MS experiment of the same sample yielded 77 peptide identifications, of which 66 were unique (see Supporting Information for Microsoft Excel "online middle-down.xls" file from ProSightHT). Of the 66 peptide forms, 31 were unique to the online run.</p><p>In the most complex region of the chromatogram, 20–50 accurate mass targets were typically identified per well. Of the 147 unique peptides, 29 were modified with 25 of these being N-terminal acetylation. At 12 kDa, one exhaustive Lys-C peptide was particularly large (Figure 4), and the fragmentation data suggested two forms of the protein hnRNP A2/B1 (P22626) from the ProSight database. The hnRNP A2/B1 was known to harbor a monomethylation at Arg203, partially characterized in this study at ∼25% occupancy (Figure 4c) without any other modifications on this 130-residue segment of the protein.</p><!><p>For top-down, target abundances directed the system to choose between 25, 50, or 100 fragmentation scans. This, in turn, sets the overall data acquisition times (along with AGC and maximum injection time settings), which ranged from 2–13 min for intact protein samples. The data for a tray of 73 sample wells collected from the online LC—MS run took ∼15 h of instrument time to attempt 535 top-down MS/MS experiments. For middle-down, target abundances directed the system to choose between 10, 25, or 50 fragmentation scans. This resulted in MS/MS spectral acquisition times per target ranging from 1–5 min, translating to 36 h of instrument time to run the 1233 targets from the 80 fractions noted above. The duty cycle for this platform in the offline mode—the fraction of time the instrument is either accumulating or detecting ions for high-resolution MS1 or MS2 data acquisition—is typically over 90%.</p><!><p>There is no system currently available capable of acquiring ultrahigh-resolution tandem mass spectra with the sensitivity of an ion trap. Custom data acquisition systems have previously been developed but only for online bottom-up proteomics with low-resolution instruments (e.g., triple quadrupoles).45,46 Therefore, we have constructed an online—offline data acquisition system representing a significant advance toward using high-value mass spectrometer time automatically and more efficiently, with the midrange goal of increasing the number of unique proteins and peptides identified and characterized in complex mixtures. The AUTOMATION WAREHOUSE database functions as a high-resolution exclusion and inclusion list to support an entire proteome project.</p><p>The power of the automated system is demonstrated by comparison to more established online experiments for both top-down and middle-down human proteomics. This is illustrated with Venn diagrams in Figure 5. For top-down, the automated system identifies proteins from approximately twice as many genes than an online zoom mapping experiment (Figure 5a). However, when all protein forms were counted, the automated system identifies nearly 4 times as many (Figure 5b), exemplifying the superior characterization power of the platform. For middle-down, the automated system identifies well over twice as many peptide forms as a traditional data-dependent double-play experiment (Figure 5c).</p><!><p>The data acquisition system has a number of features uncommon in modern commercial software on both the source and mass spectrometer side. On the source side, the COM library included in Advion ChipSoft version 8 facilitates constant monitoring and dynamic control of electrospray conditions. Users can manually adjust the electrospray voltage and gas pressure in real time, but more importantly, the software automatically checks the electrospray current against user-specified thresholds before every scan. When the spray current is not within the user-specified range, the system executes a predefined sequence of actions until the current is restored to an acceptable level. This sequence of actions includes momentarily maximizing the gas pressure, obtaining more sample, retrieving a new tip, using a new nozzle, and finally skipping the current well, in that order. This represents an important advance in offline nanospray that is only possible due to the tight integration of the TriVersa NanoMate and the LTQ FT Ultra.</p><p>On the mass spectrometer side, the software performs a custom workflow to ensure the data acquisition system minimizes time wasted on targets unlikely to produce an identification. Before normal data acquisition begins, the system acquires a low number (usually one) of preview isolation scans on every target selected for offline interrogation. The purpose of this preview scan event is twofold: it allows the system to determine if the target detected online is present offline at sufficient absolute signal abundance to warrant further acquisition and to determine how many isolation and fragmentation scans should be acquired.</p><p>Both of the solutions above are by no means foolproof, but represent a significant advance toward emulating manual, human-controlled offline data acquisition. The overall outcome is a fully automated acquisition system at offline run time, operating continually for several days without intervention at the current stage of development.</p><!><p>Expectation values for peptides that are ≤10−5 allow direct and error-tolerant identification of a protein without resorting to decoy/reverse database construction and searching47 or identifying multiple peptides from the same protein. Therefore, future comparisons of proteome coverage obtained by different data acquisition strategies will be interesting, as obtaining high-resolution MS/MS is contrasted with the lower-resolution MS/MS experiments that now dominate data acquisition for shotgun proteomics.</p><!><p>In performing the bulk of the data acquisition offline, the increased spray time can be used to average multiple scans with more ions accumulated before detection. An offline mode of operation also allows the prior information of the entire chromatographic run to be available in determining what to fragment; therefore, more intelligent decisions can be made in terms of when precursors should be fragmented and which charge state should be selected for MS/MS. Data analysis is also simplified because each target is acquired in a separate data file.</p><p>Although the abundance of precursors is typically reduced versus the maximal instantaneous concentration during elution (due to dilution and possibly sample degradation in sample wells), automated offline acquisition is still able to collect high-quality data often far superior to online LC—MS/MS for targets identified in both modes. The greatly increased time available during offline acquisition yields improved fragmentation through averaging spectra, as demonstrated in Figure 6 with human high mobility group protein 17 (expectation value online 10−3 versus offline 10−98). Additionally, automated offline acquisition expands the depth of the proteome's dynamic range accessible with the characterization power of FTMS.</p><p>The ability to average scans offline also allows for routine multiplexed identifications. This occurred in 6% of the offline middle-down experiments attempted. When present at low abundance, 3–50 kDa precursor ions are particularly challenging to identify and characterize with MS/MS data obtained on a chromatographic time scale. Until mass spectrometers can produce such high-quality data sets at the resolution of a FT but with the speed and sensitivity of an ion trap, the current system now stands as a viable option for large-scale proteome projects.</p><!><p>A critical development planned for the future is the linking of the ProSightHT database and the AUTOMATION WAREHOUSE database. Currently this feedback loop connecting prior database hits to future data acquisition runs must be performed manually. This enhancement will facilitate automated population of the AUTOMATION WAREHOUSE database with confident protein/peptide identifications, enabling it to function as a true high-resolution exclusion list for a proteome project. Well-characterized species in the database will be low priorities in the target selection stage, furthering the goal of increased proteome coverage using top-down and middle-down strategies.</p><p>Although the current system represents the state-of-the-art for automated offline mass spectrometry, there are numerous opportunities for improvement. One concept that has successfully been applied to similar platforms in the past is automated determination of fragmentation parameters based on empirical data.22,31,40 An even more sophisticated possibility is automated dynamic adjustment of those parameters based on data surveyed in real time. Also promising is decision-making based on real-time spectral analysis in order to determine when averaging more scans is producing diminishing returns in terms of the number of new fragment ions or significant improvement in database retrieval scores,22 further optimizing the use of instrument time. Stahl et al. pioneered this concept using either the total ion current (TIC) of the most recent product ion spectrum or "spectrum reproducibility," based on the abundance of the top three fragment peaks, depending on the sample levels.39</p><p>In the future, it is critical that this system be compared to another promising route, the use of "smart" LC—MS/MS using data acquisition software that makes sophisticated decisions on-the-fly. Although commercial instrument firmware has progressed greatly in recent years, making concepts such as data-dependent acquisition, inclusion/exclusion lists, and neutral-loss experiments routine, there are several other advanced strategies to be implemented to better use high-value instrument time. Recent development of "decision tree" proteomics, where the fragmentation method is determined in real time based on precursor m/z and charge state, represents a significant first step toward this goal.48 In the future, such rapid experimental logic could be extended, for example, by querying a proteome project-wide database before deciding on fragmentation targets. The advantage of such a platform would mean workflows would be left relatively unchanged from current LC—MS/MS, although there is undoubtedly a limit to the proteomic depth achievable with online MS/MS alone, particularly with top-down and middle-down using contemporary instrumentation. For some applications, the recently introduced concept of a "replay" run may be a feasible alternative that lies between a completely offline or completely online approach.49</p><!><p>Although our focus is clearly top-down and middle-down analysis of proteomic samples with Fourier transform ion cyclotron resonance (FTICR) MS, the platform functions interchangeably with the Thermo Scientific LTQ Orbitrap or even standalone LTQ linear ion trap instruments. Additionally, the system could be adapted for other types of samples, such as small molecules or small peptides (i.e., bottom-up proteomics; Luo et al. recently noted the limitations of data-dependent acquisition in a shotgun experiment50), without much effort. We introduce this automated online—offline engine as a general approach to acquire high-quality, information-rich tandem mass spectra for species not identified or characterized on a chromatographic time scale.</p>

PubMed Author Manuscript

Activated T cells exhibit increased uptake of silicon phthalocyanine Pc 4 and increased susceptibility to Pc 4-photodynamic therapy-mediated cell death

Photodynamic therapy (PDT) is an emerging treatment for malignant and inflammatory dermal disorders. Photoirradiation of the silicon phthalocyanine (Pc) 4 photosensitizer with red light generates singlet oxygen and other reactive oxygen species to induce cell death. We previously reported that Pc 4-PDT elicited cell death in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro, and furthermore that Jurkat cells were more sensitive than A431 cells to treatment. In this study, we examined the effectiveness of Pc 4-PDT on primary human CD3+ T cells in vitro. Fluorometric analyses of lysed T cells confirmed the dose-dependent uptake of Pc 4 in non-stimulated and stimulated T cells. Flow cytometric analyses measuring annexin V and propidium iodide (PI) demonstrated a dose-dependent increase of T cell apoptosis (6.6\xe2\x80\x9359.9%) at Pc 4 doses ranging from 0\xe2\x80\x93300 nM. Following T cell stimulation through the T cell receptor using a combination of anti-CD3 and anti-CD28 antibodies, activated T cells exhibited increased susceptibility to Pc 4-PDT-induced apoptosis (10.6\xe2\x80\x9381.2%) as determined by Pc 4 fluorescence in each cell, in both non-stimulated and stimulated T cells, Pc 4 uptake increased with Pc 4 dose up to 300 nM as assessed by flow cytometry. The mean fluorescence intensity (MFI) of Pc 4 uptake measured in stimulated T cells was significantly increased over the uptake of resting T cells at each dose of Pc 4 tested (50, 100, 150 and 300nM, p<0.001 between 50 and 150nM, n=8). Treg uptake was diminished relative to other T cells. Cutaneous T cell lymphoma (CTCL) T cells appeared to take up somewhat more Pc 4 than normal resting T cells at 100 and 150nm Pc 4. Confocal imaging revealed that Pc 4 localized in cytoplasmic organelles, with approximately half of the Pc 4 co-localized with mitochondria in T cells. Thus, Pc 4-PDT exerts an enhanced apoptotic effect on activated CD3+ T cells that may be exploited in targeting T cell-mediated skin diseases, such as cutaneous T cell lymphoma (CTCL) or psoriasis.

activated_t_cells_exhibit_increased_uptake_of_silicon_phthalocyanine_pc_4_and_increased_susceptibili

Introduction<!>Human subjects<!>Cell Isolation<!>Cell Culture<!>Apoptosis Assay<!>Pc 4 uptake into T-cells<!>Confocal Microscopy<!>Activated T cells incorporate higher levels of Pc 4 than resting T cells<!>Activated T cells exhibit greater susceptibility to Pc 4-PDT-induced cell death than resting T cells<!>Activated T cells incorporate more total Pc 4 than resting T cells<!>Activated T cells are larger and contain proportionally increased levels of Pc 4 in mitochondria<!>Discussion

<p>Photodynamic therapy (PDT) uses visible light to activate a photosensitizing compound to generate cytotoxic reactive oxygen species that damage target and surrounding cells through apoptosis as well as other cell death processes. This technique has been demonstrated to be effective in non-melanoma skin cancers (NMSCs) (1–5) as well as other non-oncogenic skin conditions such as acne (6–8) and warts (9–11). Silicon phthalocyanine (Pc) 4 is a second-generation PDT photosensitizer discovered and developed at Case Western Reserve University (5) that has been recently shown to be safe in Pc 4-PDT-treated patients with skin neoplasms and to produce clinical improvement in cutaneous T cell lymphoma lesions (12–14). Pc 4 offers several advantages for PDT: 1.) It can be synthesized in high purity; 2.) The far-red absorption peak of Pc 4 has a high extinction coefficient, allowing efficient absorption of light at depth; 3.) Favorable pharmacodynamics allow for rapid clearance from the skin, minimizing post-treatment cutaneous photosensitivity. The use of Pc 4-PDT in clinical trials for psoriasis and cutaneous T-cell lymphoma has demonstrated this modality to be safe (12), with a maximum tolerated dose identified in CTCL, but not reached in psoriasis.</p><p>Pc 4 has been shown to preferentially bind to mitochondrial, lysosomal and endoplasmic reticulum (ER) membranes in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro (15, 16) as well as in numerous other cancer cell lines (reviewed in ref. 15). However, whether or not there is differential uptake and function as PDT on primary human, resting, activated, and malignant T cells is unknown. Therefore, we examined the effectiveness of Pc 4-PDT on such primary human CD4+ T cells in vitro. In this paper, we found that Pc 4 is preferentially taken up by activated and malignant T cells, which are rich in mitochondria, with a resultant associated increased Pc 4-PDT mediated apoptosis.</p><!><p>All studies of human subjects were approved by the Institutional Review Board of University Hospitals Case Medical Center (Cleveland, OH). Peripheral blood samples and/or punch biopsies were obtained from volunteer healthy controls and CTCL patients with Sezary Syndrome following informed consent.</p><!><p>Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood by Histopaque (Sigma-Aldrich), according to the manufacturer's instructions. CD3+ or CD4+ cells were separated from PBMCs by negative selection on MACS LS separation columns (CD3+/CD4+ T cell isolation kit; Miltenyi Biotec), according to the manufacturer's instructions. CD4+ cells were used for regulatory T cell (Treg) uptake experiments (see Figure 3). For Treg specific experiments, after overnight incubation, CD4+ T cells were incubated with phycoerythrin (PE)-labeled anti-CD25 mAb (BD Biosciences). Based on isotype comparators and expression levels, cells were then sorted into CD25high, CD25mid and CD25− subsets using a BD FACSAria cell sorter (Case Comprehensive Cancer Center Imaging and Cytometry Core Facility).</p><!><p>For all experiments, cells were cultured in RPMI 1640 (HyClone) supplemented with 10% fetal bovine serum (Lonza), 1% L-glutamine (HyClone), 1% penicillin-streptomycin and 0.2 mM beta-mercaptoethanol (Sigma-Aldrich). To activate the sorted CD25high, CD25mid and CD25− cells, 24-well cell culture plates (Costar) were coated with 0.5–5 μg/mL anti-CD3 mAb (BD Pharmingen). Cells were cultured for 3–5 days in 500 μL complete medium in the presence or absence of 1 μg/mL soluble anti-CD28 mAb (BD Pharmingen).</p><!><p>CD25high, CD25mid and CD25− cells were cultured at 3–4 × 105 cells per well with or without CD3/CD28 stimulation for 48 h. After resting an additional 24 h, cells were incubated with varying doses of Pc 4 (50–300 nM) in RPMI complete medium for 2 h, followed by irradiation with red light using a light-emitting diode array (EFOS, Mississauga, Ontario, Canada) at a fluence of 200 mJ/cm2 (1 mW/cm2, λmax ~670–675 nm) at room temperature. After 4h, cells were harvested and stained with fluorescein isothiocyanate (FITC)-labeled anti-Annexin V mAb and PI), according to the manufacturer's instructions (Annexin V-FITC Apoptosis Detection Kit I; BD Biosciences). Flow cytometric analysis was performed using the BD FACSAria Flow Cytometer.</p><!><p>T cells were cultured at 3–4 × 105 cells per well with or without CD3/CD28 stimulation for 48 h. After resting an additional 24 h cells were incubated with the indicated concentration of Pc 4 (50–300 nM) in complete medium for 2 h. Cells were then harvested and washed with Hanks' Balanced Salt Solution (1 mL) twice. Flow cytometric analysis was performed using a LSR II Flow Cytometer (Case Comprehensive Cancer Center Imaging and Cytometry Core Facility). Pc 4 was excited by a broadband UV laser (335–365 nm) and fluorescence emission was collected with a 650-nm long-pass filter. Autofluorescence of control cells was subtracted. In a subset of cells, cells were harvested, washed with Hanks' solution (1 mL) twice and then lysed in sodium dodecyl sulfate (SDS; 2 mL of 0.5%). The SDS concentration was above the critical micelle concentration (SDS CMC = 0.24%). Cell lysates were collected, and fluorescence was measured (Vector X3 Spectrophotometer, Perkin Elmer). A standard curve was constructed from cells lysed without Pc 4 to which known concentrations of Pc 4 (0–300 nM) were added.</p><!><p>T cells were cultured at 2 × 106 cells/mL with stimulation for 72 h. After this period, cells were incubated with the indicated concentration of Pc 4 (0–300 nM) in complete medium for 2 h. Cells (100 μL) were incubated with 50 nM MitoTracker Green (Invitrogen) for 30 min at 37°C, then counterstained with 10 μg/mL Hoechst 33342 (Sigma-Aldrich) for an additional 15 min at 37°C. Five to 10 μL of stained cells were then placed on a slide with a glass coverslip and confocal images were acquired using an UltraVIEW VoX spinning disk confocal system (PerkinElmer) mounted onto a Leica DMI6000B microscope (Leica Microsystem, Inc.) equipped with a HCX PL APO 100X/1.4 oil immersion objective. Confocal images of Pc 4 fluorescence were collected using a solid state diode 640 nm laser and a 705(W90) band pass filter. Images of MitoTracker Green fluorescence and Hoechst 33342 were collected using a solid state diode 488 nm laser with a 527(W55) band pass filter and 405 nm laser with a 445(W60) band pass filter, respectively.</p><!><p>CD3+ T cells were cultured with or without anti-CD3/CD28 stimulation and then exposed to various concentrations of Pc 4 in complete medium for 2 h. Cells were then harvested and washed to remove the medium and any associated non-internalized Pc 4. Internalized Pc 4 was quantified by flow cytometry as described in Methods. Activated T cells incorporated up to 4-fold higher levels of Pc 4 compared to resting T cells (Figs. 1A & B). Cumulative data for Pc 4 uptake are indicated in Fig. 1C. T cells incorporated Pc 4 in a dose-dependent manner; at each Pc 4 concentration, activated cells incorporated more Pc 4 than resting cells (p≤0.001 at each of 50, 100 and 150nM Pc 4, n=8). Note the double peak in the activated samples. There was variation in the degree of activation in this population such that, the larger peak correlated with more activated cells, while the smaller correlated with less activated cells.</p><!><p>We next sought to determine whether or not the increased uptake of Pc 4 translated into more effective cell killing following photoirradiation of the cells. CD3+ T cells were cultured with or without anti-CD3/CD28 stimulation for 48 h. Following stimulation, cells were incubated with Pc 4 (50–300 nM) in complete medium for 2 h, followed by irradiation with 200 mJ/cm2 of 675 nm red light. Four h after irradiation, T cells were harvested and stained with fluorescein isothiocyanate (FITC)-labeled anti-Annexin V monoclonal antibody and PI (Fig. 2A, quadrant 2). Activated T cells exhibited substantial apoptosis (Annexin V) and cell death (PI uptake) at or above 100nM Pc 4 PDT, whereas resting T cells were relatively resistant to Pc 4 PDT at the 50, 100 and 150 nM doses (p< 0.01, n=8). At 300nM Pc 4, the differential cytotoxicity was lost (Fig. 2B). To confirm that apoptosis was the preferential cell killing method induced by Pc 4, we measure Caspase-3 induction in activated versus regulatory T cells from healthy controls (Fig. 2C, n=2). We next determined whether activated T cells preferentially uptake Pc 4 relative to regulatory T cells, potentially targeting pathogenic T cells while sparing T regulatory cells. Indeed, as shown in Figure 3 representative Treg samples from healthy control individuals demonstrate that both CD4+ CD25neg and CD4+ CD25mid cell populations (recently activated cells), take up more Pc 4 than their corresponding Treg (CD4+ CD25+) population.</p><!><p>Only single Pc 4 monomers have significant fluorescence, while aggregated Pc 4 molecules are essentially non-fluorescent. Thus, the measurement of cellular Pc 4 by fluorescence may not account for all of the intracellular photosensitizer. Therefore, the total amount of Pc 4 in cells (Fig. 4A) was determined in the same cell populations described above by lysing an aliquot of cells in SDS, which solubilizes and monomerizes all Pc 4, and measuring the fluorescence emission after excitation of Pc 4 at 610 nm. The level of total Pc 4 increased in a dose dependent manner (Fig. 4A, B), as observed in the intact cell flow cytometry based assays (Fig. 1C); this observation rules out the possibility that differential Pc 4 fluorescence by flow cytometry is due to differences in aggregated monomeric Pc 4 when it is present and distributed in living T cells.</p><!><p>Activated or resting cells were treated with 150 nM Pc 4 in complete medium for 2 h. Prior to imaging, 50 nM MitoTracker Green and 10 μg/mL Hoechst 33342 were loaded into the cells for 15 min at 37°C. As indicated in Fig. 5, activated T cells were larger and appeared to have internalized more Pc 4 compared to unstimulated cells. Intracellular Pc 4 demonstrated striking co-localization with mitochondria at a higher rate in activated compared to non-activated T cells, suggesting that once internalized, Pc 4 is associated primarily with the mitochondrial membrane of T cells (Fig. 5). It has been reported that oncogenes and tumor suppressors can modulate signaling pathways that regulate mitochondrial dynamics and that mitochondrial mass and function vary between tumors and individuals (17–19). Therefore, the efficacy of Pc 4 PDT in CTCL patients may be related to enhanced uptake of Pc 4. At 50 and 100nM Pc 4, resting CTCL T cells appeared to take up more Pc 4 than resting T cells isolated from healthy control individuals (Supplementary Figure 1A, B), with an average of 953 MFI in CTCL versus 529 MFI in controls (50nM) and 1280 MFI in CTCL versus 893 MFI in controls (100nM), in 2 paired patient and control samples. Upon activation, the difference in Pc 4 uptake between CTCL T cells and T cells from healthy control individuals is lost.</p><!><p>Pc 4 has been found to be highly effective against numerous human cancer cells in vitro and model tumor systems (20–25). PDT with Pc 4 is currently in clinical trials for psoriasis and CTCL (14, 26, 27), and other applications are being developed. Pc 4 has been shown to bind preferentially to mitochondrial, lysosomal and ER membranes (28, 27, 16). Interestingly, recent publications have suggested that PDT leads to an antigen-specific immune response (29), which, taken together with our data indicating less Pc 4 uptake by Treg cells than by activated T cells, suggests a potential for rebalancing the ratio of Treg to Teff. For example, if Pc 4-PDT were applied during an ongoing effector T cell-mediated immune response, it is likely that more Tregs would be found at the response site due to less apoptosis associated with phototherapy due to less uptake of Pc 4. Therefore, in addition to having less effector T cells due to increased Pc 4 uptake and PDT-dependent apoptosis, there would be a higher ratio of Treg:Teff cells and a net suppressive environment which would result in a more effective suppression of a responding effector T cell response. Whether this will be demonstrable in autoinflammatory disorders and cancer remains to be seen, as a decrease in Treg in situ in esophageal cancer following PDT with Photofrin was not observed (30). Extensive investigations using various PDT photosensitizers have previously revealed preferential uptake of photosensitizers by 1.) leukemic cells (31–35); and 2.) activated lymphocytes compared to resting cells (36–39). The use of PDT for treatment of skin cancers has been slowly increasing with advances in light delivery systems and improved photosensitizers (40). The use of PDT in non-oncogenic skin disorders, such as the treatment of psoriatic plaques, has been evaluated > 25 years ago, and recent meta analyses have concluded that PDT may not be effective for psoriasis (41). One possible explanation for this lack of efficacy may be the uptake of the photosensitizer aminolevulinic acid (ALA) predominantly by keratinocytes of the skin (42), in essence acting as a sink for the photosensitizer following oral delivery. However, these same authors also demonstrated that oral delivery of the ALA also induced T lymphocyte apoptosis in psoriasis patients (43). Given new information regarding the potential target cells and immune response in psoriasis (e.g., Th17) differential uptake by activated lymphocytes compared to regulatory T cells, and the potential to one day specifically target Th17 cells, may present a viable attack strategy for PDT on psoriasis.</p><p>The mechanism of action of Pc 4 involves absorption of a photon by Pc 4, resulting in Pc 4 emerging as a triplet-state photosensitizer that transfers energy to ground state oxygen (Type II photochemistry), generating singlet oxygen, a highly reactive form of oxygen that reacts with many biological molecules, including lipids, proteins, and nucleic acids, producing an oxidative stress and eventually leading to cell death (44). Cell types and states of activation vary significantly in their susceptibility to oxidative stress, which may account for our previous observation that a human acute T-cell leukemia cell line (Jurkat) was far more susceptible to Pc 4-PDT-induced cell death than was a human epidermoid carcinoma cell line (A431) (15). Interestingly, within populations of normal human T cells, we observed a hierarchy in which the highest uptake of Pc 4 occurred in activated T cells with large numbers of mitochondria, the next highest uptake was in resting T cells, with the lowest Pc 4 uptake occurring in Treg. The high uptake of Pc 4 by activated T cells suggests that reactive T cells in chronic inflammatory conditions or neoplastic T cells in T cell lymphomas/leukemias may be highly susceptible to Pc 4-PDT-induced cell death, with a potential therapeutic window where resting naïve and memory T cells are left alive, along with sparing of Treg cells that may be able to restore quiescence once the auto-reactive inflammatory population numbers are reduced.</p><p>To quantify the susceptibility of primary resting versus activated T cells to Pc 4-PDT, we negatively selected CD3+ T cells from normal peripheral blood samples, rested half of the T cells for 24 h and stimulated the other half with CD3/CD28. Resting or activated T cells were then treated with various doses of Pc 4 for 2 h followed by photoirradiation. In response to PDT, we observed a Pc 4 dose-dependent increase in the percentage of cell death in both resting and activated T cells. Interestingly, activated T cells exhibited greater sensitivity than resting cells, a difference that was statistically significant at all except the highest dose of Pc 4.</p><p>Because activated T cells appear to be more susceptible to Pc 4-PDT than their resting counterparts, confirming observations by other investigators (34), we then determined whether there was any difference in the ability of activated and resting T cells to incorporate Pc 4 (45). Flow cytometric analysis of Pc 4 demonstrated that T cells incorporate Pc 4 in a dose-dependent manner and that activated cells incorporate more Pc 4 compared to resting cells. Using light scatter characteristics of T cells, it was apparent that larger, more granular cells (likely more activated) exhibited the most significant Pc 4 uptake (Fig. 1B).</p><p>This suggested that Pc 4 might localize in different sites in resting and activated T cells. Since Pc 4 preferentially binds to mitochondrial membranes, we used a mitochondrial marker (MitoTracker Green) to simultaneously visualize the mitochondria of the targeted T cells (28). Upon analysis of the confocal images, it was apparent that activated T cells are larger. Additionally, mitochondria were more numerous in activated T cells and contained proportionally increased levels of Pc 4. The increased numbers of mitochondria associated with activated T cells suggests that a mitochondrion-induced apoptosis may occur in T cells as observed for other cell types. Specifically, Pc 4-PDT has been demonstrated to damage the anti-apoptotic proteins Bcl-2 and Bcl-XL when they are bound to the mitochondrial or ER membranes, resulting in Bax translocation from the cytosol to mitochondria, and the release of cytochrome c and induction of the intrinsic apoptosis pathway that may be enhanced by lysosomal disruption (46, 15, 47–51).</p><p>In conclusion, these results suggest that a window exists in which activated normal primary T cells are more susceptible to Pc 4-PDT-induced cell death compared to their resting counterparts. Additionally, our flow cytometry and fluorometry experiments demonstrate that activated cells incorporate more Pc 4 proportionally to size and granularity. The presence of activated T cells in T cell-mediated skin diseases, such as CTCL, may provide a favorable therapeutic window in which Pc 4-PDT may act. Lastly, confocal imaging appears to indicate an increase of mitochondria and thus a proportional increase in Pc 4 levels. Our results indicate that activation of T cells provides an increased target area (mitochondria) upon which Pc 4 may act, and that apoptosis induction by Pc 4 may be useful in skin diseases where T cell proliferation is ongoing. These observations may account for the increased sensitivity of activated T cells to Pc 4-PDT.</p>

PubMed Author Manuscript

Rapid Mechanochemical Synthesis of Amides with Uronium-Based Coupling Reagents, a Method for Hexa-amidation of Biotin[6]uril

Solid-state reactions using mechanochemical activation have emerged as solventfree atom-efficient strategies for sustainable chemistry. Herein we report a new mechanochemical approach for the amide coupling of carboxylic acids and amines, mediated by combination of (1сyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) or N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate (TCFH) and K2HPO4. The method delivers a range of amides in high 70-96% yields and fast reaction rates. The reaction protocol is mild, maintains the integrity of the adjacent to carbonyl stereocenters, and streamlines isolation procedure for solid amide products.Minimal waste is generated due to the absence of bulk solvent. We show that K2HPO4 plays a dual role, acting as a base and a precursor of reactive acyl phosphate species. Amide bonds from hindered carboxylic acids and low-nucleophilic amines can be assembled within 90 min by using TCFH in combination with K2HPO4 or N-methylimidazole. The developed mechanochemical liquid-assisted amidation protocols were successfully applied to the challenging couplings of all six carboxylate functions of biotin[6]uril macrocycle with phenylalanine methyl ester, resulting in an 80% yield of highly pure hexa-amide-biotin[6]uril. In addition, fast and high-yielding synthesis of peptides and versatile amide compounds can be performed in a safe and environmentally benign manner, as verified by green metrics.

rapid_mechanochemical_synthesis_of_amides_with_uronium-based_coupling_reagents,_a_method_for_hexa-am

INTRODUCTION<!>Current Work<!>RESULTS AND DISCUSSION<!>Scheme 2. Optimization Experiments<!>AE =<!>Scheme 4. Plausible Mechanistic Pathways Leading to Amide Product<!>Scheme 5. Mechanochemical Coupling of Hindered Carboxylic Acids and Poor Nucleophilic Amines

<p>Amide bond is widespread in both natural compounds and artificial materials. It occurs in molecules fundamental to life, such as peptides and proteins, as well as in synthetic polymers and in a massive array of pharmaceuticals. In fact, amide preparation from carboxylic acids and amines represents the most frequently applied chemical transformation in drug production and comprises about 25% of the current medicinal chemistry synthetic toolbox. 1 As a consequence of its wide usage, the development of sustainable amidation methods was listed among the top green chemistry research priorities by the American Chemical Society Green Chemistry Pharmaceutical Roundtable (ACS GCIPR) in 2007 2 and has been retained in the recent revision. 3 Although direct condensation of carboxylic acids and amines with water as a single by-product can be considered a "green" landmark in the field, it remains impractical because of the process's harsh reaction conditions (T > 100 °C) and limited substrate scope. [4][5][6] A robust method of amide synthesis commonly requires prior activation of a carboxylic function to replace OH with a better leaving group. [7][8][9] Notably, this is also the case in biosynthetic pathways, including the translation process and non-ribosomal enzymatic transformations. [10][11][12][13] For laboratory and industrial use, vast numbers of amide coupling reagents, performing in situ activation of carboxylic acid, have been developed in the quest for faster, milder, and high-yielding amidation protocols. 14,15 Low atom economy of these reagents and accompanying safety issues are their major drawbacks, which has incited the development of alternative approaches. [16][17][18][19] Important advancements have thus far followed traditional solution-based approaches; however, solvent is actually responsible for 80−90% of mass consumption in a typical chemical process and also plays a major role in overall toxicity. 20 In this way, solvent greatly outperforms the contributions of reagents themselves. Hazardous solvents like DMF and DCM are preferred in industrial amide synthesis, reinforcing both environmental and safety concerns. 17,21 Therefore, the application of solvent-free techniques represents an efficient way to improve the overall process mass intensity and to prevent generation of hazardous waste.</p><p>Recent advances in mechanochemistry and its related fields have established solvent-free reactions as environmentally friendly tools to perform chemical transformations that are no less efficient than the conventional solution-based chemistry. [22][23][24] In the area of amide synthesis, the benefits of solvent-free techniques have not remained unnoticed and have been previously demonstrated in numerous studies (Scheme 1). [25][26][27] For example, mechanosynthesis of various amides and peptides has been performed from a series of activated carboxylic acid derivatives, such as N-carboxyanhydrides; 28,29 N-hydroxysuccinimide esters; 30 Nacyl benzotriazoles. 31 N-Acyl imidazoles 32 and acyloxytriazine esters 33 have been produced mechanochemically from carboxylic acids prior to reacting with amines. Notably, even papain enzyme can catalyze the formation of peptides from the corresponding amino acid building blocks under solvent-free conditions. 34,35 In addition, direct coupling of amines with carboxylic acid has been demonstrated by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a coupling reagent. 36,37 In general, EDC-mediated transformations have shown remarkably short reaction times (typically within 10-30 min), high yields, and simple work-up protocols.</p><!><p>Following these prominent earlier contributions, [24][25][26][27][28][29][30][31][32][33] we aimed to further expand the scope and synthetic utility of the mechanochemical amidation methods. The current research was impelled by three objectives: First, most of the amide coupling reagents are simply not efficient enough for a range of substrates, 8 which require expansion of the established one-step mechanochemical amidation protocols beyond the previously applied EDC; for that purpose, in this work we mapped the coupling efficiency of uronium-type reagents (COMU and TCFH, Scheme 1) on several carboxylic acid/amine pairs. Second, the scope of previously published mechanochemical approaches was evaluated based mainly on peptide synthesis, while the challenging couplings of sterically hindered carboxylic acids and low-nucleophilic amines remained virtually unproven; here we demonstrated that such difficult amide bonds can also be assembled under solvent-free conditions. Implementation of the two objectives mentioned above was required as a prerequisite for the third objective as our ultimate goal. Due to the interest of our group in the synthesis and supramolecular applications of macrocyclic host molecules, [38][39][40][41][42][43] we required a robust procedure for amide-functionalization of biotin [6]uril macrocycle (1), [44][45][46] to access the family of modified biotin [6]uril hosts. Despite the apparent ease of such a transformation, it also presented a substantial challenge: six-fold stepwise amidation of carboxylate groups in 1 is inevitably accompanied by accumulation of the "failed" underfunctionalized products if incomplete coupling occurs at each step. Limited solubility of 1 in the common organic solvents dictates additional practical inconvenience of the traditional solution chemistry; in fact, only dipolar aprotic solvents like DMF can be used. Here we showed that application of solvent-free techniques, additionally reinforced with the reactive uronium-type amide coupling reagents, allows the desired functionalization of 1 in a high-yielding, scalable, and sustainable manner, avoiding harmful solvents or significant reagent excess.</p><!><p>Development of Mechanochemical Amidations with Uronium-Type Reagents. At the outset, amide coupling of Cbz-protected L-phenyl alanine (2) and ethyl 4-aminobenzoate (benzocaine, 3), mediated by COMU as a representative "green" uronium-type amide coupling reagent, [47][48][49] was selected as a model process (Scheme 2). We aimed to screen and compare the results of various reaction conditions, including the evaluation of coupling efficiency for different coupling reagents beyond the COMU itself, to reveal the most promising hits in terms of product yield and green chemistry requirements. The choice of aromatic amine 3 was dictated by its reduced nucleophilicity in comparison with aliphatic amines, additionally attenuated by an electron-withdrawing ethoxycarbonyl group. We expected that suppressed reactivity of 3 in the carbonyl addition reactions would facilitate more reliable differentiation of various coupling conditions. Use of phenyl alanine derivative 2 as coupling counterpart provided an additional opportunity to examine the resistance α-stereocenter towards its possible epimerization, as commonly encountered in peptide synthesis. 9,15</p><!><p>The test reactions were run in a Form-Tech Scientific FTS1000 shaker mill operating at 30 Hz by using 14 mL zirconia-coated milling jars, 3 × 7 mm zirconia milling balls and typical solid reactants loading around 0.3−0.4 g (including 0.2 mmol of amine 3 as a limiting substrate). After 30 min milling time, a sample of the crude reaction mixture was treated with CDCl3, followed by separation of insoluble inorganic materials. The conversion of amine 3 into amide 4 was determined by 1 H NMR analysis (see Supporting Information for the details). The amide coupling reagent, base, and amount of liquid additive needed to assist the grinding process (Scheme 2) were identified as the three most crucial parameters affecting the yield of amide 4, as described below.</p><p>The addition of a small volume of liquid constitutes an efficient method to enhance the performance of solvent-free mechanochemical reactions, known as liquid-assisted grinding (LAG). 22,24 The ratio of the volume of liquid (μL) added to the amount of solid present (mg) is denoted as η (μL/mg). 50 A value of η = 0 generally corresponds to dry grinding, but in a typical LAG process, η is usually between 0 and 1. 24 Although LAG cannot be described as a totally solvent-free technique, it requires a minimal amount of liquid, especially advantageous if a green solvent is used. Among the latter, 20,51,52 ethyl acetate appears to be the most promising and chemically compatible candidate to act as a LAG additive in COMU-mediated amide coupling. In our experiments (Scheme 2, Chart 1), the addition of ethyl acetate indeed showed a pronounced effect on the yield of amide 4, generated in the mixture of solid reactants 2 and 3, with COMU reagent and sodium carbonate (ca. 10 equiv.) as a base. Although dry grinding provided a rather modest outcome (44% conversion), LAG resulted in a markedly improved reaction performance, with the optimal η value in a range of 0.14−0.24 μL/mg, while the further increase of η led to slightly diminished conversion values.</p><p>The choice of base is also important in amide coupling. State-of-the-art solution approaches commonly apply non-nucleophilic tertiary amines, e.g. N,N-diisopropylethylamine (DIPEA). 15 However, the use of cheap and non-toxic inorganic salts, e.g. NaHCO3, K2CO3, NaH2PO4, 28,30,33,37 insoluble in common organic solvents, can be considered as an additional advantage of mechanochemical reactions. In our hands (Scheme 2, Chart 2), replacement of DIPEA with Na2CO3 gave similar conversion values (72% vs 75%). For further process optimization, a range of readily available phosphate salts, with notably distinct pKa values, were screened. Among them, potassium pyrophosphate K4P2O7 and dipotassium phosphate K2HPO4 provided the best outcomes, especially the latter (96% conversion). Generally, the performance of phosphate salts does not correlate with Brønsted basicity of the respective anions. Although the poor outcome with KH2PO4 (only 18% conversion) in comparison with K2HPO4 (96%) could be probably connected with the significantly reduced base strength of the former (respective pKa values 2.12 vs 7.21; pKa of RCO2H is typically about 4−5 in aqueous media), 53 much more basic K3PO4 (pKa 12.32) also afforded amide 4 with reduced efficiency (72%). Surprisingly, the counter-cation effect (Na + vs K + ) also had a prominent impact on reaction outcome (37% vs 96%, for Na2HPO4 and K2HPO4 respectively). These results clearly indicate that the effect of an inorganic base on a solid-state reaction is more intricate than trivial proton transfer.</p><p>Finally, amide coupling reagents are essential for attaining high yields. The selection of coupling reagent was governed by considering chemical (substrate scope, reactivity); safety; and environmental issues. Uronium salts are advantageous because of their prominent reactivity and efficient reaction rates, 8,14 but the most commonly applied triazole-based reagents, such as HBTU and HATU, possess dangerous explosive properties 54 and pose significant health risks. 55 COMU was introduced as a safe and "greener" replacement. 47,48,56 To our delight, COMU also noticeably exceeded the coupling efficiencies of HATU and EDC in our experiments (Scheme 2, Chart 2), delivering a high 96% conversion. TCFH can be considered as an even more reactive alternative with better atom economy, affording a high 97% yield of amide 4 within only 10 min. The mechanochemical amidation with COMU/K2HPO4 was also rapid, reaching the maximal conversion within 20 min (Figure 1; see the Supporting Information for further detail), far surpassing the rate of the solution-based process (in DMF-d7, Figure 1). The latter reached the maximal 70% conversion after approximately 20 h (see the Supporting Information). Concurrently, about 30% of COMU reagent degraded due its well-known hydrolytic instability in DMF solutions, which is often referred as the main disadvantage of COMU. 57,58 Evidently, this drawback can be fully eliminated under solvent-free conditions.</p><p>After achieving these results in the optimization experiments, we formulated the optimal experimental procedure as follows: COMU or TFCH (1.1 equiv.) as coupling reagents; K2HPO4 (3 equiv.) as base; ethyl acetate as LAG additive, and 20 min milling time. The amount of solid base (3 equiv.) was adjusted to keep η within the optimal range (~ 0.2 μL/mg), but not less than 2 equiv. required according to the reaction stoichiometry. Furthermore, an additional equivalent of K2HPO4 was required to release free amine when ammonium salt was used as the starting material. Isolation of pure amide 4 was achieved with a high 96% yield by simple water wash and filtration since all by-products are water soluble. No detectable racemization of the chiral center in 4 occurred during the synthesis, as was established by the chiral phase HPLC chromatography (see the Supporting Information).</p><p>Green Chemistry Metrics Comparison. The advantages and drawbacks of the developed mechanochemical amidation methods were further revealed and compared with the solution-based reaction by analyzing the respective green metrics (Table 1). The metrics were calculated and assessed by marking them with red, orange, or green flags by following the Clark's unified metrics toolkit (see Supporting Information). 59 Atom economy (AE), reaction mass efficiency (RME), and process mass intensity (PMI) are defined as follows: 59</p><!><p>molecular weight of product total molecular weight of reactants × 100 RME = mass of isolated product total mass of reactants × 100 PMI = total mass in a process mass of product First, isolated yields and product purity were much better in mechanochemical reactions, due to the higher conversion and more facile isolation procedure discussed above. Atom economy was a bit higher for the TCFH-mediated reaction because of lower molecular weight of TCFH. RME reflects both product yield and atom economy issues and was lower for the solution-based reaction.</p><p>Comparison of PMI values clearly shows that mechanochemical reactions produce far less waste.</p><p>Excluding mass-extensive work-up procedures, solvent occupied 84% of PMI for the solutionbased reaction and only about 15% (LAG additive) for the mechanochemical conditions.</p><p>Furthermore, sustainable solvents like water and ethyl acetate were used in the latter, in contrast with toxic DMF. To determine the safety risks, a combination of physical, health, and environmental threats must be assessed, which can be done with the help of MSDS 60 and further available safety data. 54 DMF, for instance, is a flammable (H226), acute toxic (H312, 332), as well as a reproductive toxin (H360) and can thus be cited as the main hazard contributor for the solution-based process, which therefore received a red flag. For the mechanochemical reactions, the TCFH-mediated process was given a red flag due to the production of tetramethylurea by-product (reproductive toxin, H360). On the other hand, exothermic decomposition with a thermal onset of 127 °C can be considered as the main hazard of COMU, according to a recent study. 54 However, this property produced an orange flag, since COMU-mediated mechanochemical amidation protocol operates at room temperature.</p><p>To conclude, although the developed mechanochemical amidation conditions cannot be considered totally safe, the risks are minimal because of its room temperature operation and relatively low amount of produced waste, as opposed to the solution-based reaction (see Supporting Information for additional safety considerations).</p><p>Substrate Scope for mapping reactivity with COMU and TCHF. Having established the optimal conditions, substrate scope and limitations was briefly examined on a range of amine and acid coupling partners (Scheme 3). The substrate scope included, besides other, N-and C-protected amino acids and pharmaceutically relevant starting materials (e.g. (S)-naproxen, (S)-ibuprofen, benzocaine 3, N-Boc-protected piperazine). In addition to the foremost example of Cbz-masked amide (S)-4 comprehensively described above, its Fmoc-protected analogue (R)-5 was obtained in a high 95% yield by using the COMU-mediated reaction. Following the same protocol, dipeptides 6 and 7 with sterically hindered amino acid residues (phenylalanine and valine) were flawlessly prepared in high yields. No detectable epimerization of the stereocenters was noted in these cases. Coupling of (S)-(+)-6methoxy-α-methyl-2-naphthaleneacetic acid [(S)-naproxen] with amine 3 provided a more demanding test for stereochemical integrity, since 2-arylpropionic acids are prone to easy epimerization. [61][62][63][64] The amide product (S)-8 was obtained from (S)-naproxen with a high 86% yield and excellent stereochemical purity (>99% ee). This was also the case in the TCFH-mediated reaction, which showed high reactivity and a subtle amount of epimerization for (S)-9. On the other hand, amidation of (S)-ibuprofen (98% ee) produced (S)-10 with slightly degraded optical purity (93-94% ee). Crude amides 10, 12, 15 appeared as oils immediately following the milling, which eventually enabled a chromatographic isolation for these cases (see Supporting Information).</p><p>One advantage of TCFH over COMU-mediated amide coupling is the higher reactivity of the former reagent, which makes it more suitable for less reactive substrates. This property was explicitly revealed during the amidation of sterically hindered ortho-substituted benzoic acids.</p><p>Thus, coupling of benzoic acid with benzocaine 3 proceeded well under the COMU-mediated protocol, furnishing amide 11 in a 93% yield after 20 min of milling time. Conversely, 2,4,6trimethylbenzoic acid under the same conditions produced only 22% of the target amide 14, without any further improvement, even when a longer milling time (up to 60 min) was applied.</p><p>After the brief optimization studies (see Supporting Information), we found that a slight excess (1.3 equiv.) of more reactive TCFH and at least 60 min of milling time are required to attain a high 89% yield of 14. Moreover, chromatographic purification of 14 was necessary to separate mesitoic anhydride impurity. Diminished reactivity was also observed for 2,6-difluorobenzoic acid, furnishing amides 15 and 16 in reactions with N-Boc piperazine and low-nucleophilic amine 3 in acceptable yields after milling times of 40 and 60 min, respectively. On the other hand, coupling of the same amines with benzoic and 3,5-bis(trifluoromethyl)benzoic acids proceeded flawlessly, producing amides 12 and 13 with excellent yields and brief reaction times.</p><p>Activating Effect of Phosphate Salts. During the optimization studies, the enhancement of yields with dipotassium phosphate and potassium pyrophosphate was especially notable (Scheme 2, Chart 2). We speculated that phosphate salts could additionally contribute to the activation of the carboxyl substrate 2 via the formation of acyl phosphate intermediates containing a "high-energy" phosphoester bond, prone to easy nucleophilic amine attack. 65,66 Interestingly, the same pathway is also involved in the ATP-dependent biosynthesis of amide bond-containing biomolecules. 11 The plausibility of our assumption is further supported by existing literature showing that acyl phosphates can be indeed generated in solution by the DCC-mediated coupling of carboxylic acids with phosphate salts. [67][68][69] To confirm the credibility of our hypothesis, mechanochemical synthesis of acyl phosphates from carboxylic acids and phosphate salts, mediated by COMU and TCFH, was attempted.</p><p>As expected, a 20-min ball milling of COMU (1.1 equiv.) with acetic acid (1 equiv.) and K2HPO4</p><p>(3 equiv.) yielded 60% of acetyl phosphate 17, which was confirmed by NMR analysis of the freshly obtained reaction mixture in D2O solution (Figure 2). Acetyl phosphate 17 displayed a singlet signal at δ = −2.1 ppm in 31 P NMR, which rapidly disappeared after the addition of morpholine, both in D2O solution and in the solid state (see Supporting Information). In the 13 C NMR spectrum, carbonyl group 17 showed a doublet signal at δ = 168.1 ppm (JCP = 8.8 Hz), due to its coupling with the neighboring phosphorus. 65 Significantly lower yields of 17 were attained with K3PO4 or with TCFH as coupling reagent (Figure 2). The reaction of acetic acid with K4P2O7 produced acetyl pyrophosphate 18 in a 50% yield, according to 31 P-NMR analysis. As a result of the non-equivalence of phosphorus atoms in 18, a pair of doublet signals appeared in 31 P NMR, at δ = −5.0 and −17.9 ppm (d, JPP = 21.7 Hz), thus confirming its structure. 67 As an extra example, the generation of acyl phosphate 19 (50% yield, δ = −7.6 ppm in 31 P NMR) was also successful from Cbz-masked phenyl alanine 2, which was similar to the acetic acid outcome. For example, K3PO4 produced a rather low 15% yield of acetyl phosphate 17 (Figure 2), which also agrees with the lower conversion to amide 4 in the comparison with K2HPO4. The acyl phosphate pathway probably contributes less in the case of the more reactive TCFH reagent, which also produced a rather low 30% yield of 17 (Figure 2). The exact mechanistic sequence leading to acyl phosphates C from COMU, RCO2H and K2HPO4 remains unclear but may include the reaction of acyl uronium intermediate A with HPO4 2anion (Scheme 4) or, alternatively, the initial formation of uronium phosphate 71 by the reaction of COMU with K2HPO4.</p><!><p>Challenging Amide Bond Formation. As shown above, the coupling of low nucleophilic amine 3 with sterically hindered mesitoic acid could be efficiently mediated by the TFCH/K2HPO4 reagent system (Scheme 3). In accordance with existing literature, 61,72 we selected the coupling of electrondeficient 4-aminobenzonitrile 20 with 2-methyl-2-phenylpropanoic acid 21 (Scheme 5), an even more arduous way to test the performance of mechanochemical amidation protocols. Brief screening of various coupling conditions was undertaken, and conversion to amide product 22 was determined by 1 H NMR analysis after 60 min of milling time (Scheme 5).</p><!><p>The use of EDC alone, 36 or the COMU/K2HPO4 system, yielded only a low ~10% conversion.</p><p>Combination of TCFH and K2HPO4 delivered a noticeably better outcome but still failed to raise the conversion above 22%. According to the recent study of Beutner et al., 61 N-methylimidazole (NMI) and TCFH reagent combined provided a high yield of 22 in solution, due to in situ generation of reactive N-acyl imidazolium ions. To our gratification, same combination of reagents also worked well under the solvent-free conditions, affording respectable 84% conversion after a 60-min reaction time. Finally, a slight excess (1.3 equiv.) of TCFH reagent, along with a bit longer milling time (90 min), allowed us to obtain pure amide 22 in 92% isolated yield after an aqueous work-up (see Supporting Information). Following the same reaction protocol, the coupling of 21 with sterically hindered 2,4,6-trimethylaniline was performed and furnished the corresponding amide 23 with a 92% yield. Notably, high yields of amides 22, 23 were attained in a rather efficient reaction time of 1.5 h, in significant contrast with the solution-based reaction (21 h for amide 22). 61 Surprisingly, the same highly reactive combination of reagents failed to render amide 14 from mesitoic acid with yields exceeding 20%. This was also the case in the CD3CN solution (see Supporting Information). We found that the reaction was stopped due to the formation of sterically bulky and therefore non-planar N-acyl imidazolium D, which, in contrast to the analogous species produced from benzoic acid, was totally inert towards the subsequent reaction with amine 3 (see Supporting Information for further detail). Inertness of D could be explained by the efficient steric shielding of the carbonyl group with both neighboring mesityl and imidazolyl moieties, preventing attack of a nucleophile along the Bürgi-Dunitz trajectory (Scheme 5). This stands in sharp contrast to the successful TCFH/K2HPO4-mediated transformation, where the less sterically crowded intermediate species are expected to form (e.g. mesitoyl chloride, uronium or phosphate).</p><p>Amide Coupling of Biotin [6]uril. As a part of our ongoing efforts towards the development of new chiral supramolecular receptors, [38][39][40][41][42][43] we needed an expedient synthetic procedure for derivatization of biotin [6]uril (1), 44 easily available in multigram quantities by HCl-catalyzed condensation of formaldehyde with D-biotin. The starting macrocyclic molecule, notable for its anion binding properties, common for the cucurbituril family, [73][74][75][76][77] satisfies 6 carboxylic functions, which could be conveniently coupled with various amines, thus providing facile access to a library of diversely functionalized chiral macrocyclic receptors. Although amide coupling of carboxylates in 1 might appear simple, unencumbered by any steric or electronic influence, full amidation of 1 is challenging because it proceeds via six consecutive steps. For example, if a high 97% yield were produced during each step, the fully functionalized product would eventually generate only a (0.97) 6 •100% = 83% yield, while the rest of the produced material would contain a set of "failed" underfunctionalized molecules, thus necessitating time-consuming, laborious and mass-inefficient chromatographic purification. The situation resembles the synthesis of oligopeptides and oligonucleotides, in which an extremely high coupling efficiency (>99% per coupling step) is required to attain reasonable yields and high purity of long-chain oligomers, and it is customarily achieved by using an excess of highly reactive coupling reagents. 56 The low solubility of 1 in the environmentally benign and volatile organic solvents, compatible with the conventional amidation protocols (e.g. ethyl acetate), constitutes an additional restriction of the solution-based chemistry.</p><p>We believed that the high coupling efficiency observed under the solvent-free conditions would allow us to perform the desired functionalization in a high-yielding, and scalable manner without using an excess of reagents, toxic solvents, or laborious purification.</p><p>As a convenient model reaction for this study, we selected the amide coupling of 1 with methyl ester of phenyl alanine 24 (used as HCl salt, see Scheme 6). At its outset, this task required us to explore the performance of different amide coupling conditions. Only a slight excess of amine 24 and a coupling reagent (7-7.8 equiv., which is 1.16-1.3 equiv. per CO2H group of 1) were applied in the optimization experiments. It was expected that more reactive combinations of reagents would deliver higher yields of the hexa-amide product 25. Based on our previous findings, the order of coupling efficiency for the different reagent systems can be roughly plotted as follows: EDC ~ COMU/K2HPO4 < TCHF/ K2HPO4 << TCHF/NMI. Although such generalizations must be made with care since the coupling performance is substrate-dependent 8,78 and exceptions are possible (e.g. case of amide 14 above), this preliminary reactivity plot provided a helpful guide. Outcomes of the test reactions were analyzed by HPLC (Figure 3, see Supporting Information for further detail) and quantified by calculating HPLC area percentage for the hexa-amide product 25 (Srel, Table 2), relative to underfunctionalized compounds. These initial experiments (Table 2, entries 1-4) clearly indicated that complete hexafunctionalization of 1 is difficult to perform. Thus, both the COMU and TCFH/K2HPO4 systems produced a mixture of phenylalanine-derivatized biotin [6]urils, containing all possible products from mono to hexa-amide 25, the latter displaying a rather low 16% contribution (entries 1 and 2; Figure 3A). The use of EDC/DMAP combination (entry 3), 36 was more successful in this case, primarily producing a mixture of penta-and hexa-amides (Figure 3B). The highly reactive TCFH/NMI combination (entry 4) generated hexa-amide 25 as its main reaction product, but it was noticeably contaminated with underfunctionalized compounds (52% HPLC area, Figure 3C). Notably, at least 90-min milling times had to be applied, since samples taken after 30 and 60 min still showed incomplete conversion (see the Supporting Information). Although the FTS1000 shaker mill could hypothetically achieve long milling, we considered any time longer than 1.5 h as impractical; therefore, our next goal was to adjust the reaction parameters accordingly, in order to reach at least 90% conversion within a 1.5-h reaction time.</p><p>Applying a slightly greater excess of TFCH (1.2 equiv. per carboxylate) and NMI (3.5 equiv. per carboxylate) noticeably improved the yield of the target product, 25 (86% HPLC area, entry 5), and also shortened the reaction time. For further improvement, a screening of optimal η and LAG additive was performed. Since NMI is a liquid, and liquid tetramethylurea is produced, the addition of solid NaCl was attempted to reduce the initial η to 0.16 μL/mg. However, this distinctly reduced the yield of the product (73% HPLC area, entry 6). On the other hand, the addition of a few drops (ca. 35-50 μL) of solvents noticeably improved the outcome (entries 7 to 9) and was best when polar solvents like DMF or EtOAc were added (entries 7 and 9). These results clearly indicate that the nature of LAG additive plays an important role 37 and can substantially increase reaction rate, a probable result of the favorable interactions of the polar reactants with the mobile surface layers of LAG additive and improved mass transfer. 24 The outcome with EtOAc was especially remarkable, providing 25 with the best purity (98% HPLC area, Figure 3D). Since the reaction mixture visibly liquefied as the reaction progressed (due to the generation of tetramethylurea), slurry stirring was also tried instead of the ball milling (entry 10) and resulted in slightly reduced coupling efficiency.</p><p>Solution-based amide couplings were performed in DMF (entries 11 and 12) for the comparison with mechanosynthesis. Homogeneous solutions were obtained with an amount of solvent (ca. 0.5 mL) comparable to the weight of solid reactants (ca. 0.24 g), what kept η at around 2 μL/mg. In the DMF solution, HATU was another frequently used and highly reactive uronium-based amide coupling reagent that produced a rather modest outcome (entry 11). Conversely, the coupling efficiency of the TCFH/NMI combination in DMF solution (entry 12) was virtually the same as in the DMF-free transformation to a solid state (entry 9). Importantly, a bulk amount of harmful solvent was fully avoided in the latter.</p><p>Under the optimal reaction conditions (entry 9), the desired hexa-amide product 25 was isolated in a nearly quantitative yield and 95% HPLC-purity (relative to all other peaks) after the simple water wash and filtration. Purity of product was further increased (99% according to HPLC) by following the simple purification protocol (filtration of chloroform solution via Celite ® , and then precipitation with hexane from EtOAc solution [see Supporting Information]). The same amide coupling reaction was also successful at loadings that were 3 times higher (150 mg of 1 per milling jar, 300 mg total), creating 25 in 80% isolated yield and 99% HPLC purity, albeit with a longer milling time (90 min).</p>

ChemRxiv

Bifluoride-catalysed sulfur(VI) fluoride exchange reaction for the synthesis of polysulfates and polysulfonates

Polysulfates and polysulfonates possess exceptional mechanical properties making them potentially valuable engineering polymers. However, they have been little explored due to a lack of reliable synthetic access. Here we report bifluoride salts (Q+[FHF]\xe2\x88\x92, where Q+ represents a wide range of cations) as powerful catalysts for the sulfur(VI) fluoride exchange (SuFEx) reaction between aryl silyl ethers and aryl fluorosulfates (or alkyl sulfonyl fluorides). The bifluoride salts are significantly more active in catalysing the SuFEx reaction compared to organosuperbases, therefore enabling much lower catalyst-loading (down to 0.05 mol%). Using this chemistry, we are able to prepare polysulfates and polysulfonates with high molecular weight, narrow polydispersity and excellent functional group tolerance. The process is practical with regard to the reduced cost of catalyst, polymer purification and by-product recycling. We have also observed that the process is not sensitive to scale-up, which is essential for its future translation from laboratory research to industrial applications.

bifluoride-catalysed_sulfur(vi)_fluoride_exchange_reaction_for_the_synthesis_of_polysulfates_and_pol

<!>Results and discussion<!>Conclusion<!>Process for the bulk preparation of polysulfate P-1

<p>Click chemistry features the formation of carbon–heteroatom bonds to generate molecules with desired functionality using highly efficient, selective, modular and benign reaction processes1. From a synthetic perspective, polymer chemistry depends on many of the same basic principles as click chemistry —modularity and efficiency are inherent in practical polymer syntheses2, which explains the growing impact of click chemistry on materials science over the past decade3, especially in the field of polymer preparation and modification4,5.</p><p>Recently, we introduced sulfur(VI) fluoride exchange (SuFEx) as another embodiment of click chemistry6. The SuFEx click chemistry allows the activation of SVI–F bond with a 'Si+' species in the presence of organosuperbase catalysts, such as 1,8-diazabi-cyclo[5.4.0]undec-7-ene (DBU) and 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP, a phosphazene base—full name cited from Reaxys). With these catalysts, the reaction between aryl fluorosulfates (or aryl sulfonyl fluorides) and aryl silyl ethers exclusively affords diaryl sulfates (or diaryl sulfonates). The SuFEx reaction has been successfully applied to the synthesis of aromatic polysulfates and polysulfonates by our group7,8, and to post-polymerization modifications by others9–11.</p><p>Polysulfates and polysulfonates with their sulfur(VI) linkages (–SO2–) possess excellent mechanical properties and high chemical stability, as needed for engineering polymers12–15. For instance, the bisphenol A (BPA) polysulfate has slightly higher tensile modulus, similar yield stress and significantly lower oxygen permeability compared to its polycarbonate counterpart7. Nevertheless, they have been rarely used for industrial applications due to a lack of reliable and scalable synthetic access. Our SuFEx process represents the first efficient protocol for the preparation of these polymers. Considering the availability of bisphenols (BPA has an annual production of millions of tonnes, most of which are used for polycarbonates and epoxy resins industry) and sulfuryl fluoride gas (SO2F2, widely used as fumigant) as commercial feed-stocks, it is attractive to further explore this process for potential industrial applications.</p><p>However, several disadvantages of the base-catalysed SuFEx process have hampered its scale-up. First, the catalyst loading is quite high. For a typical polymerization reaction, no less than 10 mol% of DBU has to be used. One limitation of high catalyst-loading is that a tedious purification procedure is required to remove catalyst residue prior to polymer processing. Second, BEMP requires only 1.0 mol% of catalyst-loading, but is quite expensive and lacks bulk accessibility. Third, the strong basic nature of the catalysts restricts the substrate scope of the process. For instance, alkyl sulfonyl fluoride monomers are not well compatible with this process due to the facile deprotonation of their α-H when subjected to strong bases16,17.</p><p>Addressing these issues, we report a new set of catalysts for SuFEx-based polysulfate and polysulfonate synthesis, namely the bifluoride salts (Q+[FHF]−, where Q+ refers to a wide range of organic and inorganic cations)18,19. The bifluoride species has been little appreciated by the synthetic community. Its presence is nearly certain wherever a fluoride ion is exposed to any potential proton sources20. The 4-electron-3-centered anion ([FHF]−) has one proton trapped between two fluoride anions via the strongest hydrogen bond ever recorded21. Such a formation gives the bifluoride ion modest acidity (pH ≈ 3.0 for the saturated aqueous solution of potassium bifluoride at room temperature)22, moderate nucleophilicity23 and excellent stability21. While the strongly basic fluoride sources, such as cesium fluoride, only exhibited moderate activity in our SuFEx reaction7, we hypothesized that the acidic bifluoride species could facilitate SuFEx reactivity.</p><!><p>We first investigated potassium bifluoride (K+[FHF]−) as catalyst for the polycondensation of A-1 and B-1 in N-methyl-2-pyrrolidone (NMP) at 130 °C (Fig. 1a). The polymerization reaction catalysed by DBU (10 mol%) and BEMP (1 mol%) served as controls (Fig. 1b; catalyst loadings are given in molar ratio to the total amount of both monomers). The number-average molecular weight (Mnps) (ps, polystyrene) and polydispersity (PDI) were determined by gel permeation chromatography (GPC), which was calibrated with commercial polystyrenes. Our results indicated that 2.0 mol% of potassium bifluoride was indeed able to promote the polysulfate formation. But it was much slower compared to the reaction catalysed with DBU or BEMP (17 h versus 1 h), affording the polysulfate P-1 with a low molecular weight (Mnps = 28 kDa). However, the molecular weight of P-1 could be dramatically improved to as high as 100 kDa by combining 2.0 mol% of potassium bifluoride with 1.0 mol% of tetrabutylammonium chloride or 18-crown-6 ether as catalyst. In all entries, the PDI remained narrow (1.4~1.6, Fig. 1c).</p><p>Subsequently, we investigated the impact of cations on the activities of the bifluoride catalysts (Q+[FHF]−, Fig. 1d). Various onium bifluoride salts were prepared through ion-exchange of their chloride/bromide precursors (Q+X−, X = Cl, Br) with silver bifluoride (Ag+[FHF]−), and were used as 0.1 M stock solution in acetonitrile (Supplementary Section 2-3-1). We found that a wide variety of onium bifluorides were highly active at promoting the reaction, requiring only 0.1 mol% loading in most cases (the minimum amount required to produce P-1 with Mnps > 20 kDa). Upon the addition of the bifluoride catalyst, the reaction was triggered instantly with the release of tert-butyldimethylsilyl fluoride (TBSF, b.p. 89 °C)24, and yet not much heat was released. Within one hour P-1 was obtained with Mnps ranging from 30 kDa to 100 kDa (PDI = 1.4~1.7). We found that tris(dimethylamino)sulfonium bifluoride (Q-5)25 was the most effective catalyst—at a 0.05 mol% catalyst loading it was able to generate P-1 with a molecular weight up to 100 kDa. Hexamethyl guanidium bifluoride (Q-6) also exhibited remarkable activity at 0.08 mol% catalyst loading (PDI = 2.0). However, a confined, five-membered cyclic version of the cation (Q-7) had decreased activity (0.25 mol% loading).</p><p>Inspired by the extraordinary activity of bifluoride salts in catalysing the SuFEx polymerization reaction, we investigated whether poly(hydrogen fluoride) salts (Q+[HnFn+1]−, n > 1, Fig. 1e) could promote the transformation as well18. The dihydrogentrifluoride salt Q-11 was prepared via ion-exchange of its chloride precursor with excess K+[FHF]− (ref. 26). Thereafter, equilibrations of Q-11 with hydrofluoric acid (48 wt.% in H2O) in acetonitrile gave catalysts Q-12–Q-14 in situ, which were used directly without the removal of water (Supplementary Section 2–3). We found Q-11–Q-13 were excellent catalysts for P-1 formation, although slightly higher catalyst-loadings were required (0.5 mol% in most cases). Intriguingly, we confirmed the formation of P-1 with a moderate molecular weight (Mnps = 20 kDa) even when the HF:F− ratio was 9:1 (Q-14), suggesting that the polymerization reaction has an excellent tolerance for the presence of a few per cent of water and HF in the system. In contrast, we only obtained a mixture of oligomers and partly hydrolysed monomers when triethylamine trihydrofluoride, pyridinium poly(hydrogen fluoride)27, or hydrofluoric acid were employed as catalysts (see Supplementary Section 2–4 for more information on catalyst evaluation).</p><p>To evaluate the fidelity of the bifluoride catalytic system, we examined monomers with diverse functional groups (Table 1). The polysulfates P-2, P-5 and P-6 had previously been prepared under the DBU-catalysed polycondensation reaction7. When the bifluoride catalyst Q-5 was used, we obtained these polysulfates with comparable molecular weights and polydispersities. For instance, the Mnps/PDI of P-2 was 46 kDa/1.5 with the DBU catalyst versus 84 kDa/1.6 with the Q-5 catalyst. Under the same reaction conditions, monomers A-3/B-3, A-4/B-4 and A-8/B-8 were converted to polysulfates with molecular weights ranging from 36 to 52 kDa. Even the sterically hindered monomer B-9 underwent cross-condensation with A-1 giving polymer P-9 with high molecular weight and good polydispersity (Mnps = 58 kDa, PDI = 1.7), alternatively A-9 reacted with B-1 giving P-9 with a slightly lower molecular weight (Mnps = 23 kDa, PDI = 1.4). The orthogonal nature of the SuFEx click reaction to many other standard chemical reactions enables us to incorporate latent functional groups on the emergent polymer backbones for post-polymerization modification. For example, propargyl groups in P-12 can serve as handles for the CuAAC-mediated functionalization5. Alternatively, the methoxy groups in P-11a were converted into fluorosulfates (P-11b) to undergo further SuFEx modification11.</p><p>We have also examined Q-5 (0.1 mol%) catalysed polycondensation of AB-1, in which the two functional groups, –OSO2F and –OTBS, were residents in a single molecule (AB-1 is bench-stable for at least one year at room temperature). Under the same reaction conditions, polysulfate P-1 was obtained with a Mnps of 65 kDa (PDI = 1.5), which is comparable to polymers obtained from the A-1/B-1 polycondensation reaction (Fig. 2a). Intriguingly, we were also able to realize the direct polycondensation of A-1 with SO2F2 gas catalysed by Q-5, affording P-1 with moderate molecular weight (Mnps = 22 kDa, PDI = 1.4, Fig. 2b). This strategy is generally applicable to other monomers like A-7, with which polysulfate P-7 was prepared in near quantitative conversion (Mnps = 28 kDa, PDI = 1.5, Fig. 2c). In these cases, aryl fluorosulfate intermediates (–C6H4OSO2F) were generated in situ via the reaction between aryl silyl ether (–C6H4OTBS) and sulfuryl fluoride gas catalysed by Q-5, which underwent further condensation with another molecule of aryl silyl ether to extend the backbone and finally resulted in the polysulfate.</p><p>When DBU or BEMP was employed as the catalyst, the reactions of aryl silyl ether with alkyl sulfonyl fluoride afforded a mixture of oligomers and decomposition adducts of both monomers and oligomers, which might be due to the deprotonation of the α-H of the sulfonyl fluoride group17. Hydrogen fluoride catalysts, however, provide an acidic reaction environment that is compatible with those alkyl sulfonyl fluorides22. To demonstrate this, we combined the aliphatic bis(sulfonyl fluorides) B-13, B-14 and B-15 respectively with A-1 in NMP in 1:1 ratio and in the presence of 5 mol% catalyst Q-5 at 130 °C. The resulting polysulfonates P-13, P-14 and P-15 were afforded in quantitative yields and moderate molecular weights (Table 2). On the other hand, the polycondensation of the aromatic bis(sulfonyl fluoride) B-17 required lower catalyst-loading (0.5 mol%) and gave polysulfonate with a higher molecular weight. The bifluoride-catalysed SuFEx polycondensation reaction is also applicable to the synthesis of the fluoropolymer P-16 from the perfluorobutane-1,4-disulfonyl fluoride B-16 and the silyl ether A-1 (Mnps = 23 kDa, PDI = 1.3). Fluoropolymers often possess excellent physical and chemical properties, such as low dielectric constants and coefficients of friction28.</p><p>For laboratory use of the bifluoride-catalysed SuFEx polymerization or ligation, there are several options for catalyst preparation summarized in Supplementary Section 2–3. For industrial interests, however, bulk supplies of high-quality catalysts at low cost is required. Fortunately, a reliable protocol for the large-scale preparation of Q-5 from commercially available SF4 has already been reported (Fig. 3a)25,29. In addition, there is a practical access to other bifluoride salts which involves the ion-exchange of onium halides (Q+X−, X = Cl, Br, I) with anhydrous hydrogen fluoride (Fig. 3b). Anhydrous HF and many onium halides are bulk chemicals, therefore practical industrial scale syntheses of bifluoride catalysts is expected. Demonstrating this, we carried out a 5-gram scale preparation of 1-ethyl-3-methylimidazolium bifluoride (EMI+[FHF]−) (modified procedure based on refs 30,31). (Caution! Anhydrous HF is dangerous for its strong corrosiveness and toxicity, and must be manipulated in specific apparatus under safety guidance. See Supplementary Section 2-3-6).</p><p>Having established the optimal conditions for the bifluoride-catalysed SuFEx polymerization reaction, as well as the protocols for catalysts preparation, we were further interested in enlarging the synthesis of polysulfate P-1 to 100-gram scale in our laboratory. A bulk polymerization with 0.2 mol of A-1 and 0.2 mol of B-1 was carried out at ~120 °C (internal temperature) in NMP (50 ml), using only 0.05 mol% of catalyst Q-5 (Fig. 3c). Polysulfate P-1 was obtained as white fibrous materials in near quantitative yield (112.7 g, Fig. 3d). GPC analysis gave a Mnps of 110 kDa (PDI = 1.7, Supplementary Fig. 8), which was even higher than the P-1 product obtained at the 2.0 mmol scale (Mnps = 92 kDa, PDI = 1.7, Fig. 1d). Differential scanning calorimetry and thermal gravimetric analysis results (Supplementary Section 2–8) were comparable with the data obtained from the BEMP-catalysed bulk polycondensation7, indicating these two batches of polymers had the same quality. We also examined the end-capping effect, which is a standard strategy for molecular weight-control (Supplementary Section 2–6)32. The only by-product of the SuFEx-polymerization process is the TBSF (Fig. 3e). Nevertheless, we found it can be used in place of tert-butyl-dimethylsilyl chloride for the production of the silyl ether monomer A and hence recycled (Supplementary Section 2–9).</p><!><p>In summary, we have developed bifluoride salts as a new class of catalysts for the SuFEx click reaction, and have applied this chemistry to the preparation of polysulfates and polysulfonates. Compared to organosuperbase catalysts (for example, DBU and BEMP), the bifluoride ion [FHF]− is acidic. It allows the SuFEx polymerization reaction to be extended to a broader substrates scope, such as aliphatic sulfonyl fluorides. Most importantly, the bifluoride catalyst is significantly more active, therefore the polymerization reaction requires much lower catalyst-loading, which is essential for cost-effective industrial scale production. Our large-scale experiment further confirms the high fidelity of the bifluoride catalytic process for scale-up. Moreover, we have also provided practical solutions for catalyst preparation and for by-product recycling (TBSF). This work sets a foundation for future translation of polysulfate synthesis from laboratory research to industrial applications.</p><!><p>Monomers A-1 (91.36 g, 0.2 mol) and B-1 (78.48 g, 0.2 mol) were combined in a dry 1 l three-neck round-bottom flask equipped with a Teflon-coated magnetic stir bar, a thermometer and a reflux condenser (Fig. 3c). The flask was purged with nitrogen gas, into which dry NMP (50 ml) was then added. Next, it was placed in a pre-heated oil bath (130 °C, oil temperature). With stirring, monomers gradually dissolved in NMP, and the internal temperature stabilized at 123 °C. Under vigorous stirring, catalyst Q-5 (0.1 M in dry CH3CN, 2.0 ml, 0.0002 mol) was added. The reaction initiated instantaneously. The internal temperature rose to 135 °C, then quickly dropped to 100–110 °C due to the refluxing of TBSF. The reaction was allowed to run for 1 h, during which the reaction mixture turned highly viscous and the internal temperature stabilized at around 120 °C. The flask was subsequently cooled to room temperature. TBSF was then distilled out from the reaction mixture as colourless liquid at 80 °C (diaphragm pump; 49.1 g product, Fig. 3e). After the distillation, 250 ml DMF was added to the reaction flask to dissolve the polymer at 120 °C. The warm DMF solution was then slowly poured into 2 l methanol with vigorous mechanical stirring at room temperature. White fibres of P-1 crashed out and aggregated like a bird's nest in methanol, which was collected and dried at 70 °C for 12 h to give 112.7 g dry polymer (Mnps = 110 kDa, PDI = 1.7, Fig. 3d). Full experimental details and characterization of compounds are given in Supplementary Information.</p>

PubMed Author Manuscript

Decreases in Plasma Membrane Ca2+-ATPase in Brain Synaptic Membrane Rafts from Aged Rats

Precise regulation of free intracellular Ca2+ concentrations [Ca2+]i is critical for normal neuronal function, and alterations in Ca2+ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca2+]i is the plasma membrane Ca2+-ATPase (PMCA), the high affinity transporter that fine tunes the cytosolic nanomolar levels of Ca2+. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In the present study, we isolated raft and non-raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised of all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess CaM to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age-related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.

decreases_in_plasma_membrane_ca2+-atpase_in_brain_synaptic_membrane_rafts_from_aged_rats

Introduction<!>Materials<!>Animals<!>Isolation of SPMs and SPM rafts from rat brains<!>Cholesterol and GM1 analysis<!>Immunoblot Analysis<!>Capillary liquid chromatography- mass spectrometry (LC-MS/MS) analysis of PMCA isoforms<!>Measurement of PMCA activity<!>Statistical Analyses<!>Characterization of lipid rafts isolated from rat brain SPMs<!>Effects of aging on PMCA protein levels and PMCAactivity in raft microdomains<!>Discussion

<p>Transient increases in free intracellular Ca2+ serve as signals in critical neuronal processes such as neurotransmitter release, energy metabolism, synaptic plasticity, and gene transcription essential for learning and memory (Berridge 1998; Blackstone and Sheng 2002). The cytosolic Ca2+ must be rapidly returned to normal resting levels following Ca2+ transients and maintained in the nanomolar concentration range through the activities of Ca2+ transporters and binding proteins. Aberrant cellular Ca2+ overload has been implicated in neuronal dysfunction occurring in brain aging (Huidobro et al. 1993; Khachaturian 1994; Camandola and Mattson 2011) and in neurodegenerative disorders such as Alzheimer's disease (Khachaturian 1994; Mattson 2007; Camandola and Mattson 2011), and Parkinson's disease and stroke (Mattson 2007). The aging process in brain is accompanied by a higher overall Ca2+ content in brain neurons and synaptic terminals and a slower clearance of Ca2+ from nerve endings following a depolarizing stimulus (Martinez et al. 1988; Michaelis et al. 1992).</p><p>The calmodulin (CaM)-activated plasma membrane Ca2+-ATPase (EC 3.6.3.8) or PMCA is the major high-affinity, low-capacity Ca2+ extrusion system at the plasma membrane. Four isoforms of PMCA have been identified and form a multi-gene family with numerous splice variants that are selectively expressed in various tissues and presumably control diverse and highly regulated functions within a given type of cell (Strehler and Zacharias 2001). By pumping excess cytosolic Ca2+ into the extracellular space, PMCA is partly responsible for the long term regulation of resting free intracellular Ca2+ concentrations and reversing transient increases that occur during Ca2+ signaling (Strehler and Zacharias 2001; Clapham 2007). The PMCA levels and ATPase activity in brain synaptic membranes have been found to decrease with brain aging, an observation that could, at least partially, explain the age-associated elevations in intra-neuronal free Ca2+ (Gibson and Peterson 1987; Martinez et al. 1988; Michaelis et al. 1992) and the slower decay of Ca2+ transients in intact synaptosomes (Martinez-Serrano et al. 1992) and in hippocampal neurons (Landfield and Pitler 1984) from aged F344 rats. We had previously observed that both Ca2+-ATPase and the ATP-dependent Ca2+ transport activity in synaptic plasma membranes (SPMs) prepared from brains of aged Fisher 344 rats was significantly lower than that in young animals (Michaelis et al. 1984; Michaelis 1989). We further confirmed the progressive age-dependent decrease in PMCA activity in the longer-lived Fisher 344/BNF1 hybrid rat strain. The Vmax for activity in SPMs isolated from 34 month old rats was 48% lower than that at 5 mos, and this decrease was only partially due to a reduction in PMCA protein levels in the SPMs (Zaidi et al. 1998). In addition to the 20% reduction in PMCA levels, the amount of CaM present in the membranes was reduced by approximately 30%. However, the addition of saturating amounts of exogenous CaM did not overcome the progressive decrease in the PMCA activity measured in SPMs from the aged rats.</p><p>Several lipids are known to regulate PMCA activity, and the activating effect of acidic phospholipids is even more pronounced than that of CaM (Di Leva et al. 2008). The activity of PMCA is quite sensitive to the biophysical properties of the surrounding lipid environment, and the transporter exhibits much higher activity when it is localized in regions of ordered lipids (Duan et al. 2006; Tang et al. 2006). A portion of the PMCA in SPMs partitions into lipid 'rafts' (Sepulveda et al. 2006; Jiang et al. 2007), highly ordered micro-domains within the plasma membrane that are enriched in cholesterol, saturated phospholipids, and sphingolipids such as GM1 ganglioside (Brown and London 1997; Lucero and Robbins 2004). The rafts are involved in signal transduction by acting as a scaffold for protein sorting and promoting protein-lipid and protein-protein interactions (Simons and Toomre 2000) as exemplified in synaptic membranes (Hering et al. 2003; Gil et al. 2006). Cholesterol plays a key role in supporting the tendency of saturated phospholipids and sphingolipids to segregate from unsaturated phospholipids in the bilayer membrane (Silvius 2003), suggesting that altered cholesterol levels likely influence the activity of proteins such as PMCA within the raft domains.</p><p>The aims of the present study were: (1) to assess the partitioning of PMCA isoforms into raft and non-raft domains isolated from brain SPMs, (2) to determine the specific activity of the PMCA associated with the raft vs that present in the non-raft domains, (3) to assess the effects of aging on the specific activity of the PMCA within the raft domains of the SPMs, and (4) to assess the levels of CaM, cholesterol and ganglioside GM1 in the rafts with increasing age as possible contributors to the age-dependent decrease in PMCA activity seen in intact SPMs. In the present study, we used quantitative liquid chromatography-mass spectrometry (LC-MS/MS) to assess the relative enrichment of PMCA isoforms in raft domains isolated from synaptic membranes. We also examined the constants (Vmax and Kact) for the activity of PMCA in raft and non-raft domains and determined the presence and activity of CaM remaining tightly bound to the lipid rafts. Following such characterization of the lipid rafts and of the PMCA associated with these lipid microdomains, we focused on the effects of increasing age on cholesterol, GM1, PMCA, and CaM content in rafts, and on the activity of PMCA associated with rafts.</p><!><p>Sources for the various primary antibodies were as follows: anti-Flotillin-1 (FLT-1) and anti-Thy-1 (BD Biosciences), anti-CaM (Millipore), pan anti-PMCA (Affinity Bioreagents). Alkaline phosphatase-conjugated secondary antibodies, ficoll, ouabain, thapsigargin, oligomycin, ATP, Brij 98, and horseradish peroxidase-coupled cholera toxin subunit B (CTXB) were purchased from Sigma. The Amplex Red cholesterol assay kit and gradient gels (8–16%) were obtained from Invitrogen, the bicinchoninic acid protein assay reagents from Pierce Biotechnology, and the polyvinylidene fluoride (PVDF) membranes from Millipore. The protease inhibitor cocktail III and purified bovine brain CaM were purchased from Calbiochem.</p><!><p>Fisher 344/Brown Norway hybrid (F344/BNF1) male rats at 6, 23, and 34 months of age were obtained from the National Institute on Aging colony maintained by Harlan Industries. All protocols were implemented in accordance with NIH guidelines and approval by the University of Kansas Institutional Animal Care and Use Committee (IACUC). The rats were quarantined for 2 weeks after their arrival, following which they were used for the studies.</p><!><p>Fisher 344/BNF1 rats were anesthetized by CO2 inhalation according to the IACUC guidelines. Rats were decapitated using a guillotine and the brains recovered quickly. The brains from one rat at each of the 3 ages were processed in parallel. Argon was bubbled through all solutions to eliminate O2. Each whole brain was homogenized and processed immediately for the isolation of synaptosomes by discontinuous ficoll density gradient centrifugation as previously described (Michaelis et al. 1983; Jiang et al. 2010). The synaptosomes recovered from the gradient were lysed in a hypotonic buffer (3mM Tris-HCl, pH 8.5) containing a cocktail of protease inhibitors and centrifuged at 64,200 × g for 15 min. The SPM pellet was homogenized in buffer containing 10 mM Tris-HCl, 50 μM MgCl2, and 0.32 M sucrose, pH 7.4. Lipid rafts were isolated from SPMs using discontinuous sucrose density gradient centrifugation essentially as described (Jiang et al. 2007). Briefly, the SPMs from each animal were solubilized in an equal volume of solubilization buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5, containing 2% Brij 98), to achieve 1% Brij 98 as the final concentration, and incubated on ice for 30 min. The suspension was mixed 1:1 with a 90% sucrose solution and the resultant mixture overlaid with 35% and 5% sucrose solutions. Ultracentrifugation was performed for 18 h at 98,300 × g in a Beckman Optima Max centrifuge in an MLS-50 rotor. Ten fractions (0.4 ml each) were collected from the top to the bottom of each tube. However, due to the negligible amount of protein in fraction 1 and the small pellet of fraction 10 at the bottom of the tube, only fractions 2 – 9 were used in these experiments. Protein concentrations of the fractions were determined using the bicinchoninic acid assay according to the manufacturer's instructions. In order to compare the raft fractions with the non-raft domains, we pooled the fractions into 3 groups. Based on the relative levels of cholesterol, GM1, and Flotillin-1, fractions #2 – 4 were pooled as the lipid raft microdomains. Fractions #5–6 and #7–9 were considered to be the higher density non-raft components of the SPMs.</p><!><p>Cholesterol was measured using the Amplex Red cholesterol assay kit, and GM1 ganglioside levels were determined by immunoblotting using cholera toxin subunit B (CTXB) at 300 μg/ml. Briefly, an 8 μg protein aliquot from each fraction was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, blocked with 1% bovine serum albumin (BSA) for 30 min, and exposed to CTXB for 2 h. Color was developed by incubation in a mixture of 1.4 mM 3,3′-diaminobenzidine tetrahydrochloride, 200 mM NiCl and 6.2 mM H2O2. Based on the levels of cholesterol and GM1 ganglioside associated with the various fractions, the contents of specific fractions were pooled into 3 groups: fraction #2–4, #5–6, and #7–9. These 3 groups were analyzed.</p><!><p>Proteins were separated by SDS-PAGE and transferred to PVDF membranes as we have described previously (Jiang et al. 2007). Non-specific interactions were blocked with 5% (w/v) milk solution for 1 h at 25°C and the membranes incubated overnight with the indicated concentrations of primary antibodies. Alkaline phosphatase-conjugated secondary antibodies (1:1000) were added for 2 h at 25°C. Immunoblots were developed using the substrate 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium. Blots were scanned using a Kodak Image Station 4000MM PRO and quantified using Carestream Molecular Imaging Software 5.x.</p><!><p>Protein samples from pooled fractions # 2–4, #5–6, and #7–9 were run on SDS-PAGE gels and stained with Coommassie blue. Gel pieces at the molecular weight range of PMCA were excised, the proteins within the slices reduced with dithiothreitol, alkylated with iodoacetamide, and digested (37 °C, overnight) with 0.25 μg of trypsin in 0.2 M NH4CO3 - 5% acetonitrile (ACN). Ten microliter peptide-containing samples were analyzed by LC-MS/MS using a NanoAcquity chromatographic system (Waters Corp.) coupled to an LTQ-FT mass spectrometer (ThermoFinnigan). The peptides were separated on a C18 column (5cm, 300 μm I.D.; Thermo Acclaim PepMap300; flow rate 10 μL/min) using a gradient 1 – 40% of solvent B (99.9% ACN, 0.1% formic acid) developed over 50 min, ramped to 95% solvent B over 4 min, and held at 95% for 5 min. Solvent A was 99.9% H2O, 0.1% formic acid. A NanoAquity UPLC Console (Waters Corp., version 1.3) was used to execute the injections and gradients. The ESI source was operated with spray voltage of 2.8 kV, a tube lens offset of 160 V, and a capillary temperature of 200 °C. All other source parameters were optimized for maximum sensitivity of the YGGFL peptide MH+ ion at m/z 556.27. The instrument was calibrated using an automatic routine based on a standard calibration solution. Data acquisition via LTQ-FT was set up using Xcalibur software (ThermoElectron Corp., version 2.0.7). Full MS survey scans were acquired at a resolution of 50,000 with an Automatic Gain Control (AGC) target of 5×105. The LTQ-FT Selected Ion Monitoring (SIM) scan sequence was adapted from that described (Olsen and Mann 2004).</p><p>MS/MS spectra were analyzed using Mascot (Matrix Science, 2.3), Sequest (Thermo Fisher Scientific, 27, rev. 12), and X! Tandem (The GPM, thegpm.org; CYCLONE (2010.12.01.1)). Search engines were set up to search the IPI_rat database (v. 3.87, 39925 entries) or Swiss-Prot database (only rattus taxonomy), assuming digestion by trypsin and allowing for 2 missed cleavages. Fragment ion mass tolerance of 0.20 Da and a parent ion tolerance of 20 ppm were used. The iodoacetamide derivative of cysteine was specified as a fixed modification, while oxidation of methionine was designated a variable modification. Scaffold software (Scaffold_3_4, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. The SIM acquisition method for the LTQ-FT was adopted based on the results of LC-MS/MS experiments to achieve better sensitivity and to focus on the four selected, PMCA isoform-specific peptides with significant sequence homology: GIIDSTVSEQR for PMCA1, GIIDSTHTEQR for PMCA2, GIIDSTTGEQR for PMCA3, GIIDSNIGEQR for PMCA4 (see also Table 1, Supplementary Material). FT SIM experiments were set to scan the m/z range of 626.30–631.30 from 10.0 to 12.6 min, of 586.30–591.30 from 12.6 to 15.2 min, of 600.32–605.320 from 15.2 to 17.2 min, and of 598.82–603.82 from 17.2 to 20.2 min. FT SIM scans were acquired at a resolution of 100,000 with an AGC target of 2×105 or maximum ion time of 1.2 s. The chromatographic peaks were integrated using Qual Browser of Xcalibur software. FT SIM peak areas were used for reporting quantitative stoichiometric ratios of PMCA isoforms in samples thus analyzed.</p><!><p>The PMCA activity was determined at 37oC by monitoring the generation of Pi from the hydrolysis of ATP as described previously (Zaidi et al. 1998; Jiang et al. 2007). The assay was conducted in a final volume of 100 μL containing 3μg sample protein, 25 mM Tris-Cl, pH 7.4, 50 mM KCl, 1 mM ATP, 1 mM MgCl2, 0.1 mM ouabain, 4 μg/mL oligomycin, 0.1 μM thapsigargin, 200 μM EGTA, and CaCl2 at different concentrations added to yield the final free Ca2+ concentrations indicated in the figures. The final free Ca2+ concentration was calculated using the software calcium.com that calculates the multiple equilibria between all ligands in solution. During their preparation, the synaptic membranes were not exposed to chelators to extract the endogenous CaM bound to them. Thus the PMCA activity was measured in the presence of the CaM already associated with the membranes except in the experiments in which it was noted that a saturating amount of exogenous CaM was added to the assay. After a 5 min pre-incubation, the reaction was initiated by the addition of ATP, continued for 30 min at 37°C, and stopped by addition of the Malachite Green dye solution (Lanzetta et al. 1979). The absorbance was measured immediately at 650 nm. The specific activity of PMCA was defined as the nmoles of Pi liberated per mg of protein per min.</p><!><p>The statistical significance of differences between samples were determined by Student's t- test for unpaired samples or by ANOVA, whichever was more appropriate for the type of data being analyzed.</p><!><p>Lipid rafts were isolated from SPMs prepared from the individual brains of 6, 23, and 34 mos F344/BNF1 rats using the non-ionic detergent Brij 98 at a final concentration of 1% (v/v). Only the brains of 6 month-old rats were used in the initial characterization of the rafts and the partitioning of the PMCA in raft and non-raft domains. After the sucrose density gradient centrifugation, fractions 2–9 were collected from the top to the bottom of the tube as noted in the Methods. Cholesterol, one of the major lipid markers of rafts, was significantly enriched in the low-density fractions, #2–4. The cholesterol content in these fractions represented 77.4 ± 0.9% of the total cholesterol in the membranes applied to the gradient (Fig. 1A). Cholesterol was substantially more enriched in fractions #2–4 than proteins were. These fractions contained 31.6 ± 1.2% of the total protein in the SPMs. An additional lipid raft marker, GM1 ganglioside, was highly enriched in fractions #2–4, as were two raft protein markers, Thy-1 and FLT-1 (Fig. 1B), consistent with our previous observations (Jiang et al. 2010). Figure 1B also shows the distribution of PMCA in the raft and non-raft fractions based on an immunoblot analysis with the pan-PMCA antibody. Densitometric analyses of the total PMCA immunoreactivity in fractions #2 to #9 indicated that a greater percentage of the total amount of PMCA was associated with the detergent-resistant, low density fractions #2–4 than with fractions #5–6 or #7–9 (data not shown).</p><p>To quantify the levels of total PMCA and of each of the four isoforms of the enzyme in the three pooled fractions, we performed quantitative MS analyses of these fractions as described under Methods. Liquid chromatography-MS/MS analysis of the in-gel digested samples followed by database searches identified the presence of all four PMCA isoforms, as well as that of many other proteins, in the gel slices excised from the region corresponding to the molecular sizes of the PMCA isoforms. Sequence coverage for each PMCA isoform was in the range of 15–20% and a total number of 45 peptides were assigned to PMCA isoforms with greater than 50% probability as specified by the Peptide Prophet algorithm (Keller et al. 2002). There were 8, 12, 5, and 12 isoform-specific peptides (including the ones with probability less than 50%) detected by MS/MS and assigned to PMCA isoforms 1, 2, 3, and 4, respectively (Tables 2–5 Supplementary Material). PMCA isoform sequence alignment (Scheme 1, Supplementary Material) indicated a region of similarity within four isoform-specific peptides (Table 1, Supplementary Material). These peptides have 8 out of 11 residues in common across the four isoforms and were selected for further quantitative FT SIM MS experiments. The analysis of pooled samples from fractions #2–4, #5–6, and #7–9, using FT SIM MS allowed us to increase sensitivity for the PMCA representative peptides relative to full scan FT MS experiments. Stoichiometric ratios of PMCA isoforms were calculated by normalizing the FT SIM isoform-specific peptide signal to the sum of signals from the four selected peptides. This was achieved by focusing the mass spectrometer on a particular ion during the expected time.</p><p>The results of such analyses were averaged and are presented in figure 2 and Table 1. The data obtained indicated that ~60% of the total PMCA partitioned into the pooled raft fractions #2–4, and ~21% and ~19% partitioned into pooled fractions #5–6 and #7–9, respectively (Fig. 2A). The content of PMCA proteins in raft fractions (~60%) was nearly twice that of the total membrane protein distributed in those fractions (~31%, see Fig. 1A), an indication of selective association of PMCA with rafts. Quantitative MS analyses also revealed that of the four PMCA isoforms, PMCA 1, 2, and 4 were the most abundant isoforms in all of the fractions as determined by quantitative MS (Fig. 2B). The stoichiometric ratios of the four isoforms in each of the three pooled fractions are shown in Table 1.</p><p>As the final step in characterizing the raft and non-raft fractions, we determined the Kact and Vmax values of the basal PMCA activity across Ca2+ concentrations in each of the 3 pooled fractions isolated from SPMs of 6 mos old rats. These studies were performed in the absence of exogenous CaM additions. We found that the specific activity of the enzyme was significantly higher in the raft fractions than in either of the pooled non-raft fractions (Fig. 3). The estimated Vmax in the rafts was ~2.6-fold (p<0.001, Student t-test) and ~4.6-fold (p<0.001) higher than the Vmax values in fractions #5–6 and #7–9, respectively (Table 2). The calculated values for the estimated Kact for Ca2+ for fractions #2–4 did not differ significantly from those for fractions #5–6 (p=0.35) and #7–9 (p=0.52). The Kact values for all fractions were similar to those observed previously for isolated SPMs (Zaidi et al. 1998) and were indicative of a high affinity state of PMCA for free Ca2+.</p><p>The high affinity for Ca2+ in these preparations might be indicative of substantial amounts of CaM remaining bound to the three pooled fractions isolated from synaptic membranes. The present measurements were obtained using preparations that were not treated with chelators, such as EDTA, to extract endogenous CaM; thus the Vmax and Kact estimates probably reflected those of PMCA activated by any CaM remaining in association with the isolated fractions. The addition of exogenous CaM (300 nM) had no significant effect (p>0.10, t-test comparisons) on either the estimated Vmax or the Kact for Ca2+-induced activation of PMCA (Table 2). The lack of an effect by excess exogenous CaM on PMCA activity would be expected if substantial amounts of CaM remained bound to these fractions.</p><p>To assess the contribution of endogenous CaM to the activity of PMCA measured in the raft fractions, we pursued two approaches. The first was to pretreat the rafts with either of two CaM inhibitors, calmidazolium (10 μM) or trifluoperazine (50 μM). The second was to treat synaptosomes and SPMs with EDTA prior to isolation of the raft and non-raft fractions in order to extract CaM that might be tightly bound to components of the isolated fractions. Both types of treatments caused a reduction in PMCA activity in rafts of approximately 30–40%. This is demonstrated in figure 4A for the effect of calmidazolium on the PMCA activity of the pooled fractions #2–4. Calmidazolium caused a significant decrease (p=0.005, t-test) in the Vmax of PMCA activity in the raft fractions and a 2.7-fold, but not significant (p=0.098), increase in the Kact for Ca2+ (Fig. 4A). The effects of calmidazolium on both Vmax and Kact were completely reversed by the addition of 300 nM CaM (Fig. 4A). The effects of inhibitors of CaM on the PMCA activity in the raft fractions were indicative of the presence of endogenous CaM remaining associated with these preparations. When 3 mM EDTA was introduced during the lysis of synaptosomes and the isolation of synaptic membranes, it led to the extraction of ~48% of the CaM that was associated with the raft fractions but did not completely remove all CaM from those fractions (Fig. 4B). Because of the residual CaM in these fractions even following Ca2+ chelation, the precise level of contribution of CaM to the overall PMCA activity in rafts could not be accurately estimated, but it was obviously significant.</p><!><p>Analysis of the levels of GM1 in SPM rafts from rats at 6, 23, and 34 mos revealed no significant age-related changes in this marker of raft domains in membranes. The total protein in the three pooled fractions as well as the levels of the protein raft markers, Thy-1 and FLT-1, also did not change with increasing brain age (data not shown). On the other hand, there was a small but statistically significant and progressive increase in the cholesterol to protein ratio in the rafts from the 23 mos and the 34 mos compared to the 6 mos old animals as shown in Fig. 5A.</p><p>The levels of the PMCA protein in rafts (fractions #2–4) were compared at the three ages in order to determine if there was any significant change in PMCA in rafts. To address the possibility that changes in raft PMCA levels during the aging process might result from altered distribution of PMCA in membranes, i.e., decreases in PMCA associated with the rafts and increases in PMCA in the non-raft domains, we also examined the effects of the aging process on the levels of PMCA in fractions #5–6 and #7–9. As shown in figure 5B, we observed a significant age effect on PMCA levels in rafts (p=0.03, ANOVA) and significant decreases for both the 23 and 34 mos raft fractions compared with the levels at 6 mos (p<0.03, t-test).</p><p>The same as described above for 6 mos old rats analyzed by quantitative MS and immunoblots (e.g., Fig. 2), the overall levels of PMCA in rafts isolated from 6 and 23 mos old rat brains were significantly higher (p<0.01, t-test) than those in fractions #5–6 and #7–9 (Fig. 5B). It should be noted that there was no significant age effect on the PMCA levels in the non-raft fractions. Therefore, it was unlikely that the decreases in PMCA levels in rafts were the result of re-distribution of the protein from the raft to the non-raft domain during the aging process. Because of the lack of a change in PMCA levels in fractions #5–6 and #7–9, the focus of studies related to PMCA activity was primarily on the effects of aging on the raft fractions.</p><p>When the activity of PMCA was determined across Ca2+ concentrations in rafts isolated from SPMs of rats at 6, 23, and 34 mos of age, the estimated Vmax decreased progressively and significantly with increasing age of the rats (Fig. 6). The Vmax at 23 mos was 23% lower than that at 6 mos (p=0.014, t-test), and the Vmax at 34 mos was 39% below that at 6 mos (p=0.012, t-test), with no significant (p>0.50) changes in the Kact for Ca2+ (Table 3). The reductions in the Vmax values for activity were considerably greater than the reductions in the levels of the PMCA protein associated with the rafts at the three different ages. These data are quite similar to those we obtained previously with aging SPMs that were not fractionated into rafts and non-rafts (Zaidi et al. 1998).</p><p>The PMCA activity shown in figure 6 was determined without the addition of exogenous CaM. As indicated previously, the rafts contain tightly bound CaM; therefore it seemed possible that a partial loss of CaM from the aged rafts could lead to the lower Vmax of the PMCA activity we observed in those rafts. To address this issue, we used immunoblots and densitometric analyses to determine the relative levels of CaM present in the rafts obtained from rats at the 3 ages. The amount of CaM present in the raft fractions showed no significant age-related decrease. The densitometric units for CaM in rafts isolated from the brains of 6 mos-old rats were 3.4 (± 0.17) ×107, that for rafts from 23 mo-old was 3.0 (± 0.05) ×107, and those from 34 mo-old 3.1 (± 0.15) ×107.</p><p>To determine if the addition of exogenous CaM could overcome the age-dependent decrease in PMCA activity in rafts, we measured the PMCA activity of rafts following the addition of exogenous CaM (300 nM) to preparations from brains of rats 6, 23, and 34 mos old. The Vmax values for the raft PMCA are shown in Table 3. The magnitude of the reductions in the Vmax values in the aging rafts was not reversed by the CaM addition. Thus the age-dependent decrease in PMCA activity in the rafts did not appear to be due to inadequate levels of CaM, an observation consistent with our results from SPMs (Zaidi et al., 1998). The presence of CaM in the enzyme assays also brought about modest but non-significant (p=0.10, t-test) decreases in the Kact for Ca2+ (Table 3).</p><!><p>The hypothesis that Ca2+ dysregulation is a fundamental contributor to the progressive decline in cognitive function with age is supported by numerous studies demonstrating changes in several protein systems responsible for neuronal Ca2+ signaling as reviewed recently (Kumar et al. 2009). Clearly our observations on the effects of aging on the PMCA system represent only one component of a very complex process, but the marked reduction in the activity of this critical regulator of free intracellular Ca2+ concentrations in the synaptic region is likely to be quite significant for overall cognitive performance. The importance of PMCA in regulating Ca2+ levels at the synapse is surmised from the demonstration in previous studies of the localization of PMCA isoforms and of specific splice variants of these isoforms in the plasma membrane of dendrites (Marcos et al. 2009; Kenyon et al. 2010) and, importantly, at the subsynaptic web of neuronal synapses (Burette et al. 2010). Furthermore, activation of synaptic Ca2+ entry into neurons through glutamate receptors leads to long-term effects on PMCA activity that differ markedly from those induced by general depolarization of neurons (Ferragamo et al. 2009). The studies presented here provide a more detailed picture of the properties of the PMCA in synaptic membranes and support the assertion that the protein does exist in both raft and non-raft domains, that approximately 60% of synaptic membrane PMCA protein is within raft domains, which is nearly twice the amount of the estimated total synaptic membrane protein distributed in rafts, and, moreover, that the most active pool of PMCA is that present in the rafts. The V max for Ca2+-activated PMCA in rafts was 2.6 and 4.6 times the values in non-raft fractions #5–6 and #7–9 respectively in the young rat brains. The reasons for the high levels of PMCA that is associated with the raft domains in synaptic membranes, and for the even higher activity of the enzyme than could be accounted by the differential distribution of the protein, are not known.</p><p>It seems quite likely that the higher specific activity in the rafts is due to the more highly ordered lipid environment in which the protein is embedded. The high cholesterol content and the sphingolipids with saturated acyl chains create a lipid domain that is more ordered than that found in the phospholipid bilayers with unsaturated acyl chains. This higher degree of order has been reported to enhance significantly the PMCA activity of the human ocular lens (Tang et al. 2006) and the red blood cell PMCA reconstituted in lipid vesicles (Zhang et al., 2005). These authors speculate that the enhanced activity is due to the lipid stabilization of the active conformation of the enzyme formed during the catalytic cycle. The sub-plasmalemmal actin filaments that promote lipid-lipid, lipid-protein, and protein-protein interactions within the rafts (Simons and Gerl, 2010) may also contribute to the markedly higher PMCA in these micro-domains.</p><p>The present studies, for the first time, have precisely quantified total PMCA as well as the PMCA isoform distribution in raft and non-raft domains of SPMs. Previous studies reported that raft domains in SPMs isolated from pig cerebellum contained only a single isoform of PMCA, the PMCA4 isoform (Sepulveda et al. 2006). This conclusion was based on the relative immunoreactivities of SPM subfractions with PMCA antibodies. The same technique was also used to demonstrate the presence of PMCA4 in raft domains in intestinal smooth muscle (El-Yazbi et al. 2008). However, in MDCK kidney cells, the localization of PMCA2 in apical membranes has been traced to the association of the PMCA2 isoform with lipid raft domains (Xiong et al. 2009). In our studies with rafts isolated from whole rat brains, we unequivocally identified all four isoforms of PMCA in raft domains, as well as in non-raft domains, using both LC-MS/MS as well as immunoblot methodologies. Furthermore, using quantitative MS techniques we were able to estimate the stoichiometric ratios for the four isoforms and to demonstrate that the most abundant forms of PMCA were PMCA4, PMCA2, and PMCA1, with PMCA3 lagging behind the other isoforms in terms of overall levels in rafts and non-raft domains. Therefore, there was no apparent exclusivity for PMCA4 localization in raft domains.</p><p>An interesting observation made in the present studies was the tight binding of endogenous CaM to the rafts isolated following treatment of SPMs with the Brij98 detergent and subsequent subfractionation on sucrose density gradients. Whereas treatment of synaptosomes and SPMs with 3 mM EDTA did bring about a decrease in raft-associated CaM and a decrease in PMCA activity, it did not deplete completely the raft-bound CaM. The interaction of raft-bound CaM with the PMCA in these lipid domains was confirmed by the fact that high concentrations of either calmidazolium or trifluoperazine partially inhibited the activity of PMCA and that such inhibition could be completely overcome by the addition of excess exogenous CaM. The residual activity of PMCA in the presence of inhibitors may be the result of remaining high affinity interactions between CaM and PMCA or of the relative abundance of PMCA2 in rafts, an isoform which is minimally affected by the presence of CaM (Brini and Carafoli 2009).</p><p>When we assessed the effects of aging on the PMCA activity in the raft fractions, we observed a statistically significant reduction in the Vmax values of the activity in the rafts at both 23 and 34 months, a reduction in activity that was in excess of the age-associated decreases in the levels of the PMCA protein in the rafts. The Kact for Ca2+ did not change with age. Furthermore, the amount of CaM associated with the rafts at the 3 ages showed no significant changes, and the addition of exogenous CaM did not reverse the reduction in activity in the rafts at 34 mos. As lipid raft microdomains are considered to be sites within which receptors and transport carriers are assembled to form important platforms for membrane signaling and molecular trafficking (Lingwood and Simons 2010), the age-associated decreases in SPM raft-associated PMCA levels and activity may strongly influence neuronal handling of Ca2+ transients.</p><p>In addition to the significant reduction in the Vmax values of the PMCA activity, we did observe a relatively small but statistically significant increase in the cholesterol to protein ratio in the rafts. This progressive increase from 6 mos to 23 mos, and to 34 mos represents an alteration that would be expected to increase the order within the lipid environment. Cholesterol is present in both leaflets of the membrane bilayer, with 85% distributed into the cytofacial leaflet (Wood et al. 2011). It fills the space under the head groups of sphingolipids or extends the interdigitating fatty acyl chain in the apposing leaflet (Simons and Ikonen 1997). Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid rafts for stability and organization based on the interaction of cholesterol with different membrane lipids (Silvius 2003). If the brain neurons do increase the cholesterol in the aging rafts, it is conceivable that the cells are attempting to make some compensatory response that might increase order and keep the PMCA more active as other age-related processes lead to a significant loss of enzyme activity.</p><p>As mentioned in the Introduction, several different mechanisms may contribute to the reduction in PMCA activity without a comparable reduction in levels of the protein. Given our observations that PMCA is uniquely sensitive to inactivation under conditions of oxidative stress (Zaidi and Michaelis 1999), it is quite possible that the protein per se could be oxidatively modified and thereby operate much less efficiently. Subtle alterations in the lipid milieu, oxidation of some of the lipids, disruption of interactions between PMCA and some of its numerous protein binding partners (DeMarco and Strehler 2001; Sgambato-Faure et al. 2006), or the state of phosphorylation of specific isoforms such as the tyrosine phosphorylation of PMCA4 (Ghosh et al. 2011) could also influence the enzymatic activity with increasing age. Detailed analyses of possible oxidative modifications of the PMCA and its binding partners in synaptic membranes and of the presence and activity of tyrosine kinases in these membrane fractions are currently underway as we attempt to determine why the contribution of this critical transporter is compromised in the aging brain.</p>

PubMed Author Manuscript

Hydrogen sulfide anion regulates redox signaling via electrophile sulfhydration

An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3\xe2\x80\xb2,5\xe2\x80\xb2-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS\xe2\x88\x92) regulates the metabolism and signaling actions of various electrophiles. HS\xe2\x88\x92 reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS\xe2\x88\x92 biosynthesis. Cardiac HS\xe2\x88\x92, in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS\xe2\x88\x92 on myocardial infarction\xe2\x80\x93associated heart failure. Thus, this study reveals HS\xe2\x88\x92-induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling.

hydrogen_sulfide_anion_regulates_redox_signaling_via_electrophile_sulfhydration

<!>HS\xe2\x88\x92-producing enzymes involved in electrophile metabolism<!>Denitration and sulfhydration of 8-nitro-cGMP by HS\xe2\x88\x92<!>HS\xe2\x88\x92-mediated sulfhydration with diverse electrophiles<!>Metabolism of sulfhydrated electrophiles and 8-SH-cGMP<!>HS\xe2\x88\x92 formation in cells and myocardial tissues<!>Electrophilic H-Ras activation is regulated by HS\xe2\x88\x92<!>Electrophilic H-Ras signaling regulated by HS\xe2\x88\x92<!>DISCUSSION<!>RNAi screening<!>Reaction of electrophiles with HS\xe2\x88\x92\nin vitro<!>Determination and quantitation of cellular 8-SH-cGMP formation<!>Measurement of cellular production of HS\xe2\x88\x92 and intracellular thiol derivatives<!>Animals and surgery<!>Immunological measurement of 8-nitro-cGMP production in vivo<!>Purification of H-Ras<!>Pulldown assay and western blotting<!>Identification of S-guanylation sites in H-Ras<!>Isolation of cardiac cells and measurement of cell senescence<!>Transthoracic echocardiography and cardiac catheterization<!>Statistical analysis<!>Other methods

<p>Endogenous formation of the gaseous signaling mediator hydrogen sulfide (H2S) has been demonstrated in mammalian cells and tissues1,2, but its chemical nature and physiological functions remain poorly defined. Although reactive oxygen species (ROS) are typically viewed as toxic mediators of oxidative stress in aerobic organisms3, ROS are now appreciated to mediate signal transduction events during both basal metabolism and inflammatory responses4–6. Electrophilic products can be formed via enzymatic oxidation reactions or reactions of ROS, nitric oxide and their secondary products7–12. Biological electrophiles can lend additional specificity to redox-dependent signal transduction via the nucleophilic substitution or Michael addition of electrophiles with cysteine sulfhydryls of various sensor or effector proteins to form their S-alkylation or S-arylation adducts7,8. Redox signal transduction reactions include those mediated by electrophilic byproducts of redox reactions4,7, such as the electrophilic nucleotide 8-nitro-cGMP and multiple unsaturated fatty acid–derived oxo and nitro derivatives7– 12. Aerobic cells rely on various oxidant-scavenging enzyme systems and low-molecular-weight scavengers to defend against the vicissitudes of oxidative stress3. Other than reactions with glutathione (GSH), however, the reactions modulating potential signaling and pathogenic actions of electrophilic species remain undefined.</p><p>Herein, we demonstrate that HS−, rather than H2S, regulates the metabolism and signaling functions of endogenous electrophiles. The nucleophilic properties of HS− support a reaction with various electrophiles in cells via direct chemical sulfhydration (that is, electrophile sulfhydration). In vivo treatment of mice with HS− ameliorated chronic heart failure after myocardial infarction, and improvement was partially a consequence of the sulfhydration of 8-nitro-cGMP generated in excess in cardiac tissues after myocardial infarction. These potent beneficial pharmacological effects stemmed from the sulfhydration of electrophilic 8-nitro-cGMP, which resulted in the suppression of a cellular senescence response induced by electrophile- dependent H-Ras activation in cardiomyocytes and cardiac tissues. This H-Ras activation involved downstream signaling pathways, including the Raf-extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways, which led to activation of p53 and retinoblastoma protein (Rb). These data support that HS−-induced sulfhydration of electrophile species is a mechanism for terminating electrophile-mediated signaling and suggest a new therapeutic strategy for treating oxidative inflammation-related diseases3,5–9.</p><!><p>To clarify the mechanisms regulating the metabolism and signaling actions of various electrophiles, we performed RNA interference (RNAi) screening that focused on cysteine metabolism and its redox-related metabolic pathways (Supplementary Methods and Supplementary Results, Supplementary Table 1). This method was based on the modulation of protein S-guanylation in A549 (human lung epithelial adenocarcinoma) and other cultured cells after treatment with small interfering RNA (siRNA) for various genes (Fig. 1a,b). This RNAi screening revealed the substantial impact of endogenous HS− generation on 8-nitro-cGMP metabolism. In particular, two key enzymes of HS−-H2S biosynthesis, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (Supplementary Fig. 1a), had a significant impact on the metabolism of cellular 8-nitro-cGMP in cultured mammalian cell lines, including A549, HepG2 (human hepatoblastoma) and C6 cells (rat glioblastoma) (P < 0.05 or P < 0.01 for each assay; Fig. 1a,b and Supplementary Fig. 1). Protein S-guanylation caused by exogenously administered 8-nitro-cGMP was markedly augmented after knockdown of CBS or CSE in A549, HepG2 and C6 cells (Fig. 1a,b and Supplementary Fig. 1b–d). Moreover, the basal extent of protein S-guanylation induced by endogenous 8-nitro-cGMP generation was also elevated by CBS or CSE knockdown (Fig. 1a,b). These data reveal that the HS−-generating enzymes (CBS and CSE) are critical for 8-nitro-cGMP metabolism. This metabolism was linked with the release of nitrite (NO2 −) via a mechanism dependent on CBS and CSE (Fig. 1a,b and Supplementary Figs. 1 and 2), supporting a reliance on the nucleophilic qualities of H2S (pKa 6.7), especially anionic HS−, which predominates in neutral biological solutions (Fig. 1c)13. The extent of NO2 − generation was linked with 8-nitro-cGMP degradation in cells, which in turn depended on HS− formed from CBS or CSE (Supplementary Fig. 2b,c). These findings support that enzymatically generated HS− mediates 8-nitro-cGMP metabolism.</p><!><p>To clarify how HS−, rather than gaseous H2S, could undergo an SH-addition reaction (that is, electrophile sulfhydration), we analyzed cell-free reactions of 8-nitro-cGMP with sodium hydrosulfide (NaHS), used as an HS− donor, via reverse-phase HPLC (RP-HPLC) and LC/MS. The electrophilic nitro moiety underwent nucleophilic substitution with HS− to yield the new product, 8-SH-cGMP (Fig. 1c,d and Supplementary Fig. 3a). Transition metal ions such as iron and manganese added to the reaction mixture greatly increased 8-SH-cGMP formation, whereas copper addition showed no or minimal effects (Fig. 1e). Metal complexes and metalloproteins, such as hemin, Cu,Zn-superoxide dismutase (Cu,Zn-SOD), Mn-SOD, horseradish peroxidase (HRP) and catalase, also promoted sulfhydration reactions, with HRP having the greatest effect. This observation supports a potent catalytic activity of endogenous transition metals—and metalloproteins—in sulfhydration. Sulfhydryls act as ligands for metal ions; thus, HS−-containing metal complexes may acquire added stability and catalyze sulfhydration (Fig. 1c). H2S can be oxidized to sulfur oxides such as thiosulfate (S2O32−), sulfite (SO32−) and sulfate (SO42−) under aerobic conditions or via cell metabolism2,14. These sulfur oxides, which may be formed via oxidation of H2S/HS−, did not react with 8-nitro-cGMP to induce sulfhydration (Supplementary Fig. 3b), confirming the specificity of HS− in electrophile sulfhydration. We observed clear pH-dependent, HS−-induced sulfhydration of 8-nitro-cGMP (Supplementary Fig. 3c). The efficacy of 8-SH-cGMP formation increased linearly as the pH increased from 5 to 8, which correlated well with the reported pH-dependent equilibrium of H2S/HS− (ref. 13). The electrophile sulfhydration potential of HS− is superior to that of GSH-dependent nucleophilic modification of 8-nitro-cGMP within a physiologically relevant pH range of the reaction mixture containing cysteine and HRP (Supplementary Fig. 3c). This finding also indicates a substantial contribution of HS− to electrophile metabolism via sulfhydration in comparison with electrophile metabolism with GSH and other sulfhydryl compounds such as cysteine and homocysteine (HCys) with a relatively high pKa (>8).</p><!><p>Sulfhydration by HS− occurred with a broad array of biological electrophiles that have either cell signaling or toxicological properties (Fig. 2 and Supplementary Fig. 4). These electrophiles include the cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), 4-hydroxy-2-nonenal (HNE), acrolein and fatty acid nitroalkene derivatives. LC/MS indicated that the common primary reaction with these electrophilic lipid derivatives was nucleophilic addition or substitution of HS− at the electrophilic carbon (Figs. 2 and 3 and Supplementary Fig. 4). Another reaction was the Michael addition of HS− to electrophiles such as 1,2-naphthoquinone (1,2-NQ) and other related quinones (Supplementary Figs. 5 and 6). The reaction of 1,2-NQ with HS− yielded 1,2-NQ-SH and (1,2-NQ)2S.</p><p>Reactions occurring after sulfhydration varied with individual electrophiles. In many cases, secondary reactions with parent electrophiles yielded bis-thio derivatives, as was observed with nitrooleic acid (OANO2), which underwent S-bridged dimerization (Figs. 2a and 3a and Supplementary Fig. 4a,b), and with 1,2-NQ, 1,4-NQ and tert-butylbenzoquinone (Supplementary Fig. 5). One exception was 8-nitro-cGMP; RP-HPLC and LC/MS detected only the sulfhydrated product, 8-SH-cGMP. Similarly to 8-nitro-cGMP, 15d-PGJ2 was readily sulfhydrated to form a transient unstable SH adduct that rapidly converted to sulfonated 15d-PGJ2-SO3H, the sole final product (Figs. 2b,c and 3a and Supplementary Figs. 4c and 6). This interpretation is supported by the fate of 8-SH-cGMP treated with various oxidants, specifically treatment resulting in a rapid oxidative desulfhydration as described below (Fig. 3). Certain lipid-derived endogenous electrophiles, such as HNE and acrolein, degraded rapidly after reacting with HS−, without forming any appreciable products detected by RP-HPLC (Fig. 2c). Michael adducts of HNE with cysteine and histidine, HNE-His and HNE-Cys, were resistant to the same HS− reaction, suggesting that both HNE and acrolein undergo efficient HS−-induced decomposition in a manner dependent on their strong electrophilicity (Supplementary Fig. 4d). Consistent with these findings, the Michael adduction of cellular proteins by 1,2-NQ or acrolein was markedly enhanced when CBS was knocked down in A549 cells (Fig. 2d). These data support the regulation of not only electrophilic S-guanylation but also S-alkylation or S-arylation of cellular proteins by HS− reactions with the parent electrophile. The relative reactivities of electrophiles with HS− was estimated by the rate of consumption of electrophiles in the presence of HS−. Acrolein showed the highest reactivity with HS−, with relative reactivities decreasing in the order OANO2 > HNE > 15d-PGJ2 > 8-nitro-cGMP, whereas the reactivities of these electrophiles with GSH were similar4,7,8.</p><!><p>The metabolic fate of these sulfhydrated derivatives of electrophiles allowed us to sort the electrophiles into three groups (Fig. 3a and Supplementary Fig. 6). In group 1, because the SH derivative was so stable, no other reaction occurred except for oxidative modifications of SH by ROS and other reactive species. Group 2 involved relatively stable bis product formation. Group 3 may include other highly reactive electrophiles, such as HNE and acrolein, for which additional metabolism and secondary chemical reactions followed SH addition.</p><p>Endogenous HS−-dependent sulfhydration occurred in A549, HepG2 and C6 cells, as evidenced by 8-SH-cGMP formation from exogenously added 8-nitro-cGMP (Fig. 3b and Supplementary Fig. 7). CBS knockdown markedly inhibited 8-SH-cGMP formation in A549 cells, a finding confirming CBS as a major source of endogenous HS−. Because 8-SH-cGMP contains sulfhydryls, it is presumably oxygen labile or susceptible to further oxidization. To test this possibility, we treated 8-SH-cGMP with hydrogen peroxide (H2O2) and reactive nitrogen oxide species (RNOS) including nitrogen dioxide (NO2) and peroxynitrite (ONOO−), which stem from the concurrent generation of NO and superoxide (Fig. 3c,d). Notably, the sole detectable product of this reaction was cyclic GMP (cGMP) rather than oxidized derivatives of 8-SH-cGMP, such as 8-sulfenyl-, 8-sulfinyl- and 8-sulfonyl-cGMP or 8-OH-cGMP (Fig. 3c,d), thus representing what is to our knowledge the first identification of oxidant-induced desulfhydration of SH-containing compounds. Remarkably, the oxidant-labile nature of several sulfhydrated products generated from electrophiles, such as 8-SH-cGMP, 15d-PGJ2-SH and even sulfhydrated adducts of HNE and acrolein, may reflect their high nucleophilic potential. Thus, secondary redox reactions of nucleophiles derived from HS−-dependent electrophile sulfhydration are expected to affect the biological stability and detection of these sulfhydrated adducts once they are formed in cells. For example, the amounts of 8-SH-cGMP formed in A549 and HepG2 cells were smaller than expected on the basis of the amounts of HS− produced in the same cells, as assessed by LC-ESI-MS/MS described below (Figs. 3b and 4a and Supplementary Fig. 7c); C6 cells, however, did not show the same result (Supplementary Fig. 7d). The small amounts of 8-SH-cGMP that were detected compared with the amount of HS− generated may thus be due to instability induced by the nucleophilicity of 8-SH-cGMP. This view is substantiated by the finding that cell treatment with PEG-derivatized SOD (PEG-SOD) and catalase (PEG-catalase) significantly improved the recovery of 8-SH-cGMP and simultaneously reduced cGMP formation from 8-nitro-cGMP administered to A549 cells in culture (P < 0.05; Fig. 3e). Because cGMP, a desulfhydrated product of 8-SH-cGMP, is metabolized by a diversity of cellular phosphodiesterases (PDEs), oxidative desulfhydration by HS− may also contribute to physiological decomposition of 8-nitro-cGMP.</p><!><p>The electrophilic fluorogenic reagent monobromobimane, typically used to analyze thiols, undergoes HS−-dependent sulfhydration to yield a bis-S-bimane derivative15. We capitalized on this bimane reaction for sensitive and specific HS− measurement using LC-ESI-MS/MS (Supplementary Fig. 7a) and detected remarkable endogenous HS− generation in A549 and HepG2 cells and cells in primary culture, specifically rat cardiomyocytes and cardiac fibroblasts (Fig. 4). CBS knockdown greatly suppressed the extent of HS− generation in A549 cells (Fig. 4a). Other cell lines, including HepG2 and C6 cells, also showed suppressed HS− generation after CBS knockdown (Supplementary Fig. 7c,d).</p><p>A technical advantage of bimane sulfhydration analysis by LC-ESI-MS/MS is the ability to measure in parallel all low-molecular-weight sulfhydryls: not only HS− but also cysteine, HCys and GSH. For example, the amounts of intracellular cysteine and GSH were not altered by cell treatment with CBS siRNA (Fig. 4b). CBS siRNA treatment of cells reduced only amounts of HS− and instead increased the intracellular level of HCys, a substrate for CBS that accumulated after CBS knockdown (Fig. 4a,b), suggesting the critical involvement of HS− rather than other cysteine-related derivatives in electrophile metabolism, particularly for 8-nitro-cGMP. This suggestion is supported by the fact that the pKa values of these cysteine-related compounds (8.3, 10.0 and 8.8 for cysteine, HCys and GSH, respectively16,17) are much higher than that of H2S (6.7), which indicates higher nucleophilic reactivity of HS− compared with other compounds at more physiological pH values, as its nucleophilicity is determined by its low pKa18.</p><p>Bimane sulfhydration analysis also showed much lower HS− production in primary cultures of cells (for example, rat cardiomyocytes and cardiac fibroblasts) compared with cultures of all tumor cell lines examined (Fig. 4c). Lower CBS and CSE expression in these primary cell cultures was also evident, as compared with that in mouse hepatocytes and tumor cell lines (A549, HepG2 and C6 cells) (Fig. 4d). The finding that myocardial cells and tissues produced less HS− than the other cells studied suggests that the amount of endogenous HS− in the heart may be limiting in the context of the amounts of endogenous electrophiles that can be generated during both basal metabolism and inflammatory responses. In other words, myocardial cells may be relatively sensitive to endogenous electrophiles, particularly when they are generated excessively during inflammatory processes.</p><p>Notably, HS− is a ubiquitous constituent of many buffers and cell culture media and is not just a product of the cysteine biosynthetic enzymes CBS and CSE13,19. For example, fresh DMEM contained an appreciable amount of HS− (Supplementary Fig. 7e). Therefore, HS− rather than H2S gas may be widely distributed endogenously, in equilibrium with environmental sources and as a byproduct of the decay of more complex thiols. Because HS− can act partly as a potent quencher of various electrophiles, even trace amounts of this ubiquitous mediator will affect the detection and actions of endogenously generated electrophiles in biological systems.</p><!><p>Recent studies of oxidative inflammatory reactions suggest that an H-Ras oncogenic cellular response can be induced by NO− or RNOS-derived species and the electrophile 15d-PGJ2 (refs. 20,21), which in turn may activate p53-dependent cellular senescence22,23. We examined the effect of exogenously administered or endogenous HS− in this context, focusing on an electrophilic signaling pathway mediated by H-Ras and p53 that may be activated by 8-nitro-cGMP or other electrophiles formed in cells.</p><p>Because less HS− was produced—that is, endogenous HS− may have been in short supply relative to the amount of endogenous electrophiles in myocardial cells and tissues (Fig. 4)—we evaluated pharmacological activities of HS− in cardiac cells and in an in vivo model of cardiac inflammatory injury. To clarify the physiological functions of HS−, we induced myocardial hypertrophy and allied chronic inflammatory tissue responses in mouse models of myocardial infarction and pressure overload generated via transverse aortic constriction (TAC) (Supplementary Fig. 8). Chronic heart failure after myocardial infarction is a major cause of morbidity and mortality worldwide24, and both myocardial infarction and TAC models manifest chronic heart failure. Inflammatory reactions that evoke oxidative stress and nitrative stress induced by NO and ROS have been implicated in the genesis of chronic heart failure25,26, findings supported by our immunohistochemical detection of nitric oxide synthase (NOS)-dependent 8-nitro-cGMP formation in the TAC-induced hypertrophic heart and in non-infarcted heart lesions after myocardial infarction (Fig. 5a and Supplementary Fig. 8a–c). Substantial 8-nitro-cGMP production, which depended on inducible NOS (iNOS) expression and activity, was strongly inhibited by NaHS treatment of mice after myocardial infarction (Fig. 5a and Supplementary Fig. 8d). NaHS treatment had no appreciable effects on the tyrosine nitration reaction occurring in cardiac tissues, as assessed by immunohistochemistry and HPLC-based electrochemical detection analysis for 3-nitrotyrosine (Supplementary Fig. 9a)4. Continuous administration of NaHS to mice after myocardial infarction led to elevated plasma HS− (4.9 ± 0.4 μM (n = 4; vehicle control) versus 7.1 ± 1.3 μM (n = 7; NaHS-treated)), determined via an LC/MS/MS bimane assay at 4 weeks after myocardial infarction (P < 0.05; Student's t-test). Other metabolic pathways related to cGMP biosynthesis and degradation, such as those involving soluble guanylate cyclase and PDEs, were not affected by the same treatment (Supplementary Fig. 9b,c). This observation confirms pronounced pharmacological actions of HS− and subsequent electrophile sulfhydration in vivo in cardiac cells and tissues.</p><p>We then investigated H-Ras activation in cardiac tissue in which increased iNOS expression and concomitant 8-nitro-cGMP production occurred. Affinity capture analyses were used to detect H-Ras activation in the tissues. Western blotting of proteins involved in myocardial infarction– and TAC-induced chronic heart failure showed H-Ras activation and simultaneous S-guanylation of activated H-Ras pulled down from hypertrophic cardiac tissue (Fig. 5b and Supplementary Fig. 8e). NaHS treatment also fully inhibited H-Ras activation and concomitant H-Ras S-guanylation in mouse hearts after myocardial infarction (Fig. 5b and Supplementary Fig. 8f).</p><p>NaHS treatment in vivo after myocardial infarction greatly improved dilation of the left ventricle and limited its dysfunction in mice, although ischemic scars were equally developed in hearts of both NaHS- and vehicle-treated mice (Fig. 5c and Supplementary Table 2). A critical downstream cellular response to Ras activation is mediated by ERK, p38 MAPK and phosphatidylinositol-3-kinase (PI3K), which have a central role in cardiac hypertrophy27. Sustained activation of ERK and p38 MAPK then induces activation of p53 and Rb, which have a critical function in cellular senescence22,23 and the transition from hypertrophy to heart failure28,29. NaHS treatment strongly suppressed the activation of ERK and p38 MAPK. Furthermore, phosphorylation of p38 MAPK, ERK, p53 and Rb increased in mouse hearts after myocardial infarction, with NaHS suppressing their activation (Fig. 5d and Supplementary Fig. 8g). These results confirm that HS− attenuates left ventricle dilation and dysfunction after myocardial infarction, in part by suppressing myocardial cell S-guanylation–dependent activation of H-Ras and its downstream signaling pathways.</p><p>We identified high HS− production (Fig. 4) and constitutive expression of different NOS isoforms30,31 in A549 cells and identified concomitant baseline protein S-guanylation by endogenous 8-nitro-cGMP, as evidenced by western blot analysis (Fig. 1a,b). Strong H-Ras activation was induced by CBS knockdown in A549 cells, with simultaneous S-guanylation of activated H-Ras pulled down from the same cell lysates (Supplementary Fig. 10a). Such elevated S-guanylation and activation of H-Ras induced by CBS knockdown were completely nullified by addition of NaHS. The same enhancement of H-Ras activation by S-guanylation was observed when 8-nitro-cGMP was added to cultured A549 cells (Supplementary Fig. 10b). This observation confirmed the substantial contribution of elevated endogenous 8-nitro-cGMP, generated by CBS knockdown, to H-Ras activation. Although an involvement of endogenous HNE formation was not evident, marked activation of H-Ras via S-alkyl adduction occurred in HNE-treated A549 cells and was inhibited by CBS-derived HS− (Supplementary Fig. 10c). These data reveal that HS− can regulate cellular electrophilic signaling involving H-Ras via electrophile sulfhydration reactions.</p><!><p>iNOS-dependent H-Ras S-guanylation concomitant with H-Ras activation was confirmed with rat cardiac fibroblasts in culture with or without iNOS knockdown after lipopolysaccharide (LPS) stimulation to generate endogenous 8-nitro-cGMP (Fig. 6a and Supplementary Fig. 11a). Suppression of iNOS expression by the siRNA resulted in almost complete abrogation of H-Ras S-guanylation and activation in the cardiac fibroblasts. Site-specific H-Ras S-guanylation and its inhibition by HS− were verified in a cell-free reaction mixture via western blotting and proteomic analysis of recombinant H-Ras and its C184S mutant treated with 8-nitro-cGMP (Fig. 6b and Supplementary Fig. 11b). LC/MS/MS sequencing analysis revealed that only Cys184 of H-Ras was S-guanylated (Fig. 6b). Thus, H-Ras Cys184 is a highly susceptible nucleophilic sensor for 8-nitro-cGMP–induced protein S-guanylation.</p><p>Oxidative stress–related nucleotides that induce DNA and nucleotide damage and activation of oncogenic Ras can induce cellular senescence via p53 and Rb tumor suppressor pathways22,23 (Supplementary Fig. 12). Thus, we investigated how 8-nitro-cGMP formation may promote cardiac cell senescence. Exogenously administered 8-nitro-cGMP, but not NO or cGMP, caused growth arrest and remarkably increased the senescence of cultured rat cardiac fibroblasts, with NaHS treatment limiting these responses (Supplementary Fig. 11c,d). The 8-nitro-cGMP–related senescence responses of the cultured cardiomyocytes were comparable to those of cells expressing RasG12V, a constitutively active form of Ras (Supplementary Fig. 11e). Therefore, the impact of site-specific H-Ras Cys184 S-guanylation was evaluated in the context of downstream signaling reactions leading to cellular senescence of cultured rat cardiac fibroblasts. Enhanced senescence of fibroblasts occurred after 8-nitro-cGMP treatment or after stimulation with LPS to generate endogenous 8-nitro-cGMP (Fig. 6c–e and Supplementary Figs. 11 and 13). Both LPS and ATP induce iNOS expression and NO synthesis in cardiac cells32. Here, ATP addition activated cultured cardiomyocytes to generate 8-nitro-cGMP, the amounts of which corresponded to the degree of protein S-guanylation (Supplementary Fig. 13a). This generation of 8- nitro-cGMP induced cellular senescence (Fig. 6c and Supplementary Fig. 11f, g). NaHS treatment markedly attenuated 8-nitro-cGMP formation and protein S-guanylation, which resulted in suppression of cellular senescence induced by electrophilic stimulation in these cultured rat cardiac cells (Fig. 6c and Supplementary Fig. 13). The senescence occurred only when cells expressed wild-type H-Ras or the H-RasC118S mutant but not the H-RasC184S mutant (Fig. 6d,e and Supplementary Fig. 11h).</p><p>Treatment of cultured rat cardiomyocytes with 8-nitro-cGMP also induced H-Ras activation with simultaneous H-Ras S-guanylation, which was strongly inhibited by NaHS treatment (Fig. 7a and Supplementary Fig. 14a). Moreover, activation of H-Ras, but not of the H-RasC184S mutant, was induced in the membrane preparation of rat cardiac fibroblasts after treatment with 8-nitro-cGMP (Fig. 7b and Supplementary Fig. 14b). Inhibition of ERK, p38 MAPK and PI3K greatly suppressed 8-nitro-cGMP–induced cardiac senescence (Supplementary Fig. 14c,d). In contrast, treatment of cardiomyocytes with 8-nitro-cGMP induced sustained activation of ERK, p38 MAPK, p53 and Rb but not of Akt, a downstream effector of class I PI3K, which was nullified by the NaHS treatment (Fig. 7c and Supplementary Fig. 14e). We conclude that Cys184 of H-Ras is a functionally critical sensor of endogenous electrophilic species, such as 8-nitro-cGMP, in their H-Ras–dependent downstream signal transduction.</p><p>Among Ras isoforms, only H-Ras contains two palmitoylation sites (Cys181 and Cys184), and palmitoylation of Ras proteins has a key role in their localization and activity33. Monopalmitoylation of Cys181, but not Cys184, was sufficient to target H-Ras to the plasma membrane, and GDP-bound H-Ras was detected predominantly in lipid rafts (Supplementary Fig. 12)34,35. GTP loading of H-Ras released H-Ras from the rafts to become more diffusely distributed in the plasma membrane, an event necessary for efficient activation of Raf 34. Although the GFP-fused H-Ras and the DsRed-fused Ras-binding domain of cRaf (cRaf-RBD) proteins did not colocalize in cardiac fibroblasts, treatment with 8-nitro-cGMP induced association of H-Ras with cRaf-RBD near the plasma membrane (Fig. 8a,b and Supplementary Fig. 16a,b). Such 8-nitro-cGMP–dependent colocalization of H-Ras and Raf was not observed in the cells transfected with the H-RasC184S mutant (Fig. 8a,b and Supplementary Fig. 16a,b). This observation indicates that 8-nitro-cGMP promotes activation of H-Ras in the plasma membrane. Because Cys184 palmitoylation leads to the correct GTP-regulated lateral segmentation of H-Ras between lipid rafts and nonraft microdomains33–35, S-guanylation of H-Ras at Cys184 is expected to redistribute H-Ras from rafts into the bulk plasma membrane. Remarkably, application of 8-nitro-cGMP to the lipid raft fraction isolated from adult rat hearts induced dissociation of GDP-bound H-Ras from rafts, and this H-Ras, no longer associated with a raft and concomitantly S-guanylated, preferentially interacted with cRaf-RBD (Fig. 8c and Supplementary Fig. 16c). The GDP-bound H-Ras without S-guanylation was left in rafts. NaHS treatment completely suppressed 8-nitro-cGMP–induced colocalization of H-Ras with cRaf-RBD and H-Ras dissociation from rafts. These results indicate that S-guanylation of H-Ras at Cys184 releases H-Ras from lipid rafts and that released H-Ras binds Raf, which leads to activation of downstream signaling pathways (Fig. 8d and Supplementary Fig. 12).</p><!><p>This study reveals that enzymatically generated HS− regulates the metabolism and signaling actions of various electrophiles and that HS−-induced electrophile sulfhydration regulates electrophile-mediated redox signaling. Although H2S is proposed to have anti-inflammatory and antioxidant effects, NaHS treatment produced no appreciable suppression of iNOS and NADPH oxidase (Nox2) expression and 3-nitrotyrosine formation in a mouse model of ischemic heart injury (Supplementary Figs. 9a and 15a). NaHS treatment also did not affect LPS-induced ROS production and ATP-induced RNOS generation in cardiac cells and C6 cells in culture (Supplementary Fig. 15b,c). In view of the modest rate constants for the reaction of H2S with ROS and RNOS such as H2O2 and ONOO− (ref. 36), HS− is not expected to directly scavenge oxygen or NO-derived reactive species, unless they have a substantial electrophilic character. Moreover, some potential HS− targets such as the ATP-sensitive K+ (KATP) channels37 and PDEs38 may be responsible for cardioprotection observed with HS− treatment. However, inhibition of KATP channels, protein kinase A or protein kinase G did not affect NaHS-induced suppression of cardiac cell senescence caused by 8-nitro-cGMP (Supplementary Fig. 14f,g). Also, NaHS treatment had no effect on cGMP metabolic pathways in vivo and in vitro (Supplementary Fig. 9b,c). Therefore, an unambiguous cause-and-effect relationship in terms of HS− regulation of electrophile-evoked cellular stress responses leading to cellular senescence exists. H2S, behaving as an anion (HS−) rather than as a gaseous molecule, reacts as a nucleophile to induce the sulfhydration of the electrophile. This results in cardioprotection in a model of heart failure after myocardial infarction by suppressing oxidative stress–induced or electrophile-mediated (for example, by 8-nitro-cGMP) cellular senescence.</p><p>Ras proteins have three isoforms, which generate distinct signal outputs despite interacting with a common set of effectors33. These biological differences can be accounted for by the 25 C-terminal amino acids of the hypervariable domain, which may contain a protein structure required for Ras to associate with the inner membrane. Cys184 of H-Ras is one of two palmitoylation sites located at its C-terminal domain. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-Ras to the plasma membrane35. Although Cys184 is not essential for targeting H-Ras to the plasma membrane, it is required for control of GTP-regulated lateral segmentation of H-Ras between lipid rafts and nonrafts, which is necessary for efficient activation of Raf34,35. In addition, inhibition of Cys184 palmitoylation efficiently delivers H-Ras to the plasma membrane with little Golgi pooling35, suggesting that S-guanylation of H-Ras at Cys184 promotes H-Ras plasma membrane localization and association with Raf by causing its dissociation from lipid rafts. Cys184 of H-Ras may be chemically modified not only by 8-nitro-cGMP but also by HNE and 15d-PGJ2, and this H-Ras, thereby modified, may increase interaction with cRaf-RBD (Supplementary Fig. 16d–g). Moreover, the electrophilic adduction causes precise structural alterations such that H-Ras is accessible to the effector molecule Raf, which, in turn, readily transduces electrophile-mediated signaling to downstream phosphorylation signaling pathways (Supplementary Fig. 12)22,39. Although the GTP-bound Ras also bound class I PI3K, S-guanylation of H-Ras never activated Akt (Fig. 7c). However, inhibition of PI3K strongly suppressed 8-nitro-cGMP–induced cardiac cell senescence (Supplementary Fig. 14), which suggests that another class of PI3K may be involved in H-Ras–mediated cardiac senescence. Electrophile adduction of H-Ras at Cys184 induced cellular senescence through kinase-dependent signaling pathways, and thus Cys184 is a facile redox sensor for any endogenous and exogenous electrophiles and their regulation by endogenous HS− via sulfhydration (Supplementary Fig. 12).</p><p>8-SH-cGMP still has cGMP activity, in that it effectively activates PKG (Supplementary Fig. 9d). Moreover, it can acquire PDE resistance, which will increase its pharmacological effects as a cGMP analog (Supplementary Fig. 9e). Because cGMP itself reportedly has a potent cardioprotective effect, sulfhydration of 8-nitro-cGMP may thus alter its chemical properties so that it can benefit not only chronic heart failure after myocardial infarction but also various other disease processes. Thus, although 8-nitro-cGMP formed in excess in the heart after myocardial infarction may accelerate heart remodeling, HS− generation in cells and tissues may rectify the pharmacological actions of this electrophilic cGMP analog, for example, by converting the pathological effects of 8-nitro-cGMP into beneficial effects associated with a PDE-resistant cGMP homolog. A similar HS−-mediated bioconversion of an electrophile to a nucleophile would apply to other electrophiles, such as 15d-PGJ2 (Fig. 3a).</p><p>p53-dependent G1 cell cycle arrest aids DNA repair of injured cells to prevent oxidative stress–related genetic mutation and genotoxicity. Abundant GTP in the cellular nucleotide pool seems to be nitrated initially and thus may function as a sensor for nucleotide modification caused by RNOS12; this step is believed to be crucial for a cellular oxidative stress response. Also, 8-nitro-GTP becomes an endogenous mutagen when incorporated into DNA40,41. Therefore, soluble guanylate cyclase not only contributes to electrophilic 8-nitro-cGMP signaling but also can suppress 8- nitro-GTP and limit mutagenic responses (Supplementary Fig. 12). Thus, 8-nitro-cGMP regulation by HS− limits nitrative modification via sulfhydration, supporting positive genomic and signaling functions and conferring protection against ROS- and RNOS-induced genotoxicity.</p><p>In conclusion, our present data reveal an important metabolic relationship between HS− and oxidative inflammation–derived electrophilic signaling mediators. This identification of HS−-induced electrophile sulfhydration as a mechanism for terminating electrophile-mediated signaling provides a fundamental new way of understanding the regulation of redox cellular signaling and therapeutic strategies for inflammation-related diseases.</p><!><p>To clarify factors contributing to regulation of 8-nitro-cGMP signaling, we performed RNAi screening, as described in Supplementary Methods. Detailed protocols and siRNAs used are described in Supplementary Methods and Supplementary Table 1.</p><!><p>8-Nitro-cGMP (100 μM) was reacted with various concentrations of NaHS (the HS− donor) in 100 mM sodium phosphate buffer (pH 7.4) containing 100 μM diethylenetriaminepentaacetic acid (DTPA) at 37 °C for 5 h. The reaction of 8-nitro-cGMP (100 μM) with NaHS (1 mM) was also carried out in the absence or presence of additives including cysteine (100 μM), metals (150 μM) and metal-containing compounds and proteins (10 μM). Reaction products of 8-nitro-cGMP with NaHS in the absence or presence of additives were analyzed by using RP-HPLC and LC/MS as described in Supplementary Methods. 15d-PGJ2 (1–10 μM) was reacted with NaHS (0–1,000 μM) in 100 mM phosphate buffer (pH 7.4) at 37 °C for 2 h. The reactions of 1,2-NQ (100 μM), 1,4-NQ (100 μM), tert-butylbenzoquinone (100 μM) or OANO2 (100 μM) with NaHS (50–800 μM) were carried out in 200 mM potassium phosphate buffer (pH 7.5) at 25 °C for 1 h.</p><!><p>A549, HepG2 and C6 cells treated with CBS siRNA or untreated cells were incubated with 8-nitro-cGMP (200 μM) in serum-free DMEM at 37 °C for 6 h. 8-SH-cGMP that formed in those cultures was quantified by means of LC-ESI-MS/MS with the use of 8-34SH]-cGMP as an internal standard. Details are in Supplementary Methods.</p><!><p>Cellular production of HS− was quantified by means of LC-ESI-MS/MS with monobromobimane derivatization. This protocol allowed us to simultaneously quantify HS− and other low-molecular-weight thiols including cysteine, HCys and GSH, as described in Supplementary Methods.</p><!><p>All protocols using mice and rats were approved by the Animal Care and Use Committee, Kyushu University. Mice with a homozygous deletion of the gene encoding iNOS were purchased from Jackson Laboratory. The left anterior descending coronary artery (LAD) was ligated (with 6-0 silk suture) near its origin between the pulmonary outflow tract and the edge of the left atrium. Acute myocardial ischemia was deemed successful when the anterior left ventricle wall became cyanotic and the electrocardiogram showed obvious ST segment elevation. Sham-operated mice were subjected to the same procedure, except that the suture around the LAD was not tied. TAC surgery was performed on 6-week-old male C57BL/6J mice. A mini-osmotic pump (Alzet) filled with vehicle (PBS) or NaHS (50 μmol kg−1 d−1) was implanted intraperitoneally into 8-week-old male C57BL/6J mice 1 d after LAD ligation. Plasma concentrations of HS− were determined by means of LC-ESI-MS/MS with monobromobimane as described in Supplementary Methods.</p><!><p>Paraffin-embedded left ventricle sections (5 μm thick) were stained with 8-nitro-cGMP (1G6)–specific antibody (1:1,000), followed by visualization with Alexa Fluor 488 rabbit-specific IgG (clone no. A11008) and Alexa Fluor 546 mouse-specific IgG (clone no. A11003) antibodies (1:1,000; Invitrogen). Digital photographs were taken at 600× magnification with a confocal microscope (FV10i, Olympus).</p><!><p>Recombinant human H-Ras was prepared and purified as described in Supplementary Methods.</p><!><p>Endogenous active H-Ras was obtained by incubating supernatants with glutathione S-transferase–fused cRaf-RBD in the presence of GSH-Sepharose beads, followed by western blotting as described in Supplementary Methods.</p><!><p>S-Guanylation sites were identified by means of MS with trypsin-digested peptide fragments of H-Ras. Details are in Supplementary Methods.</p><!><p>Cardiomyocytes and cardiac fibroblasts were prepared from ventricles of 1- to 2-d-old Sprague-Dawley rats. Cardiac fibroblasts were transfected with control vector or vectors expressing H-Ras (wild-type or carrying the C118S or C184S mutation) via electroporation (1,100 V, 10 ms × 4; NeonTM Transfection System, Life Technologies Corporation). Cardiomyocytes were infected with recombinant adenoviruses expressing LacZ control or RasG12V at 100 multiplicity of infection 1 h after serum starvation. Twenty-four hours later, cells were pretreated with NaHS (100 μM) for 24 h and then were treated with ATP (100 μM), LPS (1 μg ml−1) or 8-nitro-cGMP (10 μM) for 3 h, after which they were cultured for 4 d in 0.5% (v/v) serum-containing medium. Cardiac cells plated on 35-mm glass-bottom dishes were fixed with 4% (w/v) paraformaldehyde neutral buffer solution, and cellular senescence was assessed by measuring endogenous β-galactosidase (β-gal) activity using a senescence-associated β-gal (SA-β-gal) staining kit (Cell Signaling). Digital photographs were taken at 200× magnification with a Biozero microscope (BZ-8000; Keyence), and the number of β-gal–positive cells (n > 100 cells) was calculated by using the BZ-II Analyzer (Keyence), with colorimetric intensity adjusted as the percentage of basal senescent cells approached 1.</p><!><p>Echocardiography was performed in anesthetized mice (50 mg per kg body weight pentobarbital sodium) via the Nemio XG echocardiograph (Toshiba) equipped with a 14-MHz transducer. A 1.4-French micronanometer catheter (Millar Instruments) was inserted into the left carotid artery and advanced retrograde into the left ventricle. Hemodynamic measurements were recorded when the heart rate was stabilized within 500 ± 10 beats per min.</p><!><p>Results are presented as mean ± s.e.m. of at least three independent experiments unless specified. Statistical comparisons were made with two-tailed Student's t-test or one-way analysis of variance followed by the Student-Newman-Keuls procedure, with significance set at P < 0.05.</p><!><p>Detailed information is available in the Supplementary Methods.</p>

PubMed Author Manuscript

A Cost-Effective Semi-Ab Initio Approach to Model Relaxation in Rare-Earth Single-Molecule Magnets

We discuss a cost-effective approach to understand magnetic relaxation in the new generation of rare-earth single-molecule magnets. It combines ab initio calculations of the crystal field parameters, of the magneto-elastic coupling with local modes, and of the phonon density of states with fitting of only three microscopic parameters. Although much less demanding than a fully ab initio approach, the method gives important physical insights into the origin of the observed relaxation. By applying it to high-anisotropy compounds with very different relaxation, we demonstrate the power of the approach and pinpoint ingredients for improving the performance of single-molecule magnets.

a_cost-effective_semi-ab_initio_approach_to_model_relaxation_in_rare-earth_single-molecule_magnets

<!>Author Present Address<!>

<p>The intense research on the new generation of 4f-based single-molecule magnets (SMMs)1,2 with strong axial anisotropy3−16 has recently culminated in the identification of dysprosocenium complexes,17−19 which display blocking temperatures that surpass liquid nitrogen temperatures. This sudden jump could open the possibility to reach even higher working temperature if the main ingredients behind such performances are well understood. It is evident that the anisotropy barrier Ueff is not the only figure of merit,20 since 4f-based molecules with similarly high barriers can display very different relaxation dynamics.21−24 Hence, a sound and efficient method explaining the very different phonon-induced relaxation in molecular crystals of different high-barrier molecules is needed to make a step forward.20,25,26 A fully ab initio approach27,28 would yield the maximum insight into the origin of magnetic relaxation of specific compounds, but the huge amount of computational resources needed would strongly limit the applicability of such a method to only a small number of compounds.</p><p>To design a cost-effective theoretical approach, we need to balance the amount of predictive power with the resources required for numerical calculations. Here we describe such an approach and show that it explains why molecular crystals of different 4f SMMs with similar anisotropy barriers are characterized by very different phonon-induced relaxation. Remarkably, the approach proposed yields physical insights that cannot be obtained with the phenomenological models widely used for fitting relaxation rates, whose parameters are not connected to microscopic Hamiltonians. At the same time, it is not as demanding as fully ab initio calculations since it takes advantage of these techniques only to calculate the key ingredients of the Hamiltonian. As a preliminary step, we recently applied this approach to a protypical dysprosocenium system ([Dy(C5H2tBu3-1,2,4)2][B(C6F5)4] (1, Figure 1a)).29 Here we finally demonstrate the reliability and power of this method by successfully applying it to two benchmark Dy-based compounds, both of which are characterized by high anisotropy barriers but have very different and much faster relaxation than 1: the pentagonal bipyramidal [Dy(tBuO)Cl(THF)5][BPh4]·2THF complex (2, Figure 1b)30 and the five-coordinate [Dy(Mes*O)2(THF)2Br] complex (Mes* = 2,4,6-tri-tert-butylphenyl) (3, Figure 1c).24 Crystalline batches of 2(30) and 3(24) were synthesized according to literature procedures to make pure samples; single-crystal XRD was used to check the unit cells of multiple crystals to confirm that the identities of these samples matched literature data (see CCDC 1450752 and 1978054). Complementary characterization data were provided by the preparation of the structurally analogous diamagnetic Y(III) analogues of 2 (2-Y)30 and 3 (3-Y), which were additionally studied by 1H and 13C NMR spectroscopy to assess the bulk purity of samples. Yttrium complexes are often used to provide diamagnetic matrices for doping studies of late lanthanide SMMs, where increased distances between paramagnetic ions reduce dipolar interactions and help distinguish magnetic relaxation mechanisms. Full synthetic and crystallographic details are compiled in the Supporting Information.</p><p>Molecular structures of (a) 1, (b) 2, and (c) 3; hydrogen atoms have been omitted for clarity.</p><p>An important ingredient of our approach is the phonon density of states (pDOS), ρ(E), which we obtain ab initio. To put this calculation to the test, we probed it with Inelastic Neutron Scattering (INS), finding excellent agreement. Then, via our approach we calculate the temperature-dependence of the relaxation rates, reproducing the experimental data with a very good agreement for all the compounds. Furthermore, we highlight the main ingredients that make the Raman mechanism much more efficient in 2 and 3 and thus considerably worsen their performance as SMMs with respect to that of 1.</p><p>The Hamiltonian describing molecular crystals of these SMMs is1where the first term contains the crystal field (CF) splitting ∑k,qBkqOkq of the J = 15/2 angular momentum multiplet for each Dy3+ ion in the crystal, with Okq being Steven's operators equivalents.31 The Hph term in eq 1) is the phononic Hamiltonian, and HJp(a,o) describes magnetoelastic coupling with acoustic and optical phonons (parametrized by ζa and ζo). The CF parameters can be reliably calculated ab initio using complete active space self-consistent field spin–orbit (CASSCF-SO) calculations with MOLCAS 8.0,24,30,32 and Hph can be obtained via DFT (PBE) calculations using Quantum Espresso.33 The most demanding parts of ab initio calculations are magnetoelastic interactions. However, this huge task is not really necessary. On the one hand, Raman processes are dominated by dispersive "acoustic" modes, and HJp(a) can be reasonably approximated by applying a "rotational Debye model"34 and using the DFT (PBE) pDOS.29,35−37 Indeed, the energy integral relevant for Raman processes washes out details of individual phonon modes (see below, eq 2). On the other hand, only high-energy nondispersive optical phonons are involved in the high-temperature Orbach regime38,39 and HJp(o) can thus be approximated by simpler calculations in the gas phase (see the Supporting Information for more details). It is important to stress that in molecular crystals of SMMs such as 1, 2, and 3, many low-energy optical modes typically correspond to collective vibrations with little bond stretching and are significantly dispersive (see the Supporting Information for more details). Thus, the effective upper energy limit for the dispersive modes described via the rotational Debye model, ℏωD, is here treated as a fitting parameter.</p><p>Indeed, in our approach only three free Hamiltonian parameters (ζa, ζo, and ωD) are needed to model phonon-induced relaxation and are determined by a comparison with relaxation measurements. All the other quantities in eq 1 are efficiently calculated ab initio, with the pDOS and lowest CF energies also being independently validated by the comparison with targeted inelastic neutron scattering experiments. At last, the low-temperature relaxation of 4f-based SMMs is generally dominated by temperature-independent quantum tunneling processes, which can therefore be modeled by a constant relaxation rate. Therefore, we will focus on phonon-driven relaxation in the intermediate and high temperatures regimes.</p><p>The intermediate temperature range is crucial in determining blocking temperatures.29 In this regime, the system dynamics is restricted to the lowest Kramers doublet, and excited CF states only contribute via nonresonant Raman processes. The corresponding relaxation rate is proportional to29,35,402where ρ(E) is the pDOS computed by DFT, Δ is the (practically negligible) Zeeman gap between the two states of the ground doublet, and M contains matrix elements of the magnetoelastic term HJp(a,o). Although the integral in eq 2 is calculated over the whole phonon spectrum, due to the form of its integrand, the Raman relaxation is driven mainly by low-energy dispersive modes (in the present model, those with energy E ≤ ℏωD).29 By numerical trapezoidal integration, we obtain from eq 2 the Raman contribution to the relaxation rate. This, in general, does not follow a power-law T-dependence apart from a rather narrow intermediate temperature range (see Figure 3b and comments below). Nonetheless, extracting "effective" power-law behaviors in specific temperature regimes can be useful for a quick comparison with the experimental literature, where often such a power-law fit is adopted to interpret the data. Hence, in the following we calculate the Raman contribution to the relaxation rate from eq 2 and use it to compare the experimental data in the whole T range. In addition, we fit the calculation with an effective power-law T-dependence τRaman–1 = CTn in a properly narrow temperature range. This allows us to directly extract the two parameters governing the relaxation rate in the Raman regime: the prefactor C and, most importantly, the exponent n, which is mainly determined by the pDOS and the lowest CF energy gap.29</p><p>In the high-temperature range, excited doublets of the J = 15/2 multiplet start to be populated. Hence, in this regime we adopt a master equation approach, accounting for all the possible transitions.41,42 Calculations show that relaxation is here governed by a single rate following an Arrhenius-like behavior τOrbach–1 ≃ τ0–1e–Ueff/KBT, where the coefficient τ0 and the effective barrier Ueff are directly evaluated with our approach. More details are given in the Supporting Information.</p><p>Before applying our approach to relaxation, we have investigated the pDOS ρ(E) and low-temperature CF excitations in molecular crystals of 2 and 3 with the thermal-neutrons spectrometer MERLIN at ISIS43 (see the Supporting Information for more details). As expected, a single magnetic excitation was detected for compound 2 at ≃62 meV, which is in agreement with the ab initio calculations (52 meV). The observed wave-vector transfer (Q)-dependence of the measured intensity clearly identifies the peak at 62 meV as the only magnetic excitation (see Figure S13 in the Supporting Information). A similar energy gap between the two lowest Kramers doublets was also predicted for 3 (54 meV) but was not detectable, being covered by phonon modes around 60 meV.44 In Figure 2a and b, we report the neutron-weighted pDOS (nwDOS) measured on 2 and 3, respectively. To reproduce these INS data, we performed DFT (PBE) simulations to calculate the atom-projected DOS and reconstruct the nwDOS by applying the one-phonon incoherent approximation (see the Supporting Information for more details). The comparison reported in Figure 2a and b shows an excellent agreement between our calculations and the INS data for 2 and 3, demonstrating the reliability of the calculated pDOS. The pDOS entering the model for relaxation is the one without the weighting for the neutron cross-section and is reported in Figure 2c for 2 and 3, compared with the pDOS of 1 (for the pDOS over the full energy range, see Figure S11 in the Supporting Information).</p><p>Measured (symbols) and calculated (red line) nwDOS for (a) compound 2 and (b) compound 3. The neutron-weighted DOS was measured at T = 5 K with different incident neutron energies Ei and resolutions and are reported here with different colors. The fwhm of the calculated nwDOS varies accordingly to the experimental configuration. Error bars are of the order of the symbols size. (c) DFT (PBE) low-energy pDOS ρ(E) of compounds 2 and 3 (blue and red lines, respectively) compared to the ρ(E) of 1 (dashed black line) obtained with a single fwhm = 0.29 meV. For the sake of comparison, each pDOS has been normalized to the number of molecules in the unit cell. Vertical lines pinpoint the threshold energy ℏωD.</p><p>Measured relaxation rates τ–1 are shown in Figure 3a.24,30 The presence of two different regimes is evident: an intermediate-temperature Raman region and a high-temperature Arrhenius regime. Lines in Figure 3a are the result of our calculations and were obtained without any assumption on the functional form of the relaxation rate. It is evident that the different behaviors of the three compounds are well-reproduced in the whole temperature range. A comparison between calculated curves and experimental points is the best figure for the merit of our approach. Conversely, the derivation of effective relaxation coefficients is often not univocal because these coefficients are often strongly correlated and temperature-dependent. In our approach, Raman and Orbach mechanisms are disentangled by separately computing their respective contributions. The former, as shown in Figure 3b, exhibits a power-law behavior (with a single exponent n) only in a narrow temperature region, and the related coefficients strongly depend on the choice of such a range. In particular, the value n varies from 2 to 9 when going from the high temperature limit to the low temperature limit. Conversely, in the present systems the Orbach parameters Ueff and τ0 are stable and can be obtained by focusing on the high-temperature range. Fitted Hamiltonian parameters and effective relaxation coefficients determined from calculations for 1, 2, and 3 are summarized in Table 1. We note that the trends in the Raman exponent n and in the effective barrier Ueff extracted from the phenomonological fitting of experimental data (see the Supporting Information and refs (17), (24), and (30) for 1, 2, and 3, respectively) are in agreement with our model. Indeed, in both cases compound 1 shows the largest Ueff value and the smallest n value , complex 3 is intermediate, and 2 is characterized by the smallest Ueff value and the largest n value.</p><p>axiality = |⟨mJ|ψi⟩|2, where |ψi⟩ is the CF eigenstate and |mJ⟩ is the state with the largest component averaged over all the doublets of the J = 15/2 multiplet.</p><p>Relaxation rate τ–1 for 2 (blue scatters) and 3 (red scatters) compared with relaxation rates of 1 (gray scatters). Blue, red and gray lines are the full simulation of τ–1 comprising Raman and Orbach contributions. Inset: comparison of the relaxation rate τ–1 of 1 calculated with our model and different exponent n for the Raman power-law: original values (black line), value of 2 (blue line), value of 3 (red line). The dashed line highlights the value τ–1 = 10–2 s–1, for the extrapolation of TB as the temperature at which the relaxation time is 100 s. (b) Calculated Raman relaxation rates for compound 2 and 3 (continuous lines) and corresponding fits to extract effective Raman coefficients C and n (dashed lines) using a power-law T-dependence in the 15–35 K (15–45 K) range for complex 2 (3).</p><p>Compound 1 has several characteristics that contribute to the suppression of Raman mechanisms (eq 2). The large CF splittings and the relatively small coupling ζo with nondispersive modes make optical phonons ineffective for Raman relaxation. Moreover, the pDOS slowly increasing with energy and the rather small value of ℏωD yield a weak temperature dependence, with calculated n ≃ 2 in the Raman region, which is in agreement with experimental findings. Conversely, the steeper shape of the pDOS of 2 and 3 at low energies and the larger ℏωD values lead to more efficient Raman processes with higher exponents n = 3.6 and 3.0, respectively. This in turn leads to a much faster relaxation in 2 and 3, which are characterized by relaxation rates several orders of magnitude larger than that of 1 in the intermediate temperature range (τ–1 ∼ 101–102 s–1 vs 10–3 s–1).</p><p>Our approach also reproduces the multistep Orbach regime and yields calculated energy barriers Ueff = 1786, 1093, and 1127 K for 1, 2, and 3, respectively. An analysis of CF coefficients and eigenstates shows that 1 displays the largest average axiality (i.e., the ground and excited CF doublets are practically characterized by a single |mJ| component). Our calculations also demonstrate that the lower value of Ueff in 2 and 3 reflects the energy of the respective second-excited CF doublets, which are the lowest in energy to lose axiality (see Table S8 in the Supporting Information). Moreover, the larger coupling ζo with optical modes of 2 and 3 leads to shorter τ0 and faster relaxation in the Orbach regime.</p><p>Figure 3 shows that suppressing the Raman mechanism is fundamental to increase the blocking temperature of rare-earth SMMs toward the high-temperature (Orbach) range. Indeed, if the temperature dependence of the relaxation rate of 1 is recalculated by replacing the exponent n (depending on the pDOS and CF splittings, see above) with that of 2 or 3, a steeper power-law for τ–1(T) and a large overall increase of the relaxation rates are obtained (see inset of Figure 3a).</p><p>Raman relaxation also strongly depends on the coupling with dispersive modes ζa. Our calculations point to a small value of ζa for 3, consistently with 3 being a neutral molecule, whereas a counterion is present in 1 and 2 (see Figure 1). Indeed, in 1 and 2 we expect stronger modulations of the CF (and a larger value for ζa), because low-energy dispersive modes induce the motion of charged objects (ion and counterion). Despite having a much weaker coupling ζa with dispersive modes, 3 is characterized by a larger calculated value of C (see Table 1). Indeed, our calculations show that this parameter is largely affected by the average axiality of the lowest eigenstates, which is much lower for 3. Moreover, the pDOS at low energy of 3 is steeper than in 2 (see Figure 2c) and also contributes to the larger C value. Thus, the CF and the form of the pDOS compete with the spin–phonon coupling ζa in determining the prefactor C for the Raman power-law.</p><p>axial ground and excited doublets, which could be achieved by increasing the axial symmetry and the ratio of axial to equatorial ligand donor strength. These ingredients are important to limit Raman relaxation (through a reduction of both C and n) as well as to increase the effective barrier Ueff by hindering short-cuts in the relaxation path.</p><p>large CF gaps, which could also be achieved by increasing the ratio of the axial to equatorial ligand donor strength. A large gap between ground and low excited doublets is important to prevent Raman-like resonant two-step processes (which would effectively increase n in a narrow temperature range, see ref (29)), while the overall CF splitting affects the value of Ueff in the Orbach process.</p><p>making it not too steep, i.e., slowly increasing with energy (thus reducing the prefactor C).</p><p>displaying a rather small ℏωD to reduce the value of n and approach the high-temperature limit n = 2.</p><p>pDOS at energies corresponding to CF excitations, which must be as small as possible to increase τ0, thus quenching the resonant (Orbach) mechanism.27 This means reducing the number of vibrational modes at those energies, moving most of them out-of-resonance with the most likely CF transitions. This is hard to control a priori but can be reliably calculated for candidate molecular structures,27 so in principle designs can be tested.</p><p>Keeping magneto–elastic coupling with local modes close in energy to CF gaps (ζo) as small as possible, again to increase τ0. This means that phonon modes at those energies must not induce a significant modulation of the Dy ligand cage. Again, this can be reliably calculated for SMM candidates.27</p><p>A small magneto–elastic coupling with dispersive modes (ζa) to suppress the value of C. This could be achieved by reducing the mixing between acoustic and optical modes45 or by choosing neutral molecules. Indeed, it was recently shown45 that ζa is largely dominated by the optical component of the modes. This can be reduced, for instance, by moving low-lying optical modes to higher energies. Moreover, the Coulomb interaction between two close charged objects (magnetic core and counterion) can lead to a large modulation of the CF and thus to a large ζa.</p><p>The Raman exponent n is controlled by the axiality and gaps of the (lowest) CF doublets (point 1) and by the shape of the pDOS at low energy (point 2). n close to 2 can be achieved by axial CF doublets, large CF gaps, and a small ℏωD values.</p><p>The Raman prefactor C is limited by fulfilling points 1, 2(a), and 5.</p><p>Long τ0 are obtained by satisfying points 3 and 4.</p><p>Large barriers Ueff are achieved in the presence of large CF gaps and strongly axial ground and excited doublets (point 1). Indeed, in the examined cases we find that Ueff roughly corresponds to the gap between the ground and the first nonaxial doublet, which activates through-barrier relaxation.</p><p>In conclusion, we have demonstrated the power of our effective approach for the calculation of the magnetic relaxation by applying it to three high-barrier Dy-based SMMs characterized by very different relaxations. The application of the method to these compounds pinpoints the crucial role played by the Raman mechanism in the new generation of 4f SMMs and highlights the main ingredients controlling it. This comparative analysis thus supplies new hints for the recipe to design new SMMs. In addition, this study also provides new tools for the investigation of the relaxation dynamics of other molecular systems, such as molecular qubits, whose coherence times can be influenced by phonon-induced relaxation.45,46</p><p>Synthesis of the samples, details of the neutron scattering experiment, ab initio calculations, theoretical procedures, and supplementary results (PDF)</p><p>jz1c02367_si_001.pdf</p><!><p>∇ Department of Inorganic and Organic Chemistry, University of Barcelona, Barcelona 08007, Spain</p><!><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Measurement of the binding parameters of annexin derivatives-erythrocyte membrane interactions

Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled Annexin V (ANV) derivatives to these membranes were studied by titration with proteins and with calcium. Whereas pre-addition of EDTA to reaction mixtures totally prevented membrane binding, Ca++-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca++ dependent binding which became partially Ca++ independent. Data derived from saturation titration with ANV derivatives poorly fit simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into protein-Ca++-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was about 4-fold lower than that of ANV at 1.2\xe2\x80\x932.5 mM Ca++. We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.

measurement_of_the_binding_parameters_of_annexin_derivatives-erythrocyte_membrane_interactions

INTRODUCTION<!>Expression, purification, and FITC labeling of recombinant ANV and ANV-6L15<!>Preparation of erythrocyte ghosts<!>Confocal microscopy of preserved blood cells and erythrocyte ghosts<!>Flow cytometric analysis of preserved blood cells and erythrocyte ghosts<!>Saturation binding of ANV derivatives to erythrocyte ghosts<!>Effects of Ca++ and EDTA on the binding of ANV-FITC and ANV-6L15-FITC to erythrocyte ghosts<!>Calcium titration assay: The binding model<!>Comparison of the binding of ANV-FITC to preservative-treated blood cells and erythrocyte ghosts<!>Determination of maximum membrane binding sites [m] on erythrocyte ghosts<!>Effects of Ca++ and EDTA on the binding of ANV-FITC and ANV-6L15-FITC to erythrocyte ghosts<!>Saturation binding of ANV-FITC and A6L15-FITC to erythrocyte ghosts<!>Calcium titration of the binding of ANV-FITC and A6L15-FITC to erythrocyte ghosts<!>DISCUSSION<!>Imaging of ANV-FITC-treated blood cells and ghosts using confocal microscopy<!>Flow cytometric analysis of preserved blood cells and blood cell ghosts<!>Determination of maximum membrane binding sites [m] on erythrocyte ghosts<!>Effects of Ca++ and EDTA on the binding of ANV-FITC and ANV-6L15-FITC to erythrocyte ghosts<!>Saturation binding of ANV-FITC and ANV-6L15-FITC to erythrocyte ghosts<!>Calcium titration of the binding of ANV-FITC and ANV-6L15-FITC to erythrocyte ghosts<!>

<p>Phosphatidylserine (PS3) is one of the four major phospholipids in the plasma membranes of mammalian cells, comprising 8–15 % of the total phospholipids content. In normal cells, PS is exclusively sequestered in the inner layer of the plasma membrane together with most of the phosphatidylethanolamine (PE), whereas the outer layer is mainly composed of phosphatidylcholine (PC) and sphingomyelin [1–3]. Apoptotic cell death, cell senescence, cell activation, oxidative stress, and cell damage can all lead to exposure of PS on cell membrane surfaces and shed microparticles. Ability to detect PS exposure on pathological cells can provide important information in the diagnosis and treatment of various diseases [1].</p><p>The Annexins are a family of proteins that share the property of calcium-dependent binding to PS-expressing membranes [4–6]. Annexin V (ANV) is a typical member of the family which has been widely used for detection of PS-expressing cells by confocal microscopy and flow cytometry [7–11]. ANV is also being developed as a diagnostic agent to detect cell death in vivo in cancer chemotherapy, organ transplant rejection, and myocardial infarction [12–15]. Early work showed that ANV bound to model membranes containing 20 % PS-80 % PC with an estimated dissociation constant (Kd) of < 10−10 M at physiological concentration of Ca++ [16,17]. Subsequent studies of ANV binding to various cell types by classical saturation titration assays had produced widely different Kd values ranging from 2.1 × 10−11M to 2.5 × 10−8 M [18–22]. Thus, the real affinity of ANV binding to PS-expressing cells remained imprecisely defined. Tait et al. recently developed a calcium titration method for the measurement of the affinity and cooperativity of ANV-Ca++-membrane binding [23]. The binding of ANV to preservative-treated blood cells was titrated with Ca++ such that < 3% of the membrane binding sites was occupied throughout the titration. This experimental approach circumvented the problems of classical saturation titration in which heterogeneous binding events might occur due to acidic phospholipid segregation [24–26], protein clustering [27,28], and alterations in membrane shape and rigidity [29,30] at high membrane occupancy. Using this method, Tait et al. obtained a drastically different set of binding parameters by non-linear least squares fit of the equilibrium binding equation. However, this original calcium titration method estimated the membrane-bound ANV after washing of cells and treatment with EDTA to release the bound ANV. It was not clear whether cell washing significantly perturbed the binding equilibrium and whether EDTA released the bound ANV completely. In an effort to establish valid methods for quantifying the affinity constants of various ANV derivatives for cell membranes, we revisited the issues and investigated the binding of ANV derivatives to erythrocyte ghosts by classical saturation titration assay and by a modified calcium titration method. We found that erythrocyte ghosts prepared from fresh blood appeared to offer significant advantages over other cell systems since these membranes express PS at higher levels and in a more stable fashion. We discovered that Ca++ dependent binding of ANV derivatives to erythrocyte ghosts was abolished by co-treatment with EDTA, but was only partially reversed by post-treatment with EDTA. This new finding necessitated a modification of the original calcium titration method to measure the membrane-bound ANV derivatives. We further showed that saturation titration data poorly fit simple protein-membrane equilibrium binding equation. In contrast, calcium titration at low membrane binding site occupancy (≤ 2% saturation) provided excellent fit of the ANV-Ca++-membrane equilibrium binding equation and allowed us to calculate various binding parameters. Using this new assay system, we compared the binding parameters of ANV with those of ANV-6L15, a fusion protein consisting of an ANV domain and a Kunitz-type protease inhibitor domain that inhibited tissue factor/factor VIIa with high potency [31]. We found that the Kd for ANV-6L15 was about 4-fold lower than that of ANV at physiological concentrations of ionized Ca++, suggesting that ANV-6L15 bound to PS-expressing cells with stronger affinity than ANV.</p><!><p>E. coli BL21(DE3)pLysS and the expression vector pET20b(+) (Novagen, Medison, WI) were used for the expression of recombinant ANV and ANV-6L15, and the recombinant proteins were purified as described before [31]. The purified proteins were labeled with FITC (Pierce, Rockford IL) by the following protocol: ANV or ANV-6L15 (50 μM) was incubated with FITC (250 μM) for 1 h at room temperature (r.t.) in 100 mM Na-borate, pH 9.0. The reaction mixture (1 ml) was quenched by adding 0.1 ml of 1 M glycine and dialyzed extensively against TBS buffer (20 mM Tris, pH 7.4, 150 mM NaCl). The labeled proteins were quantitated by Bradford protein assay (BioRad, Hercules CA) using unlabeled proteins as standards and the amount of fluorescein quantitated by absorbance reading at 494 nm using E494 =80,000. This procedure resulted in FITC:protein (F:P) labeling ratios of 0.37 and 0.76 mol/mol for ANV-FITC and ANV-6L15-FITC, respectively, and the conjugates were designated by subscripts as ANV-FITC0.37 and ANV-6L15-FITC0.76. ANV-FITC with higher F:P ratios (1.3 and 2.4) were obtained by labeling ANV with 1000 μM FITC or N-hydroxysuccinimide-fluorescein. Calcium titrations using ANV-FITC1.3 yielded similar binding parameters as those using ANV-FITC0.37. However, titrations using ANV-FITC2.4 showed higher Kd values, suggesting that the membrane binding affinity decreased when the F:P ratio increased to 2.4. ANV-FITC0.37 and ANV-6L15-FITC0.76 were used for calcium titration in this study.</p><!><p>Erythrocyte ghosts were prepared from ACD blood or preserved blood (4C Plus Control or 5C control, Beckman-Coulter, Miami, FL) by repeated hypo-osmotic lysis. Ten ml of blood was dispensed into 2-ml microfuge tubes in 0.5 ml aliquots and centrifuged at 13,000 rpm for 5 min at r.t. The supernatant plasma was discarded. De-ionized water (1.8 ml) was then added to each tube containing the cell pellet. The tubes were vortexed for 50 sec., incubated in a 37°C bath for 45 min, and centrifuged at 13,000 rpm for 5 min at r.t. TBSA (TBS buffer containing 1 mg/ml BSA and 0.02% NaN3) was added to each cell pellet to a final volume of 2 ml to re-suspend the cells. The tubes were vortexed, incubated at 37°C, and centrifuged as above. De-ionized water (1.8 ml) was added to each cell pellet to lyse the cells again. The tubes were vortexed, incubated at 37°C, and centrifuged as above. TBSA (1.8 ml) was then added to each cell pellet to re-suspend the cells. The cells were then left at 4 °C overnight. After centrifugation, the cell ghosts were pooled, re-suspended in a final volume of 10 ml TBSA, and stored at 4°C. A Coulter Z1 dual threshold model was used to determine cell count using the setup [TU35fl, TL30fl, >TU] for red cell ghosts. Stocks of erythrocyte ghosts (2.8 × 108 cells/ml) were stored at 4 °C in TBSA. The cell counts of the stocks did not change over a 3-month storage period.</p><!><p>4C Plus Control, 5C Control, and erythrocyte ghosts were diluted 200 fold into HBSA buffer [10 mM Hepes, pH 7.4, 137 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 0.5 mM NaH2PO4, 0.1 % D(+)-glucose, 0.02 % NaN3, 0.1 % BSA] containing 2.5 mM CaCl2 and 50 nM ANV-FITC. The mixtures were incubated at r.t. for 1 h followed by centrifugation at 13,000 rpm for 3 min to pellet the cells. The cell pellets were washed once by suspending in an equal volume of HBSA-2.5 mM CaCl2. The washed cell samples were drop onto Lab-Tek 8-chamber slides (Nunc Inc. Rochester, NY) and sealed with a coverslip and nail polish. The samples were examined under a Olympus confocal laser scanning microscope (FV1000) using a 20× objective lens and excitation/emission filters 488/510–560 nm.</p><!><p>5C Control cells or erythrocyte ghosts were diluted to about 5 × 106 cells/ml in HBSA buffer containing 2.5 mM CaCl2 and 50 nM ANV-FITC. The samples were incubated at r.t. for 30 min and aliquots were aspirated into a FACSCalibur (Becton-Dikinson) for flow cytometric analysis (excitation 488 nm, emission filter 530±30 nm).</p><!><p>A 1:200 dilution of the ghosts was mixed with 1–256 nM FITC-labeled ANV derivatives at 2-fold increasing concentrations in 1.5-ml microfuge tubes containing 0.4 ml of TBSA buffer supplemented with 1.25 mM, 2.5 mM or 5 mM CaCl2. A separate set of microfuge tubes contained 0.4 ml of the same mixtures but were supplemented with 50 μl of 0.5 M EDTA to prevent the binding of FITC-labeled proteins to the erythrocyte ghosts. After incubation for 40 min at r.t., all the reaction mixtures were centrifuged at 13,000 rpm for 10 min to separate the free- and the cell-bound FITC-labeled proteins. Duplicate samples of supernatants (160 μl from mixtures with Ca++ and 180 μl from mixtures with EDTA) were transferred into a black 96-well plate. The wells with 160 μl supernatants were added 20 μl of 0.5 M EDTA so that all the sample wells had the same buffer composition. Fluorescence measurements were carried out using a fmax microplate reader (Molecular Device, Sunnyvale, CA) with excitation/emission at 485/538 nm. Fluorescence was read 4 times and the average values were used for calculating the concentrations of free- and bound- FITC-labeled ligands.</p><p>The binding reaction between the proteins and the membrane binding sites was analyzed according to the following model: eq. (1)Protein+membrane↔Protein∗MembraneKd=[Protein][Membrane]/[Protein∗Membrane] where Kd was the equilibrium constant of the dissociation reaction.</p><p>Equation (1) could be transformed into equation (2)</p><p> eq. (2)[Protein]bound={Bmax×[Protein]free}/(Kd+[Protein]free) where [Protein]bound was the concentration of protein bound to the membrane, [Protein]free was the concentration of free protein, and Bmax was the maximum concentration of membrane-bound protein at saturation. Non-linear least-squares analysis using the Solver function of Microsoft Excel [32] was performed to determine the fit of equation (2).</p><!><p>The distribution of fluorescence after incubation of erythrocyte ghosts with ANV-FITC or ANV—6L15-FITC was determenied as follows. Erythrocyte ghosts (7.1×106 cells/ml in TBSA-1.25mM CaCl2) were centrifuged at 13,000 rpm for 10 min. Aliquot of the supernatant (380 μl) was mixed with 20 μl of 40 nM ANV-FITC or ANV-6L15-FITC so that the final concentration of ANV-FITC was 2 nM. Duplicate samples (160 μl) of the supernatant were each mixed with 20 μl of 0.5 M EDTA for fluorescence reading. The reading was taken as 100% fluorescence in solution phase control. ANV-FITC or ANV-6L15-FITC (2 nM) and erythrocyte ghosts (7.1×106 cells/ml) in 400 μl TBSA-1.25 mM CaCl2 was mixed with 50 μl of 0.5 M EDTA, incubated for 40 min at r.t., and centrifuged at 13,000 rpm for 10 min. Duplicate samples (180 μl) of the supernatant were taken for fluorescence reading. The reading was taken as the fluorescence remained in the solution phase after incubation in the presence of EDTA. A mixutre of ANV-FITC or ANV-6L15-FITC (2 nM) and erythrocyte ghosts (7.1×106 cells/ml) in 400 μl of TBSA-1.25 mM CaCl2 was incubated at r.t. for 40 min (c), 80 min (d), 4 h (e), and 22 h (f), respectively. The reaction mixtures were centrifuged as above and duplicate samples (160 μl) of the supernatant were each mixed with 20 μl of 0.5 M EDTA for fluorescence reading. The reading was taken as the unbound flourescence. Each cell pellet was re-suspended in 400 μl TBSA plus 50μl 0.5 M EDTA, incubated and centrifuged as above. Duplicate samples (180 μl) of the supernatant were taken for fluorescence reading. The reading was taken as the flourescence in the EDTA-extracted phase. The flourecence in the EDTA-resistant membrane phase was calulated by sustracting the flourescence in solution phase and flourescence in EDTA extracted phase from the 100% fluorescence in solution phase control.</p><!><p>The binding reaction between calcium ions, ANV derivatives, and membrane binding sites was analyzed according to the model described by Tait et al. [23].</p><p> eq. (3)nCa+Protein+Membrane↔Protein∗Membrane∗CanK=[Ca]n[Protein][Membrane]/[Protein∗Membrane∗Can] where K was the equilibrium constant of the dissociation reaction.</p><p>When the concentrations of free and bound protein were equal, and Equation (3) reduced to</p><p> eq. (4)K=EC50n[Membrane] eq. (5)pK=−logK=−(nlogEC50+log[Membrane]) where EC50 is the free calcium concentration at which half of the protein was bound to the membrane and [Membrane] was the concentration of membrane binding sites [m]. [m] could be estimated by measuring the amount of ANV derivatives bound at saturating concentration of Ca++ (15 mM). If the reaction were highly cooperative with respect to calcium, the Hill coefficient (N) would be nearly the same as the calcium binding stoichiometry n. The binding parmeters EC50 and N could be determined by fitting the calcium titration data to the following equation, which could be derived from Equations (3) and (4): eq. (6)B/Bmax=[Ca]N/([Ca]N+EC50N) where B is the amount of protein bound at a given calcium concentration, Bmax is the amount of protein bound at saturating calcium concentrations. Fits were performed by non-linear least-squares analysis using the Solver function of Microsoft Excel [32]. The apparent dissociation constant at a fixed calcium concentration, Kapp, can be calculated from the following equation: Kapp=K/[Ca++]N.</p><p>Calcium titration was carried out as follows. Stock solutions of TBSA, TBSA-8 mM CaCl2, TBSA-40 nM ANV- FITC or TBSA-40 nM ANV-6L15-FITC, and TBSA-erythrocyte ghosts were mixed in appropriate proportions in sixteen 1.5-ml microfuge tubes such that each reaction mixture contained 0.4 ml TBSA, 2 nM FITC-proteins, 7.1 × 106 cells/ml erythrocyte ghosts, and varied concentrations of CaCl2 (0–3 mM). Under this reaction condition, less than 2.1 % of membrane binding sites [m] were occupied throughout titration. A separate set of microfuge tubes contained 0.4 ml of the same mixtures but were supplemented with 50 μl of 0.5 M EDTA to prevent the binding of FITC-labeled proteins to erythrocyte ghosts. After incubation for 40 min at r.t., all the reaction mixtures were centrifuged at 13,000 rpm for 10 min to separate the free- and the cell-bound FITC-labeled proteins. Duplicate samples of supernatants (160 μl from mixtures with Ca++ and 180 μl from mixtures with EDTA) were transferred into a black 96-well plate. The wells with 160 μl supernatants were added 20 μl of 0.5 M EDTA so that all the sample wells had the same buffer composition. Fluorescence measurements were carried out using a SpectraMax® M5 microplate reader (Molecular Device, Sunnyvale, CA) with excitation/emission/cutoff at 485/538/530 nm). Fluorescence was read 4 times and the average values were used for calculating the B/Bmax values according to the following equation: B/Bmax=(FE−FCa)/(FE−Fm) where FE was the fluorescence intensity remained in the supernatant of EDTA-supplemented reaction mixtures; FCa was the fluorescence intensity remained in the supernatants of reaction mixtures with varying concentration of Ca++; and Fm was the fluorescence intensity remained in the supernatants of reaction mixtures that showed maximal binding. Under the above experimental condition, maximal binding was reached at 1.5–3 mM Ca++ for both ANV-FITC and ANV-6L15-FITC.</p><!><p>A preservative-treated human blood, 4C Plus Cell Control (Beckman-Coulter) was proposed for routine study of ANV-Ca++-membrane binding [23]. We compared the binding of ANV-FITC to 4C Plus Control, 5C Control (Beckman-Coulter), and erythrocyte ghosts prepared from preservative-treated blood and freshly collected ACD blood using fluorescence confocal microscopy. Fig. 1(A) showed that relatively few blood cells in un-expired 4C Plus Control were fluorescently labeled by ANV-FITC, suggesting that only a small number of erythrocytes in the sample expressed PS. Fig. 1(B) showed that a larger percentage of erythrocytes were fluorescently labeled by ANV-FITC after 5C Control had been stored at 4 °C for 6 months. Fig. 1(C) showed that erythrocyte ghosts prepared from 5C Control were fluorescently labeled by ANV-FITC more intensely than those in Fig. (A) and (B). However, many fluorescently labeled microparticles, possibly derived from fragmented cells, were present. Fig. 1(D) showed that virtually all the erythrocyte ghosts prepared from fresh ACD blood were fluorescently labeled by ANV-FITC, and very few cell fragments were present. Flow cytometric analysis of various preparations of blood cells and ghosts was also carried out. Fig. 2(A) showed that 5C Control that had been aged 11 months had a normal FSC-H/SSC-H plot (erythrocytes in R1; lymphocytes in R2; neutrophils in R3) [panel a], and had moderate cell-associated fluorescence in 2.5 mM Ca++ [panel b] and low cell-associated fluorescence in 1.25 mM Ca++ [panel c]. Fig. 2(B) showed that cell ghosts prepared from 5C Control that had been aged 1 month had large amount of cell fragments in FSC-H/SSC-H plot [panel a], and had high cell-associated fluorescence in 2.5 mM Ca++ [panel b] and moderate cell-associated fluorescence in 1.25 mM Ca++ [panel c]. Fig. 2(C) showed that cell ghosts prepared from 5C Control that had been aged 11 month had even larger amount of cell fragments in FSC-H/SSC-H plot [panel a], and had moderate cell-associated fluorescence in 2.5 mM Ca++ [panel b] and very low cell-associated fluorescence in 1.25 mM Ca++ [panel c]. Fig. 2(D) showed that ghosts prepared from fresh ACD blood had very few cell fragments in FSC-H/SSC-H plot [panel a], and had higher cell-associated fluorescence [panels b and c] than those in Fig. 2(A), (B), and (C). Thus, results from fluorescence confocal microscopy and flow cytometry analysis together indicated that erythrocyte ghosts prepared from fresh ACD blood expressed PS to higher levels and were less heterogeneous in size and binding of ANV-FITC compared with preserved blood cells and ghosts prepared from them. Therefore, erythrocyte membranes prepared from fresh blood appeared better suited for ANV binding studies.</p><!><p>FITC-labeled proteins were incubated with erythrocyte ghosts at varying ratios in TBSA-15 mM Ca++ to determine the concentration of membrane binding sites on the erythrocyte ghosts Following incubation and centrifugation, the amount of labeled proteins bound to erythrocyte ghosts was calculated from the decrease of fluorescence in the supernatants compared with the control. Fig. 3 showed the concentration of bound ANV-FITC (A) and ANV-6L15-FITC (B) as a function of protein/cell ratio. The protein/cell ratio corresponding to 100% occupancy of the binding sites on erythrocyte ghosts was determined from the ratio at which the ascending line intersected with the horizontal line. Saturation was reached at ratios of approximately 7.1 ×106 and 8.5 × 106 molecules/cell, respectively for ANV-FITC (A) and ANV-6L15-FITC (B). The average [m] values of the erythrocyte ghosts (7.1 × 106 cells/ml) were determined to be 76 ± 7 nM and 91 ± 9 nM, respectively for ANV-FITC and ANV-6L15-FITC (n = 7).</p><!><p>ANV-FITC or ANV-6L15-FITC (2 nM) was incubated with erythrocyte ghosts (7.1×106 cells/ml) in TBSA buffer containing 1.25 mM CaCl2 in the presence and absence of excess EDTA for 40 min at r.t. followed by EDTA extraction of the cell pellets. The percentages of unbound FITC-labeled proteins in the solution phase, the EDTA-extracted phase, and the EDTA-resistant membrane phase were calculated. Fig. 4(A) showed the percentage distribution of ANV-FITC in various phases. Column (a) was 100% fluorescence in solution phase control obtained by adding ANV-FITC to the cell-free, ANV-FITC-free supernatant of the mixture. Column (b) was the percentage distribution of fluorescence after incubation of ANV-FITC with erythrocyte ghosts in the presence of excess EDTA. Note that pre-addition of EDTA in the reaction mixture completely prevented the binding of ANV-FITC to erythrocyte ghosts since 100% of fluorescence was recovered in the solution phase as compared to that in column (a). Columns (c), (d), (e) and (f) were percentage distributions of fluorescence in solution phase, EDTA-extracted phase, and EDTA-resistant membrane phase after incubation of ANV-FITC and erythrocyte ghosts for 40 min, 80 min, 4 h, and 22 h, respectively, followed by EDTA extraction of the cell pellets. Note that significant fractions (20–36%) of the fluorescence initially added to the reaction mixtures remained bound to membranes (EDTA-resistant membrane phase) and were not released by EDTA treatment. The EDTA resistant fraction was larger when ANV-FITC was bound to the membranes for a prolonged period (4 h and 22 h). As shown in Fig. 4(B), similar results were obtained when the experiment was carried out using ANV-6L15-FITC instead of ANV-FITC. Thus, these experiments clearly indicated: a) Ca++-mediated the binding of ANV and ANV-6L15 to erythrocyte ghosts; b) an excess of EDTA over Ca++ completely prevented the binding of these proteins to the erythrocyte ghosts; and c) once membrane-bound in the presence of Ca++, ANV and ANV-6L15 were only partially released by EDTA treatment.</p><!><p>Erythrocyte ghosts (1:200 dilution of the preparation) was incubated with increasing concentrations of FITC-labeled ANV or ANV-6L15 in TBSA containing 1.25 mM, 2.5 mM, or 5 mM CaCl2. After incubation, the concentrations of free- and membrane bound- labeled proteins were quantitated by fluorescence measurement as described in Material and Methods. Figure 5(A) showed that the binding of ANV-FITC to the erythrocyte ghosts reached increasing saturation levels with increasing Ca++ concentration. The full-range saturation titration data, however, poorly fit the equilibrium binding equation, [Protein]bound = {Bmax × [Protein]free}/(Kd + [Protein]free), derived for simple binding reaction between a protein and a membrane. Inset to Fig. 5(A) showed that nonlinear least-squares regression analysis using the first 6 titration points produced a reasonable fit (R2 = 0.991, Bmax=177 nM, Kd=32 nM) of the equation (solid and dotted lines). This predicted a much higher saturation level than the actual saturation data points (solid triangles). The result suggested negative cooperativity of ANV-FITC binding to the membrane when the membrane binding sites were increasingly occupied. Negative cooperativity of binding at high protein density on membranes had been demonstrated for protein kinase C and other Ca++-dependent phospholipid-binding proteins [33]. These proteins induced clustering of acidic phospholipids in membranes and reduced the membrane's ability to bind later-binding proteins [25]. The results of saturation titration study suggested a sequential binding model described previously in which the affinity of proteins for membranes progressively decreased with increasing occupancy of membrane binding sites [33]. Therefore, the classical saturation titration method could not be used for determination of the equilibrium binding constant with precision. Fig. 5(B) showed that the binding of ANV-6L15-FITC to the erythrocyte ghosts also reached increasing saturation levels with increasing Ca++ concentration. At each Ca++ concentration, however, saturation level was higher for ANV-6L15-FITC than ANV-FITC [compare Fig. 5(A) and (B)]. Similarly, saturation titration data for ANV-6L15-FITC did not fit the equilibrium binding equation.</p><!><p>Titration of the binding of ANV-FITC or A6L15-FITC to erythrocyte ghosts with increasing concentration of Ca++ was carried out as described in Methods. The experimental titration data were fit to the binding equation B/Bmax = [Ca]N/( [Ca]N + EC50N) by nonlinear least-squares regression analysis [32] to determine the binding parameters, EC50 (the [Ca++] at which half of the protein is bound to the membranes), N (Hill coefficient), and R2 (correlation coefficient). Fig. 6 showed excellent non-linear least-squares fits for the binding of ANV-FITC (A) and ANV-6L15-FITC (B) to the erythrocyte ghosts. Solid lines were the fits of the experimental data points (solid circles). Dotted lines were the 95% confidence intervals around the fits. Table 1 summarized the binding parameters for ANV-FITC and ANV-6L15-FITC. Note that the Hill coefficient (N) was approximately 4 for both ANV-FITC and ANV-6L15-FITC, suggesting that about 4 Ca++ ions participated in the binding of each ANV-FITC or ANV-6L15-FITC molecule to the membranes. The mean values of N and EC50 from six calcium titrations were used for calculation of the dissociation constants (Kd, pK) and apparent dissociation constants (Kapp) at 1.2 mM and 2.5 mM Ca++ concentrations. The Kapp values at 1.2 mM and 2.5 mM Ca++ were approximately 4-fold lower for ANV-6L15-FITC compared to those for ANV-FITC, suggesting that ANV-6L15 had higher binding affinity for the erythrocyte ghosts than ANV.</p><!><p>ANV has been extensively used for detection of PS-expression on the membrane surfaces of cells such as activated platelets and leukocytes, abnormal red cells, and a variety of cells undergoing programmed cell death. Extent of PS expression is cell type-dependent and varies with time and experimental conditions. This makes it difficult to precisely define the binding parameters and to compare the Ca++-dependent binding of ANV derivatives to different cell membranes. Tait et al. proposed to use aged 4C Plus Cell Control (Beckman-Coulter) for routine comparative study of the binding of ANV derivatives [23]. However, we found that only a small fraction of red blood cells in the unexpired preserved blood bound ANV-FITC and the expression of PS on erythrocytes varied over time on prolonged storage. These irregularities render the assay ineffective for side-by-side and longitudinal comparisons of the binding properties of ANV derivatives. Erythrocyte ghosts prepared from preserved blood expressed increased amount of PS but such preparations contained numerous cell fragments. In contrast, erythrocyte ghosts prepared from fresh ACD blood described here offered a number of advantages compared with other membrane systems: (a) Most of the erythrocyte membranes bound ANV-FITC and the preparation contained fewer cell fragments; (b) PS expression on the erythrocyte ghosts was highly stable and could be used for calcium titration experiments over weeks with consistent results; (c) The erythrocyte ghosts were roughly the same size (diameter ~6 μm) as the erythrocytes in normal blood. Simple microcentrifugation afforded easy separation and quantification of free- and membrane bound- ligands; and (d) Erythrocyte ghosts might simulate membranes of pathological cells more closely than artificial phospholipids vesicles. We propose that erythrocyte ghosts prepared by the procedure outlined in this study may be used as a reference material for the study of PS exposure on stored, sickle and thalassemia red blood cells, and for routine quantitative assessment of the affinities of phospholipid-binding proteins to biological membranes.</p><p>Calcium-dependent binding to PS-expressing membranes is a characteristic property of annexins. It has been widely assumed that EDTA chelation of Ca++ completely released the membrane-bound ANV. The experiments summarized in Fig. 4 contradicted such assumption. ANV-FITC and ANV-6L15-FITC bound to erythrocyte ghosts in the presence of 1.2 mM Ca++ were not completely released into solution phase after treatment with EDTA. This suggested the existence of two pools of bound proteins - an EDTA-releasable pool; and an EDTA-resistant pool. The percentage of the bound proteins released by EDTA decreased when the proteins were allow to stay membrane-bound for longer periods, suggesting that there was a transition of part of the proteins from the EDTA-releasable to the EDTA-resistant pool. However, when the proteins were incubated with erythrocyte ghosts in the presence of EDTA, the binding of the proteins to the membranes was prevented completely. To account for the partial release of ANV derivatives by EDTA treatment, we proposed a two-step binding model: Initial binding of the proteins to the PS-exposed membranes was dependent on the formation of protein-calcium-phosphate chelates; this was followed by increased interactions between the proteins and the phospholipid bilayer, the latter being resistant to dissociation by EDTA. This model would be consistent with a previous isothermal microcalorimetry study [34] in which protein-calcium-phosphate chelates was found to accounted for about 70 % of the free energy of binding while dehydration of the hydrophobic region of the protein surface as they entered the interfacial region contributed to the rest of overall binding energy. Hydrophobic interaction might contribute to the EDTA resistance observed in the present study.</p><p>In previous studies, the affinity constant of ANV-membrane interaction was mostly determined by saturation titration method in which a fixed concentration of membranes was titrated with increasing concentrations of ANV at a specific Ca++ concentration [16,18–22]. Our study showed that saturation titration data poorly fit the equilibrium binding equation and suggested heterogeneity of binding events over the full range of titration (Fig. 5). Tait et el. developed the original calcium titration method in which ANV derivatives and preservative-treated erythrocytes were titrated with increasing concentrations of Ca++ such that the membrane binding site occupancy was 1–3% throughout the titration [23]. In theory, this would minimize the heterogeneity of binding events and allow measurement of the parameters of a single binding equilibrium. However, this method involved washing cells with buffers followed by releasing cell-bound ANV derivatives by treatment with EDTA. Cell washing could significantly disturb the binding equilibrium, and EDTA was found to release membrane-bound ANV derivatives incompletely (Figure 4). Thus, Tait's original calcium titration method might not yield true equilibrium binding parameters. In our modified calcium titration assay, no cell washing was involved and cell-bound ANV derivatives was directly derived from the difference between the total and the free without EDTA release step. This approach allowed equilibrium to be maintained throughout the assay and more accurate equilibrium binding parameters might be obtained. Our modified calcium titration method yielded Hill coefficients (N) of 3.9 ± 0.3 and 3.8 ± 0.3, respectively, for the binding of ANV-FITC and ANV-6L15-FITC to erythrocyte ghosts (Table 1). This results correlated well with the structure of the core domains of ANV which were composed of a 4-fold repeat of conserved amino acid sequence, with each repeat containing a type II Ca++ binding site that was thought to mediate Ca++-dependent binding to the membrane by a 'Ca++-bridge' mechanism [35]. In contrast, Tait et al reported N ~8 for the binding of ANV to 4C Plus Cell Control [23]. Furthermore, the Kd value for the binding of ANV to erythrocyte ghosts as determined by our modified method was 3.4 × 10−20 (Table 1), in contrast to 10−30 reported by Tait et al.[23]. The large discrepancies in binding parameters may be accounted for by methodological differences of the assays.</p><p>The number of Ca++ involved in the binding of annexins to membranes had been extensively investigated by others before, yet the reported stoichiometries had differed widely ranging from 3 to 12 [35,36 and references therein]. Based on data from crystal structures, 45Ca++ copelleting assay, and isothermal titration calorimetry study, Patel et al. proposed a molecular model to account for up to 12 Ca++-binding sites on ANV and annexin XII that mediated the binding to vesicles containing 2:1 PS-PC [35]. In this model, the footprint of ANV monomer would cover approximately 26 phospholipids on the monolayer. A high-affinity type II Ca++ binding site and two "low affinity" carboxylate side chains on each of the 4-fold repeat of annexin core domain were postulated to form "Ca++-bridges" with the phospholipids in a complementary manner, so 12 of the 26 phospholipids were anchored to the protein via Ca++ bridges. However, since the PS content of human erythrocyte was only approximately 15% of total phospholipids [37], the erythrocyte ghost membranes might not be able to form 12 Ca++-bridges under each ANV monomer. In stead, 3.9 Ca++-bridges (26 phospholipids × 15 %) per ANV monomer could be involved, assuming random distribution of phospholipids across the bilayer of the ghost membranes. Thus, our experimentally determined Hill coefficient (N) of approximately 4 for the binding to erythrocyte ghosts was consistent with Patel's model, albeit with lower Ca++ stoichiometry because of much lower PS content compared with 2:1 PS-PC vesicles. According to the equilibrium binding equation (3), N value affected the dissociation constant exponentially. Decreases of Ca++ concentration and N value could translate into greatly diminished binding affinity. PS expressions on the membrane surface of pathological cells were generally in the range of 0–15% of total phospholipids. In contrast, vesicles containing 20–100% PS were commonly used for study of ANV-Ca++-phospholipid interactions [17,33,36,38]. This might be a major reason for the large discrepancies of reported binding parameters. ANV has been used for detection of PS expression during platelet activation, cell senescence, pathological changes, and apoptosis. Thrombin-treated platelets and cells in early stages of apoptosis have been shown to express low levels of PS which was poorly detected by ANV at physiological concentration of Ca++, but was easily detected by the Ca++-independent PS binding protein, lacadherin [39]. Thus, the sensitivity of detecting PS-expressing cells by ANV was critically dependent on the exoplasmic PS content and the extracellular Ca++ concentration.</p><p>The affinity of ANV binding to erythrocyte ghosts declines sharply over a narrow range of Ca++ concentration with Kapp increased 15-fold from 4.78 × 10−10 M to 7.15 × 10−9 M when the ionized Ca++ decreased from 2.5 mM to 1.2 mM. In vitro imaging of ANV binding is usually made at 2.5 mM Ca++. Under such condition, the binding signal can be detected using 10–100 nM concentrations of labeled ANV without difficulties. For in vivo imaging applications, suboptimal detection sensitivity and low signal-to- background ratio have been important problems that continue to hamper the progress of ANV imaging [15.16]. ANV binding occurs at a Ca++ concentration of 1.2 mM (the typical ionized Ca++ in circulating plasma) under in vivo conditions in which the binding affinity is not very high. Furthermore, pathological cells may express lower levels of PS in vivo. It is also possible that PS-exposed sites may also be partly occupied by endogenous ANV in circulating blood [40] and released locally from apoptotic, injured, and activated cells. Thus, a relatively high blood concentration of labeled ANV may be needed to allow sensitive detection of the target cells. This in turn may compromise the signal-to-background ratio. ANV-6L15, an ANV derivative with a 4-fold lower Kapp at physiological concentration of Ca++, may displace endogenous ANV from the PS sites more effectively and afford detection of PS-exposed cells with greater sensitivity in in vivo imaging applications. This will be the subject of a future study.</p><p>The mechanism for the increased binding affinity of ANV-6L15 for erythrocyte membranes compared to ANV is currently unknown. Calcium titration study indicated that it was not due to increased Ca++-bridging since the Hill coefficient for the binding of ANV-6L15 was almost identical to that of ANV. ANV-6L15 was previously found to require much lower concentration of Ca++ than ANV to bind to homogenized E. coli membranes. PS is virtually absent in E. coli, whereas PE constitutes about 69 % of the total phospholipids [41]. Therefore, the PE co-expressed on the erythrocyte membranes might contribute importantly to the increased affinity of ANV-6L15 compared to ANV.</p><!><p>Preserved blood cells and ghosts were treated with 50 nM ANV-FITC in HBSA-2.5 mM CaCl2 and examined under a Olympus confocal laser scanning microscope (FV1000) using a 20× objective lens and excitation/emission filters 488/510–560 nm as described in Materials and Methods. (A) 4C Plus Control; (B) 5C Control, aged 6 months at 4°C; (C) Ghosts prepared from 5C Control that had been aged 1 month at 4° after expiration date; and (D) Ghosts prepared from fresh ACD blood. The scale was 30-μm.</p><!><p>5C Control and blood cell ghosts were diluted to concentrations of about 5 ×106 cells/ml in HBSA buffer containing 50 nM ANV-FITC and 2.5 mM CaCl2 or 1.25 mM CaCl2. The samples were incubated at r.t. for 30 min and aliquots were aspirated into a FACSCalibur (Becton-Dikinson) for flow cytometric analysis (excitation 488 nm, emission filter 530±30 nm). (A) 5C Control cells aged for 11 months at 4°C; (B) Erythrocyte ghosts prepared from 5C Control that had been aged for 1 month at 4°C; (C) Erythrocyte ghosts prepared from 5C Control that had been aged for 11 month at 4°C; and (D) Erythrocyte ghosts prepared from fresh ACD blood. Panel a, forward scatter (FSC-H) vs. side scatter (SSC-H) plot. Panels b and c, histograms of cells incubated with 50 nM ANV-FITC in HBSA-2.5 mM CaCl2, and HBSA-1.25 mM CaCl2, respectively.</p><!><p>ANV-FITC or ANV-6L15-FITC (10 nM) and erythrocyte ghosts (8.88 × 104–1.14 × 107 cells/ml) were mixed in 0.4 ml of TBSA supplemented with 15 mM CaCl2 to provide the indicated protein/cell ratio. The reaction mixtures were incubated at r.t. for 40 min. Following centrifugation at 13,000 rpm for 5 min, duplication samples (160 μl) of the supernatants were mixed with 20 μl of 0.5 M EDTA for fluorescence reading. No-binding controls were prepared by mixing 50 μl of 0.5 M EDTA with 0.4 ml of the above reaction mixtures, incubated and centrifuged as above. Duplicate samples (180 μl) of the supernatants were taken for fluorescence reading. The concentrations of the membrane-bound FITC-proteins were calculated by the following equation: [(FT − FS)/FT] × 10 nM, where FT is the relative fluorescence units of the no-binding control; FS is the relative fluorescence units of the supernatant of the reaction mixture. (A) Concentration of bound ANV-FITC as a function of ANV:cell ratio. (B) Concentration of bound ANV-6L15-FITC as a function of ANV-6L15:cell ratio. The protein-cell ratios corresponding to 100 % saturation of binding sites as determined from the intersecting lines were approximately 7.1 × 106 ANV-FITC molecules/cell (A), and 8.5 × 106 ANV-6L15-FITC molecules/cell (B), respectively.</p><!><p>(A) Distribution of fluorescence after incubation of erythrocyte ghosts with ANV-FITC and extraction of cell pellets with EDTA. Column (a), 100% fluorescence in solution phase control. Erythrocyte ghosts (7.1×106 cells/ml in TBSA-1.25mM CaCl2) were centrifuged at 13,000 rpm for 10 min. Aliquot of the supernatant (380 μl) was mixed with 20 μl of 40 nM ANV-FITC so that the final concentration of ANV-FITC was 2 nM. Duplicate samples (160 μl) of the supernatant were each mixed with 20 μl of 0.5 M EDTA for fluorescence reading. Column (b), distribution of fluorescence after incubation of erythrocyte ghosts with ANV-FITC in the presence of excess EDTA. ANV-FITC (2 nM) and erythrocyte ghosts (7.1×106 cells/ml) in 400 μl TBSA-1.25 mM CaCl2 was mixed with 50 μl of 0.5 M EDTA, incubated for 40 min at r.t., and centrifuged at 13,000 rpm for 10 min. Duplicate samples (180 μl) of the supernatant were taken for fluorescence reading. Columns (c), (d), (e) and (f) were distributions of fluorescence in solution phase (gray), EDTA-extracted phase (dotted), and EDTA-resistant membrane phase (open) after incubation of ANV-FITC (2 nM) and erythrocyte ghosts (7.1×106 cells/ml) in 400 μl of TBSA-1.25 mM CaCl2 at r.t. for 40 min (c), 80 min (d), 4 h (e), and 22 h (f), respectively, followed by EDTA extraction of the cell pellets. The reaction mixtures were centrifuged as above and duplicate samples (160 μl) of the supernatant were each mixed with 20 μl of 0.5 M EDTA for fluorescence reading. Each cell pellet was re-suspended in 400 μl TBSA plus 50 μl 0.5 M EDTA, incubated and centrifuged as above. Duplicate samples (180 μl) of the supernatant were taken for fluorescence reading. (B) Distribution of fluorescence after incubation of erythrocyte ghosts with ANV-6L15-FITC followed by extraction of cell pellets with EDTA. The experimental protocols were identical to (A).</p><!><p>Saturation binding was carried out as described in Materials and Methods. Erythrocyte ghosts (1:200 dilution of the ghost preparation) were mixed with 1-256 nM ANV-FITC (A) or ANV-6L15-FITC (B) at 2-fold increasing concentrations in TBSA buffer supplemented with 1.25 mM (●), 2.5 mM (■) or 5 mM (▲) CaCl2. A separate set of tubes containing the same reaction mixtures were supplemented with EDTA to prevent the binding of FITC-labeled proteins to the erythrocyte ghosts as controls. After incubation for 40 min at r.t., all the reaction mixtures were centrifuged at 13,000 rpm for 10 min to separate the free- and the cell-bound FITC-labeled proteins. Supernatants were taken for measurement of florescence with excitation/emission wavelengths at 485 nm/538 nm. The concentrations of the free- and membrane bound- proteins were then calculated from the fluorescence reading. Inset to Fig. 5(A): Non-linear least-squares regression analysis of the binding of ANV-FITC to erythrocyte ghosts in TBSA buffer supplemented with 5 mM CaCl2 using the first 6 titration data points (0–32 nM ANV-FITC) to fit the saturation binding equation, [Protein]bound = {Bmax × [Protein]free}/(Kd + [Protein]free). Solid line was the fit based on the first 6 experimental data points. Dotted lines were the 95% confidence intervals around the fit. The correlation coefficient of the fit based on the 6 data points was R2=0.991 with Bmax=177 nM and Kd=32 nM.</p><!><p>Calcium titration was carried out as described in Materials and Methods. The binding parameters, EC50 (the [Ca++] at which half of the protein is bound to the membrane), N (Hill coefficient), and R2 (correlation coefficient) were determined by fitting the experimental calcium titration data to the following equation: B/Bmax = [Ca]N/([Ca]N + EC50N). Fits were performed by nonlinear least-squares analysis with the Solver function of Microsoft Excel33. Solid lines were the fits of the experimental data points (solid circles). Dotted lines were the 95% confidence intervals around the fits. (A) Calcium titration of the binding of ANV-FITC to erythrocyte ghosts. The following parameters were obtained from this titration: N = 3.954; EC50 = 0.704; and R2 = 0.995. (B) Calcium titration of the binding of ANV-6L15-FITC to erythrocyte ghosts. The following parameters were obtained from this titration: N = 4.157; EC50 = 0.441; and R2 = 0.997.</p><!><p>Parameters for the binding of ANV-FITC (A) and ANV-6L15-FITC (B) to erythrocyte ghosts.</p><p>N, EC50, and R2 were mean ± SD (n=6) from calcium titrations performed over a 4-week period using the same preparation of erythrocyte ghosts stored at 4°.</p><p>Kd, pK, and Kapp were calculated values based on the mean values of N and EC50 determined by calcium titration using the following equations: Kd = (EC50)N × [m]; pK = − log K = − (N log EC50 + log [m]); and Kapp = Kd/[Ca]N</p>

PubMed Author Manuscript

Direct C-C Coupling of Ethylene and \xce\xb1-Olefins with Diols, Ketols or Hydroxy Esters via Osmium(0) Catalyzed Transfer Hydrogenation

Osmium(0) complexes derived from Os3(CO)12 and XPhos (2-dicyclohexylphosphino-2\xe2\x80\xb2,4\xe2\x80\xb2,6\xe2\x80\xb2-triisopropylbiphenyl) catalyze the C-C coupling of \xce\xb1-hydroxy esters 1a-1i, \xce\xb1-ketols 1j-1o or 1,2-diols dihydro-1j-1o with ethylene 2a to form ethylated tertiary alcohols 3a-3o. As illustrated in couplings of 1-octene 2b with vicinally dioxygenated reactants 1a, 1b, 1i, 1j, 1k, 1m, higher \xce\xb1-olefins are converted to adducts 4a, 4b, 4i, 4j, 4k, 4m with complete levels of branched regioselectivity. Oxidation level independent C-C coupling is demonstrated by the reaction of 1-octene 2b with diol dihydro-1k, \xce\xb1-ketol 1k and dione dehydro-1k. Functionalized olefins 2c-2f react with ethyl mandelate 1a to furnish adducts 5a-8a as single regioisomers. The collective data, including deuterium labeling studies, are consistent with a catalytic mechanism involving olefin-dione oxidative coupling to form an oxa-osmacyclopentane, which upon reductive cleavage via hydrogen transfer from the secondary alcohol reactant releases the product of carbinol C-alkylation with regeneration of the ketone. Single crystal X-ray diffraction data of the dinuclear complex Os2(CO)4(O2CR)2(XPhos)2 and the trinuclear complex Os3(CO)11(XPhos) are reported. These studies suggest increased \xcf\x80-backbonding at the stage of the metal-olefin \xcf\x80-complex plays a critical role in facilitating alkene-carbonyl oxidative coupling, as isostructural ruthenium(0) complexes, which are weaker \xcf\x80-donors, do not catalyze the transformations reported herein.

direct_c-c_coupling_of_ethylene_and_\xce\xb1-olefins_with_diols,_ketols_or_hydroxy_esters_via_osmium

Introduction<!>Research Design and Methods<!>Mechanism<!>Conclusions<!>General Information<!>General Procedure A<!>General Procedure B<!>Ethyl 2-hydroxy-2-(4-(methylthio)phenyl)acetate (1g)<!>Ethyl 2-hydroxy-2-phenylbutanoate (3a).24a<!>Ethyl 2-(4-bromophenyl)-2-hydroxybutanoate (3b)<!>Ethyl 2-hydroxy-2-(4-methoxyphenyl)butanoate (3c)<!>Ethyl 2-hydroxy-2-(4-(trifluoromethyl)phenyl)butanoate (3d)<!>Ethyl 2-hydroxy-2-(3-(trifluoromethyl)phenyl)butanoate (3e)<!>Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-2-hydroxybutanoate (3f)<!>Ethyl 2-hydroxy-2-(4-(methylthio)phenyl)butanoate (3g)<!>Ethyl 2-(furan-2-yl)-2-hydroxybutanoate (3h)<!>Ethyl 2-hydroxy-2-(thiophen-2-yl)butanoate (3i)<!>2-Ethyl-2-hydroxy-2,3-dihydro-1H-inden-1-one (3j)<!>2-Ethyl-2-hydroxyacenaphthylen-1(2H)-one (3k)<!>2-Ethyl-2-hydroxy-3,4-dihydronaphthalen-1(2H)-one (3l).24b<!>3-Ethyl-3-hydroxychroman-4-one (3m)<!>3-Ethyl-3-hydroxy-2,2-dimethylchroman-4-one (3n)<!>2-Ethyl-2-hydroxycyclohexan-1-one (3o).24c<!>Ethyl 2-hydroxy-3-methyl-2-phenylnonanoate (4a)<!>Ethyl 2-(4-bromophenyl)-2-hydroxy-3-methylnonanoate (4b)<!>Ethyl 2-hydroxy-3-methyl-2-(thiophen-2-yl)nonanoate (4i)<!>2-Hydroxy-2-(octan-2-yl)-2,3-dihydro-1H-inden-1-one (4j)<!>2-Hydroxy-2-(octan-2-yl)acenaphthylen-1(2H)-one (4k)<!>3-Hydroxy-3-(octan-2-yl)chroman-4-one (4m)<!>Ethyl 2-hydroxy-3-methyl-2,4-diphenylbutanoate (5a)<!>Ethyl 2-hydroxy-2-phenyl-3-(pivaloyloxy)butanoate (6a)<!>Ethyl 2-hydroxy-2-phenyl-2-(tetrahydrofuran-2-yl)acetate (7a)<!>Ethyl 4-acetoxy-2-hydroxy-3-methyl-2-phenylbutanoate (8a)<!>2-(4-bromophenyl)-2-hydroxy-3-methylnonyl 4-bromobenzenesulfonate (4b Derivative)<!>2-hydroxyacenaphtylen-1(2H)-one-2-d (deuterio-1k)<!>2-(ethyl-1,2-d2)-2-hydroxyacenaphthylen-1(2H)-one (deuterio-3k)

<p>α-Olefins are the most abundant petrochemical feedstock beyond alkanes.1 Despite their ubiquity and low cost, the use of α-olefins in the commercial manufacture of commodity chemicals is largely restricted to polymerization,2 hydroformylation3 and alkene metathesis.4 The discovery of alternate classes of byproduct-free catalytic C-C couplings that convert α-olefins to value-added products remains an important yet elusive goal. For example, while intermolecular alkene hydroacylation is attractive, decarbonylation of acylmetal intermediates to form inactive metal carbonyl complexes mandates use of esoteric reactants with chelating groups.5,6 Similarly, intermolecular Prins or carbonyl ene reactions do not extend to the coupling of α-olefins with unactivated aldehydes.7a–c Finally, whereas nickel(0) catalyzes the coupling of α-olefins with simple aldehydes, superstoichiometric quantities of TESOTf and Et3N are required.7d</p><p>In connection with the development of C-C bond forming hydrogenations and transfer hydrogenations beyond hydroformylation,8 we recently found that zerovalent ruthenium complexes generated in situ from Ru3(CO)12 and various phosphine ligands catalyze the C-C coupling of vicinally dioxygenated hydrocarbons (1,2-diols, α-ketols, α-hydroxy esters) with diverse π-unsaturated reactants, including dienes,9a–d acrylates9e and alkynes.9g,h,j As in related ruthenium(0) catalyzed Pauson-Khand reactions of vicinal dicarbonyl compounds described by Chatani and Murai,10 these processes are initiated through C=C/C=O oxidative coupling to furnish oxaruthenacycles. Catalytic turnover is achieved via transfer hydrogenolysis of the metalacycle by the alcohol reactant to release product and regenerate the carbonyl partner (Figure 1, top). Based on this mechanism, a ruthenium(0) catalyzed coupling of α-olefins was developed (Figure 1, middle).9f This process, however, was restricted to the use of 3-hydroxy-2-oxindoles, which may be attributed to the exceptional reactivity of the transient isatins. In continuing efforts to broaden the scope of transfer hydrogenative α-olefin coupling, we now demonstrate that osmium(0) catalysts overcome this limitation, enabling the direct C-C coupling of ethylene11 and higher α-olefins with diverse diols, α-ketols and α-hydroxy esters (Figure 1, bottom).</p><!><p>The limitations evident in ruthenium(0) catalyzed C-C couplings of α-olefins9f were believed to stem from a high energetic barrier to oxidative coupling. Guided by Hoffmann's theoretical analysis of the conversion of metal bisolefin complexes to metalacyclopentanes,12 and a large body of experimental evidence,13a the facility of oxidative coupling should be influenced by the degree of backbonding in the preceding metal-olefin π-complex.14 Backbonding confers nucleophilic character to the bound olefin and, in the limiting case, may be viewed as an oxidative addition to the C=C π-bond to form a metalacyclopropane. The Kulinkovich reaction,15 wherein titanium(II)-olefin complexes behave as vicinal dianions, represents a dramatic illustration of this effect. Hence, it was posited that a more strongly reducing metal center should facilitate C=C/C=O oxidative coupling to broaden substrate scope in transfer hydrogenative C-C couplings of α-olefins.</p><p>As borne out by the carbonyl stretching frequencies of isostructural ruthenium and osmium complexes HClM(CO)(PPh3)3, M = Os, νco = 1906 cm−1; M = Ru, νco = 1922 cm−1, osmium is a stronger π-donor than ruthenium.16 Indeed, osmium(0) catalysts are effective in couplings of activated secondary alcohols with vinyl acetates in cases where ruthenium(0) catalysts are not.9i For this reason, osmium(0) complexes were assayed in the coupling of racemic ethyl mandelate 1a with ethylene 2a with the goal of generating the ethylated tertiary alcohol 3a (Scheme 1). It was found that monodentate or bidentate triaryl phosphine ligands were ineffective. However, the osmium(0) catalyst modified by PCy3 (tricyclohexylphosphine) provided the desired adduct in 57% yield. Given this promising result, the osmium(0) complex modified by XPhos was eventually identified as the optimal catalyst, delivering the product of carbinol C-H ethylation 3a in 78% yield. Notably, under all conditions evaluated, the corresponding ruthenium(0) catalysts were unable to promote formation of adduct 3a.</p><p>Under these optimal conditions, aryl- and heteroaryl-substituted α-hydroxy esters 1a-1i were coupled to ethylene 2a to form products of carbinol C-H ethylation 3a-3i (Table 1). As illustrated by the conversion of ethyl 4-bromomandelate 1b to adduct 3b, the osmium(0) catalyst is tolerant of aryl halide functional groups. The transformation of ethyl 4-methoxymandelate 1c and ethyl 4-(trifluoromethyl)mandelate 1d to adducts 3c and 3d, respectively, highlight tolerance of electron rich and as well as electron deficient aryl groups. Substituents at the meta-position of the aryl ring are tolerated, as shown in the formation of 3e and 3f, respectively. Finally, sulfur containing α-hydroxy ester 1g and heteroaromatic α-hydroxy esters 1h and 1i groups are converted to adducts 3g, 3h and 3i, respectively. ortho-Substituted mandelates and alkyl-substituted α-hydroxy esters such as ethyl lactate, were inefficient partners for C-C coupling under these conditions.</p><p>α-Hydroxy esters 1a-1i react by way of transient α-ketoesters for which the vicinal dicarbonyl moieties are electronically differentiated. In corresponding reactions of non-symmetric α-ketols, the vicinal dicarbonyl intermediates are quite similar electronically, rendering the control of regioselectivity uncertain. In the event, application of optimal conditions to the coupling of ethylene 2a with α-ketols 1j-1o delivered the ethylated tertiary alcohols 3j-3o in good to excellent yield (Table 2). Further, in the coupling of α-ketols 1j, 1l-1n, which proceed by way of nonsymmetric diones, the adducts 3j, 3l-3n form as single regioisomers. In addition to the influence of electronic effects on the regioselectivity of oxidative coupling as described by Hoffman12 and in prior work from our laboratory,9e steric effects also play an important role. That is, oxidative coupling will occur such that the osmium center is placed distal to the site of greatest steric demand. α-Ketol 1n is an exception due to the electronic effect associated with the mesomeric effect of the ortho-oxygen atom.</p><p>The coupling of ethylene 2a with α-hydroxy esters 1a-1i or α-ketols 1j-1o to form adducts 3a-3o are redox-neutral transformations. In contrast, the reaction of ethylene 2a with 1,2-diols dihydro-1j-1o represent oxidative processes in which one equivalent of H2 is evolved or transferred to an acceptor (Table 3). The feasibility of such an oxidative process finds precedent in the work of Shvo, who demonstrates that zero-valent ruthenium catalysts derived from Ru3(CO)12 promote oxidative esterifications in which tolane (diphenyl acetylene) serves as H2-acceptor,17 as well as work from our laboratory on oxidative diol-diene [4+2] cycloadditions.9c Initial attempts at the coupling of ethylene 2a with 1,2-diols dihydro-1j-1o using the osmium(0) catalyzed modified by XPhos led to only modest yields of adducts 3j-3o. Given the ability of carboxylic acids to catalyze the hydrogenolysis13 and transfer hydrogenolysis9e of oxametalacycles, these reactions were conducted in the presence of adamantane carboxylic acid (10 mol%). To our delight, the yields of adducts 3j-3o improved considerably and, as observed in couplings conducted from the α-ketol oxidative level, compounds 3j, 3l-3n were again generated as single regioisomers.</p><p>To evaluate the applicability of these conditions to higher α-olefins, the coupling of 1-octene 2b with α-hydroxy esters 1a, 1b and 1i and α-ketols 1j, 1k and 1m was attempted (Table 4). Although corresponding reactions of ethylene 2a proceed efficiently in the absence of a carboxylic acid cocatalyst, couplings of 1-octene 2b required the presence of adamantane carboxylic acid (10 mol%) to increase conversion. Additionally, higher concentrations were beneficial, so the reactions were conducted neat. The α-hydroxy esters 1a, 1b and 1i were converted to adducts 4a, 4b and 4i, respectively, with complete levels of branched regioselectivity and good levels of diastereoselectivity. Relative stereochemistry for adducts 4a, 4b and 4i was determined by single crystal X-ray diffraction analysis of a derivative of 4b. A stereochemical model is provided (Scheme 2). α-Ketols 1j, 1k and 1m were converted to adducts 4j, 4k and 4m in a completely regioselective fashion, but with diminished levels of diastereoselectivity.</p><p>The present transfer hydrogenative couplings of α-olefins can be conducted in oxidative, redox-neutral or reductive modes. While redox-neutral couplings are most efficient, oxidative and reductive transformations are preparatively useful. The following transformations illustrate this unique capability (eq. 1–3). In the oxidative coupling of 1-octene 2b with diol dihydro-1k, wherein 1-octene serves as hydrogen acceptor, adduct 4k forms in 70% yield (eq. 1). The redox-neutral coupling of 1-octene 2b with α-ketol 1k proceeds in 95% yield (eq. 2). Finally, using 1,4-butanediol as terminal reductant,18 the reductive coupling of 1-octene 2b with the 1,2-dione dehydro-1k proceeds in 68% yield (eq. 3). Such redox-economy allows one to bypass discrete manipulations otherwise required for the adjustment of oxidation level.19</p><p>To determine the scope of the alkene partner, the coupling of olefins 2a-2f with ethyl mandelate 1a was explored (Table 5). Beyond the previously described couplings of ethylene 2a and 1-octene 2b to form adducts 3a and 4a, respectively, allyl benzene 2c participates in C-C coupling to form tertiary alcohol 5a. For carboxy- and alkoxy-substituted alkenes 2d and 2e, the indicated regioisomers 6a and 7a are formed exclusively. Here, omission of XPhos and adamantane carboxylic acid is required to suppress metalacycle fragmentation en route to products of vinyl transfer (not shown).9i Finally, allyl acetate 2f participates in C-C coupling to form adduct 8a with complete levels of branched regioselectivity. This result is remarkable in view of the fact that ionization of allyl acetate 2f to form π-allyl species in the presence zero-valent osmium does not override the transfer hydrogenative C-C coupling pathway.</p><!><p>With regard to the catalytic mechanism, a simple working model has been proposed as a basis for further refinement (Scheme 2). It is unclear whether the catalyst is mononuclear versus dimetallic or trimetallic. Upon heating toluene solutions of Os3(CO)12 with XPhos in the presence and absence of adamantane carboxylic acid (RCO2H), crystals of the dinuclear complex Os2(CO)4(O2CR)2(XPhos)2 and the trinuclear complex Os3(CO)11(XPhos), respectively, were isolated and characterized by X-ray diffraction (Figure 2). Additionally, the reaction of Os3(CO)12 with 2-(dicyclohexylphosphino)-1-(2-methoxyphenyl)-imidazole, a monophosphine that is structurally related to XPhos, provides the trinuclear osmium complex, Os3(CO)8L2.20b Alternatively, intervention of a mononuclear catalyst finds support in the reaction of Ru3(CO)12 with dppe, bis-(diphenylphosphino)ethane, to provide Ru(CO)3(dppe),20a and the reaction of Ru3(CO)12, 1-adamantanecarboxylic acid and dppp, bis-(diphenylphosphino)propane, to form the catalytically active mononuclear complex Ru(CO)(dppp)(O2CR)2.9e Oxidative coupling of the α-oxoester, dehydro-1a, with 1-octene 2b mediated by zero-valent osmium delivers the oxaosmacycle I.9,10 Related ruthenium(0)-mediated carbonyldiene oxidative couplings deliver isolable metalacycles that are catalytic active and have been shown to form in a reversible manner.9d The oxoester, dehydro-1a, required in the first turnover of the catalytic cycle may be generated via alcohol-olefin hydrogen transfer.17 Direct protonation of oxaosmacycle I by ethyl mandelate 1a to form the osmium alkoxide III requires a 4-centered transition structure and is postulated to be slow compared to protonation of oxaosmacycle I by 1-adamantanecarboxylic acid to form the osmium carboxylate II, which can proceed by way of a 6-centered transition structure.13 Exchange of the carboxylate ligand with 1a to form osmium alkoxide III also may proceed by way of a 6-centered transition structure.13 β-Hydride elimination converts osmium alkoxide III to the osmium alkyl hydride complex IV, which upon C-H reductive elimination releases product 4a and regenerates the osmium(0) catalyst. Beyond the aforesaid electronic effects,9e,12 steric interactions between the n-hexyl side chain of 1-octene 2b and the crowded osmium center contribute to branched regioselectivity.</p><p>To challenge the veracity of the proposed mechanism, the following isotopic labelling experiment was performed (eq. 4). The deuterated acenaphthylene ketol deuterio-1k was exposed to ethylene under standard conditions. The pattern of deuterium incorporation in the adduct deuterio-3k was established by 1H and 2H NMR, as well as HRMS analysis. Deuterium incorporated occurs exclusively at the methyl group (22% 2H). The transfer of deuterium from the carbinol position of deuterio-1k to the methyl group of deuterio-3k is consistent with the proposed mechanism (Scheme 2). The relatively low levels of deuterium incorporation may be attributed to exchange with adventitious water or with the hydroxylic proton deuterio-1k.21</p><!><p>In summary, the ability to transform abundant hydrocarbon feedstocks to value-added products in the absence of stoichiometric byproducts is a characteristic shared by nearly all large volume chemical processes. Hence, the discovery and development of byproduct-free transformations applicable to ethylene and α-olefins represents an important objective. Toward this end, we have shown that osmium(0) complexes derived from Os3(CO)12 and XPhos catalyze the transfer hydrogenative C-C coupling of ethylene and higher α-olefins with diverse vicinally dioxygenated hydrocarbons. Coupling may be conducted in a redox-neutral mode using α-ketols or α-hydroxy esters as reactants, or in oxidative or reductive modes using 1,2-diols or 1,2-diones as reactants, respectively. The collective data suggest increased π-backbonding at the stage of the osmium(0)-olefin π-complex plays a critical role in facilitating alkene-carbonyl oxidative coupling, as does the use of transient vicinal dicarbonyl partners, which have relatively low-lying LUMO energies. A challenge associated with the design of transfer hydrogenative coupling of α-olefins with simple primary alcohols will reside in the identification of metal catalysts that are sufficiently electron rich so as to promote oxidative coupling, and whose low-valent forms are accessible through alcohol mediated reduction of the high-valent ions. Indeed, intermolecular catalytic reductive couplings of α-olefins with unactivated carbonyl compounds remain an unmet challenge in chemical research.22</p><!><p>All reactions were run under an atmosphere of argon. Os3(CO)12, XPhos, 1-adamantanecarboxylic acid, alkenes 2a-2f, α-hydroxy ester 1a, α-ketol 1o, diol dihydro-1o, and dione dehydro-1k were purchased from commercial suppliers and used as received. α-Hydroxy esters 1b-1i23a were prepared in accordance with the literature procedure. α-Ketols 1j,23b 1k23c 1l,23b 1m,23b 1n23d and diols dihydro-1j,23e dihydro- 1k,23f dihydro-1l,23b 1m23g and dihydro-1n23g were prepared using the cited literature procedures. Pressure tubes were flame dried followed by cooling in a desiccator. Toluene was dried over sodium metal-benzophenone and was distilled immediately prior to use. Anhydrous solvents were transferred by oven-dried syringes. Analytical thin-layer chromatography (TLC) was carried out using 0.25 mm commercial silica gel plates. Infrared spectra were recorded on a Perkin-Elmer 1600 spectrometer. High-resolution mass spectra (HRMS) are reported as m/z (relative intensity). Accurate masses are reported for the molecular ion (M+H, M+Na) or a suitable fragment ion. 1H Nuclear magnetic resonance spectra were recorded using a 400 MHz spectrometer. Coupling constants are reported in Hertz (Hz) for CDCl3 solutions, and chemical shifts are reported as parts per million (ppm) relative to residual CHCl3 δH (7.26 ppm). 13C Nuclear magnetic resonance spectra were recorded using a 100 MHz spectrometer for CDCl3 solutions, and chemical shifts are reported as parts per million (ppm) relative to residual CDCl3 δC (77.16 ppm).</p><!><p>A resealable pressure tube (15 × 100 mm, 13 mL or 15 × 125 mm, 16 mL) was charged with Os3(CO)12 (5.5 mg, 0.006 mmol, 2 mol%), XPhos (17.1 mg, 0.036 mmol, 12 mol%) and the reactant alcohol (0.30 mmol, 100 mol%). The tube was sealed with a rubber septum and purged with ethylene. Toluene (0.15 mL, 2.0 M) was added and the rubber septum was quickly replaced with a screw cap. The reaction was allowed to stir at the indicated temperature for the stated period of time. After cooling to room temperature, the mixture was evaporated under reduced pressure and the residue was subjected to flash column chromatography (SiO2) under the conditions noted to afford the indicated product.</p><!><p>A resealable pressure tube (13 × 100 mm, 9 mL) was charged with Os3(CO)12 (3.7 mg, 0.004 mmol, 2 mol%), XPhos (11.4 mg, 0.024 mmol, 12 mol%), AdCO2H (3.6 mg, 0.02 mmol, 10 mol%) and the reactant alcohol (0.20 mmol, 100 mol%). The tube was sealed with a rubber septum and purged with argon. 1-Octene (112.2 mg, 1.0 mmol, 500 mol%) was added via syringe and the rubber septum was quickly replaced with a screw cap. The reaction was allowed to stir at the indicated temperature for the stated period of time. After cooling to room temperature, the mixture was evaporated under reduced pressure and the residue was subjected to flash column chromatography (SiO2) under the conditions noted to afford the indicated product.</p><!><p>To a flame-dried 50 mL round-bottom flask charged with ethyl 2-hydroxy-2-(4-(methylthio)phenyl)acetate (1.1 g, 4.9 mmol), was added ethanol (25 mL, 0.2 M). NaBH4 (200 mg, 5.3 mmol) was added portionwise. The reaction mixture was allowed to stir at ambient temperature until the suspension became colorless. Distilled water was added and the reaction mixture was allowed to stir until bubbling stopped. The mixture was extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were washed with brine (1 × 50 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated under reduced pressure. The residue was subjected to column chromatography (SiO2: 20% ethyl acetate in hexanes) to give the title compound (0.93g, 4.1 mmol) in 84% yield as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.34 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 5.11 (d, J = 8.0 Hz 1H), 4.22 (m, 2H), 3.42 (d, J = 8.0 Hz, 1H), 2.48 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 173.6, 138.9, 135.2, 127.0, 126.5, 72.5, 62.3, 15.7, 14.0; HRMS (ESI-MS) Calcd. for C11H14O3S [M+Na]+: 249.0556, Found: 249.0557; FTIR (neat): 3438, 2979, 1726; MP: 91 °C.</p><!><p>In accordance with general procedure A, 1a (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 2–5% ether/hexanes) provided the title compound (48.7 mg, 0.23 mmol) as a yellow oil in 78% yield. 1H NMR (400 MHz, CDCl3): δ 7.62–7.59 (m, 2H), 7.37–7.32 (m, 2H), 7.30–7.26 (m, 1H), 4.32–4.16 (m, 2H), 3.78 (d, J = 0.4 Hz, 1H), 2.24 (dqd, J = 14.4, 7.2, 0.8 Hz, 1H), 2.07–1.98 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 175.5, 142.0, 128.3, 127.7, 125.7, 78.7, 62.5, 32.8, 14.2, 8.2; HRMS (ESI) Calcd. for C12H16O3, [M+Na]+: 231.0992, Found: 231.0998; FTIR (neat): 3504, 2980, 1721.</p><!><p>In accordance with Procedure A, 1b (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 2–4% ether/hexanes) provided the title compound (63.7 mg, 0.22 mmol) as a yellow oil in 74% yield. 1H NMR (400 MHz, CDCl3): δ 7.51–7.44 (m, 4H), 4.32–4.15 (m, 2H), 3.80 (d, J = 0.5 Hz, 1H), 2.24–2.12 (m, 1H), 1.97 (dq, J = 14.7, 7.4 Hz, 1H), 1.27 (t, J = 7.1 Hz, 3H), 0.99 (dd, J = 9.5, 5.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 175.0, 141.0, 131.4, 127.7, 121.9, 78.4, 62.8, 32.9, 14.2, 8.1; HRMS (ESI) Calcd. for C12H15BrO3, [M+Na]+: 309.0097, 311.0077, Found: 309.0104, 311.0085; FTIR (neat): 3499, 2980, 1723.</p><!><p>In accordance with Procedure A, 1c (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 50–100% dichloromethane/hexanes to 5% ethyl acetate/hexanes) provided the title compound (43.6 mg, 0.18 mmol) as a yellow oil in 61% yield. 1H NMR (400 MHz, CDCl3): δ 7.54–7.48 (m, 2H), 6.90–6.85 (m, 2H), 4.31–4.14 (m, 2H), 3.80 (s, 3H), 3.73 (s, 1H), 2.26–2.15 (m, 1H), 1.99 (dq, J = 14.6, 7.4 Hz, 1H), 1.27 (t, J = 7.1 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 175.7, 159.1, 134.2, 126.9, 113.6, 78.4, 62.5, 55.4, 32.8, 14.3, 8.2; HRMS (ESI) Calcd. for C13H18O4, [M+Na]+: 261.1097, Found: 261.1099; FTIR (neat): 3511, 2970, 1721.</p><!><p>In accordance with Procedure A, 1d (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 1–5% ether/hexanes) provided the title compound (63.8 mg, 0.23 mmol) as a yellow oil in 77% yield. 1H NMR (400 MHz, CDCl3): δ 7.79–7.72 (m, 2H), 7.64–7.57 (m, 2H), 4.35–4.17 (m, 2H), 3.87 (s, 1H), 2.23 (dq, J = 14.5, 7.2 Hz, 1H), 2.01 (dq, J = 14.5, 7.4 Hz, 1H), 1.28 (t, J = 6.2 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 174.8, 145.9, 130.0 (q, J = 32.0 Hz), 126.3, 125.2 (q, J = 4.0 Hz), 124.3 (q, J = 271.0 Hz), 78.6, 63.0, 33.1, 14.2, 8.0; 19F NMR (376 MHz, CDCl3): δ -62.6; HRMS (ESI) Calcd. for C13H15F3O3, [M+Na]+: 299.0866, Found: 299.0871; FTIR (neat): 3510, 2985, 1726.</p><!><p>In accordance with Procedure A, 1e (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 2–5% ether/hexanes) provided the title compound (63.0 mg, 0.23 mmol) as a yellow oil in 76% yield. 1H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H), 7.86–7.79 (m, 1H), 7.55 (dd, J = 7.7, 0.6 Hz, 1H), 7.46 (dd, J = 7.8, 7.8 Hz, 1H), 4.34–4.19 (m, 2H), 3.90 (d, J = 0.5 Hz, 1H), 2.29–2.18 (m, 1H), 2.01 (dq, J = 14.7, 7.4 Hz, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 174.9, 143.1, 130.7 (q, J = 32.0 Hz), 129.3, 128.8, 124.6 (q, J = 3.7 Hz), 124.3 (q, J = 271.0 Hz), 122.9 (q, J = 4.0 Hz), 78.5, 63.0, 33.2, 14.2, 8.1; 19F NMR (376 MHz, CDCl3): δ -62.6; HRMS (ESI) Calcd. for C13H15F3O3, [M+Na]+: 299.0866, Found: 299.0873; FTIR (neat): 3513, 2985, 1725.</p><!><p>In accordance with Procedure A, 1f (0.2 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 290 mol%) in toluene (2.0 M) at 140 °C for a 40 hour period. Flash column chromatography (SiO2: 3–5% ether/hexanes) provided the title compound (30.8 mg, 0.12 mmol) as a colorless oil in 61% yield. NOTE: Os3(CO)12 (3.6 mg, 0.004 mmol, 2 mol%) and XPhos (11.4 mg, 0.024 mmol, 12 mol%). 1H NMR (400 MHz, CDCl3): δ 7.13–7.05 (m, 2H), 6.77 (dd, J = 7.5, 1.1 Hz, 1H), 6.01–5.92 (m, 2H), 4.33–4.14 (m, H), 3.75 (s, 1H), 2.16 (dq, J = 14.4, 7.2 Hz, 1H), 1.96 (dq, J = 14.7, 7.4 Hz, 1H), 1.27 (t, J = 7.2 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 175.5, 147.7, 147.1, 136.1, 119.1, 107.9, 106.7, 101.2, 78.5, 62.6, 32.9, 14.3, 8.1; HRMS (ESI) Calcd. for C13H16O5, [M+Na]+: 275.0890, Found: 275.0899; FTIR (neat): 3507, 2971, 1722.</p><!><p>In accordance with Procedure A, 1g (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 2–5% ether/hexanes) provided the title compound (30.8 mg, 0.18 mmol) as a yellow oil in 61% yield. 1H NMR (400 MHz, CDCl3): δ 7.57–7.48 (m, 2H), 7.26–7.20 (m, 2H), 4.37–4.10 (m, 2H), 3.76 (s, 1H), 2.48 (s, 3H), 2.25–2.15 (m, 1H), 2.04-1.95 (m, 1H), 1.28 (t, J = 7.1 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 175.2, 138.7, 137.8, 126.2, 126.1 78.3, 62.4, 32.6, 15.7, 14.1, 7.9; HRMS (ESI) Calcd. for C13H18O3S, [M+Na]+: 277.0869, Found: 277.0878; FTIR (neat): 3507, 2979, 1721.</p><!><p>In accordance with Procedure A, 1h (0.2 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 290 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 3–5% ether/hexanes) provided the title compound (25.0 mg, 0.13 mmol) as a yellow oil in 64% yield. 1H NMR (400 MHz, CDCl3): δ 7.36 (d, J = 1.1 Hz, 1H), 6.33 (s, 2H), 4.36–4.14 (m, 2H), 3.82 (s, 1H), 2.21–2.07 (m, 2H), 1.25 (t, J = 7.0 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 173.7, 154.5, 142.5, 110.4, 106.8, 75.5, 62.8, 29.8, 14.3, 7.7; HRMS (ESI) Calcd. for C10H14O4, [M+Na]+: 221.0784, Found: 221.0790; FTIR (neat): 3511, 2970, 1728.</p><!><p>In accordance with Procedure A, 1i (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 30–50% dichloromethane/hexanes) provided the title compound (45.0 mg, 0.22 mmol) as a colorless oil in 70% yield. 1H NMR (400 MHz, CDCl3): δ 7.22 (dd, J = 5.1, 1.2 Hz, 1H), 7.09 (dd, J = 3.6, 1.2 Hz, 1H), 6.97 (dd, J = 5.1, 3.6 Hz, 1H), 4.35–4.21 (m, 2H), 4.05 (d, J = 0.8 Hz, 1H), 2.26–2.16 (m, 1H), 2.06 (dq, J = 14.7, 7.4 Hz, 1H), 1.31 (t, J = 7.1 Hz, 3H), 0.94 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 174.5, 147.1, 127.1, 124.9, 124.1, 77.7, 62.9, 34.4, 14.2, 8.1; HRMS (ESI) Calcd. for C10H14O3S, [M+Na]+: 237.0556, Found: 237.0563; FTIR (neat): 3499, 2979, 1724.</p><!><p>(Using ketol) In accordance with Procedure A, 1j (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 140 °C for a 40 hour period. Flash column chromatography (SiO2: 5–15% ethyl acetate/hexanes) provided the title compound (44.4 mg, 0.25 mmol) as a yellow oil in 84% yield. (Using diol) In accordance with Procedure A, H2-1j (0.15 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 390 mol%) in toluene (1.5 M) at 140 °C for a 48 hour period. Flash column chromatography (SiO2: 5–15% ethyl acetate/hexanes) provided the title compound (18.8 mg, 0.11 mmol) as a yellow oil in 71% yield. NOTE: Os3(CO)12 (2.7 mg, 0.003 mmol, 2 mol%), XPhos (8.5 mg, 0.018 mmol, 12 mol%). 1H NMR (400 MHz, CDCl3): δ 7.77–7.72 (m, 1H), 7.61 (ddd, J = 7.5, 7.5, 1.2 Hz, 1H), 7.45–7.41 (m, 1H), 7.40–7.34 (m, 1H), 3.27 (d, J = 17.0 Hz, 1H), 3.14 (d, J = 17.0 Hz, 1H), 2.89 (s, 1H), 1.81–1.64 (m, 2H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 208.4, 151.7, 135.9, 134.4, 127.9, 126.7, 124.7, 80.3, 39.8, 31.6, 8.0; HRMS (ESI) Calcd. for C11H12O2, [M+Na]+: 199.0730, Found: 199.0736; FTIR (neat): 3413, 2967, 1709.</p><!><p>(Using ketol) In accordance with Procedure A, 1k (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 48 hour period. Flash column chromatography (SiO2: 5–15% ethyl acetate/hexanes) provided the title compound (40.1 mg, 0.18 mmol) as a yellow solid in 63% yield. (Using diol) In accordance with Procedure A, H2-1k (0.15 mmol, 100 mol%) was reacted with ethylene (15 × 125 mm pressure tube, 0.71 mmol, 480 mol%) in toluene (1.5 M) at 140 °C for a 48 hour period. Flash column chromatography (SiO2: 5–15% ethyl acetate/hexanes) provided the title compound (21.3 mg, 0.11 mmol) as a yellow solid in 70% yield. NOTE: Os3(CO)12 (2.7 mg, 0.003 mmol, 2 mol%), XPhos (8.5 mg, 0.018 mmol, 12 mol%) and AdCO2H (2.7 mg, 0.015mmol, 10 mol%). 1H NMR (400 MHz, CDCl3): δ 8.13 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 7.0 Hz, 1H), 7.89 (dd, J = 7.9, 1.2 Hz, 1H), 7.74 (dd, J = 8.1, 7.1 Hz, 1H), 7.71–7.62 (m, 2H), 2.86 (s, 1H), 2.17–1.99 (m, 2H), 0.76 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3−): δ 206.2, 141.8, 139.5, 132.1, 131.2, 130.8, 128.9, 128.4, 125.4, 122.0, 120.5, 80.9, 31.6, 8.2; HRMS (ESI) Calcd. for C14H12O2, [M+Na]+: 235.0730, Found: 235.0737; FTIR (neat): 3369, 2970, 2931, 1716; MP: 92.7–93.1 °C</p><!><p>(Using ketol) In accordance with Procedure A, 1l (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 140 °C for a 40 hour period. Flash column chromatography (SiO2: 5–7% ethyl acetate/hexanes) provided the title compound (47.4 mg, 0.25 mmol) as a brown oil in 83% yield. NOTE: AdCO2H (5.4 mg, 0.03mmol, 10 mol%). (Using diol) In accordance with Procedure A, H2-1l (0.15 mmol, 100 mol%) was reacted with ethylene (15 × 125 mm pressure tube, 0.71 mmol, 480 mol%) in toluene (1.5 M) at 140 °C for a 48 hour period. Flash column chromatography (SiO2: 5–7% ethyl acetate/hexanes) provided the title compound (20.3 mg, 0.11 mmol) as a brown oil in 71% yield. NOTE: Os3(CO)12 (2.7 mg, 0.003 mmol, 2 mol%), XPhos (8.5 mg, 0.018 mmol, 12 mol%) and AdCO2H (2.7 mg, 0.015mmol, 10 mol%). 1H NMR (400 MHz, CDCl3): δ 8.01 (dd, J = 7.8, 1.2 Hz, 1H), 7.51 (ddd, J = 7.5, 7.5, 1.4 Hz, 1H), 7.33 (dd, J = 7.6, 7.6 Hz, 1H), 7.27–7.21 (m, 1H), 3.81 (s, 1H), 3.15–2.94 (m, 2H), 2.34 (ddd, J = 13.5, 5.1, 2.3 Hz, 1H), 2.21–2.10 (m, 1H), 1.78–1.60 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3−): δ 202.1, 143.6, 134.1, 130.4, 129.1, 128.0, 127.0, 75.9, 33.7, 28.5, 26.6, 7.3; HRMS (ESI) Calcd. for C12H14O2, [M+Na]+: 213.0886, Found: 213.0892; FTIR (neat): 3488, 2931, 1681.</p><!><p>(Using ketol) In accordance with Procedure A, 1m (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 48 hour period. Flash column chromatography (SiO2: 2–4% ethyl acetate/hexanes) provided the title compound (49.6 mg, 0.26 mmol) as a yellow oil in 86% yield. (Using diol) In accordance with Procedure A, H2-1m (0.15 mmol, 100 mol%) was reacted with ethylene (15 × 125 mm pressure tube, 0.71 mmol, 480 mol%) in toluene (1.5 M) at 140 °C for a 48 hour period. Flash column chromatography (SiO2: 2–4% ethyl acetate/hexanes) provided the title compound (17.3 mg, 0.09 mmol) as a yellow oil in 60% yield. NOTE: Os3(CO)12 (2.7 mg, 0.003 mmol, 2 mol%), XPhos (8.5 mg, 0.018 mmol, 12 mol%) and AdCO2H (2.7 mg, 0.015mmol, 10 mol%). 1H NMR (400 MHz, CDCl3): δ 7.89–7.85 (m, 1H), 7.51 (ddd, J = 8.4, 7.2, 1.8 Hz, 1H), 7.06 (ddd, J = 8.0, 7.2, 1.0 Hz, 1H), 6.97 (dd, J = 8.4, 0.6 Hz, 1H), 4.39 (d, J = 11.3 Hz, 1H), 4.16 (d, J = 11.3 Hz, 1H), 3.62 (s, 1H), 1.80 (q, J = 7.5 Hz, 2H), 0.94 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 196.9, 161.5, 136.7, 127.6, 121.9, 118.5, 118.0, 73.1, 72.9, 27.8, 7.0; HRMS (CI) Calcd. for C11H12O3, [M+H]+: 215.0679, Found: 215.0686; FTIR (neat): 3466, 2973, 2936, 1691, 1607.</p><!><p>(Using ketol) In accordance with Procedure A, 1n (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 140 °C for a 40 hour period. Flash column chromatography (SiO2: 2–4% ether/hexanes) provided the title compound (56.2 mg, 0.26 mmol) as a yellow oil in 85% yield. (Using diol) In accordance with Procedure A, H2-1n (0.15 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 390 mol%) in toluene (1.5 M) at 140 °C for a 48 hour period. Flash column chromatography (SiO2: 2–4% ethyl acetate/hexanes) provided the title compound (31.1 mg, 0.14 mmol) as a yellow oil in 94% yield. NOTE: Os3(CO)12 (2.7 mg, 0.003 mmol, 2 mol%), XPhos (8.5 mg, 0.018 mmol, 12 mol%). 1H NMR (400 MHz, CDCl3): δ 7.78 (dd, J = 7.8, 1.7 Hz, 1H), 7.48 (ddd, J = 8.6, 7.2, 1.8 Hz, 1H), 6.98 (dt, J = 12.0, 2.5 Hz, 1H), 6.89 (dd, J = 8.4, 0.5 Hz, 1H), 3.89 (s, 1H), 1.92–1.80 (m, 2H), 1.52 (s, 3H), 1.26 (s, 3H), 0.68 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 197.8, 159.3, 136.7, 126.9, 121.1, 118.7, 118.3, 84.6, 78.7, 25.3, 22.2, 20.4, 7.3; HRMS (ESI) Calcd. for C13H16O3, [M+Na]+: 243.0992, Found: 243.0993; FTIR (neat): 3484, 2976, 1690.</p><!><p>(Using ketol) In accordance with Procedure A, 1o (0.3 mmol, 100 mol%) was reacted with ethylene (15 × 100 mm pressure tube, 0.58 mmol, 190 mol%) in toluene (2.0 M) at 130 °C for a 48 hour period. Flash column chromatography (SiO2: 5–10% ether/hexanes) provided the title compound (31.1 mg, 0.22 mmol) as a colorless oil in 73% yield. (Using diol) In accordance with Procedure A, H2-1o (0.15 mmol, 100 mol%) was reacted with ethylene (15 × 125 mm pressure tube, 0.71 mmol, 480 mol%) in meistylene (2.0 M) at 150 °C for a 48 hour period. Flash column chromatography (SiO2: 5–10% ether/hexanes) provided the title compound (10.7 mg, 0.15 mmol) as a colorless oil in 50% yield. NOTE: Os3(CO)12 (4.1 mg, 0.0045 mmol, 3 mol%), XPhos (13.2 mg, 0.027 mmol, 18 mol%) and AdCO2H (4.1 mg, 0.023 mmol, 15 mol%). 1H NMR (400 MHz, CDCl3): δ 3.93 (s, 1H), 2.51–2.40 (m, 2H), 2.18 (ddd, J = 13.1, 5.8, 3.0 Hz, 1H), 2.14–2.03 (m, 1H), 1.91 (dq, J = 14.7, 7.4 Hz, 1H), 1.84–1.54 (m, 5H), 0.90–0.75 (m, 3H); 13C NMR (100 MHz, CDCl3): δ 214.6, 79.4, 40.7, 38.2, 30.2, 28.0, 22.9, 7.0; HRMS (ESI) Calcd. for C8H14O2, [M+Na]+: 142.0994, Found: 142.0994; FTIR (neat): 3485, 2938, 1707.</p><!><p>In accordance with Procedure B, 1a (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 2% ether/hexanes) provided the title compound (31.1 mg, 0.22 mmol, d.r. = 5:1) as a colorless oil in 62% yield. 1H NMR (400 MHz, CDCl3): δ (major) 7.67–7.62 (m, 2H), 7.37–7.30 (m, 2H), 7.29–7.24 (m, 1H), 4.33–4.13 (m, 2H), 3.68 (d, J = 0.6 Hz, 1H), 2.47–2.35 (m, 1H), 1.51–1.16 (m, 13H), 0.90 (dd, J = 8.9, 4.9 Hz, 3H), 0.68 (d, J = 6.8 Hz, 3H). (minor) 7.67–7.62 (m, 2H), 7.37–7.30 (m, 2H), 7.29–7.24 (m, 1H), 4.33–4.13 (m, 2H), 3.74 (d, J = 0.6 Hz, 1H), 2.47–2.35 (m, 1H), 1.51–1.16 (m, 13H), 0.97 (d, J = 6.6 Hz, 3H), 0.82 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (major) 176.0, 141.5, 128.1, 127.5, 126.1, 81.7, 62.5, 40.8, 31.9, 31.8, 29.5, 27.7, 22.8, 14.3, 14.2, 12.8. (minor) 175.9, 141.3, 128.1, 127.5, 126.2, 81.6, 62.6, 40.6, 31.9, 29.6, 29.3, 27.6, 22.7, 14.2, 14.1, 12.8; HRMS (ESI) Calcd. for C18H28O3, [M+Na]+: 315.1931, Found: 315.1940; FTIR (neat): 3514, 2928, 2857, 1721.</p><!><p>In accordance with Procedure B, 1b (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 20–35% dichloromethane/hexanes) provided the title compound (45.4 mg, 0.12 mmol, d.r. = 4:1) as a colorless oil in 61% yield. 1H NMR (400 MHz, CDCl3): δ (major) 7.54–7.50 (m, 2H), 7.48–7.43 (m, 2H), 4.33–4.14 (m, 2H), 3.67 (s, 1H), 2.38–2.28 (m, 1H), 1.48–0.97 (m, 13H), 0.89 (d, J = 6.9 Hz, 3H), 0.66 (d, J = 6.8 Hz, 3H). (minor) 7.54–7.50 (m, 2H), 7.48–7.43 (m, 2H), 4.33–4.14 (m, 2H), 3.73 (s, 1H), 2.38–2.28 (m, 1H), 1.48–0.97 (m, 13H), 0.95 (d, J = 6.6 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (major) 175.5, 140.6, 131.3, 128.1, 121.7, 81.5, 62.8, 40.9, 31.9, 31.7, 29.5, 27.6, 22.8, 14.3, 14.2, 12.8. (minor) 175.4, 140.6, 131.3, 128.1, 121.7, 81.4, 62.9, 40.7, 31.9, 29.6, 29.3, 27.6, 22.7, 14.2, 14.1, 12.8; HRMS (ESI) Calcd. for C18H27BrO3, [M+Na]+: 393.1036, Found: 393.1043; FTIR (neat): 3507, 2927, 2856, 1723.</p><!><p>In accordance with Procedure B, 1i (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 20–40% dichloromethane/hexanes) provided the title compound (47.8 mg, 0.16 mmol, d.r. = 5:1) as a colorless oil in 80% yield. 1H NMR (400 MHz, CDCl3): δ (major) 7.21 (dd, J = 5.2, 1.2 Hz, 1H), 7.09 (dd, J = 3.6, 1.2 Hz, 1H), 6.98 (dd, J = 5.2, 3.6 Hz, 1H), 4.38–4.19 (m, 2H), 3.95 (d, J = 0.5 Hz, 1H), 2.33–2.20 (m, 1H), 1.48–1.05 (m, 14H), 0.91–0.83 (m, 2H), 0.81 (d, J = 6.8 Hz, 3H). (minor) 7.22 (dd, J = 5.2, 1.2 Hz, 1H), 7.09 (dd, J = 3.6, 1.2 Hz, 1H), 6.99–6.97 (m, 1H), 4.38–4.19 (m, 2H), 4.00 (d, J = 0.5 Hz, 1H), 2.33–2.20 (m, 1H), 1.48–1.05 (m, 14H), 0.93 (d, J = 6.6 Hz, 3H), 0.91–0.83 (m, 2H); 13C NMR (100 MHz, CDCl3): δ (major) 175.0, 146.9, 127.1, 124.8, 124.3, 81.0, 62.8, 42.6, 31.9, 31.6, 29.5, 27.6, 22.8, 14.22, 14.15, 12.8. (minor) 174.9, 146.7, 127.1, 124.9, 124.4, 80.9, 62.9, 42.5, 31.9, 29.6, 29.4, 27.7, 22.7, 14.2, 14.0, 12.8; HRMS (ESI) Calcd. for C16H26O3S, [M+Na]+: 321.1495, Found: 321.1502; FTIR (neat): 3502, 2929, 2857, 1725.</p><!><p>In accordance with Procedure B, 1j (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 2–3% ether/hexanes) provided the title compound (44.3 mg, 0.17 mmol, d.r. = 1:1) as a colorless oil in 85% yield. 1H NMR (400 MHz, CDCl3): δ (A) 7.75 (d, J = 7.7 Hz, 1H), 7.61 (dd, J = 10.8, 4.1 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.37 (dd, J = 7.5, 7.5 Hz, 1H), 3.28 (d, J = 17.4 Hz, 1H), 2.98 (d, J = 17.4 Hz, 1H), 2.51 (s, 1H), 1.93–1.72 (m, 15H), 1.48–0.99 (m, 9.5H), 0.87 (t, J = 6.8 Hz, 3H), 0.67 (d, J = 6.9 Hz, 3H). (B) 7.75 (d, J = 7.7 Hz, 1H), 7.61 (dd, J = 10.8, 4.1 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.37 (dd, J = 7.5 Hz, 7.5 Hz, 1H), 3.28 (d, J = 17.4 Hz, 1H), 2.98 (d, J = 17.4 Hz, 1H), 2.53 (s, 1H), 1.93–1.72 (m, 1.5 H), 1.48–0.99 (m, 12.5H), 0.82 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (A) 209.1, 152.8, 135.8, 135.4, 127.8, 126.6, 124.5, 82.3, 40.6, 36.8, 32.0, 30.5, 29.6, 27.8, 22.8, 14.5, 13.5. (B) 209.0, 152.6, 135.8, 135.5, 127.8, 126.7, 124.5, 82.3, 40.6, 37.1, 31.9, 31.5, 29.4, 27.7, 22.7, 14.22, 14.16; HRMS (ESI) Calcd. for C17H24O2, [M+Na]+: 283.1669, Found: 283.1679; FTIR (neat): 3447, 2926, 1709.</p><!><p>(Using dihydro-1k) In accordance with Procedure B, dihydro-1k (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 5–7% ethyl acetate/hexanes) provided the title compound (41.5 mg, 0.14 mmol, d.r. = 2:1) as a light green solid in 70% yield. (Using 1k) In accordance with Procedure B, 1k (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 5–7% ethyl acetate/hexanes) provided the title compound (56.3 mg, 0.19 mmol, d.r. = 2:1) as a light green solid in 95% yield. (Using dehydro-1k) In accordance with Procedure B, dehydro-1k (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 5–7% ethyl acetate/hexanes) provided the title compound (40.3 mg, 0.14 mmol, d.r. = 2:1) as a light green solid in 68% yield. NOTE: The reaction was conducted in the presence of 1,3-butane diol (36.0 mg, 0.4 mmol, 200 mol%). 1H NMR (400 MHz, CDCl3): δ (major) 8.11 (dd, J = 8.1, 0.5 Hz, 1H), 7.92 (ddd, J = 4.0, 2.0, 2.0 Hz, 1H), 7.90–7.85 (m, 1H), 7.72 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 7.68–7.61 (m, 2H), 2.84 (d, J = 2.4 Hz, 1H), 2.27–1.94 (m, 1H), 1.49–0.96 (m, 10H), 0.91–0.83 (m, 3H), 0.58 (t, J = 6.2 Hz, 3H). (minor) 8.11 (dd, J = 8.1, 0.5 Hz, 1H), 7.92 (ddd, J = 4.0, 2.0, 2.0 Hz, 1H), 7.90–7.85 (m, 1H), 7.72 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 7.68–7.61 (m, 2H), 2.84 (d, J = 2.4 Hz, 1H), 2.27–1.94 (m, 1H), 1.49–0.96 (m, 13H), 0.79 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (major) 207.2, 142.5, 138.4, 132.01, 131.99, 130.7, 128.7, 128.3, 125.4, 121.7, 121.6, 83.1, 41.6, 32.0, 30.2, 29.6, 27.9, 22.8, 14.4, 14.2. (minor) 207.2, 142.4, 138.7, 132.0, 131.9, 130.8, 128.7, 128.3, 125.4, 121.6, 121.4, 82.9, 41.4, 31.8, 31.3, 29.2, 27.5, 22.6, 14.1, 13.3; HRMS (ESI) Calcd. for C20H24O2, [M+Na]+: 319.1669, Found: 319.1678; FTIR (neat): 3423, 2924, 1708; MP: 79.8–81.1 °C.</p><!><p>In accordance with Procedure B, 1m (0.2 mmol, 100 mol%) was reacted with 1-octene (13 × 100 mm pressure tube, 0.15 mL, 1.0 mmol, 500 mol%) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 2–3% ether/hexanes) provided the title compound (34.8 mg, 0.13 mmol, d.r. = 1:1) as a pale yellow solid in 63% yield. 1H NMR (400 MHz, CDCl3): δ (A) 7.84 (dd, J = 7.8, 1.7 Hz, 1H), 7.56–7.47 (m, 1H), 7.05 (ddd, J = 8.2, 1.9, 1.0 Hz, 1H), 6.96 (ddd, J = 8.4, 3.0, 0.6 Hz, 1H), 4.58 (dd, J = 21.0, 11.7 Hz, 1H), 4.06 (dd, J = 11.7, 5.6 Hz, 1H), 3.56 (s, 1H), 1.97–1.87 (m, 1H), 1.76–1.64 (m, 0.5H), 1.49–0.94 (m, 12.5H), 0.88 (dd, J = 8.4, 5.0 Hz, 3H). (B) 7.84 (dd, J = 7.8, 1.7 Hz, 1H), 7.56–7.47 (m, 1H), 7.05 (ddd, J = 8.2, 1.9, 1.0 Hz, 1H), 6.96 (ddd, J = 8.4, 3.0, 0.6 Hz, 1H), 4.58 (dd, J = 21.0, 11.7 Hz, 1H), 1.76–1.64 (m, 0.5H), 1.49–0.94 (m, 9.5H), 0.80 (t, J = 7.0 Hz, 3H), 0.74 (d, J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (A) 209.1, 152.8, 135.8, 135.4, 127.8, 126.6, 124.5, 82.3, 40.6, 36.8, 32.0, 31.5, 29.6, 27.7, 22.8, 14.5, 14.2. (B) 209.1, 152.6, 135.8, 135.4, 127.8, 126.7, 124.5, 82.3, 40.6, 37.1, 31.9, 30.5, 29.4, 27.7, 22.7, 14.2, 13.5; HRMS (ESI) Calcd. for C17H24O3, [M+Na]+: 299.1618, Found: 299.1623; FTIR (neat): 3453, 2927, 1684; MP: 67.8–68.0 °C.</p><!><p>In accordance with Procedure B, 1a (0.2 mmol, 100 mol%) was reacted with 2c (13 × 100 mm pressure tube, 0.13 mL, 1.0 mmol, 500 mol%) in toluene (2.0 M) at 130 °C for a 40 hour period. Flash column chromatography (SiO2: 30–60% dichloromethane/hexanes) provided the title compound (34.6 mg, 0.11 mmol, d.r. = 5:1) as colorless oil in 58% yield. NOTE: AdCO2H (30 mol%). 1H NMR (400 MHz, CDCl3): δ (major) 7.71–7.65 (m, 2H), 7.38–7.26 (m, 5H), 7.22 (d, J = 7.3 Hz, 3H), 4.33–4.15 (m, 2H), 3.84 (d, J = 0.5 Hz, 1H), 2.82–2.67 (m, 2H), 2.57 (dd, J = 13.4, 10.5 Hz 1H), 1.33 (t, J = 7.1 Hz, 3H), 0.62 (t, J = 6.8 Hz, 3H). (minor) 7.81–7.77 (m, 2H), 7.42 (dd, J = 10.5, 4.9 Hz, 2H), 7.38–7.26 (m, 2H), 7.18 (dd, J = 11.2, 4.3 Hz, 2H), 7.05 (d, J = 7.1 Hz, 2H), 4.33–4.15 (m, 2H), 3.87 (d, J = 0.6 Hz, 1H), 2.82–2.67 (m, 1H), 2.50 (d, J = 13.7 Hz, 1H), 2.21 (dd, J = 13.6 Hz, 11.6 Hz, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (major) 175.8, 141.3, 141.0, 129.4, 128.4, 128.3, 127.6, 126.1, 126.0, 81.3, 62.8, 43.2, 38.6, 14.3, 12.7. (minor) 175.5, 141.4, 141.1, 129.2, 128.4, 128.3, 127.8, 126.2, 125.9, 81.2, 62.8, 43.4, 36.4, 14.2, 13.6; HRMS (ESI) Calcd. for C19H22O3, [M+Na]+: 321.1461, Found: 321.1466; FTIR (neat): 3505, 2976, 2361, 2342, 1715.</p><!><p>In accordance with Procedure B, 1a (0.2 mmol, 100 mol%) was reacted with 2d (13 × 100 mm pressure tube, 0.09 mL, 0.6 mmol, 300 mol%) in toluene (2.0 M) at 130 °C for a 24 hour period. Flash column chromatography (SiO2: 2–4% ethyl acetate/hexanes) provided the title compound (51.8 mg, 0.25 mmol, d.r. = 1.5:1) as a pale yellow oil in 84% yield. NOTE: XPhos and AdCO2H was omitted. 1H NMR (400 MHz, CDCl3−): δ (major) 7.47–7.28 (m, 5H), 6.09 (q, J = 5.2 Hz, 1H), 5.19 (s, 1H), 4.25–4.06 (m, 2H), 1.53 (d, J = 5.2 Hz, 3H), 1.23–1.17 (m, 3H), 1.11 (s, 9H). (minor) 7.47–7.28 (m, 5H), 5.84 (q, J = 5.2 Hz, 1H), 5.14 (s, 1H), 4.25–4.06 (m, 2H), 1.44 (d, J = 5.3 Hz, 3H), 1.23–1.17 (m, 3H), 1.21 (s, 9H); 13C NMR (100 MHz, CDCl3): δ (major) 178.3. 170.6. 136.4. 128.7. 128.6. 127.3. 95.5. 79.0. 61.5. 38.9. 27.0. 20.9. 14.2. (minor) 178.3, 170.1, 136.0, 129.0, 128.8, 127.5, 95.0, 79.3, 61.4, 39.0, 27.1, 20.8, 14.1; HRMS (ESI) Calcd. for C17H24O5, [M+Na]+: 331.1516, Found: 331.1520; FTIR (neat): 2979, 1789, 1174.</p><!><p>In accordance with Procedure B, 1a (0.2 mmol, 100 mol%) was reacted with 2e (13 × 100 mm pressure tube, 0.05 mL, 0.6 mmol, 300 mol%) in toluene (2.0 M) at 140 °C for a 24 hour period. Flash column chromatography (SiO2: 2–5% ethyl acetate/hexanes) provided the title compound (39.0 mg, 0.23 mmol, d.r. = 1:1) as a pale yellow oil in 78% yield. NOTE: XPhos and AdCO2H was omitted. 1H NMR (400 MHz, CDCl3): δ (A) 7.44 (ddd, J = 7.8, 7.8, 1.3 Hz, 2H), 7.39–7.27 (m, 3H), 5.36 (d, J = 3.6 Hz, 1H), 5.26 (s, 1H), 4.25–4.06 (m, 2H), 4.01–3.94 (m, 1H), 3.89–3.83 (m, 1H), 2.19–1.80 (m, 4H), 1.21 (t, J = 7.1 Hz, 3H). (B) 7.44 (ddd, J = 7.8, 7.8, 1.3 Hz, 2H), 7.39–7.27 (m, 3H), 5.17 (d, J = 4.4 Hz, 1H), 5.09 (s, 1H), 4.25–4.06 (m, 2H), 3.89–3.83 (m, 1H), 3.81–3.77 (m, 1H), 2.19–1.80 (m, 4H), 1.21 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (A) 171.4, 137.2, 128.5, 128.4, 127.2, 102.7, 75.8, 67.5, 61.3, 32.6, 23.3, 14.2. (B) 171.4, 136.6, 128.64, 128.56, 127.4, 103.2, 77.4, 67.7, 61.1, 32.5, 23.6, 14.2; HRMS (ESI) Calcd. for C14H18O4, [M+Na]+: 273.1097, Found: 273.1108; FTIR (neat): 2981, 1747.</p><!><p>In accordance with Procedure B, 1a (0.2 mmol, 100 mol%) was reacted with 2f (13 × 100 mm pressure tube, 0.11 mL, 1.0 mmol, 500 mol%) in toluene (2.0 M) at 140 °C for a 40 hour period. Flash column chromatography (SiO2: 5–7% ethyl acetate/hexanes) provided the title compound (33.6 mg, 0.12 mmol, d.r. = 1.6:1) as a pale yellow oil in 60% yield. 1H NMR (400 MHz, CDCl3): δ (major) 7.62 (ddd, J = 3.4, 1.9, 1.9 Hz, 2H), 7.35 (ddd, J = 11.8, 4.6 Hz, 3H), 4.33–4.15 (m, 3H), 4.07 (dd, J = 11.0, 6.5 Hz, 1H), 3.80 (s, 1H), 2.96–2.86 (m, 1H), 2.04 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H), 0.70 (d, J = 6.9 Hz, 3H). (minor) 7.66 (ddd, J = 3.4, 1.9, 1.9 Hz, 2H), 7.31–7.25 (m, 3H), 4.33–4.20 (m, 2H), 3.90–3.81 (m, 2H), 3.80 (s, 1H), 2.96–2.86 (m, 1H), 1.81 (s, 3H), 1.29 (t, J = 7.0 Hz, 3H), 1.04 (d, J = 6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (major) 175.3, 170.8, 140.8, 128.3, 127.8, 126.0, 78.7, 66.0, 62.7, 40.0, 21.0, 14.1, 11.6. (minor) 174.7, 171.1, 140.2, 128.4, 127.9, 125.9, 79.6, 65.5, 62.9, 40.1, 20.8, 14.2, 12.6; HRMS (ESI) Calcd. for C15H20O5, [M+Na]+: 303.1203, Found: 303.1215; FTIR (neat): 3494, 2981, 1727, 1231.</p><!><p>An ethereal solution (5 mL) of 4b (1.39 g, 3.7 mmol) was added dropwise to a 100 mL round-bottom flask charged with an ethereal (30 mL, 0.12 M) suspension of LAH (709 mg, 18.7 mmol) at 0°C. The reaction was removed from the ice-bath and was allowed to stir for a 1 hour period. Distilled water (1 mL) was added slowly. Distilled water (3 ml) and 15% NaOH aqueous (1 mL) were added to the reaction mixture. To the vigorously stirred solution was added portions of MgSO4 until the reaction mixture solidified. The reaction mixture was filtered through a fritted glass funnel with the aid of ether. The filtrate was evaporated under reduced pressure and was used in the next step without further purification. To the crude diol (1.16 g, 3.5 mmol) was added dichloromethane (30 ml, 1.1 M), 4-bromobenzenesulfonyl chloride (996 mg, 3.9 mmol), DMAP (42 mg, 0.35 mmol) and Et3N (1 mL, 7.1 mmol). The reaction was allowed to stir at ambient temperature for a 1 hour period. NaHCO3 (10 mL) and distilled water (10 ml) were added to the reaction mixture. The mixture was transferred to a separatory funnel and extracted with ethyl acetate (3 × 20 mL). The combined organic extracts were washed with brine (1 × 50 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated under reduced pressure. The crude 4b derivative residue was subjected to column chromatography (SiO2: 20% ethyl acetate/hexanes) to give the title compound (1.7 g, 3.3 mmol) in 90% yield as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.65 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.39. (d, J = 8.6, 2H), 7.12 (d, J = 8.6 Hz, 2H), 4.34 (s, 2H), 2.11 (s, 1H), 1.86–1.76 (m, 1H), 1.55–1.46 (m, 1H), 1.37–1.05 (m, 8H), 0.91–0.80 (m, 4H), 0.74 (d, J = 6.9 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 140.3, 134.4, 132.6, 131.1, 129.2, 127.6, 74.7, 40.5, 31.8, 30.2, 29.4, 27.6, 22.6, 14.0, 13.8; HRMS (ESI-MS) Calcd. for C22H28Br2O4S [M+Na]+: 568.9967, Found: 568.9983; FTIR (neat): 3610, 2927, 1727, 1577; MP: 98–100 °C.</p><!><p>To a flame-dried 50 mL round-bottom flask charged with 2H-spiro[acenaphthylene-1,2′-[1,3]dioxolan]-2-one (891 mg, 3.9 mmol) was added ethanol (20 ml, 0.2 M). NaBD4 (180 mg, 4.3 mmol) was added portionwise. The reaction mixture was allowed to stir at ambient temperature for 30 min. Distilled water was added and the reaction mixture was allowed to stir until bubbling stopped. The mixture was extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were washed with brine (1 × 50 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated under reduced pressure. Without further purification the crude alcohol residue was added ethanol (20 mL) and 6.0 M HCl aqueous (15 ml). The reaction mixture was allowed to stir at ambient temperature for the stated time. Distilled water was added and the mixture was extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were washed with brine (1 × 50 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated under reduced pressure. The crude solid was subjected to column chromatography (SiO2: 15% ethyl acetate in hexanes) to give the title compound (0.69 g, 3.7 mmol) in 95% yield as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.15 (d, J = 8.1 Hz, 1H), 7.98 (d, J = 7.1 Hz, 1H), 7.92. (dd, J = 8.1, 1.1 Hz, 1H), 7.79–7.66 (m, 3H), 3.12 (s, 1H); 2H NMR (77 MHz, CHCl3): δ 5.37 (s, 1D); HRMS (ESI-MS) Calcd. for C12H7DO2 [M+Na]+: 208.0479, Found: 208.0460; FTIR (neat): 3400, 3064, 1702; MP: 160–162 °C.</p><!><p>In accordance with Procedure A, deuterio-1k (0.2 mmol, 100 mol%) was reacted with ethylene (15 × 125 mm pressure tube, 0.82 mmol, 410 mol%) in toluene (2.0 M) at 130 °C for a 48 hour period. Flash column chromatography (SiO2: 5–15% ethyl acetate/hexanes) provided the title compound (18.0 mg, 0.08 mmol) in 42% yield as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.14 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 7.0 Hz, 1H), 7.91 (dd, J = 7.9, 1.2 Hz, 1H), 7.79–7.64 (m, 3H), 2.72 (s, 1H), 2.19–2.01 (m, 2H), 0.78 (t, J = 7.5 Hz, 2.78H); 2H NMR (77 MHz, CHCl3): δ 0.77 (s, 0.22H); HRMS (ESI-MS) Calcd. for C14H11DO2 [M+Na]+: 236.0792, Found: 236.0781; FTIR (neat): 3370, 2973, 2927, 1716; MP: 92–93 °C.</p>

PubMed Author Manuscript

Ultrasensitive Detection of Pb2+ Based on a DNAzyme and Digital PCR

In this study, an ultrasensitive detection method for aqueous Pb2+ based on digital polymerase chain reaction (dPCR) technology and a Pb2+-dependent DNAzyme was developed. In the presence of Pb2+, the Gr-5 DNAzyme was activated and catalyzed the hydrolytic cleavage of the substrate strand, resulting in an increase in the amount of template DNA available for dPCR and a resultant change in the number of droplets showing a positive signal. Moreover, the detection system was found to be sensitive and stable in environmental sample detection. In summary, an ultrasensitive quantitative detection method for Pb2+ within environmental substrates was established.

ultrasensitive_detection_of_pb2+_based_on_a_dnazyme_and_digital_pcr

1. Introduction<!>2.1. Oligonucleotides and Reagents<!>2.2. DNAzyme Cleavage Assay<!>2.3. Digital PCR Assay<!>2.4. Real-Time PCR Assay<!>2.5. Environmental Sample Analysis<!>3.1. Principle of the Detection System<!>3.2. Quantitative Assessment of Pb2+<!>3.3. Specificity Assessment of the Detection System<!>3.4. Detection of Pb2+ in Environmental Samples<!>4. Discussion

<p>Lead ions (Pb2+), a major heavy metal pollutant, are a widespread and highly toxic contaminant in the environment. Pb2+ can accumulate in the human body, and exposure to very small amount of Pb2+ can lead to serious damage to the human brain and central nervous system, especially in children [1–3]. Various analytical methods have been developed for Pb2+ detection, such as atomic absorption spectrometry [4], atomic emission spectrometry, inductively coupled plasma atomic emission spectrometry [4, 5], inductively coupled plasma mass spectrometry [6], and X-ray fluorescence spectrometry [7]. In recent years, many new methods for Pb2+ detection have been developed, such as colorimetric [8], electrochemical [9, 10], fluorometric [11, 12], surface-enhanced Raman scattering [13, 14], and dynamic light scattering techniques [15]. One approach is based on the catalytic activity of DNAzymes [10, 14, 16–20]. The activity of DNAzymes, which are DNA-based catalysts, requires metal ions. Once activated by cofactors, the DNAzyme can induce chemical transformations and cleave DNA specifically. DNAzymes were first discovered via an in vitro selection process for RNA cleavage in the presence of Pb2+, and they have shown excellent selectivity for Pb2+ [21]. There are many advantages of DNAzymes as metal ion sensors. First, DNAzymes can be obtained with minimal information about metal ion-binding sites. Second, DNAzyme selection is more efficient than aptamer selection because of its higher separation efficiency. Finally, DNAzymes are easier to synthesize. Consequently, many DNAzyme-based sensors have been developed in the past few years. For example, lead-specific DNAzymes are known to cleave at adenosine ribonucleotide (rA) sites on partially complementary DNA substrates in the presence of Pb2+. Typical Pb2+-specific DNAzyme-based sensors utilize the enzymatic activity of the Gr-5 and "8–17" DNAzymes on a target element to produce a fluorescent or colorimetric output [10, 22, 23]. However, DNAzyme-based methods also have inherent drawbacks, such as high detection limits and high background signals. Recently, polymerase chain reaction (PCR) has been introduced combining with DNAzymes so that PCR methods could be used to improve the sensitivity and specificity of Pb2+ detection techniques.</p><p>Droplet digital PCR (ddPCR), a biotechnological refinement of conventional PCR methods, provides high-precision absolute quantification of nucleic acid target sequences and has wide-ranging applications for both research and clinical diagnostic uses [24–26]. First, the PCR mix is partitioned into thousands of water-in-oil droplets. Each droplet contains one or fewer copies of the target DNA. After end-point PCR amplification, each partition is checked for fluorescence with a binary readout of 0 (presence of PCR product) or 1 (absence of PCR product). Finally, absolute quantification of the target DNA molecules in the original sample can be directly calculated based on the Poisson distribution [24, 27, 28]. ddPCR has several benefits for nucleic acid quantification, including unparalleled precision, increased signal-to-noise ratio, removal of PCR efficiency bias, and simplified quantification, allowing a more reliable collection and more sensitive measurement of nucleic acid amounts. The method has been demonstrated to be useful for studying variations in gene sequences and is regarded as an improved nucleic acid detection technique compared with real-time PCR [29–32]. The absolute quantification of lead ions was essential for the risk assessment of lead ions. However, in methods such as colorimetric and electrochemical techniques, the quantitative detection of target ions could only be achieved through the analysis of standard curves which may be affected by the operation or the data analysis [33]. Previous studies have shown that when Pb2+ is present in the solution, it binds to the Gr-5 DNAzyme and facilitates the cleavage of the phosphodiester bond of the internal RNA base (rA) by the enzyme strand, and the amount of Pb2+ and cleavaged DNA is about one to one ratio. Thus, through absolute quantification of cleavaged DNA by droplet digital PCR, the absolute quantitative detection of Pb2+ can be achieved.</p><p>Herein, we report an ultrasensitive detection method for Pb2+ based on a DNAzyme biosensor and digital PCR (dPCR). A Pb2+-dependent Gr-5 DNAzyme was selected and immobilized on the inner wall of a plate [34]. The addition of aqueous Pb2+ to the plate leads to cleavage of the substrate DNA at the rA site by the Gr-5 DNAzyme, resulting in the release of the template DNA. Consequently, the PCR amplification signal varies according to the amount of template available for amplification, which in turn depends on the concentration of aqueous Pb2+. The method takes advantage of the selectivity of the DNAzyme and the sensitivity of dPCR. Moreover, this method can detect as low as 500 pM Pb2+ and can be used for analyzing complex assay mixtures such as environmental water samples.</p><!><p>Oligonucleotide sequences were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences are shown in Supplemental Table 1 as described previously [35]. Enzyme-labeled plates pretreated with streptavidin were purchased from Haili Biotech Co. Ltd. (Suzhou, China). The reagents related to ddPCR were purchased from Bio-Rad (Pleasanton, CA, USA). Lead ions and mercury ions were obtained from the National Standard Substances Center (Beijing, China). Zn(NO3)2, MgCl2, FeCl2, CuCl2, KCl, and CaCl2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were of analytical grade, and double-distilled water was used throughout the experiments.</p><!><p>An enzyme-labeled plate pretreated with streptavidin was washed with PBST (10 mM PBS, pH 7.2, 0.05% Tween-20). Then, 50 µL of a mixture of Gr-5 DNAzyme and substrate DNA at the appropriate concentration was added to PCR tubes. Gr-5 DNAzyme was fixed on the plate through biotin-streptavidin interaction and hybridized with the substrate DNA at 37°C for 30 min. Next, the plate was washed with hybridization buffer (750 mM NaCl, 75 mM, C6H5Na3O7, pH 8.0), and Pb2+ was added to the plate. After incubation at 37°C for 30 min, the plate was washed with hybridization buffer, and the cleaved DNA was collected for digital PCR (dPCR).</p><!><p>Digital PCR assay was operated according to the instruction of Bio-Rad. Each reaction mixture contained 10 μL of ddPCR master mix for probe (Bio-Rad), 0.5 μM reverse and forward primers, 1 μL of template, and 7 μL of distilled water. The cleaved DNA from the DNAzyme cleavage assay was used as the template DNA and was serially diluted to ensure the proper copy number range. Then, the mixture was loaded onto a droplet generation cartridge for droplet generation. Droplets were then collected and amplified. The PCR amplification conditions were as follows: 95°C for 5 min, 40 cycles of denaturation for 10 s at 95°C, and annealing for 60 s at 60°C and finally 10 min at 98°C. The amplified droplets were detected by a dPCR system, and the data were analyzed using Right PCR software.</p><!><p>The cleavage DNA from DNAzyme cleavage assay was used as the template DNA. The final 20 μL PCR mixture contained 10 μL of SGExcel FastSYBR Mix (Sangon Biotech), 2 μL of reverse and forward primers at a final concentration of 0.5 μM, 1 μL of template, and 7 μL of distilled water. Then, the above 20 μL mixture was amplified with PCR on an ABI7500 system. The PCR amplification procedure consisted of predenaturation at 95°C for 5 min, 40 cycles of denaturation for 10 s at 95°C, and annealing for 30 s at 60°C.</p><!><p>To demonstrate the practicality of our proposed method, water samples were spiked with Pb2+ (1 nM, 5 nM, 10 nM, and 50 nM). These samples were tested with the same procedure used for Pb2+ detection. The recovery ratio was calculated based on the dPCR signals.</p><!><p>The principle of the detection system is described in Figure 1. We combined dPCR with the Gr-5 DNAzyme as the sensing system for Pb2+ detection. In the first step, biotin-modified Gr-5 DNAzyme was fixed on the inner wall of a streptavidin-coated plate via biotin-streptavidin interactions, the substrate strand was hybridized to the biotin-modified Gr-5 DNAzyme through complementary base pairing, and substrates that were not fixed were washed off. The substrate strand contains a single RNA base that is cleaved specifically by the classic Pb2+-dependent Gr-5 DNAzyme as the catalytic unit and a 50-base substrate strand extension, which is required as a template for PCR amplification. The addition of aqueous Pb2+ to the plate leads to cleavage of the substrate DNA at the rA site by the Gr-5 DNAzyme, resulting in the release of the cleaved substrate DNA. The cleaved substrate DNA was used as template DNA and detected with digital PCR. Consequently, the PCR amplification signal varies according to the amount of template available for amplification: the higher the concentration of aqueous Pb2+ was, the more the droplets with template DNA were detected. Thus, the concentration of the Pb2+ target can be quantified using dPCR.</p><!><p>To improve the performance of the detection system for Pb2+ detection, the reaction time was optimized with real-time PCR [35]. Various reaction times were investigated, and the results showed that the Ct value decreased with increasing substrate-binding time up to 30 min, indicating that sufficient binding of Gr-5 DNAzyme and substrate DNA occurred within 30 min (Figure 2(a)). In the substrate cleavage experiments in the presence of Pb2+, the Ct value decreased as the cleavage time increased up 30 min (Figure 2(b)). Based on these results, both the hybridization time of the Gr-5 DNAzyme and the substrate DNA and the cleavage time were set at 30 min. The concentration of DNAzyme was set at 100 nM, as described previously. Then, titration experiments were carried out to test whether the proposed method using a DNAzyme assay and dPCR can be used for Pb2+ quantification. There are few positive droplets in the absence of Pb2+, whereas there are many positive droplets when Pb2+ is added, suggesting that the DNAzyme was activated and catalyzed the cleavage of the substrate strand. As a result, template DNA was released, and positive droplets were generated after amplification. As shown in Figure 3, the number of positive droplets has a good linear relation with the Pb2+ content between 500 pM and 100 nM. This result indicated that our detection system has a dynamic range over 3 orders of magnitude and thus can achieve accurate detection of Pb2+ in most practical samples.</p><!><p>To evaluate the specificity of the detection system, a series of potential interference metal ions, such as Zn(II), Hg(II), Cu(II), Fe(II), K(I), and Ca(II), were tested by measuring and comparing the number of positive droplets with those in the presence of lead ions. As shown in Figure 4, only Pb2+ showed significantly high digital signals, while no substantial signal was produced by the remaining metal ions or the blank. These results indicate that our detection system exhibits excellent selectivity for Pb2+ over other environmentally relevant metal ions.</p><!><p>We further evaluated the potential application of our detection system to real samples such as environmental substrates. Lake water samples were centrifuged to remove the insoluble materials. Then, the supernatant spiked with various concentrations of Pb2+ were evaluated. As shown in Table 1, the recovery for the water samples ranged from 96.2% to 105%, and the standard deviation was less than 5%. This indicated that our detection system based on the DNAzyme biosensor and dPCR offers a convenient and sensitive approach for Pb2+ detection in environmental substrates.</p><!><p>In this study, an ultrasensitive detection method for aqueous Pb2+ based on digital polymerase chain reaction (dPCR) technology and a Pb2+-dependent DNAzyme was developed. The relationship between Pb2+ concentration and the number of positive droplets was established. Owing to the high sensitivity of dPCR, our proposed method exhibits high sensitivity for Pb2+ detection with a detection limit of 500 pM. The method also exhibits excellent selectivity due to the specificity of the Gr-5 DNAzyme. In addition, this method showed good feasibility for use in water sample detection. Previous studies have shown that many ions, such as Cu2+, Cd2+, and Na+, could be detected with DNAzymes [36–39]. It is very likely that this method, which combines a DNAzyme and dPCR, could be extended to other ions, which may improve its sensitivity for ion detection. In summary, the new sensor is a potential tool for Pb2+ detection in environmental samples.</p>

PubMed Open Access

Multiphysics Modeling of Plasmonic Photothermal Heating Effects in Gold Nanoparticles and Nanoparticle Arrays

Induced hyperthermia has been demonstrated as an effective oncological treatment due to the reduced heat tolerance of most malignant tissues; however, most techniques for heat generation within a target volume are insufficiently selective, inducing heating and unintended damage to surrounding healthy tissues. Plasmonic photothermal therapy (PPTT) utilizes light in the near-infrared (NIR) region to induce highly localized heating in gold nanoparticles, acting as exogenous chromophores, while minimizing heat generation in nearby tissues. However, optimization of treatment parameters requires extensive in vitro and in vivo studies for each new type of pathology and tissue targeted for treatment, a process that can be substantially reduced by implementing computational modeling. Herein, we describe the development of an innovative model based on the finite element method (FEM) that unites photothermal heating physics at the nanoscale with the micron scale to predict the heat generation of both single and arrays of gold nanoparticles. Plasmonic heating from laser illumination is computed for gold nanoparticles with three different morphologies: nanobipyramids, nanorods, and nanospheres. Model predictions based on laser illumination of nanorods at a visible wavelength (655 nm) are validated through experiments, which demonstrate a temperature increase of 5 \xc2\xb0C in the viscinity of the nanorod array when illuminated by a 150 mW red laser. We also present a predictive model of the heating effect induced at 810 nm, wherein the heating efficiencies of the various morphologies sharing this excitation peak are compared. Our model shows that the nanorod is the most effective at heat generation in the isolated scenario, and arrays of 91 nm long nanorods reached hyperthermic levels (an increase of at least 5 \xc2\xb0C) within a volume of over 20 \xce\xbcm3.

multiphysics_modeling_of_plasmonic_photothermal_heating_effects_in_gold_nanoparticles_and_nanopartic

INTRODUCTION<!>Geometry Models of GNPs: Nanorod, Bipyramid, and Nanosphere.<!>Optical Absorbance by GNPs.<!>Calculating Heat Source Power of a GNP.<!>Heat Transfer for GNPs in a Homogeneous Environment (Single GNPs and Arrays).<!>Synthesis of the GNPs.<!>UV\xe2\x80\x93vis Spectroscopy and Imaging of the GNPs.<!>Photothermal Heating Experiments.<!>Verification of the Computational Models.<!>Validation of the Computational Models.<!>Potential PPTT Implementation.<!>Nanoparticle Heating.<!>Ensemble Effect.<!>CONCLUSIONS

<p>The first applications of heat in tumor treatment are dated at least 3000 years back to the ancient Egyptians, where a glowing tip of a firedrill was used to treat a breast cancer tumor.1–5 In recent years, new techniques have been developed to enable hyperthermia, which is defined as heating tissue to a temperature between 41 and 47 °C for at least 20–30 min,3,6 to treat different malignant cancers. This approach is considered selective due to the reduced heat tolerance typical to most malignancies, but most methods have still been demonstrated to cause collateral damage within surrounding healthy tissue.7 Among the techniques developed and refined over the past few decades, those using light absorption have received substantial attention due to their capabilities of superior control and confinement of thermal damage within tumor tissue.3,8 The use of lasers in medicine was first introduced in the field of ophthalmology back in the 1960s,3,9 with the most commonly employed laser gain media being neodymium–yttrium aluminum garnet (Nd:YAG, 1.06 μm) and CO2 (10.6 μm) A collimated laser beam reaches a wavelength-dependent tissue depth determined by the scattering and absorption of the tissue chromophores, which has a local minimum in the near-infrared (NIR) region.10 The absorbed light generates heating of the tissue, thus inducing hyperthermia.11</p><p>The use of this technology enabled physicians to reach deep-seated targets, but it has one major disadvantage: the high power density needed to effectively ablate tumors causes damage to the healthy tissue in the path of the beam.3,12–14 One technique leveraged to overcome this issue is photodynamic therapy (PDT), which uses a photosensitizer that reacts with tissue oxygen upon exposure to a specific wavelength in the visible or NIR region. This photochemical reaction occurs at much lower light fluence levels, causing cell destruction via toxic singlet oxygen. However, these sensitizers (mainly porphyrin-based) stay in the body for long periods of time, leaving patients highly sensitive to light and likely to experience related complications while undertaking this treatment.3</p><p>As an alternative to these treatments, photothermal therapy (PTT) leverages localized photoabsorbing chromophores to achieve spatially selective heating.15 PTT typically uses chromophores with absorption peaks within the NIR range, due to the high physiological transmissivity observed at those wavelengths, minimizing undesired tissue absorption and subsequent heating.16 The agents used in this therapy include natural tissue chromophores, added dye molecules, and, more recently, nanoparticles.17 Research and advances in the field of nanotechnology have provided a variety of nanostructures with unique and tunable optical properties, providing utility to a broad range of applications in medicine. Metallic nanoparticles, when exposed to light at their resonance wavelength, experience a coherent, collective oscillation of the conduction-band electrons that leads to significant light scattering or absorption.3,18 This oscillation is known as localized surface plasmon resonance (LSPR), which yields a light absorption that is about five times larger than that offered by conventional photoabsorbing dyes.3 Gold nanoparticles (GNPs) are studied and used in a variety of applications; some of the most commonly used geometries for oncological treatment are nanospheres, nanorods, and nanoshells. The popularity of these geometries lies in their ease of preparation, their high light-to-heat ratio, and the possibility of providing an alternative treatment for chemotherapy-resistant tumors.17–19 Among the aforementioned variants, gold nanorods (GNRs) are frequently preferred due to their optical response tunability, which can be achieved by changing their aspect ratio, and efficiency in large-scale synthesis.20 The capabilities of these nanoparticles to undergo surface modification have also allowed PTT to be combined with other treatment strategies such as molecular targeting, chemotherapy, and gene therapy.21–23 It is important to note that cytotoxicity due to synthesis methods was a historic limitation to in vivo implementation of GNPs. However, advances in recent years have demonstrated that the incorporation of polyethylene glycol (PEG), dense silica (d-SiO2), and titanium dioxide (TiO2) coatings dramatically mitigates cell injury.24</p><p>There are three primary factors that affect the optimization space for wavelength selection to induce nanoparticle hyperthermia: penetration depth, off-target absorption within healthy tissues, and the optical properties of the nanoparticles (e.g., absorption). Absorption levels of tissue chromophores in the 800–1100 nm range are about 2 orders of magnitude lower than absorption in the visible range. Additionally, penetration depth at these NIR wavelengths is several folds higher than in the visible range. These properties, in addition to the tunable versatility of GNRs, make NIR light the ideal candidate for plasmonic photothermal therapy (PPTT).18,25–27</p><p>Light–nanoparticle interactions, as well as light–tissue and nanoparticle–tissue interactions, can be studied using computational modeling to further understand the effect of light diffusion in tissue and the temperature change induced by the heating of nanoparticles when excited with light.28–34 The window opened by computational modeling enables the optimization of therapies like PPTT as the size and shape of GNPs can be easily modified in search of the type of nanoparticles that will enhance the thermal effects within a target. Other computational methods, such as the one presented by Baffou et al., implement shape factors to simplify the calculation of the power absorbed by nonspherical nanoparticles and their subsequent heat generation.20,35 Another method, proposed by Hogan et al., studies the effect of multiple scattering in heat generation. This is due to the use of nanoshells with a relatively large size (~150 nm), which possess a scattering-dominated extinction cross-section.36</p><p>In this work, we take advantage of the tools provided by computational modeling and compare the effects of size and shape by creating computational models of different types of nanoparticles, more specifically gold nanorods, gold nanobipyramids (GBPs), and gold nanospheres (GNSs). The light–nanoparticle interaction can be explored using these computational models, as the power absorbed by the nanoparticles, when excited by an incident light wave, can be calculated from the solutions of Maxwell's equations. Furthermore, the subsequent nanoparticle heating due to the absorption of light and its effect on temperature change of the surrounding medium (water) can be assessed by using the power absorbed as a heat source to solve the heat equation. Our computational model uses a novel approach to calculate the power absorbed by the nanoparticles and their subsequent heat generation. It incorporates a full electrodynamics simulation to solve the light–nanoparticle interaction and couple the results into a heat transfer model without implementing shape simplifications. Additionally, we focus on nanoparticles whose extinction cross-section is dominated by absorption and exhibits relatively little scattering, making multiple scattering effects less significant in our models. These simulations are verified by comparing with an analytical solution obtained using Mie theory and validated by comparing the simulated results with those obtained experimentally.</p><!><p>Computational models of different GNPs were developed using computer-aided design (CAD) software SOLIDWORKS (Dassault Systèmes SolidWorks Corp; www.solidworks.com). The models were based on transmission electron microscopy (TEM) images of GNRs and GBPs.</p><p>Each one of the models was set up so that different sizes and aspect ratios could be analyzed by modifying specific parameters. In the case of the GNS, this parameter is the radius. For the GNR, the parameters include the length and width. Lastly, the modifiable parameters for the GBP are the length, width, and tip radius.</p><!><p>Analytical solutions to Maxwell's equations can be obtained using Mie theory in the case of spherical particles.37 However, to obtain solutions to Maxwell's equations for nonspherical geometries, numerical approaches are needed. In this study, we imported the SOLIDWORKS models of different geometries into the finite element method (FEM) simulation software COMSOL Multiphysics (COMSOL; www.comsol.com) using LiveLink and used COMSOL's RF Module to solve Maxwell's equations. Additionally, an absorbing boundary, or perfectly matched layer (PMLs), with a thickness of λ/2 (λ is the wavelength) was implemented to truncate the computational domain. Details on the mathematical model and computational setup can be found in the Supporting Information.</p><p>An example of the PMLs truncation for our setup is shown Figure 1a.</p><p>In addition to the absorbing property of the PMLs and to reduce the impact of the discretization, a scattering boundary condition was assigned to the outermost surface, meaning that the waves reaching the external boundary of the computational domain would not travel back into the domain of interest.</p><p>Illumination at the nanoparticles' LSPR could result in either light scattering or light absorption. The amount of scattered and absorbed light was determined using the cross-section calculations38 and summed up to obtain the extinction cross-section of the nanoparticles (Figure 2). However, for the scope of this work, we focused on the absorption of light and the resulting localized heating. The absorption cross-section of the particle, σabs can be calculated from the Poynting theorem as (1)σabs=WabsSin where Sin is the Poynting vector magnitude and where the power absorbed by the nanoparticle is (2)Wabs=∫∫∫VQhdV Here, Qh is defined as the total power dissipation density [Wm3] (i.e., the total losses of the system) based on the calculated EM fields. We obtained the total power absorbed by integrating over the volume, V, of the GNP.35,39</p><!><p>The absorbing properties of nanoparticles play an important role in photothermal heating. At the LSPR wavelength, the light absorbed is maximized, and so is the photothermal heating effect. To calculate the photothermal heating generated by a spherical nanoparticle,35 eq 3 yields an accurate approximation based on the absorption cross-section, the volume of the particle, and the irradiance of the light source.</p><p>This approach, however, yields inconsistent results when treating complex geometries such as the nanobipyramid or the nanorod; in these cases, a more general approach is undertaken as shown in eq 435 (4)Qabs=WabsV with Qabs being the power per unit volume generated by the nanoparticle and considering that the effect of other losses due to atomic effects is negligible. It is clear to see how the power absorbed by the GNP is readily transformed into heat.</p><!><p>COMSOL's Coefficient Form PDE module was used for our heat transfer model, due to its versatility for adding or neglecting different effects in the simulations. With a heat source, Qabs, measured in Wm3 and temperature, T, being the dependent variable, the steady state heat equation reads (5)−∇(κ∇T)=Qabs where κ is the diffusion coefficient [Wm⋅K]. Equation 5 is where the absorption calculations from the optical properties model feed into the heat transfer model. Initially, Qabs is computed using the RF module (eq 4). This value is then used as the heat source in eq 5.35,40,41</p><p>For anisotropic nanoparticles such as the GNR, their optical absorption is polarization dependent. The RF module was used to calculate the average heat power across all possible orientations and use this value as the heat generated by a nanoparticle in any orientation or even in an array where nanoparticles are randomly oriented.</p><p>Considering that we are not working with propagating waves anymore, the truncation method (PMLs) is no longer required, and so the size of the domain is not restricted. A 3 μm sphere was placed around the particle to act as a surrounding medium (water) as shown in Figure 1b.</p><!><p>Synthesis of gold nanobipyramids and gold nanorods followed seed-mediated growth protocols. For the GNRs, seeds were synthesized using a method modified from Nikoobakht et al., and the growth solution preparation was modified from Vigderman et al. To make seed solution, CTAB (5 mL, 0.2 M) was added to HAuCl4 (5 mL, 0.5 mM) in a 15 mL Falcon tube while gently stirring, creating a bright orange solution. Freshly prepared, ice cold NaBH4 solution (600 μL, 0.01 M) was then added and thus changed the solution to a pale brown color. The seed solution was set aside at room temperature for at least 2 h before use. For the growth solution, CTAB (5 mL, 0.2 M) was added to HAuCl4 (5 mL, 1 mM) in a fresh 50 mL Falcon tube. AgNO3 (70 μL, 0.01 M) was added and inverted several times to mix. Hydroquinone (500 μL, 0.1 M) was then added, followed by gently inverted mixing until the solution turned clear and colorless. Afterward, the seed solution (160 μL) was finally added to the growth solution, inverted to mix, and allowed to incubate overnight for nanorod growth.42–45 For the GBPs, synthesis was carried out as in Liu and Guyot-Sionnest.46</p><!><p>Characterization of the LSPRs was performed via UV–vis using a FLAME-CHEM-VIS-NIR spectrometer (Ocean Optics; Largo, FL) for nanoparticles in the range of 500–550 nm in order to compare the simulated results and those obtained experimentally. Images of the different nanoparticles were obtained using a TEM with the JEM-1400 Plus (JEOL USA, Inc. Peabody, MA). ImageJ was used to measure the dimensions of the nanoparticles so that the computational models accurately resemble the nanoparticles synthesized in the lab.</p><!><p>The nanoparticle heating measurements were carried out using a red laser at 655 nm wavelength and 150 mW (DL655-150, CrystaLaser; Reno, NV). We measured the heating in two samples with nanoparticles at OD = 1 and one control sample with water only. The nanoparticle samples were nanorods and nanobiopyramids synthesized to have resonance at the laser's wavelength, and the temperature measurements were taken using a Temp-300 thermocouple datalogging thermometer (Oakton Instruments; Vernon Hills, IL) until a steady state was reached (~22 min).</p><!><p>The computational models were verified by comparing the results obtained computationally with the analytical results obtained following Mie theory.47 Since the analytical solution is available only for spherical nanoparticles, we used Mie theory to calculate the spectra of various GNSs accross several wavelengths and with different surrounding media. These analytical solutions were then compared with the numerical simulations obtained when using a computational model with the same surrounding media and a spherical geometry of the same dimensions. Figure 2 shows an excellent agreement between the computational and the analytical results for spheres of two sizes: a 150 nm sphere in air and a 50 nm sphere in water. The average error is 2.3% and 4.2% for the nanoparticle in air and water, respectively. It can be noticed that, as expected, a smaller particle has a higher absorption cross-section, whereas the scattering cross-section is more predominant in the larger-sized particle.</p><!><p>To validate the computational models measuring light–nanoparticle interactions (light absorption), we compared the UV–vis absorbance spectra obtained from samples of GNRs and GBPs synthesized in our lab, with the spectra obtained from corresponding computational models of individual GNR and GBP.</p><p>TEM images of the samples were taken (left column of Figure 3), and the individual GNPs were generated based on the average dimensions (length, L; width, W; and tip radius, R) measured from the images. The experimental UV–vis spectra (center column of Figure 3) were measured in arbitrary units, and the calculated spectra (right column of Figure 3) were computed following eq 1. The optical efficiencies were obtained for the simulated spectra as the ratio between the power absorbed by the particle (σabs) and the cross-sectional area of a sphere with equivalent volume (Aeq).</p><p>UV–vis spectroscopy measures the absorbance as the amount of photons that do not pass through the sample at a given wavelength, whereas the calculated spectra indicate how strong the absorption is for a single nanoparticle. Keeping this difference in mind, the comparison made between experimental and computational spectra is in terms of the plasmon peak location. It can be noticed that the plasmon resonance wavelengths are similar between the simulated and measured spectra for both nanoparticle types. However, due to the inhomogeneity in the samples (particles of different sizes and shapes as well as additional byproducts and aggregates resulting from the synthesis process), the breadth of the peaks observed in the experimental spectra is different from the ones observed in the simulations.</p><p>The measured dimensions (avg ± Std Dev) obtained from the TEM images of the nanorod sample were L = 97 ± 20 nm, W = 25 ± 2 nm, and R = 8 ± 3 nm. The measured dimensions (avg ± Std Dev) from the bipyramid sample were L = 97 ± 20 nm, W = 25 ± 2 nm, and R = 8 ± 3 nm. Based on these measurements, the computational GNR model had dimensions of L = 91 nm, W = 26 nm, and R = 11 nm, while the dimensions of the computational GBP model were L = 158 nm, W = 58 nm, and R = 8 nm.</p><p>It is observed, however, that the calculated spectra yield a similar resonance wavelength only in the case of the nanorod sample (peak at 807 nm). The blue shift observed when comparing the experimental and simulated bipyramid (experimental peak at 850 nm and calculated peak at 820 nm) can be attributed to the fact that the nanobipyramids found in the experimental sample are heterogeneous in shape, whereas the simulations were carried out with only one particle. For the remainder of this work, we will focus on the simulation results, and thus we assume that the resonance wavelength of the GBP is 820 nm.</p><!><p>For implementation as photothermal agents, the desired GNPs must exhibit high absorptive properties in the NIR (~800 nm). For this purpose, six different computational models of nanoparticles were compared: two rods, two bipyramids, and two spheres.</p><p>The GNR and GBP samples synthesized in the lab (Figure 3) showcase resonance wavelengths appropriate for NIR implementation; however, the peaks were not close enough to conduct a one-to-one comparison between the two samples (GNR peak at 807 nm and GBP peak at 820 nm), so two new models were generated: a GNR that peaks at 820 nm and a GBP that peaks at 807. These additional models were developed by modifying only the length of the TEM-based model in order to match the desired resonance wavelength.</p><p>The spherical models were generated based on the volume measured from the TEM of the gold nanorods and the gold bipyramids. The radii of the modeled spheres were chosen so that their volume would be equal to the volume of the modeled GNR and GBP (Req = 34 nm in the case of the GBP and Req = 22 nm in the case of the GNR).</p><p>It is worth noting that both width and tip radius remain unchanged for the two nanorod models (W = 26 nm; R =11 nm) as well as for the two nanobipyramid models (W = 58 nm; R = 8 nm). For this reason, the dimensions column of Table 1 reports only the length, L, of the GNR and GBP and the diameters of the GNS</p><p>Depending on the size and shape of the nanoparticle, it will exhibit either the highest absorbance or the highest scattering when the longitudinal modes are excited at the resonance wavelength. With our model, the maximum light absorption is measured when the wave's propagation vector (k) is perpendicular to the longitudinal axis of a nonspherical nanoparticle, and the polarization of the wave (E) is parallel to the longitudinal axis. The opposite configuration yields the minimum amount of power absorbed: k parallel to the longitudinal axis of the GNP and E perpendicular.</p><p>The orientation-dependent nature of the power absorption was accounted for by calculating a mean power absorbed (QabsAvg) as the average between three configurations of light propagation: the first configuration is where E and the longitudinal axis of the particle are parallel (Qabs0°), and the other two configurations, grouped together as they yield the same value, are where E and the longitudinal axis are perpendicular (Qabs90°). This average absorbed power density can be written as (6)QabsAvg=Qabs0°+2Qabs90°3 and is reported in Table 1 for each nanoparticle.</p><p>Solving Maxwell's equations in the light–nanoparticle system allowed for the calculation of the electric field enhancement induced by the plasmon excitation in the GNPs. The magnitude of both absorbed power and field enhancement also depends on the light intensity used. Based on the typical values found in the literature,35,40,41 we chose an irradiance of 1 mW/μm2 and a wavelength of 808 nm. Given the size difference between the laser beam spot and the nanoparticle, a plane wave model was implemented, and the amplitude was calculated using the relationship between the wave's amplitude and irradiance (see eq 3 of the Supporting Information). The effect of size and shape in the electric field enhancement for a single GNR, GBP, and GNS can be observed on the left column of Figure 4, and a volume map of the total power dissipation density (power absorbed by the same particles) is shown on the right column of Figure 4. This power density was then utilized in eq 4 and ultimately in eq 6.</p><p>The results observed in both Table 1 and Figure 4 indicate that the nanorods absorb the highest amount of power when under illumination of a 808 nm laser. More specifically, the 91 nm long GNR absorbs more power than the 94 nm GNR due to the close proximity between the particle's resonance wavelength and laser's wavelength. Furthermore, it is observed that the bipyramid models show a considerable amount of absorbed power, despite being over 50% larger than the rods. Thus, we may assume that both the rod and the bipyramid geometries could be good candidates for plasmonic heating applications. However, size limitations inherent to the particular application need to be taken into consideration. Additionally, shape effects are observed as the individual spherical models do not absorb a significant amount of power despite having the same volume as the GNR and GBP models and, therefore, are not suitable for plasmonic heating in the NIR region due to their low power absorption levels at these wavelengths.</p><!><p>We have shown that both the GNRs and the GBPs absorb significant amounts of power in the NIR region, and now we will measure the change in temperature achieved by each particle due to the amount of absorbed power under 808 nm laser illumination. The average power absorbed by the individual particles will be used as a heat source in the heat transfer model (eq 5).</p><p>The maximum temperatures reached by the six nanoparticles used in this work, along with the average absorbed power used for the calculations, are reported in Table 2. The heat maps of the three nanoparticles exhibiting the highest temperature reached (within each type) are shown in Figure 5.</p><p>As expected, since the nanorods absorbed the highest amount of power, they also reach the highest temperature out of the three shapes compared, with the 91 nm nanorod being the one that reached the highest temperature (101 °C). These results correlate to the trends observed experimentally, where a 655 nm laser was used to heat three samples: GNRs, GBPs, and pure water. After 22 min, the system reached a steady state, and it was observed that the nanorods induced a temperature change of ~5 °C; however, the GBPs induced a temperature increase of ~2 °C. Water alone did not show any temperature increase under laser illumination (Figure 6).</p><p>Interestingly, both the experiments and the simulations show that the bipyramids can also induce a temperature increase. Table 2 shows that the smaller GBP reaches a higher temperature than the larger GBP. Similar to the GNR, the smaller bipyramid has a plasmon resonance wavelength closer to the wavelength of the laser; thus, the plasmonic effect is enhanced, and the subsequent heating is higher. This finding provides a sound argument to the initial thought presented earlier: both GNRs and GBPs can achieve significant heating under NIR illumination, thus making them good candidates for plasmonic heating applications. Because they exhibited the highest temperature increase, we select the 91 nm GNR as the best morphology for plasmonic heating under NIR illumination.</p><!><p>Now that we have chosen the 91 nm nanorod as the ideal geometry, we can study the effect induced by multiple particles of the same size and shape (i.e., ensemble effects) in different arrangements: a 3-dimensional array to represent a nanoparticle distribution throughout a volume and a 2-dimensional array to represent the nanoparticles deposited on a surface. Both the 2D and 3D models measure the effect posed by changing the distance between the nanoparticles (i.e., changes in the concentration). We tested two different concentrations, 1 and 22 nM: the former resulted in an interparticle spacing of ~1100 nm, whereas the latter resulted in an interparticle spacing of ~420 nm. These interparticle spacings are much larger than the nanoparticle size, so we expect that interparticle near-field interactions are insignificant. An approximation of the randomly distributed nanoparticles can be obtained by modeling a perfect cubic (3D) or square (2D) lattice array where the distance between nanoparticles is controlled by the concentration of the solution. Both arrays are composed of a finite amount of nanoparticles: 27 GNRs in the 3D array and 9 in the 2D array, allowing us to simulate the effects in the micron scale. Crucially, the heat power calculated from one nanoparticle in the nanoscale simulation (as described in the Nanoparticle Heating Section) is input into the micron-scale simulation as the new heat source power, thus bridging the two length scales in our computational model. The interparticle spacing in these systems is much smaller than the laser beam spot and the light decay length in the solution, so it can be assumed that each particle absorbs the same amount of power as its immediate neighbors.36 It is worth noting that a randomly distributed array of nanoparticles may enable increased interparticle interactions; future work is underway to evaluate this effect. For this reason, and to account for the random orientation observed experimentally, the average power density absorbed by a single 91 nm long GNR (Table 2) was assigned to each particle in the array, and the heat transfer for the different configurations was calculated using a steady state model. Figure 7 shows the temperature maps computed for the 3D model in both concentrations.</p><p>As mentioned above, the average power absorbed by the 91 nm GNR (311.52 mW/μm3) was assigned to each one of the particles in the array. Each of them contains 27 NRs arranged on a cubic lattice. The steady state heat transfer simulations from the 3D arrays at different concentrations show that the area where hyperthermia levels are reached (T > 43 °C) is similar in size for both systems: the 1 nM system induced hyperthermia levels over a region of 24.4 μm3, whereas the region affected by the heating of the 22 nM system has a volume of 28.7 μm3. Although the volume experiencing the plasmonic heating is similar, the highly concentrated system reached higher temperatures than the diluted one, indicating that a solution with higher concentration of GNPs absorbs greater amounts of light,36 thus inducing hyperthermia levels much faster while containing the effect within one micron from the center of the array. Comparing these results to those observed in the photothermal heating experiment (Figure 6), where the measured temperature increase observed in the GNR sample was about 5 °C (measured 5 mm away from the laser spot), and considering that the experimental sample had an optical density of approximately 1 at the resonance wavelength, it can be compared to the 1 nM simulated system which also shows a temperature increase of at least 5 °C around the boundaries of the simulation domain, as well as an average temperature increase of 7 °C throughout its volume.</p><p>For the 2D case, it is important to consider possible interface effects, e.g., for nanoparticles on a substrate or at the interface between two different substances or tissues.48,49 To check whether interface effects would be significant, one simulation was run with a 2D array of nanorods placed at a water–glass interface, and another simulation contained the 2D array surrounded by water only. There was a negligible difference between the optical absorbance between the array at the glass–water interface and the one surrounded by water. For this reason, and to reduce computational time, the heat maps of the 2-dimensional arrays at 1 and 22 nM are calculated without substrate and are shown in Figure 8. These maps show a behavior comparable to the one observed with the 3D array, but the regions where hyperthermia levels are reached are significantly smaller: the 1 nM system induced hyperthermia in the areas surrounding the particles but no further than 400 nm away from each particle. The 22 nM array induced hyperthermic levels within a region of ~1.5 μm, smaller than its 3D counterpart but significantly larger than the 2-dimensional array at 1 nM. This indicates that the heating effect of GNPs in 2D, similar to the 3D case, is enhanced with increased concentration.</p><!><p>We have developed computational models that successfully describe the plasmonic excitation of single gold nanoparticles, the subsequent heat generation due to power absorption from laser illumination, and its effects on temperature distribution throughout the surrounding medium. We developed these models in a FEM framework such as in COMSOL Multiphysics to provide the community with a complete and detailed set of procedures to enable a widespread of such a type of PPTT modeling. Comparing GNPs of different sizes and shapes confirmed the gold nanorod as the particle shape with the greatest heat power per unit volume; more specifically, the 91 nm GNR was found to have optimal heat generation. Additionally, due to the small size of the nanoparticle and the location of its resonance wavelength in the NIR, the GNR can be used as a photothermal agent in applications such as PPTT. Furthermore, the concentrations chosen for this study represent realistic concentrations of nanoparticles which would be used in medical applications, and it was found that arrays of nanorods can induce hyperthermic levels in the surrounding medium at low and high concentrations. Moreover, highly concentrated solutions induce hyperthermia over a larger volume. We note that our computational model showcases qualitative agreement with the experiments regarding the localized temperature increase, as well as the trend of GNRs exhibiting the highest amount of heating, followed by the GBPs. Locally concentrated GNRs, especially through methods like molecular targeting, will enhance the clustering effects and enable higher temperature increase within the target region as is the purpose of plasmonic heating applications.</p>

PubMed Author Manuscript

Bioorthogonal two-component drug delivery in HER2(+) breast cancer mouse models

The HER2 receptor is overexpressed in approximately 20% of breast cancers and is associated with tumorigenesis, metastasis, and a poor prognosis. Trastuzumab is a first-line targeted drug used against HER2(+) breast cancers; however, at least 50% of HER2(+) tumors develop resistance to trastuzumab. To treat these patients, trastuzumab-based antibody-drug conjugates (ACDs) have been developed and are currently used in the clinic. Despite their high efficacy, the long circulation half-life and non-specific binding of cytotoxic ADCs can result in systemic toxicity. In addition, standard ADCs do not provide an image-guided mode of administration. Here, we have developed a two-component, two-step, pre-targeting drug delivery system integrated with image guidance to circumvent these issues. In this strategy, HER2 receptors are pre-labeled with a functionalized trastuzumab antibody followed by the delivery of drug-loaded nanocarriers. Both components are cross-linked by multiple bioorthogonal click reactions in situ on the surface of the target cell and internalized as nanoclusters. We have explored the efficacy of this delivery strategy in HER2(+) human breast cancer models. Our therapeutic study confirms the high therapeutic efficacy of the new delivery system, with no significant toxicity.

bioorthogonal_two-component_drug_delivery_in_her2(+)_breast_cancer_mouse_models

<!>Enhancement of cellular internalization by a two-component delivery strategy.<!>Enhancement of tumor uptake of components.<!>High cellular internalization of components in vivo.<!>Enhancement of therapeutic efficacy with low toxicological effects.<!>Discussion<!>Materials & Methods<!>Formulation of components.<!>MALDI of components.<!>Human breast cancer mouse models.<!>Minimally invasive surgery and intravital imaging. Mice were injected intravenously with<!>Statistical analysis.

<p>The HER2 receptor is one of four human plasma membrane receptors from the ErbB tyrosine kinase receptor family 1 . HER2 regulates the cellular proliferation and survival of cells by dimerizing with other ErbB receptors and stimulating tyrosine kinase activity 2,3 . Approximately 20-30% of human breast cancers overexpress HER2 receptors by amplification of the HER2/neu gene, which is a marker of aggressive cancer with an unfavorable prognosis that correlates with tumorigenesis and metastasis [4][5][6][7] . The anti-HER2 humanized monoclonal antibody, trastuzumab (Tz), is a first-line biotherapeutic against HER2(+ ) breast cancer 8,9 . However, recent clinical statistics have revealed that the long-term use of trastuzumab can generate trastuzumab resistance in HER2(+ ) tumors 10 . The causes of trastuzumab resistance are not yet fully understood [10][11][12] . The HER2 receptor also exhibits a poor ability to internalize, even after the binding of bioligands, such as anti-HER2 antibodies, HER2-specific antibody fragments, aptamers, and peptides 13 . Poor internalization of HER2 is also a potential drawback to the use of this receptor as a therapeutic target. To overcome the trastuzumab resistance in HER2(+ ) tumors, trastuzumab-based antibody-drug conjugates (ADCs), such as trastuzumab-emtansine (T-DM1), have been developed with the chemotherapeutic drug mertansine directly attached to the antibody, which boosts the cell toxicity. After successful clinical trials, T-DM1 is currently used in the clinic 14,15 . However, by design, ADCs are intrinsically highly toxic and can produce severe side effects due to their long circulatory half-life and non-specific toxicity in healthy tissues 16 . Furthermore, a simple ADC does not provide a mechanism by which to enhance the cellular internalization of therapeutics to maintain a high therapeutic index 17 .</p><p>To circumvent these issues, we have designed a pre-targeting two-component, two-step drug delivery system driven by bioorthogonal click chemistry between the pre-targeting and delivery components. In this strategy, HER2(+ ) cancer cells are pre-labeled by click-reactive trastuzumab, and subsequently, click-reactive, drug-loaded albumin nanocarriers (Alb) are delivered to enhance the internalization of drug carriers after the in situ bioorthogonal cross-linking of components, as shown in Fig. 1. The pre-targeting approach was used for imaging and therapy in lung cancer [18][19][20] . We have also demonstrated the efficacy of HER2 pre-targeted therapy in isolated cells 21 . However, to the best of our knowledge, this is the first demonstration of an anti-HER2 pre-targeted therapeutic strategy in vivo.</p><p>By definition, bioorthogonal reactions are reactions that can occur in living systems at physiological conditions without interfering with regular biochemical and physiological processes. For example, the Staudinger ligation, the copper-free azide/alkyne click reaction, and the trans-cyclooctene/tetrazine cycloaddition have been explored for in vitro and in vivo imaging. Azide (Az)/difluorocyclooctene (DIFO) click chemistry has been used by Bertozzi's group for in vivo imaging in living systems 22 . Kim's group has demonstrated the in vivo imaging of mouse tumor models using Az and dibenzylcyclooctyne (DBCO) click chemistry between metabolically labeled glycans and liposomes 18 . Trans-cyclooctene (TCO)/tetrazine (Tt) cycloadditions have been extensively studied for in vivo imaging experiments because this reaction is extremely fast (3,100-380,000,000 × 10 3 M −1 s −1 ) compared to the Az/DBCO (0.9-4,000 × 10 3 M −1 s −1 ) and Az/DIFO (7.6 × 10 −2 M −1 s −1 ) bioorthogonal click reactions 23,24 . The TCO/Tt click reaction has been used to image nanoparticles in living systems 25 . We have previously used Az/DBCO bioorthogonal click chemistry in two-component drug delivery system to evaluate the strategy in vitro in HER2(+ ) BT-474 cells 21 . We observed the cluster formation and internalization of nanoclusters with confocal fluorescence imaging. Our in vitro therapeutic experiments confirmed the high therapeutic efficacy of a two-component, two-step drug delivery system. Due to its fast kinetics, TCO/Tt cycloaddition was used in this study for the in situ conjugation of the components of a two-component, two-step drug delivery system in a HER2(+ ) human breast cancer mouse model, as shown in Fig. 1. In this strategy, due to the close proximity of overexpressed HER2 receptors on cancer cells and multiple functionalizations of the pre-targeting and delivery components, multiple cross-linking reactions induce the self-assembling of cell membrane-bound nanoclusters in situ. These nanoclusters can be effectively internalized by clathrin-mediated endocytosis 26 . In the present study, we employed the new delivery system in mouse models of HER2(+ ) human breast cancer, evaluated the strategy, and determined whether this system could enhance therapeutic efficacy.</p><!><p>The cellular internalization of components by the two-component strategy was evaluated in HER2(+ ) BT-474 breast cancer cells, using a confocal fluorescence microscope (Supplementary Figure S1A). As shown in Fig. 2A, the cell surface was initially labeled by a Tz(TCO) 6 (AF-488) 4 pre-targeting component. Co-localization of the delivery component, Alb(Peg 4 -Tt) 15 (Rhod) 4 , with the pre-targeting components on the cell surface was detected immediately within 15 minutes after labeling. Upon incubation at 37 °C for 4 h, we observed a rapid internalization of co-localized nanoclusters formed by the two components (Fig. 2B). No cellular internalization was detected after incubation at 20 °C (Fig. 2A). Moreover, neither co-localization nor internalization of components was observed when reactive Tz(TCO) 6 (AF-488) 2 and non-reactive Alb(Rhod) 4 were used as the pre-targeting and delivery components, respectively (Supplementary Figure S2).</p><!><p>We evaluated the tumor uptake and plasma clearance of circulating non-specifically bound components on an in vivo Xenogen optical imaging system (Supplementary Figure S1B). Three groups of mice, including click-treated, mock-treated, and untreated-controls were used in this study. The first two treatment groups, click-treated and mock-treated, were administered pre-targeting components, including a reactive Tz(TCO) 6 (CF-680) 2 (where CF-680 is an NIR fluorophore), and a non-reactive Tz(CF-680) 2 , respectively. Animals were imaged for 12 h to measure the biodistribution of pre-targeting components. Generally, a rapid clearance of both components from the circulation was detected in our models. A significant accumulation of TCO-functionalized and non-functionalized pre-targeting components was detected in the tumors at approximately eight hours. For TCO-functionalized Tz, the degree of functionalization (DOF) of 6 was maintained. There was no significant change in the binding affinity of Tz with HER2 at DOF = 6 (Supplementary Figure S3) 27 . At this time-point, plasma was free of excess pre-targeting components; however, some amount of the component accumulated in the kidneys (Supplementary Figure S4A) and was found in urine, as well. After 24 h, the amount of pre-targeting component in the kidneys had decreased significantly and was completely cleared from the urine (Supplementary Figure S4B). At the eight-hour post-injection, the Alb(Px) 2.6 (Pe g 4 -Tt) 15 (DL-800) 2 therapeutic carrier was administered to the click-treated and mock-treated groups, where Px and DL-800 were paclitaxel and DyLight 800 NIR fluorophore, respectively, while the untreated group received saline. Animals were imaged to track the drug delivery component routinely for two days. During this imaging time-frame, pre-targeting components were still visible in tumors in both click-treated (Fig. 3-i, intensity 1,174 a.u.) and mock-treated (Fig. 3-iii, intensity 1,362 a.u.) mice, and mice in the click-treated group showed a higher tumor uptake of the Alb drug delivery component (Fig. 3-ii, intensity 6,753 a.u.) compared to mice in the mock-treated group (Fig. 3-iv, intensity 3,327 a.u.).</p><!><p>To explore the in vivo cell labeling and cellular internalization of components, we observed the tumor microenvironment using an intravital multiphoton microscope during the treatment (Supplementary Figure S1C). Mice were first administered Tz(TCO) 2 (Rhod) 2 intravenously, and, at eight-twelve hours post-injection, they were imaged after minimally invasive skin-flap surgery (Fig. 4A,B). The surface labeling of cancer cells by pre-targeting components was observed after 10 minutes and did not change significantly for twelve hours (Fig. 4C-i). At the next step, mice were administered Alb(Peg 4 -Tt) 15 (AF-488) 2 intravenously and the imaging was continued for approximately two hours post-injection. The delivery of Alb(Peg 4 -Tt) 15 (AF-488) 2 and the build-up of the fluorescence signal was detected and the signal was co-localized with the pre-targeting agent, Tz(TCO) 6 (Rhod) 2 (Fig. 4C-v). No significant auto-fluorescence was detected in the green channel used to image the drug carrier component (Fig. 4C-ii). The co-localization of components and the internalization of clusters was observed starting at 30 min post-injection, and reached a maximal level after 90 minutes (Fig. 4C-vi). We observed neither co-localization nor internalized nanoclusters in the intravital imaging experiment repeated with reactive Tz(TCO) 6 (Rhod) 2 (Fig. 4D-i) and non-reactive Alb(AF-488) 2 (Fig. 4D-ii).</p><!><p>We evaluated the therapeutic effect based on the change in relative tumor volumes (RTV = tumor volume at day t, V t /V 0 , initial tumor volume), calculated from the tumor dimensions and measured by a caliper, over 28 days, with two doses of treatments (Supplementary Figure S1D). Mice in the click-treated and mock-treated groups were injected with Tz(TCO) 6 (CF-680) 2 and Tz(CF-680) 2 , respectively, and the untreated control group received saline. After eight hours, the first two groups were administered Alb(Px) 2.6 (Peg 4 -Tt) 15 (CF-750) 2 and the mice in the untreated-control group received saline. Mice received the second dose of therapy after 14 days. During the treatment period, the changes in tumor volumes and body weights were examined for 28 days and plotted as a function of time post-injection. The mice in the click-treated group exhibited a significant inhibition of tumor growth, as confirmed by the lowest RTV over the treatment period (Fig. 5A), compared to the mock-treated and untreated-control groups. The corresponding bar chart was used for the statistical analysis of the changes in relative tumor volumes (Fig. 5B). The relative tumor volume in the click-treated group was significantly low compared to the untreated group from day 8 onward (Fig. 5B), and it was significantly low compared to the mock-treated group after day 12 onward. The Kaplan-Meier analysis curves shown in Fig. 5C were obtained using changes in the terminal RTV by a factor of four from the initial tumor size. The highest RTV was detected in the untreated-control group. Visible changes in tumor sizes in different treatment groups are shown in Fig. 5D.</p><p>We also performed a comprehensive study of the toxicological effects in mouse models of BT-474 breast cancer. Mice in the click-treated and mock-treated groups received both components at doses of 0× , 1× , 2× , and 5× following the same treatment schedule used for the therapy (Supplementary Figure S1D). Based on the representative results of the study, there was no body weight loss (Supplementary Figure S5), and only mild thrombocytopenia (Supplementary Figure S6) was detected at 24 h post-treatment dose in the high-dose (5× ) treatment group. However, in this group, the platelet count recovered by factor of five at day 28 of the therapy.</p><!><p>We chose trastuzumab as the target-specific pre-targeting ligand for HER2, since Tz has high target-specificity and binding affinity to HER2 receptors, even after chemical conjugations on its free amine groups 27 . Furthermore, Tz resistance in HER2(+ ) cancer cells does not decrease the degree of HER2 expression 28 . There is no HER2-specific endogenous ligand in the human body for competitive binding affinity with Tz. As a chemotherapeutic, Px has high therapeutic efficacy against solid tumors and can be easily modified and conjugated with drug delivery platforms without altering cytotoxicity 29 . Paclitaxel albumin conjugates are also used in chemotherapeutic regimens (Abraxan) 30 . Serum albumin was chosen as the drug carrier for several reasons. Albumin is an acidic, hydrophilic, and highly stable globular protein. It is stable in a broad pH range (pH 4-9), in 40% ethanol, and at high temperatures (60 °C) without denaturing 31 . Hydrophobic low-molecular-weight chemotherapeutics can be chemically conjugated with albumin without a significant change in the hydrophilicity of albumin in plasma. Drugs encapsulated in albumin exhibit favorable pharmacokinetics with albumin as a drug carrier. Albumin can also be accumulated in solid tumors by the enhanced permeability and retention (EPR) effect; however, our approach was intended to increase the cellular uptake of drug-loaded nanocarriers rather than to enhance the accumulation of nanocarriers in the tumor extracellular microenvironment 32 .</p><p>To synthesize drug-loaded nanocarriers, paclitaxel was first derivatized into an amine reactive sulfo-NHS analogue (sulfo-NHS-paclitaxel), and conjugated with albumin. The conjugation ratio of paclitaxel to albumin is a critical parameter that significantly decreases the hydrophilicity of the delivery component. Thus, the degree of conjugation (DOC) of paclitaxel was chosen at ~2.6 to synthesize the Alb(Px) 2.6 precursor. Paclitaxel was conjugated with albumin by an ester linkage, which is stable in vivo and enables the efficient release of drugs after internalization, followed by acidic or enzymatic cleavage. We evaluated the cell surface labeling and the internalization of nanoclusters in vitro by confocal fluorescence imaging. The poor internalization kinetics of Tz in HER2(+ ) cells increases its availability as a pre-targeting component on the cell surface for multiple click reactions with Alb-based delivery components. Multiple bioorthogonal click reactions in TCO/Tt-based delivery strategy were faster than those for previously used with Az/DBCO system 21 . The co-localization of two components on the cell surface provides efficient click reactions in physiological conditions. We also observed the formation of nanoclusters and their internalization after four hours incubation at 37 °C. The control Alb(Rhod) 4 delivery component, which lacked functional Tt groups, did not react with the pre-targeting component and none of the components were internalized upon incubation (Supplementary Figure S2).</p><p>We explored the tumor uptake of components based on the in vivo fluorescent intensities of the components. Images acquired by Xenogen IVIS optical imaging suggested a high accumulation of pre-targeting Tz components in the tumor. Interestingly, we observed higher tumor uptake of non-reactive Tz(CF-680) 2 in the mock-treated mice compared to the reactive Tz(TCO) 6 (CF-680) 2 in the click-treatment. We suggest that this observation is, in part, due to the slight decrease in the binding affinity of trastuzumab functionalized with TCO (Supplementary Figure S3). In addition, specific click reactions between the components resulted in the formation of cross-linked complexes, which may have further reduced fluorescence by quenching. Finally, cell internalization of the complexes resulted in their fast degradation and clearance of the fluorescent marker observed in the bladder of the click-treated mice (Fig. 3-i).</p><p>Clinically, a relatively long mean serum half-life was reported for unmodified Tz and Tz-based ADCs, T-DM1, of 5.83 days and 4 days, respectively 33 . Both TCO-functionalized and control pre-targeting Tz components exhibited a relatively short circulatory half-life in the plasma in our studies. While the Tz conjugation strategy did not saturate the available amino groups in the antibody, possible changes in the lipophilicity and surface charge of the component would depend on the DOF and the nature of the conjugation groups 34 and might affect the clearance and circulation time of the antibody. In addition, the nude mice used in our study have a functional complement system and mature B-cells. Therefore, humanized trastuzumab antibodies can be recognized and cleared by the host. The in vivo cell surface labeling, cluster formation, and internalization were assessed using intravital multiphoton imaging, which is a powerful technique with which to study dynamic processes in living animals 35 . This technique is particularly appropriate when observing cancer cells, the tumor microenvironment, and the microvascular architecture in tumor models in vivo 36 . Traditional dorsal window chambers are a common accessory used for intravital in vivo imaging studies due to the convenience of focusing and choosing a suitable FOV. However, these chambers allow a narrow time-window for tumor growth and imaging, and create an artificial microenvironment for the tumors. These chambers can be optimized for imaging in subcutaneous tumor models, but are difficult to use with orthotopic tumor models 37 . Therefore, in this study, we used a custom-made mouse-holder equipped with a compression window plate that can fix orthotopic tumors to allow imaging without motion artifacts. This set-up facilitates intravital imaging of cancer cells, the tumor microenvironment, and the vascular architecture after a minimally invasive skin-flap surgery (Fig. 4A). This imaging system was used for the real-time visualization of perfusion and extravasation of drug delivery components in the tumor microenvironment. Intravital imaging demonstrated high uptake and internalization of drug-loaded components in the delivery system driven by bioorthogonal click chemistry. After approximately two hours of skin-flap surgery, the rate of the blood flow in the tumor site was reduced, possibly due to the local thrombosis in the area.</p><p>Mice in the click-treated group showed a higher therapeutic response, likely due to the tumor uptake of drug-loaded nanocarriers by cluster formation and subsequent cellular internalization (Fig. 5A). The mock-treated delivery system included non-reactive variants of both components and did not facilitate tumor uptake, cluster formation, and internalization driven by bioorthogonal click chemistry. However, we did detect an improved therapeutic response in mock-treated mice compared to untreated controls, and this observation was presumably attributable to the combination of nonspecific accumulation of the cytotoxic nanocarrier through the EPR effect and the therapeutic effect of Tz. The therapeutic efficacy in the click-treated group was significantly higher than the treatment effect in the untreated group (after day 8 onward) or in the mock-treated group (after day 12 onward). At the end of the therapeutic study, the click-treated group showed significantly low tumor sizes compared to both control groups (Fig. 5B). The Kaplan-Meier curves based on the time taken by tumors to reach four times the initial size (Fig. 5C) demonstrated a significant effect of the targeted therapy compared to mock-treated and untreated controls. The typical appearance of tumors that shrank in the click-treated group (left) compared to the mock-treated group (right) is shown in Fig. 5D. The nanoclusters of pre-targeting and drug-loaded delivery components were rapidly internalized by tumor cells through a receptor-mediated endocytosis and led to controlled-release of paclitaxel by enzymatic or acidic cleavage of the linker. During the course of the treatment, we observed no significant change in body weights in any group of mice. The analysis of in vivo images also confirmed the liver and kidney uptake of a trace amount of pre-targeting components and a medium amount of drug-loaded nanocarriers at the eight hours post-injection, with no toxicological effects. The liver and kidneys are major sites for metabolism and excretion in the animal body. Drug-induced toxicity in the liver and kidneys could disrupt their functionality and alter the composition of platelets (PLT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alanine transaminase (ALT), and aspartate aminotransferase (AST) in the blood. During the treatment in this study, no significant drug-induced toxicity was detected in long liver and kidney blood panels, even at treatments with 3× and 5× doses.</p><p>Pre-targeted therapy was originally proposed for radioimmunotherapy using biotin-avidin conjugation chemistry [38][39][40][41][42] . A pre-targeting approach was also used for molecular imaging of cancer using specific antibodies and a biotin-avidin in situ multi-step conjugation 43 . Other investigators reported a pre-targeting approach using antibodies and bioorthogonal chemistry for imaging and radioimmunotherapy [44][45][46] . Compared to those previously published reports, our strategy uses a unique combination of specific pre-targeting and cluster formation by multiple click reactions between the pre-targeting and therapeutic carrier components to achieve rapid internalization and delivery of the therapeutic cargo to cancer cells. We also suggest that the cross-linking between neighboring receptors is only plausible on HER2-overexpressing tumor cells; hence, the cluster formation and internalization of drug-loaded nanocarriers are likely not possible in healthy cells. In this approach, the size, location, and stage of tumor can be determined by imaging of the pre-targeting component before the administration of the drug-loaded delivery component. Since trastuzumab is used in this delivery system only as the target-specific molecule, the efficacy of therapy could be high for trastuzumab-resistant tumors as well. This approach also provides direct delivery of therapeutics via HER2-mediated endocytosis, and thus, avoids multi-drug resistance (MDR) transporters 47 . The low pH of subcellular compartments and the enzymatic environment of the cytoplasm trigger the hydrolysis of drug-linkers and controlled-release of Px.</p><p>In summary, we have developed a two-step, two-component drug delivery system driven by bioorthogonal click chemistry and evaluated it in preclinical systems, in vivo. In this strategy, a trastuzumab-based first component was used to pre-label the HER2(+ ) tumor cells and a paclitaxel-loaded albumin carrier was subsequently delivered as the cytotoxic treatment component. This new two-component delivery system showed high accumulation of delivery components in the cancer cells and enhanced therapeutic efficacy. Image guidance can be provided by labeling the components with appropriate imaging agents, and can be used for cancer staging (location, size, and HER2 status) by tracking the low/non toxic pre-targeting component with noninvasive imaging. The results of pre-labeling can be used to make clinical decisions regarding the administration and timing of the cytotoxic drug carrier component with suitable chemotherapeutics to maximize efficacy and minimize non-specific toxicity and side effects.</p><!><p>Biotherapeutics, chemotherapeutics, and chemicals.</p><!><p>Trastuzumab (500 μL of 10 mg/mL in PBS) was treated with TCO-NHS ester (300 moles equiv. in 10-20 μL of dry DMSO) by gently stirring for one hour. Samples were purified by ultracentrifugation followed by HPLC. The resulting Tz(TCO) 6 (500 μL of 10 mg/mL in PBS) was treated with NHS-AlexaFluor 488, NHS-Rhodamine or NHS-CF-680 (10 moles equiv. in 10 μL of dry DMSO) and stirred for one hour. The degree of labeling (DOL) of the fluorophore was maintained at 2-4. The products were purified by ultracentrifugation followed by HPLC. The resultant Tz(TCO) 6 (AF-488) 4 , Tz(TCO) 6 (Rhod) 2 and Tz(TCO) 6 (CF-680) 2 were used as the pre-labeling component in vitro confocal fluorescence imaging, intravital multiphoton confocal imaging, in vivo imaging and therapeutic studies, respectively (Supplementary Figure S7A). For imaging and therapeutic experiments, albumin or drug-loaded albumin 21 (2 mL of 10 mg/mL in PBS) was treated with NHS-Peg 4 -Tt (25 moles equiv. 4.0 mg in 20 μL of dry DMSO) and gently stirred for one hour (Supplementary Figure S7B). Modified albumin was labeled with fluorophores by treating with NHS-Rhodamine, NHS-AlxaFluor 488, DyLight 800, or NHS-CF-750 (10 moles equiv. in 10 μL of dry DMSO). The final products, Alb(Peg 4 -Tt) 15 (Rhod) 4 , Alb(Peg 4 -Tt) 15 (AF-488) 2 , Alb(Px) 2.6 (Peg 4 -Tt) 15 DL-800) 2 , and Alb(Px) 2.6 (Peg 4 -Tt) 15 (CF-750) 2 , were used for in vitro confocal fluorescence imaging, intravital multiphoton confocal imaging, in vivo imaging and image-guided drug delivery studies, respectively. The molecular masses of intermediates at each step were measured by MALDI-TOF (Supplementary Figure S7C). The products were purified by ultracentrifugation followed by HPLC. For therapeutic experiments, albumin was first conjugated with paclitaxel, followed by functionalization with Peg 4 -Tt and labeling with CF-750 according to the procedure described above.</p><p>Ultracentrifugation and HPLC purification. Amicon ultra centrifugation filter units (0.5 mL, 3 kDa, and 15 mL, 30 kDa) were used to concentrate samples and to remove the unreacted low-molecular weight reagents after each step of the conjugation reactions. The samples were further purified by a Waters binary pump/dual absorbance HPLC system equipped with a YMC-Pack Diol-300 (300 × 8.0 mm I.D.; particle size, 5 μm; pore size, 30 nm) size exclusion column, using 0.1 M PBS with 0.2 M NaCl (pH 7.2) as the mobile phase.</p><!><p>The molecular weights of modified proteins were determined by MALDI-TOF (Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine). The DOF of the functional group and the DOC of paclitaxel were calculated based on the change in molecular weights (Supplementary Figure S7C). The DOL of fluorophores was determined following manufacturers' protocols.</p><p>Cell lines. The HER2-overexpressing BT-474 cell line was purchased from the American Type Culture Collection (ATCC). The cells were grown in 46-X medium supplemented with 10% FBS and 1% penicillin-streptomycin according to the manufacturer's protocol, and maintained at 5% CO 2 in a humidified incubator at 37 °C. Cells were confirmed to be free of mycoplasma infection.</p><!><p>A pellet of 17β-estradiol (0.72 mg/90 day release, Innovative Research of America) was implanted in the subdermal space of each healthy, four-to-six-week-old, female Nu/Nu mouse. After approximately 24 h, BT-474 cells at 70-80% confluency, in fresh medium for < 24 h, were collected and prepared with 5 × 10 6 in 50 μL of 46-X medium:Matrigel (1:1) for each inoculation and maintained at 4 °C. Cells were orthotopically inoculated into the 2 nd mammary fat-pads. When tumor volume reached 100-150 mm 3 , mice were used for in vivo and intravital imaging and therapeutic experiments. At the end of the experiments, mice were euthanized according to the protocol. All animal experiments were carried out in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee, and were conducted in strict compliance with all federal and institutional guidelines.</p><p>In vitro confocal fluorescence imaging. BT-474 cells at the third or fourth passage (5 × 10 5 cells/well in 0.5 mL of 46X medium) were placed in four-well chamber slides and grown for 24-48 h to 70-80 confluency. Cells were first incubated with Tz(TCO) 6 (AF-488) 4 (20 μg/mL, 130 nM) in PBS+ (PBS supplemented with 0.5% BSA) at room temperature for 20 min. Pre-labeled cells were treated with reactive Alb(Peg 4 -Tt) 15 (Rhod) 4 or non-reactive Alb(Rhod) 4 and incubated at room temperature for 15 min. Treated cells were then incubated in fresh 46-X medium for 3 h at room temperature, or 37 °C, and fixed by 4% PFA in PBS. Cells were counterstained by Hoechst 33342 (1 μg/mL in dH 2 O) and wet-mounted for confocal microscopic imaging on a Zeiss Axiovert 200 system equipped with an LSM 510-Meta confocal module.</p><p>In vivo Xenogen optical imaging. Mice were injected intravenously with either Tz(TCO) 6 (CF-680) 2 or Tz(CF-680) 2 (0.2 mg in 200 μL of sterile PBS), and imaged using a Xenogen IVIS 200 Optical Imaging system at 5, 15, 30 min, 1, 2, 4, and 8 h post-injection. At 8-10 h post-injection time, there were significant amounts of tumor uptake in the pre-targeting components, while the targeting components were below the detection limit in the systemic circulation. At this time point, mice were injected with a drug-loaded nanocarrier, Alb(Px) 2.6 (Peg 4 -Tt) 15 (DL-800) 2 (2.0 mg in 200 μL of sterile PBS), and we continued to image them to see the tumor uptake of the delivery component.</p><!><p>Tz(TCO) 6 (Rhod) 2 (0.2 mg in 200 μL of sterile PBS). After 8 h, mice were anesthetized with ketamine and acepromazine (i.p. ketamine 100 μg/g body weight; acepromazine 10 μg/g body weight) and placed on the custom-made mouse-holder in the supine position (Fig. 4A). The holder was maintained at 37 °C using a feedback-controlled heating pad. The tumor was disinfected using alcohol-prep and the dorsolateral skin of the tumor was pulled and cut using a forceps and micro surgical scissors. The "U"-shaped skin-flap, with a ~0.5 cm width, was immobilized using stitches. The skin opening treated with saline was covered with a compression plate and imaged through the window on the compression plate, which was parallel to the longitudinal axis. Intravital images were obtained under 10× or 25× magnification using an Olympus FV1000MPE multiphoton laser-scanning microscope. The drug-loaded nanocarrier, Alb(Peg 4 -Tt) 15 (AF-488) 2 or control Alb(AF-488) 2 (2.0 mg in 200 μL of sterile PBS), was injected through a catheter secured to the tail vein and was continuously observed in real-time.</p><p>Therapeutic procedure. Three groups of female Nu/Nu mice, orthotopically inoculated with BT-474 human breast cancer cells, were administered Tz(TCO) 6 (CF-680) 2 , Tz(CF-680) 2 (both intravenously at a dose of 10 mg/kg) or saline, and were considered click-treated, mock-treated control, and untreated control groups (n = 5 each group), respectively. After 12 h, mice in the click-treated and mock-treated groups were administered Alb(Px) 2.6 (Peg 4 -Tt) 15 (CF-750) 2 at a dose of 25 mg/kg, while mice in the untreated group were given saline as a control. The second therapeutic dose was given after two weeks. The sizes of the tumors were measured with a caliper every fourth day. The tumor volume was calculated using the formula (L × W 2 )π/6, where L is the longest diameter (the major axis) and W is the width dimension, which is perpendicular to the major axis.</p><!><p>The statistical analysis between treated and untreated groups was performed using JMP 12.1.0 Statistical Discovery TM from SAS. The significance of therapeutic effects in each pair was analyzed by the nonparametric, multiple comparisons Wilcoxon each pair test. A p value of less than 0.05 was considered significant (*p < 0.05, **p < 0.005).</p>

Scientific Reports - Nature

Selective inhibition of bacterial and human topoisomerases by N-arylacyl O-sulfonated aminoglycoside derivatives

Numerous therapeutic applications have been proposed for molecules that bind heparin-binding proteins. Development of such compounds has primarily focused on optimizing the degree and orientation of anionic groups on a scaffold, but utility of these polyanions has been diminished by their typically large size and non-specific interactions with many proteins. In this study N-arylacyl O-sulfonated aminoglycosides were synthesized and evaluated for their ability to selectively inhibit structurally similar bacterial and human topoisomerases. It is demonstrated that the structure of the aminoglycoside and of the N-arylacyl moiety imparts selective inhibition of different topoisomerases and alters mechanism. The results here outline a strategy that will be applicable to identifying small, structurally defined oligosaccharides that bind heparin-binding proteins with a high degree of selectivity.

selective_inhibition_of_bacterial_and_human_topoisomerases_by_n-arylacyl_o-sulfonated_aminoglycoside

<p>Topoisomerases are enzymes that wind and unwind DNA by breaking and then religating the DNA. Type II topoisomerases, such as DNA gyrase and topoisomerase IV (Topo IV), make a double stranded break in DNA and pass unbroken DNA through the break, creating a net change of two in the linking number.1 Type I topoisomerases, conversely, make single strand breaks that allow the DNA to swivel around the unbroken strand.2, 3 Topoisomerases are targets for antibacterial and anticancer agents.4, 5</p><p>Heparin, a highly sulfated human glycosaminoglycan (GAG) has been shown to inhibit topoisomerase I (Topo I), a type I topoisomerase.6 The polyanionic polysaccharide is thought to mimic the phosphate backbone of DNA and bind to the DNA binding site.6 Heparin and heparan sulfate (HS) have been shown to inhibit type I topoisomerase, while HS has an as-yet unexplained inductive effect on type II topoisomerases.7–9 Inhibition of topoisomerase with fractionated heparin mixtures demonstrated that highly sulfated heparin chains are better at inhibiting Topo I.8 Heparin was also a much more potent inhibitor of Topo I than HS, a less sulfated, though structurally similar GAG.9 Other oligosaccharide and polysaccharide GAG mimics have also been shown to inhibit topoisomerases, including suramin, a hexa-sulfated poly aromatic compound, 10, 11 a sulfated polysaccharide isolated from microalgae in red tide,12 and an unsulfonated dextran derivative that was modified to various degrees by addition of aromatic carboxymethyl-benzylamides.13</p><p>Previous work in our laboratory evaluated replacement of N-sulfo groups on heparin and other GAGs with non anionic-N-acyl groups to afford reduced-charge structures capable of competing with unmodified heparin for binding endogenous HS-binding proteins.14,15 Replacement of N-sulfo groups with select N-aryl groups afforded heparin derivatives with maintained or increased affinity for HS-binding proteins.14–16 As an intracellular target, topoisomerases are not readily accessed by sulfated saccharides. However, there is a significant degree of structural homology among topoisomerases,17 which makes topoisomerase inhibition a rigorous test for evaluating new types of GAG mimics for their ability to selectively interact with structurally similar heparin-binding proteins.</p><p>In this study, a panel of bacterial (E. coli gyrase, E. coli Topo IV) and human (hTopo I and hTopo II) topoisomerases was used to evaluate N-arylacyl O-sulfonated aminoglycoside derivatives for their ability to selectively inhibit the different topoisomerases. The goal of this work was to determine the level of selectivity that could be achieved for these novel trisaccharide and tetrasaccharide heparin mimics to inhibit function of such structurally similar heparin-binding proteins, and to test this strategy for creating novel, small heparin mimics that selectively bind heparin and modulate function of heparin-binding proteins. In addition, the identification of derivatives that inhibit topoisomerases would represent new lead structures for further elaboration as possible anticancer or antibacterial agents.</p><p>Synthesis of the O-sulfonated N-arylacyl aminoglycosides was accomplished in two steps (Scheme 1, Panel A). The aminoglycosides apramycin, kanamycin, and neomycin were first converted to their N-acetyl, N-benzoyl (N-bz), N-benzyloxycarbonyl (N-cbz), and N-phenylacetyl (N-pha) derivatives by coupling with the corresponding N-(acyl) succinimide (NHS ester) or acyl chloride under alkaline conditions. Synthesis and characterization of per N-cbz derivatives of neomycin,18 kanamycin,19 and apramycin20 have been reported. The per-N-acyl aminoglycosides were sulfonated with pyridine sulfur trioxide complex (Pyr·SO3) or chlorosulfonic acid (ClSO3H). The degree of sulfonation for each derivative was determined as previously described (see also Supporting Information for compound synthesis, purification and characterization).21 The acetyl derivatives were not assigned an average degree of sulfation because they lack a chromophore for calculating peak areas with HPLC, and are instead characterized by a range of sulfated states (e.g. "3–5" indicates the sample contains a mixture of tri-, tetra-, and penta-sulfated states).</p><p>The N-arylacyl O-sulfonated aminoglycosides were initially screened to determine inhibition of E. coli gyrase-mediated supercoiling of relaxed DNA and E. coli Topo IV-mediated relaxation of supercoiled DNA and decatenation of kinetoplast DNA (kDNA). (see, Figure 1; methods in supporting information). For inhibition of gyrase-mediated supercoiling, gyrase was incubated with relaxed DNA and either no test compound, or 200 μM of one of the following compounds: ab0, nz7, nb4.6, aa3-5, or ab4.1 (Figure 1A). In the absence of a test compound, E. coli gyrase converted relaxed DNA into supercoiled DNA (lane 2). Of the compounds screened, only nz7 (lane 4) blocked the supercoiling activity of gyrase. For Topo IV-mediated relaxation of supercoiled DNA, Topo IV was incubated with negatively supercoiled DNA in the absence (lane 2) or presence of 200 μM of one of the test compounds: ab0, nz7, nb4.6, aa3-5, or ab4.1. Topo IV-mediated relaxation of supercoiled DNA was inhibited by both nz7 (lane 4) and nb4.6 (lane 5) (Figure 1B). While nz7 inhibited both supercoiling by gyrase and relaxation by Topo IV, nb4.6 only inhibited topo-IV-mediated relaxation. For Topo IV-mediated decatenation of kDNA, E. coli Topo IV was incubated with kDNA and 200 μM of one of the test compounds (Figure 1C). Only nz7 completely blocked the decatenation reaction (lane 4), while nb4.6 partially inhibited decatenation (lane 5). Thus, Both nz7 and nb4.6 inhibited Topo IV activity in both relaxation and decatenation assays, though the inhibitory effect of nb4.6 was more pronounced in the relaxation assay.</p><p>The observed differential inhibition of bacterial type-II topoisomerases by select N-arylacyl O-sulfonated aminoglycosides may be due to the degree of sulfation, the N-arylacyl moiety, the core aminoglycoside, or a combination these structural differences. As shown in Figure 1A, nz7 inhibited the gyrase-mediated supercoiling reaction. Since more highly sulfated heparin derivatives are more potent inhibitors of topo I8, N-cbz O-sulfonated neomycin with an average degree of sulfation of 5.6 (nz5.6) was screened along with persulfated nz7 to determine concentration-dependent inhibition of gyrase- mediated supercoiling of relaxed DNA (Figure 1D). Although 100 μM of nz5.6 only partially inhibited gyrase activity (lane 6), 200 μM of nz5.6 completely inhibited the supercoiling activity of gyrase (lane 7). Comparatively, 50 μM of nz7 partially inhibited gyrase (lane 11), while 100 μM of nz7 completely inhibited gyrase supercoiling activity (lane 12). Thus, nz7 is about twice as potent as nz5.6. While this result demonstrates the importance of sulfate groups for these particular neomycin derivatives to inhibit gyrase, the overall data in Figure 1 clearly shows the influence of N-arylacyl moieties and the core aminoglycoside for selective inhibition of the bacterial type-II topoisomerases.</p><p>To more fully evaluate the role of sulfate groups, core structure and N-arylacyl group on selective topoisomerase interactions, the complete panel of N-arylacyl and N-acetyl aminoglycosides, both O-sulfonated and bearing no sulfate, were screened for inhibitory activity with four topoisomerases: E. coli gyrase, E. coli Topo IV, hTopo II and hTopo I (Table 1). None of the unsulfated N-arylacyl aminoglycosides (azo, kb0, kz0, kp0, na0, nz0, np0) inhibited any topoisomerase (data not shown). The presence of sulfate groups on the N-acyl aminoglycosides was not, alone sufficient to afford topoisomerase inhibition; neither of the N-acetyl O-sulfonated aminoglycosides (aa3-5, na1-6) inhibited any of the topoisomerases. Indeed, sulfated dextran oligosaccharides with a degree of polymerization of 14 are capable of inhibiting hTopo-I.22 Some DNA-binding and heparin-binding proteins, such as fibroblast growth factor-I, will bind persulfated disaccharides like sucrose octasulfate and sulfonated polyaromatics like suramin with weak affinity. However, most DNA-binding and heparin-binding proteins have poor affinity for sulfated polysaccharides less than octasaccharide in length.</p><p>Only select N-arylacyl O-sulfonated aminoglycoside that have both N-arylacyl groups and sulfate groups on a certain aminoglycoside scaffolds inhibited different topoisomerases to different degrees (Table 1Figure 1). This result is consistent with previous studies for , N-desulfonated, N-acyl derivatives of heparin, where only select N-arylacyl groups could be substituted in place of N-sulfo groups to maintain or increase affinity for select heparin-binding proteins.14,15,16 In addition to the aryl groups on these tri- and tetra-saccharide-sized compounds being necessary for topoisomerase inhibition, these results show that both the structure of the core aminoglycoside and the structure of the N-arylacyl moiety imparts selectivity for which of the topoisomerase(s) are inhibited. For example, among the N-cbz O-sulfonated aminoglycosides, only az4.9 showed greater than 90% inhibition of all four topoisomerases tested (Table 1). The N-cbz kanamycin derivative kz6, which is more highly sulfated than az4.9, showed 75% inhibition of gyrase and greater than 95% inhibition of hTopo I, but did not inhibit either Topo IV or hTopo II. The cognate N-cbz neomycin derivative, nz5.6 showed greater than 95% inhibition of gyrase, hTopo II, and hTopo I, but it did not inhibit Topo IV.</p><p>To better understand the mechanism of topoisomerase inhibition, the N-arylacyl O-sulfonated aminoglycosides were coadministered with ciprofloxacin to evaluate their ability to interfere with ciprofloxacin-induced DNA cleavage. Ciprofloxacin is a known topoisomerase poison that, when incubated with a bacterial type II topoisomerase and DNA, induces DNA cleavage by arresting the enzyme and its DNA substrate in a ternary complex that prevents religation of the double stranded break in the DNA.23–2523–25</p><p>Incubation of E. coli Topo IV with a plasmid DNA affords mainly supercoiled DNA, and small amounts of nicked and linear DNA (Figure 2, lane 2). Addition of ciprofloxacin stimulates the production of linear, cleaved DNA (Figure 2, lane 3). Ciprofloxacin-induced DNA cleavage is completely blocked by nz7 (Figure 2, lane 5), while ab0 and nb4.6 produce a slight decrease (Figure 2, lanes 4 and 6). No effect on ciprofloxacin-induced DNA cleavage was seen with aa3-5 or ab4.1 (Figure 2, lanes 7 and 8).</p><p>The effect of N-arylacyl O-sulfonated aminoglycosides on ciprofloxacin-induced DNA cleavage by gyrase was similarly investigated (Table 2). With gyrase, kz6 and nz5.6 completely blocked the production of ciprofloxacin-induced linear DNA while np5.9, kb6.4 and az4.9 partially blocked ciprofloxacin-induced DNA cleavage. These results indicate that, despite the structural homologies between gyrase and Topo IV17, compounds az4.9, kb6.4 and np5.9 block ciprofloxacin-induced DNA cleavage with Topo IV and not with gyrase, and thus may have different binding sites on Topo IV and gyrase. In the absence of ciprofloxacin, the N-arylacyl O-sulfonated aminoglycosides did not induce production of linear DNA, and thus they do not act as topoisomerase poisons (see Supporting Information). This unique ability of some N-arylacyl O-sulfonated aminoglycosides to block ciprofloxacin-induced DNA cleavage is unexpected and unusual.</p><p>To further test for mechanistic differences, kb6.4 and kz6, were compared for their ability to inhibit the supercoiling activity of E. coli gyrase and to block ciprofloxacin-induced DNA cleavage (Figure 3). At 20 μM neither kb6.4 nor kz6 inhibited gyrase supercoiling activity (Figure 3A, lanes 3 and 5) while at 200 μM both inhibited the supercoiling activity of gyrase (Figure 3A, lanes 4 and 6). In contrast, kb6.4 showed no apparent effect on ciprofloxacin-induced cleavage of DNA (Figure 3B, lanes 3 and 5) while kz6 blocked the effect of ciprofloxacin on gyrase-mediated DNA cleavage (Figure 3B, lanes 3 and 7). This suggests that inhibition of gyrase by kz6 and kb6.4 may occur at different steps during the gyrase--mediated supercoiling reaction. One possible explanation for this result would be that kb6.4 does not inhibit the binding of the G-segment but it inhibits either the binding of the T-segment or the binding of ATP. In contrast, kz6 and other oligosaccharides inhibit the supercoiling activity of gyrase by blocking the binding of the G-segment.</p><p>Studies are currently underway to evaluate selective binding of these novel small molecule heparin mimics with extracellular heparin-binding proteins that play a role in a number of disease states. In addition, while the different topoisomerases were employed in this study as a class of test proteins to evaluate selective interaction of test compounds with structurally similar heparin-binding proteins, select compounds possess unexpectedly unique activity. Although these N-arylacyl O-sulfonated aminoglycosides do not appear to readily enter human or bacterial cells, as evidenced by a lack of cytotoxicity at 200 μM (see supporting information), understanding how these novel agents uniquely bind and inhibit the different topoisomerases will further the design, synthesis and development of smaller, lower charge structures that might penetrate cells and have therapeutic utility as novel topoisomerase inhibitors. The apparent lack of cellular penetration and cytotoxicity for the present compounds is a positive characteristic in the context of pursuing agents to selectively bind and modulate activity of extracellular heparin-binding proteins.</p>

PubMed Author Manuscript

Nanoscale Metal-Organic Frameworks for Phototherapy of Cancer

Phototherapy involves the irradiation of tissues with light, and is commonly implemented in the forms of photodynamic therapy (PDT) and photothermal therapy (PTT). Photosensitizers (PSs) are often needed to improve the efficacy and selectivity of phototherapy via enhanced singlet oxygen generation in PDT and photothermal responses in PTT. In both cases, efficient and selective delivery of PSs to the diseased tissues is of paramount importance. Nanoscale metal-organic frameworks (nMOFs), a new class of hybrid materials built from metal connecting points and bridging ligands, have been examined as nanocarriers for drug delivery due to their compositional and structural tunability, highly porous structures, and good biocompatibility. This review summarizes recent advances on using nMOFs as nanoparticle PSs for applications in PDT and PTT.

nanoscale_metal-organic_frameworks_for_phototherapy_of_cancer

1. Introduction<!>2. PDT<!>2.1 Optimization of PSs for PDT<!>2.2 Improving the localization of PSs in tumor<!>3. nMOFs for PDT<!>3.1 The first nMOF for PDT<!>3.2 The first Chlorin-based nMOF for PDT<!>3.3 Pegylated nMOF for PDT<!>3.4 Targeted delivery of nMOF for in vitro PDT<!>3.5 nMOFs for controlled singlet oxygen generation<!>3.6 BODIPY-containing nMOF for PDT<!>3.7 nMOF for combined PDT and immunotherapy<!>4. nMOFs for PTT<!>4.1 nMOF-enabled PTT<!>4.2 nMOF-combined PTT<!>4.3 nMOFs for combined PDT and PTT<!>5. Conclusions and perspectives<!>

<p>Phototherapy dates back to ancient times [1, 2]. In 1903, Niels Ryberg Finsen was awarded the Nobel Prize in Physiology or Medicine for using short wavelength light to treat lupus vulgaris[3, 4], which was regarded as the beginning of modern phototherapy. Today, phototherapy is widely used to treat various diseases, such as atopic dermatitis [5], psoriasis [6], vitiligo [7], acne vulgaris [8, 9], and cancer [10]. However, due to the poor tissue penetration of light, pure phototherapy can only be used for superficial treatment. To enhance phototherapy, photosensitizers (PSs) are typically used to sensitize singlet oxygen generation in photodynamic therapy (PDT) and photothermal responses in photothermal therapy (PTT). Light of a specific wavelength is used to activate PSs to improve the therapeutic effects of PDT and PTT.</p><p>PDT treatment relies on three intrinsically nontoxic components: PSs, light, and tissue oxygen [11–14]. Irradiated by the light of an appropriate wavelength, a PS is promoted to the excited state, which reacts with nearby oxygen to generate reactive oxygen species (ROS), particularly singlet oxygen (1O2). By localizing both the PSs and the light exposure to tumor regions, PDT can selectively kill tumor cells while preserving surrounding normal tissues. As a result, PDT has been used to treat many different kinds of cancers, including esophageal cancer, non-small cell lung cancer, and head and neck cancer [14–18]. Porphyrin and its derivatives are the most commonly used PSs for PDT, and several of them, including PHOTOFRIN®, VERTEPORFIN®, FOSCAN®, PHOTOCHLOR®, and TALAPORFIN®, have been approved for clinical use [16, 19, 20]. Despite their excellent photochemistry properties for ROS generation, these PSs have suboptimal tumor accumulation after systemic administration and tend to aggregate in solution due to their hydrophobicity, limiting the efficacy of PDT in the clinic [21]. Therefore, there is an ongoing need for effective delivery of PSs to tumors to improve the therapeutic effects of PDT.</p><p>In PTT, a PS is activated by light to the excited state, which then releases vibrational energy as heat to elevate local temperature above 40 °C to kill targeted cells. With moderate dark toxicity and light-activated cytotoxicity, PTT is also a local therapy with the ability to selectively damage tumors over normal tissues [22]. Because PTT treatment does not relies on local oxygen, it can be used to treat hypoxic cancers, which are typically non-responsive to PDT treatment. However, when normalized on the same energy of light irradiation, PTT is less effective than PDT due to lower cytotoxicity of the generated heat in comparison to the generated 1O2 [23]. To further enhance the PTT efficacy, many powerful PSs, such as plasmonic gold nanorods [24] and phthalocyanin [25], have been developed. Selective delivery of PSs to tumor sites is also needed for more effective PTT treatment.</p><p>Nanoscience and nanotechnology have undergone rapid development over the past twenty years, with numerous types of nanomaterials now readily available. Nanoparticles (NPs) have provided a novel approach for enhanced cancer imaging and therapy due to their tunable sizes, modifiable surfaces, good biocompatibility, high agent loadings, and most importantly, the ability to preferentially deposit in tumors via the enhanced permeability and retention (EPR) effect [26–28]. The delivery of NPs to tumors can be further enhanced via active targeting of the receptors that are overexpressed on cancer cells [15, 29, 30]. Generally, nanomaterials can be broadly classified into three categories: (1) purely organic nanomaterials, such as micelles [31], liposomes [32], dendrimers [33], and polymeric hydrogel NPs [34]; (2) purely inorganic nanomaterials, such as metal NPs [24], metal oxide NPs [35], quantum dots [36], zeolites NPs [37], silica NPs [38], up-conversion NPs [39], and carbon nanomaterials [40]; (3) hybrid nanomaterials constructed via coordination bonds between inorganic and organic components, such as nanoscale metal-organic frameworks (nMOFs) and nanoscale coordination polymers (NCPs) [41–43].</p><p>MOFs are a new class of crystalline, porous hybrid materials constructed from metal-containing nodes, also known as secondary building units (SBUs), bridged by organic linkers. The archetype of MOFs dates back to Prussian blue (PB), a blue pigment with a cubic crystal structure constructed via Fe(II)-CN-Fe(III) coordination bonds [44]. MOFs have been intensely studied since the 1990s, and as a result of their tunability, nearly 20,000 MOFs with different structures have already been reported to date [43]. MOFs have been explored for diverse applications, including gas storage/separation [45–50], magnetism [51], nonlinear optics [52, 53], ferroelectricity [54], conductivity/semiconductivity [55–57], chemical sensing [58–62], catalysis [63–68], and energy conversion [69–71]. The Lin group pioneered in scaling down MOFs to the nanoscale and explored their potential in biomedical imaging [72–75] and drug delivery [41, 76–79]. In this review, we provide an overview on PDT and PTT and summarize recent advances in the applications of nMOFs in PDT and PTT of cancers.</p><!><p>PDT is an effective anticancer treatment that involves the administration of a tumor-localizing PS followed by light irradiation to generate highly cytotoxic ROS. Irradiated by light, a PS is first activated from the ground singlet state (S0) to the excited singlet state (S1). The PS at the S1 state can undergo three different processes: decay to the ground state to generate fluorescence, vibrational relaxation to generate heat, or intersystem crossing to the triplet excited state (T1) through a change in the electron spin orientation [80–82]. After intersystem crossing, the PS in the T1 state can either decay to its ground state through emitting phosphorescence or exert PDT process by reacting with nearby triplet state molecules, particularly molecular oxygen (3O2), via a Type-I process to generate radical species, including superoxide anions (O2−), or a Type-II process to generate 1O2 [13, 14, 83, 84]. Most PDT treatments are believed to go through the Type-II process.</p><p>In a PDT treatment, a PS agent is first injected into the bloodstream of a patient, which is distributed throughout the whole body but is retained longer in cancer cells than in normal cells. The tumor site is then exposed to light 24 to 72 h after PS injection, when most of the PS agents have left normal tissues but significant amounts of them still remain in cancer cells. The PS agents accumulated in the tumors absorb the light and generate ROS, mainly 1O2, to destroy the cancer cells. Clinical studies have shown that PDT can treat certain types of cancers and pre-cancers, including head and neck cancer, mesothelioma, and pancreatic cancer. Compared to conventional chemotherapy and radiation therapy (RT), PDT has several advantages: (1) PDT is highly selective and less-invasive than surgery with little to no long-term side effects; (2) by localizing both the PSs and the light exposure to tumor regions, PDT can be targeted very precisely to selectively kill tumor cells while preserving local tissues; (3) PDT treatment usually takes only a short time and can be repeated many times at the same site if needed with little or no scarring after the site heals; (4) PDT does not have cross resistance with other therapies, such as chemotherapy, RT, immunotherapy, and surgery; (5) PDT is most often performed on an outpatient basis and typically costs less than other cancer treatments.</p><p>Despite these advantages, PDT has not become a mainstream cancer therapy due to several severe drawbacks: (1) PDT cannot be used to treat large or deep-seated tumors because light cannot penetrate deeply (< 1 cm) through tissues; (2) As a local treatment, PDT cannot be used to treat cancers that have metastasized; (3) PSs used for PDT can leave patients very sensitive to light for a period of time, necessitating special precautions before and after PDT treatment. Significant efforts have been devoted to address these limitations of PDT over the past few decades, and many of these efforts were directed at improving two aspects of PDT: to discover or synthesize more powerful PSs and to improve the localization of PSs in tumors.</p><!><p>PSs have been optimized to exhibit appropriate excitation wavelengths in order to increase tissue penetration for high PDT efficacy. The tissue penetration of light is wavelength dependent with the deepest tissue penetration at 800 nm. Generally, only PSs with the excitation wavelength located in the "tissue transparent" window of 650–800 nm, which have moderate tissue penetration of 3–10 mm, are regarded as having potential for clinical use [82, 85, 86]. In addition to this excitation wavelength requirement, there are several other guidelines for the design of clinically useful PSs: (1) the PS must have a strong absorption along with a high extinction coefficient (ε) in the "tissue transparent" window; (2) the PS must have no or minimal dark toxicity; (3) the PS must be chemically stable, photostable, and soluble or well dispersible in cellular environment; (4) the PS must be preferentially taken up and retained by tumor cells. Based on these design criteria, PSs have already evolved for three generations over the past few decades. Porphyrin-based PSs have been shown to be among the best PSs for PDT in the clinic.</p><p>Porphyrins refer to a series of heterocyclic macrocycles composed of four modified pyrrole subunits interconnected at their α carbon atoms via methylene bridges [86]. The aromatic porphyrin, with a total of 26 electrons in the conjugated system, has two groups of absorption bands: a sharp intense absorption band in the range of 390–425 nm, termed as the Soret band, and a set of relatively weak absorption bands in the range of 500–700 nm, termed as the Q-bands [87, 88]. Generally, in the case of porphyrin-based PSs, the last Q-band located in "tissue transparent" window is used to absorb light for PS excitation in PDT.</p><p>The first generation PSs were represented by clinically used Photofrin®, which is also called Porfimer or hematoporphyrin Derivative (HpD). Hematoporphyrin (Hp) was discovered in 1841 and the investigation of HpD in the 1970s and early 1980s led to its clinical approval as Photofrin® for PDT in bladder cancer, esophageal cancer, and early non-small cell lung cancer [89–92]. The first generation PSs are non-ideal with relatively short wavelength excitation at 630 nm, weak absorption at 630 nm, strong and persistent skin photosensitization, low tumor uptake, and relative poor tissue penetration.</p><p>Late 1980s witnessed the development of the second generation PSs represented by 5-aminolevulinic acid (ALA) and porphyrin derivatives, including chlorins, benzoporphyrins, phthalocyanines, and naphthalocyanines [13, 92, 93]. The second generation PSs feature near-infrared absorption, which affords deeper tissue penetration and extremely high absorption, with more than an order of magnitude higher molar ε's than the first generation PSs. In addition, the second generation PSs alleviate skin photosensitization. However, the second generation PSs still suffer from suboptimal tumor accumulation and poor aqueous solubility or dispersity. Many of the second generation PSs require organic hydrotropes for administration in the clinical use, which can cause unwanted side effects. Nanotechnology has been used to deliver the third generation PSs, with the goal of improving tumor accumulation and eliminating organic hydrotropes.</p><!><p>In order to selectively localize PSs in tumors and improve the solubility/dispersity of the second generation PSs, the third generation PSs emerged in 2000's by conjugating PSs with biologic components or delivering the PSs with nanocarriers [85, 94]. Two different strategies have been used to design the third generation PSs for the targeted delivery of PDT agents. The first strategy is to conjugate PSs to biological targeting molecules, including carbohydrate [95], proteins [96–98], peptides [99, 100], and antibodies [101], to realize active targeting of PSs. The second strategy is to encapsulate the PSs into porous nanocarriers, or to load the PSs on the surfaces of nanocarriers by physical absorption or covalent interactions, to realize passive targeting of PSs through the EPR effect [102, 103]. These nanocarriers can be broadly classified into two categories: organic nanomaterials such as liposomes [104–106] and dendrimers [107, 108], and inorganic nanomaterials such as gold NPs [109, 110] and silica NPs [111–113]. Meanwhile, the surfaces of these nanocarriers can be further modified with targeting molecules to actively target cancer cells. After accumulating in tumor cells, the PSs are slowly released from the nanocarriers and then irradiated by light to exert PDT effects.</p><p>Although the third generation PSs improve tumor accumulation and aqueous solubility/dispersity of PSs, they have their own limitations. First, when in close proximity, the excited states of PSs on the NPs will self-quench, leading to diminished PDT efficacy. Second, a large fraction of the generated ROS inside NPs cannot diffuse out of the NPs to reach intracellular targets for PDT effects, due to the short ROS diffusion length (20–220 nm for 1O2 inside the cells). Third, the timing for light irradiation can be very difficult to optimize for NP-based PSs due to the necessity of balancing NP accumulation in tumors and the release of PSs from the NPs. As a result, no particle-based PSs have yet been approved for clinical use in spite of numerous efforts devoted to the development of the third generation PSs over the past decade.</p><p>To address the issues faced by NP PSs, the concept of nanophotosensitizers (nPSs) was proposed in 2011 to directly incorporate PSs into NPs as construction units rather than encapsulating PSs as cargoes [114, 115]. Compared to typical PSs delivery with NPs, nPSs do not need to release PS molecules to exert PDT effects; instead, nPSs work as a whole PS to generate ROS, and the generated ROS can diffuse out of the matrix of the nPS to reach cellular or subcellular targets. Ideal nPS should simultaneously have high PS loadings without self-quenching, enhanced ROS generation efficiency over molecular PSs, and efficient diffusion of ROS from the nPS matrix to the cellular environment to cause cytotoxicity. Although some nPSs, such as porphysome [114] and peptide self-assembled PSs [116–118], have been studied, no nPS met all of these requirements until the discovery of nMOF-based nPSs. We will discuss the design and applications of nMOFs as nPSs in PDT in the next section.</p><!><p>Constructed via coordination bonds between metal cluster secondary building units (SBUs) and bridging ligands, MOFs have emerged as a new class of hybrid materials with tunable, crystalline, and porous structures. These features make MOFs excellent candidates as nanocarriers for imaging contrast agents and therapeutic cargoes. Ideal sizes of nanocarriers should be larger than 10 nm to avoid rapid renal clearance, and smaller than 200 nm for efficient extravasation and optimum EPR effect [26–28]. Controlling the morphologies and sizes of MOF particles is a non-trial task due the intrinsic complexity of MOF compositions and typical MOF synthesis conditions. A number of factors are involved in MOF growth, including the concentrations and ratios of precursors (metal and organic linkers), the types and concentrations of modulators, solvents, temperature, water, and surfactant, making it difficult to predict and precisely control the morphologies and sizes of nMOFs. Through trials and errors, several synthetic methods, including solvothermal, surfactant-templated, and reverse microemulsion synthesis, have been demonstrated to be effective in nMOF synthesis [41]. The successful synthesis of these nMOFs provides the foundation for the rational design of nMOFs with desired structures, compositions, morphologies, and particle sizes in the future.</p><p>By judicious choices of functional SBUs or/and bridging ligands, a number of nMOFs have been designed and explored for biomedical imaging, including magnetic resonance imaging (MRI) [73, 74], computed tomography (CT) [119, 120], optical imaging [121] and sensing [122, 123], and drug delivery, including chemotherapeutic agents [124–126], PDT and PTT agents [127–154], nucleic acid [124, 155], and gas molecules such as nitric oxide [156–158]. Compared to purely organic and inorganic nanocarriers, nMOFs have several advantages: (1) high porosity of nMOFs permits a high payload of various diagnostic and therapeutic agents; (2) structural and compositional diversity of nMOFs allows for different sizes and morphologies, various chemical properties, and multiple functionalities; (3) bio-degradability of nMOFs alleviates the concern of long-term toxicity.</p><p>Compositional and structural tunability of nMOFs has also allowed for the loading of imaging or/and therapeutic cargoes through several different methods: encapsulation into the nMOF channels, attachment to the ligands or the SBUs, or direct incorporation as nMOF linkers or metal nodes. The encapsulation method is the simplest and most frequently used, but premature cargo release can be a concern. Attachment to the linkers or metal nodes provides a nice option for postsynthetic loading if the attachment strength is appropriate to balance the loading and release. The most unique feature of nMOFs in cargo delivery lies in the ability to directly incorporate the imaging or therapeutic agents into metal nodes or linkers, which is particularly important for designing nMOF PSs for PDT. The PSs can be derivatized to become organic linkers for the construction of nMOFs as nPSs. In the PDT process, the PSs do not need to be released form the matrix of nMOF to exert PDT effects. The nMOF nPSs can absorb light to generate ROS, which can rapidly diffuse out of nMOFs due to the highly porous structures.</p><p>As mentioned in the previous section, nMOFs are the most promising nPSs and have the potential of becoming the fourth generation PSs. Compared to other nPSs, the porous and crystalline structures of nMOFs isolate PSs from each other to avoid self-quenching of PS excited states. As a result, nMOFs can realize high PS loadings without suffering from self-quenching. Biodegradability of nMOFs alleviates long term toxicity whereas tunable compositions and structures allow for the optimization of nMOF nPSs for PDT applications. Since the first report on PDT with nMOFs in 2014 [127], at least 13 more papers on PDT of nMOFs have appeared in the literature [127–142, 159], highlighting tremendous interest in the use of nMOFs for PDT. Table 1 summarizes all of nMOFs that have been examined for PDT to date. We will discuss representative works in the following section.</p><!><p>In 2014, the Lin group first reported a Hf-porphyrin nMOF, DBP-Hf, as a highly effective photosensitizer for PDT of resistant head and neck cancer [127]. DBP-Hf was constructed from Hf4+ and 5,15-di(P-benzoato)-porphyrin (H2DBP) via a solvothermal method in dimethylformamide (DMF). DBP-Hf was first assigned as a typical UiO structure in the original paper based on its powder X-ray diffraction (PXRD) pattern, but was recently re-assigned as a new Hf-MOF structure with Hf12 cluster SBUs [160]. DBP-Hf displayed plate morphology of approximately 100 nm in diameter and 10 nm in thickness (Fig. 1a–b), which facilitates the diffusion of 1O2 from the interior of DBP-Hf to the cell cytoplasm to exert cytotoxic effects. DBP-Hf carried exceptionally high PS loading of 77 wt% and processed 2.8 times better 1O2 generation efficiency than H2DBP according to the Singlet Oxygen Sensor Green (SOSG) assay (Fig. 1c). Comparing to H2DBP, DBP-Hf not only overcame the aggregation issue of hydrophobic PSs, but also alleviated the self-quenching of PSs due to site isolation in the crystalline DBP-Hf structure. In addition, the highly porous structure of DBP-Hf facilitated the diffusion of generated 1O2.</p><p>Consequently, DBP-Hf exhibited greatly enhanced PDT efficacy both in vitro and in vivo, when investigated in SQ20B head and neck cancer cells. By incubating SQ20B cells with DBP-Hf (30 µg/mL) for 4 hours, up to 30% DBP-Hf was taken up by the tumor cells to afford a high intracellular DBP-Hf concentration of ~1 mg/mL, which proved DBP-Hf could be effectively accumulated in tumor cells to achieve a high PS concentration. After incubation with free H2DBP ligand and DBP-Hf at various concentrations, the cells were illuminated under 640 nm for 15 mins or 30 mins to investigate the cytotoxicity. Significant PDT efficacy was observed in the DBP-Hf treated group, while the H2DBP treated group only showed moderate PDT efficacy (Fig. 1d). Meanwhile, no cytotoxicity was observed in dark control or blank control groups. DBP-Hf also exhibited significant in vivo efficacy, as evidenced by complete tumor eradication in half of the mice receiving a single DBP-Hf dose (3.5 mg/kg, local administration) and a single light exposure (630 nm LED, 100 mW/cm2 for 30 min) (Fig. 1e).</p><!><p>Despite the excellent performance in pilot animal studies, the photophysical properties of DBP-Hf are not optimal. DBP-Hf has the lowest energy absorption of 634 nm, which is near the high-energy edge of the tissue-penetrating window (600–900 nm), and a relatively small ε of 2200 M−1·cm−1. In molecular PS design, reduction of porphyrins to chlorins has been shown to shift the absorption to a longer wavelength with a concomitant increase in ε. Based on this rationale, Lin and co-workers designed the first chlorin-based nMOF, DBC-Hf, with much improved photophysical properties and PDT efficacy [128].</p><p>5,15-di(p-benzoato)chlorin (H2DBC) was first synthesized by reduction of H2DBP with toluenesulfonhydrazide, and then treated with HfCl4 and acetic acid in DMF at 80°C to afford a new nMOF, DBC-Hf. DBC-Hf had the lowest-energy Q band of 646 nm, which was red-shifted by 12 nm relative to DBP-Hf. In addition, DBC-Hf had a ε value of 24600 M−1·cm−1 for the lowest-energy Q band, which was 11-fold greater than that of DBP-Hf (Fig. 2b). DBC-Hf adopted the same plate morphology and topology structure as DBP-Hf as shown by transmission electron microscopy (TEM) images (Fig. 2a) and powder X-ray diffraction (PXRD) pattern. The plate diameters were 100–200 nm, while the thickness varied from 3.3 to 7.5 nm, which can further facilitate ROS diffusion during PDT compared to DBP-Hf (~10 nm in thickness). Consequently, DBC-Hf was approximately 3 times as efficient as DBP-Hf in generating 1O2 based on SOSG assay (Fig. 2c).</p><p>The superior PDT efficacy of DBC-Hf was demonstrated in two colorectal cancer mouse models, HT29 and CT26. DBC-Hf, DBP-Hf, or two PS ligands were intratumorally injected into mice at a ligand does of 1 mg/kg. Upon light irradiation (650 nm LED, 100 mW/cm2 for 15 min per fraction) for four times, only DBC-Hf effectively suppressed the tumor growth in both models. With higher ligand doses (3.5 mg/kg) and light irradiation (30 min per fraction), DBC-Hf successfully eradicated tumors in the HT29 model with a single treatment and in the CT26 model with two treatments (Fig. 2d–e). Interestingly, DBC-Hf-based PDT was discovered to induce both apoptosis and immunogenic cell death to contribute to the superior PDT efficacy.</p><!><p>In 2016, Liu and coworkers reported the Hf-TCPP nMOF (where TCPP is 5,10,15,20-tetrakis(4-carboxylphenyl)-porphyrin) of MOF-525 structure and demonstrated the ability to coat Hf-TCPP with polyethylene glycol (PEG) for efficient PDT efficacy upon intravenous injection [131]. Hf-TCPP was synthesized from Hf4+ and TCPP via a solvothermal method in DMF (Fig. 3a–b), displaying spherical morphology of approximately 130 nm in diameter based on DLS. PEG-grafted poly(maleicanhydride-alt-1-octdecene) (C18PMH-PEG) was used to encapsulate hydrophobic Hf-TCPP to afford Hf-TCPP-PEG with good water dispersity. Hf-TCPP-PEG exhibited similar photophysical properties and 1O2 generation efficacy as porphyrin nMOFs, such as DBP-Hf. Hf-TCPP-PEG also showed enhanced in vitro PDT efficacy against 4T1 cells.</p><p>To understand the in vivo behaviors of Hf-TCPP-PEG after intravenous injection, blood circulation, biodistribution, and clearance were studied in mice. After i.v. injection of Hf-TCPP-PEG in healthy Balb/c mice (Hf: 12.5 mg/Kg, TCPP: 24 mg/kg), blood was drawn from the mice at different time points to determine the Hf-TCPP-PEG concentration by quantifying the TCPP fluorescence. Hf-TCPP-PEG had a blood circulation half-life of ~ 3.27 h (Fig. 3c). Biodistribution and clearance of Hf-TCPP-PEG were investigated in 4T1 tumor-bearing mice by sacrificing the mice at different time points to determine the concentrations of Hf-TCPP-PEG in different organs. Hf-TCPP-PEG had a tumor accumulation of ~ 7% ID/g at 12 h post injection (Fig. 3d) with the majority of Hf4+ cleared in 7 days.</p><p>Hf-TCPP-PEG showed in vivo PDT therapeutic effect on 4T1 breast tumor model. 20 h post intravenous injection (Hf: 12.5 mg/Kg, TCPP: 24 mg/kg), the tumor sites of the mice were irradiated with laser (661 nm, 5 mW/cm2) for 1 h. In comparison to the control group, Hf-TCPP-PEG could partially suppress the tumor growth.</p><!><p>Because ROS indiscriminately kills both diseased and normal cells, it is important to selectively accumulate the PSs in tumors to enhance the PDT efficacy. Zhou and coworkers modified the surface of Zr-based porphyrin nMOF, PCN-224, with folic acid (FA) to increase cell uptake of nMOFs and demonstrated higher in vitro PDT efficacy with FA-modified PCN-224 [134].</p><p>PCN-224 with a sphere morphology was constructed from a solvothermal reaction between Zr4+ and H4TCPP in the presence of benzoic acid. By tuning the concentration of benzoic acid from 22 to 33 mg/mL in DMF, the size of PCN-224 NP increased from 24 to 232 nm (Fig. 4a). Size-dependent cytotoxicity assay was performed on human cervical cancer HeLa cells with PCN-224 particles of 30, 60, 90, 140, and 190 nm in diameter. After incubating HeLa cells with various sizes of PCN-224 in the concentration range of 0.5 to 40 µM based on TCPP for 12 h, 90 nm-PCN-224 showed the highest cell uptake. The in vitro PDT efficacy was tested for HeLa cells under irradiation at the 420 nm Soret band. At 20 µM concentration, 90 nm-PCN-224 gave the highest cytotoxicity of 81%, while 190 nm-PCN-224 gave the lowest efficacy of 49% (Fig. 4b).</p><p>90 nm-PCN-224 was then further modified with FA, a commonly used ligand for the folate receptor (FR) that is overexpressed in many tumor cells, by coordinating interaction between carboxylate end of FA and the Zr6 SBUs. FA-PCN-224 showed slightly better cytotoxicity (~90%) than unmodified PCN-224 (~80%) on FR-positive HeLa cells (Fig. 4c), while similar cytotoxicity to unmodified PCN-224 was observed on FR-negative A549 cells. However, in vivo work is needed to confirm target-specific PDT of FA-PCN-224.</p><!><p>Zhou and coworkers further reported on controlling the generation of 1O2 using nMOFs built from photochromic molecules [130, 133]. Short-wavelength light was used to switch the photochromic molecule from the closed form to the open form to influence the ROS generation by nMOFs.</p><p>A Zn-TCPP-BPDTE MOF with a pillar-layer structure was synthesized from Zn2+, TCPP, and 1,2-bis(2-methyl-5-(pyridine-4-yl)thiophen-3-yl)cyclopent-1-ene (BPDTE) via a solvothermal reaction in DMF. In the Zn-TCPP-BPDTE MOF, paddlewheel-type Zn cluster SBUs linked TCPP to form 2-D Zn-TCPP layers, which were connected by BPDTE via the Zn-N bonds to form a 3-D MOF. By taking advantage of the photochromic property of BPDTE, Zn-TCPP-BPDTE could adopt both open and closed forms. Upon UV irradiation at λ=350 nm, the open form of Zn-TCPP-BPDTE was transformed to the closed form, which could be reversibly transformed back to the closed form upon irradiation of visible light at λ>450 nm. Photophysical studies indicated that BPDTE in the open form of Zn-TCPP-BPDTE did not affect the 1O2 generation of TCPP, while the BPDTE in the closed form of Zn-TCPP-BPDTE quenched the S1 state of TCPP to reduce 1O2 generation (Fig. 5).</p><p>The 1O2 generation efficiencies of Zn-TCPP-BPDTE in the open and closed forms were evaluated by 1,3-diphenylisobenzofuran (DPBF), which can react with 1O2 to decrease the absorption at 410 nm. Upon irradiation of 420 nm laser (6 mW/cm2) for 60 seconds, Zn-TCPP-BPDTE in the closed form generated half as much 1O2 as Zn-TCPP-BPDTE in the open form, suggesting the ability to modulate 1O2 generation with switchable MOFs.</p><p>Zhou and coworkers used a similar strategy to control the 1O2 generation by a UiO-66 nMOF doped with TCPP as the PS, and 1,2-bis(5-(4-carbonxyphenyl)-2-methylthien-3-yl)cyclopent-1-ene (BCDTE) as the photochromic switch. In the cytotoxicity study, the doped UiO-66 with BCDTE in the open form exhibited a PDT cytotoxicity of ~90%, while the doped UiO-66 with BCDTE in closed form had a PDT cytotoxicity of only 10%. This result validated the strategy of incorporating both PSs and photochromic molecules into nMOFs to modulate 1O2 generation and thus impact in vitro PDT efficacy.</p><!><p>Diiodo-substituted BODIPYs (I2-BDPs), featuring high ε and low dark toxicities, are regarded as a potential PS in addition to porphyrin-based PSs. In 2016, Xie and coworkers reported the synthesis of a BODIPY-immobilized nMOF for PDT [135].</p><p>UiO-66 nMOF was first synthesized and then treated with monocarboxyl-modified I2-BDP (carboxy-I2-BDP) via solvent-assisted ligand exchange to afford the final product, UiO-PDT (Fig. 6a). The as-synthesized UiO-PDT maintained the UiO-66 structure based on the PXRD pattern, and the doping of I2-BDP was confirmed by ultraviolet–visible spectroscopy (UV-Vis), energy-dispersive X-ray spectroscopy (EDS) mapping, 1H NMR, solid 13C NMR, and inductively coupled plasma mass spectrometry (ICP-MS). These analyses indicated that 12.5 mol% of benzenedicarboxylate ligand was exchanged by carboxy-I2-BDP to afford a PS loading of 31.4 wt%. UiO-PDT processed similar octahedral morphology as UiO-66 with a mean diameter of 70 nm. Confocal laser scanning microscopy (CLSM) studies showed that both UiO-PDT and carboxy-I2-BDP could cross the cell membrane to accumulate in the cytoplasm, while flow cytometry studies indicated that UiO-PDT had 1.54 times higher cell uptake than carboxy-I2-BDP. However, the 1O2 generation efficiency of UiO-PDT was lower than carboxy-I2-BDP due to its heterogeneous nature (Fig. 6b). In vitro PDT efficacy of UiO-PDT and I2-BDP was studied against B16F10, CT26, and C26 cell lines. Both UiO-PDT and I2-BDP exhibited good cytotoxicity and inhibited cell growth by >80% in all three cell lines with light irradiation (80 mW/cm2 for 10 min) (Fig. 6c). In spite of lower PDT efficacy of UiO-PDT than carboxy-I2-BDP, this work provided a new approach to incorporate PDT agents into nMOFs and suggested the applicability of the solvent-assisted ligand exchange approach in loading other therapeutic cargoes onto nMOFs.</p><!><p>PDT is a local treatment and generally not effective in treating metastatic tumors. Based on their observation of immunogenic cell death caused by Hf-DBC, Lin and coworkers hypothesized that nMOF-mediated PDT could be combined with immune checkpoint blockade to treat metastatic tumors. They recently reported a novel treatment strategy that combined local PDT of a new chlorin-based nMOF, TBC-Hf, with checkpoint blockade immunotherapy to achieve effective and consistent abscopal responses in mouse models of colorectal cancers [129] (Fig. 7a).</p><p>TBC-Hf, constructed from a chlorin derivative, 5,10,15,20-tetra(p-benzoato)chlorin (H4TBC) and Hf-based SBUs, was synthesized via a solvothermal reaction in DMF. TBC-Hf displayed a nanorod morphology of 50–100 nm in length and 30–60 nm in width based on TEM images. Based on PXRD pattern, TBC-Hf adopts the same structure as MOF-545 with a highly porous structure. TBC-Hf exhibited similar photophysical properties and 1O2 generation efficacy to DBC-Hf.</p><p>Checkpoint blockade immunotherapy, which uses small molecules or antibodies to stimulate the immunosuppressive microenvironment of tumors by modulating protein expressions and/or functions at dysregulated immune checkpoints, has emerged as a highly effective cancer treatment strategy. Indoleamine 2,3-dioxygenase (IDO), one such checkpoint, is often overexpressed in the tumor microenvironment. IDO causes tryptophan catabolism through the kynurenine pathway, facilitating the survival and growth of tumor cells by suppressing antitumor immune response. A small molecule IDO inhibitor (IDOi) was loaded into highly porous TBC-Hf to afford IDOi@TBC-Hf. The IDOi weight percentage after loading was determined to be 4.7% by thermogravimetric analysis (TGA) and 1H NMR. Incubation in HBSS at 37 °C showed slow release of IDOi from IDOi@TBC-Hf, reaching 83.3% release after 24 h.</p><p>IDOi@TBC-Hf exhibited superior in vivo efficacy and abscopal effects in two colorectal mouse models, CT26 and MC38. Mice bearing a large primary tumor and a small distant tumor in the bilateral models were established for testing in vivo efficacy. Only the primary tumor was treated with a single injection of H4TBC, H4TBC+IDOi, TBC-Hf, or IDOi@TBC-Hf (TBC: 20 µmol/kg and IDOi: 1.5 mg/kg) and light irradiation (650 nm, 100 mW/cm2 for 30 min). Mice treated with IDOi@TBC-Hf without light irradiation served as a dark control. At the endpoint, all the mice with primary tumors treated with IDOi@TBC-Hf or TBC-Hf and PDT therapy had tumors only 1% size of PBS treated tumors in both models. H4TBC with light irradiation and IDOi@TBC-Hf dark group failed to inhibit the tumor growth while H4TBC plus IDOi with light irradiation slightly inhibited the tumor growth. Moreover, only IDOi@TBC-Hf with light irradiation group successfully reduced the sizes of the distant tumors, suggesting that the treatment evoked systemic antitumor immunity in mice. In comparison, TBC-Hf with light irradiation group and IDOi@TBC-Hf dark control only showed slight inhibition of distant tumor growth, showing ineffectiveness of monotherapies (Fig. 7b–e).</p><p>Mechanistic studies showed that TBC-Hf mediated PDT caused immunogenic cell death of cancer cells in the primary tumors, which activated innate immune system and promoted antigen presentation. The massive stressed and dying necrotic tumor cells in the PDT-treated primary tumor sites were engulfed by the innate immune effector cells followed by presenting tumor-derived antigenic peptides to T cells, thus stimulating a tumor-specific T cell response. Meanwhile, IDOi@TBC-Hf could both release IDOi into local tumor environment and enter blood circulation to systemically inhibit IDO activity to reverse the immunosuppressive tumor environments. PDT and IDOi checkpoint blockade therapy synergized with each other to kill local cancer cells and created an immunogenic tumor microenvironment systemically, leading to durable and consistent abscopal effects (Fig. 7a).</p><!><p>Thermal therapy, such as hypothermia and thermal ablation, is an emerging cancer treatment owing to its relatively simple operation, fast recovery, and short hospital stay [161]. Clinically, radiofrequency pulse, microwave radiation, and ultrasound wave have been employed as energy sources for thermal therapy of cancer. Mechanistically, local temperature elevated above 40 °C can damage cancerous tissue directly, and sensitize tumors to radiation or chemotherapy treatment.</p><p>PTT is a thermal therapeutic treatment induced by near-infrared light energy and has attracted attention in recent years. Different from PDT, PTT is an oxygen-independent and ROS-free process mediated by photothermal agents. After being excited by light of a specific wavelength, normally within the near-infrared (NIR) range, the activated PS falls back to its ground state and releases vibrational energy to generate heat. Such a non-radiative relaxation process converts light to local heat rapidly and can regress tumors by increasing the temperature in the tumor area sufficiently.</p><p>Light absorption and photothermal conversion efficiency together determine the performance of a PTT agent. In the past decade, there have been tremendous amounts of interest in developing NP PTT agents. Various inorganic and organic nanomaterials have been examined as PTT agents for treating cancer. Inorganic NP PTT agents include plasmonic gold nanostructures (nanospheres [162], nanoshells [163], nanorods [164], nanostars [165], and nanocages [166]), carbon (graphene [167], carbon nanotubes [168], and nanodiamonds [169]), iron oxide [170], palladium (Pd) nanosheets [171], metal chalcogenide [172], and polyoxometalate [173]. Several organic polymers, such as polypyrrole [174], polydopamine [175], and semiconducting polymer [176], have been employed as surface coatings to increase blood circulation time and to reduce toxicity. With high light absorptivity and photothermal conversion efficiency, NP PTT agents can significantly increase local temperature in the tumor area compared to adjacent normal tissues, leading to anti-cancer efficacy with low side effects. Inspired by many potential biomedical applications of nMOFs, they are attracting significant attention as a platform to implement PTT and PTT-based combination therapies.</p><p>PTT as monotherapy typically cannot completely eradiate tumors due to inhomogeneous heat distribution in tumor tissues. Several strategies have been adopted to increase the anticancer efficacy of PTT and PTT-based combination therapies. First, better PTT agents with high light absorptivity in the NIR spectrum, high photothermal conversion efficiency, long blood circulation times, and enhanced tumor uptake are being sought to enhance photothermal therapy. Second, synergistic effects of PTT and other therapeutic modalities are being explored to enhance anticancer efficacy. Combination of PTT with ROS, small interference RNAs, or chemotherapeutics can drastically increase treatment efficacy. Third, image-guided PTT with theranostic agents based on multifunctional nanomaterials can also increase treatment efficacy of PTT via selective delivery of PTT agents to tumors.</p><p>With versatile tunability of nMOFs, the three strategies outlined above can be employed to design multifunctional nMOF hybrids for enhanced PTT. Both metal-cluster SBUs and bridging linkers can be functionalized to enhance photothermal conversion efficiency. Through post-synthetic modification or assembly from pre-modified linkers, functional moieties can be readily incorporated into nMOFs for combination therapies. Finally, encapsulation of functional components in nMOF cavities can further enhance the efficacy of PTT and PTT-based combination therapies. As summarized in Table 2, nMOFs have been used as PTT agents (nMOF-enabled PTT), as a platform for combining PTT with other diagnostic and therapeutic modalities (nMOF-combined PTT), and as hybrid nanomaterials for combined photodynamic therapy and photothermal therapy (nMOFs for PDT+PTT).</p><!><p>PB is mixed-valence hexacyanoferrate with a formula of FeIII4[FeII(CN)6]3·nH2O, and can be regarded as an archetypical MOF. Although devoid of large pores, PB exhibits high photothermal conversion efficiency, high photothermal stability, and good biocompatibility. PB was approved by US Food and Drug Administration (US FDA) for treating radionuclide exposure [177].</p><p>Yue and coworkers first reported PB NPs with PTT effects upon NIR light excitation [145]. Simple mixing of aqueous solutions of FeCl3 and K4[Fe(CN)6] in the presence of citric acid as surfactant afforded PB nanocubes with controllable sizes from 10 to 50 nm. PB NPs absorbed broadly in the 500 nm to 900 nm range with the peak wavelength at 712 nm, due to the charge transfer transition between Fe(II) and Fe(III) centers [178]. PB NPs had a molar ε of 1.09 × 109 M−1 cm−1 at 808 nm, comparable to that of gold nanorods (5.24 × 109 M−1 cm−1 at 808 nm). PB NPs showed much better photothermal stability than gold nanorods. With continuous exposure to an 808 nm laser, the temperature increase of a dispersion of gold nanorods gradually declined due to the melting of gold nanorods. Such a melting behavior was not observed for PB NPs. The photothermal anti-cancer effect was further demonstrated by in vitro studies.</p><p>Chen and coworkers synthesized core–shell PB@MIL-100(Fe) nanocomposites for combined MRI and PTT [150]. PB@MIL-100(Fe) was shown to be a T1–T2 dual-modal MRI contrast agent and could also be used for fluorescence optical imaging. More importantly, the PB core enabled PTT with NIR light irradiation. The as-prepared nanocomposite was also loaded with artemisinin at a loading of 848.4 mg/g. Both in vivo and in vitro data showed that artemisinin-loaded PB@MIL-100(Fe) possessed high antitumor efficacy by combing multi-modality imaging diagnosis, chemotherapy from triggered drug release, and NIR-activated PTT in a single system (Fig. 8).</p><p>Chen and coworkers further developed a nanoplatform based on a PB analog, DOX@Mn3[Co(CN)6]2@SiO2@Ag (where DOX is doxorubicin), for combined chemotherapy and PTT [149]. The PB analog core endows T1–T2 dual-modal MRI imaging due to paramagnetic Mn(II) and Co(II) ions. The Ag NPs deposited on the surface provided PTT capability. Loaded doxorubicin (DOX) could be released by local heat generated from Ag NP-enabled PTT. In vitro experiments indicated that combined PTT and chemotherapy treatment was superior to monotherapy. However, no in vivo experiments were performed.</p><p>Zhang and co-workers reported a similar PB@mSiO2–PEG nanoplatform with a high DOX loading [147]. The mesoporous silica and covalently conjugated PEG layer improved biocompatibility and photo-stability, while PB served both as a PTT agent and as a photoacoustic agent for theranostics. PB@mSiO2–PEG with loaded DOX had good antitumor efficacy and diagnostic properties by integrating MRI, photoacoustic imaging (PAI), pH/light-triggered release, and combined PTT and chemotherapy.</p><p>Dai and coworkers designed and synthesized PB-coated gold NPs for simultaneous PAI, CT imaging, and PTT treatment of cancer [146]. With intravenous administration of PB-coated gold nanoparticles and one-dose treatment of NIR-laser irradiation of 900 J·cm−2, mice bearing 100 mm3 tumors were completely cured without recurrence.</p><!><p>Photothermal agents can be incorporated into nMOFs in several ways. The PTT agents can be encapsulated in the pores of nMOFs. Alternatively, photothermal nanomaterials can be presented as the core or the shell of a core-shell nMOF nanocomposite.</p><p>Wang and coworkers reported a Pd@Au/DOX@ZIF-8 nanocomposite for pH and NIR-triggered PTT-chemotherapy in 2017 [152]. Pd nanocubes were first synthesized as seeds and covered with Au nanosheets to afford Pd@Au NPs as a photothermal converter. Zeolitic imidazolate framework (ZIF)-8, an acid-degradable MOF, was used to encapsulate Pd@Au NPs and DOX to form the Pd@Au/DOX@ZIF-8 nanocomposite. Pd@Au/DOX@ZIF-8 converted 780 nm NIR light into heat to not only promote the release of DOX from ZIF-8 but also realize combined PTT-chemotherapy. The IC50 value of DOX/Pd@Au@ZIF-8+NIR decreased more than twice compared to either Pd@Au@ZIF-8+NIR or DOX/Pd@Au@ZIF-8 in in vitro cytotoxicity studies, indicating enhanced efficacy for combined PTT-chemotherapy treatment.</p><p>Tian and coworkers reported a simple one-pot synthesis of ZIF-8/graphene quantum dot NPs for combined PTT-chemotherapy in 2016 [148]. DOX was encapsulated into micropores of ZIF-8 in situ during hydrothermal MOF synthesis by taking advantage of weak coordinating interactions between DOX and zinc ions. The hydroxyl, epoxy, and carboxyl groups on graphene oxide (GO) quantum dots were utilized for the formation of MOF/GO nanocomposites via hydrogen bonding interaction with imidazolate ligands. The PTT effect of graphene quantum dots not only caused cancer cell death, but also enhanced drug release from ZIF-8.</p><p>Many NP PTT agents, such as gold nanorods, need to keep their morphologies during PTT treatment in order to maintain their PTT efficacy. This can sometimes be difficult to achieve due to thermally induced melting and aggregation of NP PTT agents. NP PTT agents were surface modified with silica shells or coated with PEG to stabilize them against thermally induced melting and aggregation. Fang et al. recently reported ZIF-8 coated gold nanorods for enhanced PTT with good stability and biocompatibility [144]. In vitro cytotoxicity studies showed that ZIF-coated gold nanorods had a lower dark toxicity (IC50 = 157.19 µg/mL) than uncoated gold nanorods (IC50 = 23.26 µg/mL). ZIF-8 coated gold nanorods showed higher PTT cytotoxicity (IC50 = 4.45 µg/mL) than uncoated gold nanorods (IC50 = 7.39 µg/mL). The ZIF-8 coating likely protected gold nanorods from melting.</p><p>Chen et al. developed a polypyrrole@MIL-100 core-shell nanocomposite for dual-modal imaging and combined PTT and chemotherapy [143]. The polypyrrole core served as a PTT agent and an organic PAI agent for deep tissue imaging. The MIL-100 shell was employed for DOX loading and MRI T2 contrast imaging. It was proposed that Fe(III) ions mediated oxidative polymerization of pyrrole to form polypyrrole and subsequently binding of Fe(III) ions to the surface of polypyrrole facilitated the formation of MIL-100 shell on polypyrrole. The polypyrrole@MIL-100 core-shell nanocomposite enabled dual-modal imaging, light- and pH-triggered cargo release and combined PTT and chemotherapy treatment (Fig. 9).</p><!><p>nMOFs have also been explored as a versatile platform to combine PDT and PTT, two noninvasive light-induced anticancer treatments, to elicit synergistic effects and enhanced potency while reducing side effects. Yang and coworkers synthesized a therapeutic system based on a Au25 cluster-deposited MOF [137]. The as-synthesized Fe3O4/ZIF-8-Au25 nanocomposites combined PDT-PTT therapy, magnetic targeting, and MRI imaging into one single system. PDT effect was caused by 1O2 generated from Au25 clusters. Interestingly, the hyperthermia effect of magnetic Fe3O4 core was enhanced by PTT of Au25 clusters. In addition, Fe3O4 endowed magnetic targeting and MRI T2 contrast imaging. A higher anti-tumor effect of PDT-PTT therapy with magnetic targeting was supported by in vivo experiments.</p><p>Yin and coworkers reported a porphyrin-based nMOF nanocomposite for dual-modality imaging-guided PDT-PTT combination therapy [138]. They utilized Fe3O4@C as a core for both T2-weigthed MRI imaging and photothermal therapy. The Fe3O4@C@Zr-TCPP nanocomposite was assembled in situ by treating Zr(IV) ion and H4TCPP in the presence of Fe3O4@C. The PTT effect was adversely impacted by the Zr-TCPP shell, as shown in the laser-induced temperature increase data, but the photo-triggered anti-cancer potency was significantly increased in cytotoxicity tests. PDT and PTT in this system were induced by 655 nm and 808 nm lasers, respectively (Fig. 10).</p><!><p>The exploration of nMOFs in biomedical imaging and anticancer drug delivery started more than 10 years ago [73, 77]. Potential applications of nMOFs in phototherapy were demonstrated only 5 years ago. A large number of papers have already appeared on phototherapy with nMOFs in the past 3 years, highlighting the strong interest in developing potentially clinically relevant phototherapy regimens based on nMOFs.</p><p>Since the first publication in 2014, nMOFs have already emerged as a promising nPS platform for PDT. Compared to other nPSs, nMOFs have several advantages for PDT: (1) by incorporating PSs as organic linkers, nMOFs achieve very high PS loadings while the crystalline structures of nMOFs avoid self-quenching of PSs by preventing them from aggregating; (2) highly porous structures of nMOFs not only facilitate the diffusion of oxygen and ROS, but also accommodate loading of diverse diagnostic and therapeutic agents for theranostics and synergistic therapy; (3) monocarboxylic modulators capping on the nMOFs can be easily replaced by other carboxyl modified functional molecules, such as PEG and targeting ligands, to endow biocompatibility and enhance tumor accumulation. With these beneficial features, nMOFs have great potential of becoming the fourth generation PSs.</p><p>Given synthetic tunability of nMOFs, we foresee many more efforts on designing new nMOFs for PDT. As PDT is a relatively well established treatment, future efforts should be focused on examining the utility of nMOF PSs under realistic in vivo conditions. With very little in vivo data available to date, it is too early to assess the clinical relevance of nMOFs in PDT.</p><p>Combing PDT with immunotherapy provides a powerful approach to treating metastatic cancers. As a local treatment, PDT can also produce a strong inflammatory response by inducing immunogenic cell death [14, 80, 94, 179]. The promising data from Lin's group on combining nMOF-enabled PDT and IDOi inhibition for treating metastatic cancer will inspire the exploration of this strategy using other nMOFs and checkpoint inhibitors. We expect significant amounts of research efforts in combining nMOF-mediated PDT and checkpoint blockade immunotherapy in the near future.</p><p>Compared to PDT, PTT monotherapy with nMOFs has not been explored in depth. Instead, by incorporating multiple functionalities into nMOF assemblies, researchers have explored combination therapies of PTT with other imaging and therapeutic modalities (such as MRI, fluorescence, PAI, radiotherapy, chemotherapy, and immune checkpoint blockade therapy) using nMOFs. Because the involvement of multiple therapies in these studies, it is difficult to assess the contribution of PTT to the overall anticancer efficacy. The light dose for nMOF PTT studies are typically several times higher than the acceptable clinical dose of ~180 J/cm2. Several attempts have also been made to incorporate diagnostic/imaging functionalities into nMOFs with PTT capabilities, however, it is difficult to envision how such nMOF theranostics can be used in the clinic. Regulatory hurdles can also be significantly higher for nMOF assemblies with multiple components.</p><p>The synthetic tunability of nMOFs will allow fine-tuning of nMOFs for enhanced PDT and PTT efficacy. Judicious combination of nMOF phototherapy with other therapeutic modalities will likely further leverage the unique attributes of nMOFs and lead to superior antitumor efficacy. Although at their infancy, the future of nMOF-based phototherapy is bright. It is likely that nMOFs will find future applications in phototherapy or related therapeutic modalities for cancer treatment in the clinic.</p><!><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.</p>

PubMed Author Manuscript

Two Cytotoxic Stereoisomers of Malyngamide C, 8-Epi-Malyngamide C and 8-O-Acetyl-8-epi-Malyngamide C, from the Marine Cyanobacterium Lyngbya majuscula

Two new epimers of malyngamide C, 8-O-acetyl-8-epi-malyngamide C (1) and 8-epi-malyngamide C (3) have been isolated along with known compounds 6-O-acetylmalyngamide F (5), H (6), J (7) K (8), and characterized from a Grenada field collection of the marine cyanobacterium Lyngbya majuscula. The planar structures of these compounds were deduced by 1D- and 2D-NMR and mass spectral data interpretation. The absolute configurations were determined by a combination of CD-spectroscopy, chemical degradation and the variable temperature Mosher\xe2\x80\x99s method. Compounds 1\xe2\x80\x935, 7 and 8 displayed moderate cytotoxicity to NCI-H460 human lung tumor and neuro-2a cancer cell lines, with IC50 values ranging between 0.5 and 20 \xce\xbcg/mL.

two_cytotoxic_stereoisomers_of_malyngamide_c,_8-epi-malyngamide_c_and_8-o-acetyl-8-epi-malyngamide_c

1. Introduction<!>2. Results and discussion<!>3. Conclusions<!>4.1. General experimental procedures<!>4.2. Collection<!>4.3. Extraction and isolation<!>4.4. Basic hydrolysis of 8-O-acetyl-8-epi-malyngamide C (1)<!>4.5. Enzymatic hydrolysis of 8-O-acetyl-8-epi-malyngamide C (1)<!>4.6. Preparation of the (R)-MPA-Cl<!>4.7. Preparation of the R-MPA ester derivative of 8-epi-malyngamide C (3)<!>4.8. Variable temperature NMR experiments<!>4.9. Cancer cell line assays (HCT-116, NCI-460, neuro-2a)<!>4.10. Disk Diffusion Soft Agar Colony Formation Assay<!>4.11. Voltage-Gated Sodium Channel Modulation Assays<!>4.12. 8-O-acetyl-8-epi-malyngamide C (1)<!>4.13. Lyngbic acid (2)<!>4.14. 8-Epi-malyngamide C (3)<!>4.15. R-MPA ester of 8-epi-malyngamide C<!>

<p>Malyngamides are a class of secondary metabolites frequently encountered during chemical investigations of filamentous cyanobacteria, particularly Lyngbya majuscula or sea hares of the genus Bursatella and Stylocheilus (Gerwick et al., 2001; Tan, 2007). In the latter case, however, it has been shown that molluscs incorporate these compounds as a consequence of their diet of cyanobacteria (Pennings and Paul, 1993). Thus, malyngamides are considered metabolites of cyanobacterial origin. To date, more than 30 members belonging to this compound class have been isolated. Structurally, malyngamides consist of a methoxylated fatty acid tail, known as 'lyngbic acid', and a presumed amino acid derived head which are joined through an amide linkage. Whereas for the lipid part of malyngamides only five variations are known, namely lyngbic acid and its 12-, 16- and 20-carbon analog (Gerwick et al., 2001) and one 7R-epimer (Suntornchashwej et al., 2007), the amine portion of the molecule shows a remarkable structural diversity in carbon skeletons and bears a multitude of functional groups. These include unsaturations, covalently-attached chlorine atoms, and various oxygen-containing functional groups such as ketones, alcohols, epoxides and lactones. In addition to the variations in planar structure, differences in the absolute configuration have also been observed (Kan et al., 2000; Milligan et al., 2000; Suntornchashwej et al., 2007).</p><p>In the present study, a collection of L. majuscula from Grenada was chemically analyzed in order to identify the compounds responsible for the cytotoxicity observed in the crude extract. Subsequent bioassay-guided fractionation led to the isolation of two new stereoisomers of malyngamide C (1, 3), along with a suite of malyngamides previously reported, including 6-O-acetylmalyngamide F (4) and malyngamides H (5), J (6), and K (7). This paper describes the isolation, structure elucidation, and biological activity of the two new malyngamides 1 and 3.</p><!><p>Compound 1 showed an [M+H]+ peak at m/z 498.2607, consistent with the molecular formula C26H41ClNO6 by HRESITOFMS. The 1H NMR data exhibited several resonances typical of a malyngamide-type metabolite. For example, a methoxy singlet signal (δ 3.31) and its corresponding α-methoxy methine multiplet (δ 3.14), two olefinic signals (δ 5.46), three methylenes between δ 2.17 and 2.31, a methylene envelope (δ 1.26 – 1.42) and a terminal methyl (δ 0.87) were indicative of the amide of a lyngbic acid moiety in 1. In addition, the 1H and 13C NMR spectra contained resonances consistent with the presence of an exomethylene functionality possessing a vinyl chloride (δH 6.39/δC 122.7) and an exchangeable amide signal (δ 6.02), revealing further typical structural features of this compound class. Based on its mass and overall NMR features, isolate 1 was initially dereplicated as malyngamide C acetate (Ainslie et al., 1985; Wright and Coll, 1990) using the MarinLit database Blunt and Munro, 2005). However, when the spectral data were carefully compared with those reported in the literature, deviations in the specific rotation in sign and amount as well as in the 13C NMR spectra between 1 and malyngamide C acetate became apparent (Table 1). Analysis of the 2D-NMR data confirmed that 1 had the same planar structure as malyngamide C acetate, and hence, the two molecules must differ in configuration of either the stereogenic centers C-4, C-8, C-9 and C-7′ and/or the geometry of the Δ2 and Δ4′ double bonds.</p><p>Base hydrolysis of 1 yielded the acid portion, which was identical in all respects to that previously reported for 7(S)-methoxytetradec-4-(E)-enoic acid (2), thus establishing the 7′S-configuration for 1. The Z-configuration of the Δ2-double bond was evident from the observation of ROE correlations from H-1α and H-3 and NOE correlations from NH to H-3. The geometry of the Δ4′-double bond was inferred from 1H decoupling experiments. The olefinic proton signals of C-4′ and C-5′ were overlapped in CDCl3 (Table 1), but resolved to a distinct pair of multiplets in C6D6. Decoupling of the adjacent methylene protons H2-3′ and H2-6′ allowed the measurement of the 3JH4′-H5′ as 15.4 Hz, confirming an E-geometry for Δ4′ in 1.</p><p>These results focused our efforts on elucidation of the configuration of the cyclohexyl amino moiety of 1. Moreover, this was the part of the molecule where the majority of 13C NMR chemical shift differences were observed between 1 and malyngamide C acetate (Table 1). Because the empirical reversed octant rule can be applied to determine the absolute configuration of an epoxy-ketone ring system (Djerassi et al., 1965; Kan et al., 1998), the configuration of carbons C-4 and C-9 was clarified on the basis of a circular dichroism (CD) spectrum. The CD spectrum of 1 showed a negative Cotton effect by the C-5 carbonyl group at 308 nm, indicating that the absolute configuration of the epoxide was 4S and 9S, as seen in malyngamide C acetate. This result was also supported by comparison of the CD spectra of 1 with an authentic sample of malyngamide C acetate, obtained from a Lyngbya species, collected in Madagascar (See Supplementary Data).</p><p>In order to investigate the absolute configuration at C-8, 1 was deacetylated and the hydrolysis product was subsequently analyzed by single derivatization with methoxyphenylacetic acid (MPA) followed by variable temperature 1H NMR spectroscopy analysis (Latypov et al., 1998). Complete deacetylation of 1 without ring opening of the epoxide function was accomplished by use of porcine liver esterase. Calculation of the shift differences ΔδT1, T2 gave negative values for H2-6 and H2-7, and a positive value for H-9 (Figure 2), hence revealing the 8-R absolute configuration (Seco et al., 2001). Compound 1 was thus proven to be the C-8 epimer of the known compound malyngamide C acetate, and for which we propose the trivial name 8-O-acetyl-8-epi-malyngamide C.</p><p>Compound 3 was analyzed for C24H38ClNO5 by HRMS. Inspection of the 1H and 13C NMR data of 3 showed these data to be extremely similar to those of 8-O-acetyl-8-epi-malyngamide C (1), the major differences being the missing proton and carbon NMR resonances accounting for the acetyl function (Table 2). Indeed, compound 3 was identical in all respects to the hydrolysis product of 8-O-acetyl-8-epi-malyngamide C (1). Thus, compound 3 was shown to be the C-8 epimer of malyngamide C (4) and given the name 8-epi-malyngamide C. Late in the preparation of this manuscript, we became aware that Kwan et al. (2010) also recently isolated and defined the structure of compound 3. In this latter work, the absolute configuration was determined using a selective Mitsunobu inversion of C-8. The reported structural data for 3 are in good agreement with our findings and hence, corroborate the assignment of the absolute configuration. The identities of the known compounds 4–7 were readily established by direct comparison of HR-MS, 1H NMR and 13C NMR data with published data (Gerwick et al., 1987; Orjala et al., 1995; Wu et al., 1997).</p><p>All malyngamide compounds obtained during this study were tested for cytotoxicity and for modulatory activity in the mammalian Voltage-Gated Sodium Channel. Cytotoxicity (VGSC) was evaluated by testing the metabolites towards NCI-460 human lung tumor, neuro-2a mouse neuroblastoma and HCT-116 cells and in the disk-diffusion assay (Table 3). All compounds except malyngamide H (5) exhibited moderate activity with IC50 values ranging from 0.5 to 20 μg/mL. Of these, only metabolite 3 showed selectivity toward solid-tumor cell lines. 8-O-acetyl-8-epi-malyngamide C (1) and its free alcohol (3) were proven to be less active than their respective epimers malyngamide C acetate and malyngamide C, indicating the importance of the absolute configuration at this position for the interaction of malyngamides with their corresponding target structure. In the sodium channel assay (Table 4) compounds 1 and 5 were shown to have moderate blocking activity and malyngamide C and C acetate moderate activating properties.</p><!><p>In conclusion, the present work reports on two new natural products (1, 3) in the malyngamide structure class which are C-8 epimers of the reported compounds malyngamide C and malyngamide C acetate. Biosynthetically, the NH, C-1 and C-2 carbons of these malyngamide C derivatives are likely incorporated from glycine, C-3 from an HMGCoA synthase-like reaction as determined for jamaicamide A (Edwards et al., 2004), and this is followed by three rounds of acetate extension. This is interesting in that the C-8 carbon is predicted to derive from C-2 of acetate, and thus, the C-8 hydroxyl functionality is likely introduced as a post-assembly P450 oxidation. That both stereoisomers at C-8 in the malyngamide C structure class are produced in different populations is intriguing, and appears to be differentiated by the ocean of origin. Literature reports of the 8-S isomer (= malyngamide C) all derive from Pacific collections [Fanning Island by Ainslie et al. (1985) and Palmyra Atoll by Taniguchi et al. (2010)] whereas the 8-R isomer has now been reported from two Caribbean collections [Grenada in this work and Florida in the recent publication by Kwan et al. (2010)]. Thus, cytochrome P450 enzymes with opposite chiral preferences may have evolved in these different populations of L. majuscula. It is additionally intriguing that this modest structural change in the malyngamide C class (e.g. C-8 epimerization) results in an approximately 5-fold reduction in biological activity, and therefore identifies this as an important stereogenic site to be considered in any future medicinal chemistry efforts.</p><!><p>Optical rotation measurements were recorded on a Jasco P-1010 polarimeter. CD spectra were measured using a model J-720 Jasco spectropolarimeter. UV and FT-IR spectra were obtained employing Hewlett Packard 8452A and Nicolet 510 instruments, respectively. All NMR spectra were recorded on Bruker Avance DRX300, DPX400 and DRX600 spectrometers. Spectra were referenced to residual solvent signal with resonances at δH/C 7.26/77.1 (CDCl3) and δH 7.15/128.0 (C6D6). Low-resolution ESI-MS spectra were obtained on a Thermo Finnigan LCQ Advantage mass spectrometer. High-resolution ESI-TOF and CI mass spectra were recorded on Waters Micromass LCT Classic and JEOL MSRoute mass spectrometers, respectively. HPLC was carried out using a Waters system consisting of a Rheodyne 7725i injector, two 515 pumps, a pump control module and a 996 photodiode array detector. TLC grade (10–40 μm) Si gel was used for vacuum chromatography. All solvents were purchased as HPLC grade.</p><!><p>The marine cyanobacterium Lyngbya majuscula (voucher specimen available from WHG as collection no. GBI-26Jul95-07) was collected from shallow waters (1–3 m) in True Blue Bay, Grenada, on July 26, 1995 and stored in 2-propanol at −20 °C until workup. Taxonomy was assigned by microscopic comparison with the description given by Desikachary.7</p><!><p>A total of 19.1 g (dry wt.) of the cyanobacterium L. majuscula was extracted five times with CH2Cl2-MeOH (2:1, v/v) to produce 662 mg of cytotoxic crude organic extract (solid tumor selective at 15 μg/disk: U251ΔCEM = 400 units). The extract was fractionated by vacuum liquid chromatography (VLC) over silica gel using a stepwise gradient of hexanes-EtOAc and EtOAc-MeOH to give nine fractions. Fractions 5, 6 and 7, eluted with 60% and 80% EtOAc in hexanes and 100% EtOAc, respectively, showed cytotoxic effects (solid tumor selective at 15 μg/disk: C38ΔL1210 = 300 units) and were further investigated. Fraction 5 was further separated by RP-HPLC (column: YMC ODS AQ-323, 250 × 10.0 mm, 5 μm; MeOH-H2O (85:15), 2 mL/min) giving two distinct fractions. A second chromatography of the first fraction by RP-HPLC on a Phenomenex Synergi Fusion-RP 80 column (250 × 10.0 mm, 4 μm; MeOH-H2O (79:21), 2 mL/min) yielded 24.3 mg of 8-O-acetyl-8-epi-malyngamide C (1). Further separation of VLC fraction 6 by RP-HPLC (column: YMC ODS AQ-323, 250 × 10.0 mm, 5 μm; MeOH-H2O (85:15), 2 mL/min) resulted in additional quantities of compound 1 (9.4 mg) and an inseparable mixture of malyngamide H (6) and K (8) (1.9 mg). Reversed-phase HPLC (column: YMC ODS AQ-323, 250 × 10.0 mm, 5 μm; MeOH-H2O (85:15), 2 mL/min) of VLC fraction 7 yielded compounds 3 (0.9 mg), 6-O-acetyl malyngamide F (5) (1.7 mg) and additional quantities of the inseparable mixture of malyngamide H (6) and K (8) (1.0 mg). 1H NMR profiling of all VLC fractions indicated fraction 8 (eluted with 25% MeOH in EtOAc) also contained a malyngamide type metabolite. Therefore, VLC fraction 8 was first subjected to C18 HPLC (YMC ODS AQ-323, 250 × 10.0 mm, 5 μm; MeOH-H2O (90:10), 2 mL/min), and those materials eluting at 12 to 14 min were then further purified with RP-HPLC (Phenomenex Synergi Fusion-RP 80, 250 × 10.0 mm, 4 μm; MeOH-H2O (80:20), 2 mL/min) to give malyngamide J (7).</p><!><p>Compound 1 (6.9 mg) was dissolved in 2 mL solution of 10% KOH in EtOH-H2O (4:1) and the stirred mixture refluxed for 15 h. The hydrolysate was concentrated in vacuo and partitioned between H2O and CH2Cl2. The H2O layer was isolated, acidified, and extracted with CH2Cl2 to yield 1.6 mg (45.1%) of lyngbic acid (2).</p><!><p>Compound 1 (4 mg, 8.0 μmol) was dissolved in 0.2 mL MeOH and the resulting solution was added to 6 mL of 0.3 M K2HPO4 buffer (pH 8.0) containing 100 units porcine liver esterase (Sigma-Aldrich). The solution was stirred for 12 h at room temperature and then repeatedly extracted with ether (3 × 10 mL). The extracts were dried over MgSO4 and the solvent removed in vacuo to yield 8-epi-malyngamide C (3, 3.6 mg, 98.3 % yield).</p><!><p>Oxalyl chloride (51.9 μL, 0.6 mmol) was added to a mixture of R-MPA (10 mg, 0.06 mmol) and DMF (0.47 μL, 0.006 mmol) in hexanes at room temperature. After 48 h, the solvent was evaporated to dryness at reduced pressure to afford 0.06 mmol of R-MPA-Cl (11.1 mg, 100%).</p><!><p>To a solution of 3.6 mg of 8-epi-malyngamide C (3) in 800 μL CH2Cl2 were added 3 mg of DMAP and 7.3 mg of R-MPA-Cl. The reaction mixture was reacted for 24 h and then partitioned between EtOAc and 0.1 M NaHCO3, and the EtOAc layers then washed with 0.1 M HCl. The EtOAc layer was evaporated and then separated by RP-HPLC (column: Phenomenex Synergi Fusion-RP 80, 250 × 10.0 mm, 4 μm; MeOH-H2O (80:20), 2 mL/min) to yield the R-MPA ester of 3 (1.8 mg, 37.7 % yield).</p><!><p>1H NMR and 1H-1H-COSY spectra (300 MHz) of the R-MPA ester of 3 dissolved in CS2/CD2Cl2 (4:1) were recorded at T1 = 25° (298 K), T1a = −50° (223 K) and T2 = −70° (203 K). Chemical shifts (ppm) were internally referenced to the TMS signal (0 ppm). For low-temperature NMR spectroscopy, the probe temperature was cooled by a constant stream of liquid nitrogen, controlled by a standard unit (See Supplemental data) calibrated with a methanol reference. The sample was allowed to equilibrate for 15 min at each temperature before recording the spectra. ΔδT1T2 values (δT1 − δT2) of all protons surrounding the chiral secondary alcohol were calculated and applied to the corresponding model for the assignment of the absolute configuration of secondary alcohols by single derivatization, established by the Riguera group (Latypov et al. 1998; Seco et al., 2001).</p><!><p>Human colon carcinoma cells HCT-116 (Brattain et al., 1981) were grown in 5 mL culture medium (RPMI-1640 + 15% fetal bovine serum containing 1% penicillin-streptomycin, and 1% glutamine) (Moore and Woods, 1977) at 37°C and 5% CO2 from a starting cell density of 5 × 104 cells/T25 flask. On day 3, duplicate wells containing HCT-116 cells were exposed to various concentrations of the malyngamide analogs. Flasks were incubated for 120 h (5 d) at 5% CO2 and 37°C, and the cells harvested with trypsin, washed once with Hanks' balanced salt solution (HBSS), re-suspended in HBSS, and counted using a hemocytometer. The results were normalized to an untreated control. The IC50 value was determined using Prism 4.0 software (GraphPad, San Diego, CA). Cytotoxicity to NCI-H460 lung tumor cells and neuro-2a cells were run in quadruplicate using the method of Alley et al. (1988) with cell viability being determined by MTT reduction (Manger et al., 1995). Cells were seeded in 96-well plates at 5000 and 8000 cells/well in 180 μL for H460 and neuro-2a cells, respectively. Twenty-four hours later, the test malyngamide was dissolved in DMSO and diluted into medium, and then added at 20 μL/well producing concentrations between 15 and 0.5 μg. DMSO was less than 1% final concentration. After 48 h, the medium was removed and cell viability determined.</p><!><p>An in vitro cell-based assay using murine L1210 (leukemia), C38 (colon), and CFU-GM (normal) cells and human HCT116 (colon), H125 (lung), and leukemia (CEM) cells, assessed general and differential cytotoxicity of pure compounds (Valeriote et al., 2002). Samples were dissolved in 250 μL of DMSO, and duplicate 15 μL aliquots were applied to cellulose disks in agar plates containing cells. After a period of incubation, a zone of cell colony inhibition (z) was measured from the edge of each disk to the edge of colony growth, and expressed as zone units (zu), where 200 zu = 6 mm. General cytotoxic activity for a given sample was defined as an antiproliferation zone of 300 zu or greater. The differential cytotoxicity of a pure compound was expressed by observing a zone differential of 250 units or greater between any solid tumor cell (murine colon C38, human colon HCT-116, human lung H125) and either leukemia cells (murine L1210 or human CEM) or normal cells (CFU-GM).</p><!><p>The malyngamides were evaluated for their capacity to either activate or block sodium channels using the following modifications to the cell-based bioassay of Manger et. al (1995). Twenty-four h prior to chemical testing, cells were seeded in 96-well plates at 8 × 104 cells/well in a volume of 200 μL. Test chemicals, tested in quadruplicate, were dissolved in DMSO were serially diluted with medium and added at 10 μL/well resulting in concentrations of 10, 3 and 1.0 and 0.3 μg/mL. DMSO was less than 1% final concentration. Plates to evaluate sodium channel activating activity received 20 μL/well of either a mixture of 3 mM ouabain and 0.3 mM veratridine (Sigma Chemical Co.) in 5 mM HCl, or 5 mM HCl alone in addition to the test chemical. Plates were incubated for 18 h and results compared to similarly treated solvent controls with 10 μL medium added in lieu of the test chemical. The sodium channel activator brevetoxin PbTx-1 (Calbiochem) was added at 10 ng/well in 10 μL of medium and used as the positive control. Sodium channel blocking activity was assessed in a similar manner except that the ouabain and veratridine stock solution was 5.0 and 0.5 mM, respectively, and the sodium channel blocker saxitoxin (Calbiochem) was used as the positive control. Plates were incubated for approximately 22 h.</p><!><p>Clear oil; [α]26D +6 (c 0.35, EtOH); UV (EtOH) λmax 216 nm (log ε 3.99), 278 nm (log ε 3.64); IR (KBr) νmax 3305, 2925, 2854, 1726, 1718, 1652, 1541, 1435, 1373, 1233, 1093, 1043 cm−1; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 498.2607 [M+H]+ (calcd. for C26H4135ClNO6, 498.2622).</p><!><p>Brownish oil; [α]26D −6 (c 0.5, CHCl3) [lit. −5 (c 0.22, CHCl3) Kan et al., 2000; −10° (c 0.5, CHCl3), Cardellina et al., 1978); HRCIMS m/z 257.2110 [M+H]+ (calcd. for C15H29O3, 257.2117); 1H NMR (300 MHz, CDCl3): δ 0.88 (3H, t, J=6.9 Hz, H-14′), 1.27 m (10H, m, H-9′, H-10′, H-11′, H-12′ and H-13′), 1.42 (2H, m, H-8′), 2.19 (2H, m, H-6′), 2.36 (2H, m, H-2′), 2.42 (2H, d, J=5.9 Hz, H-3′), 3.15 (1H, t, J=5.8 Hz, H-7′), 3.32 (3H, s, H-15′), 5.49 (2H, m, H-4′and H-5′).</p><!><p>Clear oil; [α]26D −8 (c 0.5, EtOH); UV (EtOH) λmax 208 nm (log ε 3.59), 282 nm (log ε 1.46); IR (KBr) νmax 3322, 2927, 2855, 1714, 1651, 1537, 1432, 1265, 1092, 1071, 971, 915 cm−1; 1H and 13C NMR data, see Table 1; HRCIMS m/z 456.2506 [M+H]+ (calcd for C24H3935ClNO5, 456.2517).</p><!><p>Yellow oil; 1H NMR (300 MHz, CDCl3, See Supplemental data): δ 0.88 (3H, t, J=6.9 Hz, H-14′), 1.26 m (10H, m, H-9′, H-10′, H-11′, H-12′ and H-13′), 1.42 (2H, br t, H-8′), 1.93–2.50 (10H, m, H-6, H-7, H-2′, H-3′, H-6′, ), 3.13 (1H, t, J=5.4 Hz, H-7′), 3.31 (3H, s, H-15′), 3.40 (3H, s, MPA-OCH3), 3.47 (1H, d, J=2.6 Hz, H-9), 3.77 (1H, dd, J=15.4, 4.7, H-1a), 3.93 (1H, dd, J=15.4, 5.7, H-1b), 4.80 (1H, s, MPA-CH), 5.46 (2H, m, H-4′and H-5′), 5.54 br s (1H, br s, H-8), 5.96 (1H, br t, NH), 6.30 (1H, br s, H-3 ), 7.32–7.44 (5H, m, aromatic protons of MPA); LRESIMS m/z [M+Na]+ 626.3.</p><!><p>Structures of lyngbic acid and all malyngamide analogs obtained during this study.</p><p>Results of the variable temperature NMR study on the R-methoxyphenylacetate (MPA) derivative of 8-epi-malyngamide C. Significant shifts in the 1H NMR spectrum of the protons of interest are shown on the left panel, while the corresponding ΔδT1T2 values (ppm) are detailed on the right (R = acyl portion of lyngbic acid).</p><p>1H and 13C NMR data for 8-O-acetyl-8-epi-malyngamide C (1) and malyngamide C acetate in CDCl3.</p><p>The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.</p><p>NMR values taken from Wright and Coll, 1990.</p><p>1H and 13C NMR data for 8-epi-malyngamide C (3) and malyngamide C in CDCl3.</p><p>The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.</p><p>NMR values taken from Ainslie et al., 1985.</p><p>Cytotoxic activities of malyngamide analogs towards selected cancer cell lines in conventional cell line assays and in the disk diffusion assay.</p><p>dash = not active</p><p>n.d.= not determined</p><p>zu = zone units</p><p>Four replicates at each test concentration were averaged to construct dose-response curves; LC50 values were determined graphically.</p><p>A minimum of two measurements were made at each concentration tested</p><p>Since in this study, malyngamide H (5) and K (7) were obtained as an inseparable mixture, for testing, pure reference compounds were taken from an in-house compound library</p><p>Mammalian Voltage-Gated Sodium Channel (VGSC) modulatory activity of malyngamide analogs.</p><p>Values are averages of 4 replicates</p><p>dash = not active</p><p>n.d. = not determined</p><p>Since in this study, malyngamide H (5) and K (7) were obtained as an inseparable mixture, for testing, pure reference compounds were taken from an in-house compound library</p>

PubMed Author Manuscript

Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion with non-canonical amino acids, yet understanding of its structure and activity is incomplete. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon-independent; the anticodon does not contact the enzyme. Using standard microbiological culture equipment, we then established a new method for laboratory evolution \xe2\x80\x93 a non-continuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. We finally employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a non-canonical amino acid.

crystal_structures_reveal_an_elusive_functional_domain_of_pyrrolysyl-trna_synthetase

<!>Structure of the PylRS N-terminal domain\xe2\x80\x93tRNAPyl complex<!>PANCE, a new method for directed protein evolution<!>Evolved PylRS variants have improved enzymatic properties<!>DISCUSSION<!>Preparation of MmPylRS NTD<!>Preparation of tRNAPyl<!>Crystallization and data collection<!>Structure Determination and Refinement<!>Purification of c-Myc-chPylRS-6xHis variants<!>Western blot analysis of c-Myc-chPylRS-6xHis variants<!>LCMS analysis of intact purified proteins<!>sfGFP Assay<!>Aminoacylation Kinetics<!>General PANCE Methodology<!>PANCE of chPylRS<!>Use of mutagenesis plasmid MP6 in PANCE evolution<!>Data Availability

<p>Pyrrolysyl-tRNA synthetase is an aminoacyl-tRNA synthetase (aaRS) found in a small group of archaeal and bacterial species1. Together with its cognate substrate, tRNAPyl, PylRS variants have profoundly advanced our ability to genetically encode non-canonical amino acids (ncAAs) in live cells2–4. Thus, in the past few years, natural and engineered PylRS variants enabled the encoding of more than 100 ncAAs2 and improved encoding of its cognate substrate, pyrrolysine (Pyl, 1)4 (Supplementary Results, Supplementary Fig. 1) in a wide variety of species, ranging from bacteria to human5,6. The PylRS/tRNAPyl pair is commonly used to encode ncAAs due to the following features: (i) PylRS does not use the tRNAPyl anticodon as an identity element for recognition7, hence the tRNAPyl anticodon can be altered without loss of PylRS recognition7,8, (ii) PylRS is highly polyspecific and can utilize several distinct classes of ncAAs2,9,10, and (iii) the PylRS/tRNAPyl pair is orthogonal (i.e., does not cross-react with the host tRNAs and aaRSs) both in bacterial and eukaryotic species4. For over a decade, PylRS engineering has remained a key strategy in expanding the chemistry of protein synthesis, however our understanding of PylRS activity is still incomplete and the structural analysis of PylRS remains challenging.</p><p>In the archaeal genus Methanosarcina– one major source of PylRS variants for genetic code expansion – PylRS comprises 419–530 amino acids that are organized in two conserved domains connected by a variable linker (Supplementary Fig. 2): the tRNA-binding11 N-terminal domain (~120 aa), and the C-terminal domain (~270 aa) that also binds tRNA and harbors the catalytic site. In bacteria, PylRS is encoded by two different pylS genes4,11,12: pylSn comprises the N–terminal ~120 amino acids, while pylSc encodes the C–terminal ~280 amino acids. It was realized early that the full-length archaeal enzyme is fairly insoluble (Supplementary Table 1) and refractory to crystallization, although the C-terminal domain (CTD) of Methanosarcina mazei PylRS could be crystallized13. The CTD structure was determined in the apo form and in complexes with various ligands and ncAA substrates9,14, and with tRNAPyl (ref. 15) that has enabled the rational design of the PylRS amino acid binding pocket. However, the structure of the N-terminal domain so far has remained elusive, despite this domain being indispensable for PylRS activity in vivo12.</p><p>Here we report the crystal structure of the M. mazei PylRS N-terminal domain in complex with its cognate substrate, M. mazei tRNAPyl. We use this structure to gain insights into understanding PylRS specificity to its cognate tRNA, and to interpret the improved activity of multiple PylRS variants that were evolved through phage-assisted non-continuous evolution, a new method developed in this study.</p><!><p>To gain insight into the PylRS N-terminal domain (NTD) structure and activity, and enable its rational design, we co-crystalized the M. mazei PylRS N-terminal fragment (101 aa) with the transcript of M. mazei tRNAPyl, and determined the structure at 2.4 Å resolution (Fig. 1, Supplementary Fig. 3). We found that the PylRS N-terminal domain folds into a compact protein globule that coordinates a zinc ion (Fig. 1a). This Zn2+ ion does not directly contact the tRNAPyl and appears to stabilize the NTD fold. Our search for similar folds by using the DALI database indicated that none of the aaRSs has a similar fold to those of the PylRS NTD.</p><p>The PylRS NTD forms extensive contacts with the tRNAPyl by fitting snugly into the concave comprised of the T-loop and the authentic minimal variable loop of tRNAPyl (Fig. 1b). This tight fit between the PylRS NTD and the variable loop of tRNAPyl provides a steric explanation for the PylRS orthogonality; the larger variable arm of canonical tRNAs would impede their productive binding to PylRS (Fig. 2). Also, the structure reveals H-bond interactions of the PylRS N–terminal residues K3 and H24 with tRNAPyl and shows coordination of Zn2+ by residue H24, thereby explaining why mutations of these residues alter PylRS activity12 (discussed below).</p><p>Remarkably, the N- and C-terminal domains of PylRS bind on the opposite sides of the tRNAPyl molecule, suggesting that the complete archaeal enzyme wraps around the tRNA molecule (Fig. 1). This cooperative binding may explain why PylRS has such a high specificity for tRNAPyl and minimal cross-reactivity with other tRNAs in a wide variety of species15. Also, the tRNAPyl anticodon has no contacts with either N- or C-terminal PylRS domains (Fig. 1)7,8. This is consistent with biochemical and genetic studies showing that tRNAPyl anticodon can be mutated and utilized by PylRS to encode ncAAs with UAG, UAA, UGA, CUG, AGU, and AGGA codons2,8.</p><!><p>Simultaneously with the structure determination, we sought to evolve PylRS variants with improved recognition of non-canonical amino acids. For this purpose, we modified the effective phage-assisted continuous evolution (PACE) method16, which does not require prior structural knowledge for protein evolution and has been successfully applied to evolution of such proteins and enzymes as polymerases16, receptor binding proteins17, proteases18, and aaRSs19. One cornerstone of this technique is its customized continuous flow machine, uniquely utilizing a two-chambered system which houses Escherichia coli cells infected with M13 bacteriophage inside a secondary 'lagoon' vessel. Given our lack of a continuous flow machine, and because non-continuous experiments have been shown to produce comparable results17, we developed phage-assisted non-continuous evolution (PANCE), a simplified technique for rapid in vivo directed evolution using serial flask transfers in standard laboratory equipment (Fig. 3). This approach serially transfers evolving 'selection phage' (SP), which contain a gene of interest to be evolved, across fresh E. coli host cells, thereby allowing genes inside the host E. coli to be held constant while genes contained in the SP are continuously evolving. Serial flask transfers have long served as a widely-accessible approach for laboratory evolution of microbes, and more recently analogous approaches have been developed for bacteriophage evolution20,21.</p><p>Similar to PACE, selection by PANCE relies on linking activity of the evolving gene of interest inside the SP to the infectivity of the progeny phage (Fig. 3a, Online Methods). Here we achieved this linkage by first inserting M. mazei tRNAPylam and gIII containing one or more UAG stop codons into an accessory plasmid (AP) inside the E. coli host. This AP contains genes necessary for selection, which are maintained within the host cell to prevent the acquisition of escape mutations. The essential gIII gene was then deleted from the SP and replaced with gene of interest chpylS, and the ncAA Boc-lysine (BocK, 12) (Supplementary Fig. 1) was supplied during transfection. Production of full-length gIII, and phage survival, is thereby linked to the aminoacylation activity of the evolving chPylRS (Supplementary Fig. 4).</p><p>We first used PANCE to evolve a PylRS for improved translational incorporation of BocK (Supplementary Fig. 1). It has been shown earlier that PylRS mutagenesis frequently decreases PylRS affinity for Pyl and increases affinity for diverse ncAAs9,10 (Supplementary Fig. 5); we therefore expected similar changes to occur in PylRS after PANCE evolution. As the PylRS ancestor, we designed a novel chimeric PylRS (chPylRS, encoded by chpylS) in which the M. barkeri PylRS N–terminal domain (1–149 aa) was fused to the M. mazei PylRS C-terminal domain (185–454 aa) (Supplementary Table 2). This chimera was selected due to its improved solubility in vitro compared to the solubility of non-chimeric proteins (Supplementary Table 1). Three independent PANCE lines containing chPylRS (termed lines A, B, and C) were passaged through a host E. coli strain containing an increasing number of UAG codons in gIII. Initially, SPs were unable to grow when gIII contained more than one UAG codon. However, following 18–21 transfers in the presence of an increased mutation rate, each lineage has acquired the capacity to survive in the presence of even three UAG codons (Fig. 3b).</p><!><p>Sequencing of chpylS genes from individual plaques following growth in the presence of mutagenesis plasmid MP6 (ref. 22) revealed a highly polymorphic population. While MP6 was shown to be critical in enabling growth of SP in higher stringency conditions following relatively few serial transfers (Online Methods), we reasoned that continued growth under mutagenic conditions was no longer necessary after higher activity chPylRS variants had arisen. Thus, after 1–6 additional passages in the absence of MP6 (Online Methods), successful mutations became fixed within each lineage as each population converged upon a clonal genotype; as a result, three mutant chPylRS variants named 32A, 24B, and 25C were isolated (Fig. 3b). The mutations in each variant are listed in Supplementary Table 2. Six mutations were observed in the NTD, which is responsible for tRNA binding11, with only mutation H62Y appearing in all evolved variants. Five mutations were found in the C-terminal domain (CTD). In addition, we created the 32A-Nter variant that corresponds to wild-type chPylRS but carries only the four major mutations (D2N, K3N, T56P, H62Y) of the NTD. Read-through analysis of sfGFP containing one or three amber stop codons confirmed increased activity of evolved chPylRS variants in live cells (Supplementary Fig. 6a,b).</p><p>Western blot analysis (Supplementary Fig. 7a,b) of chpylS gene expression revealed that wild-type chpylS initiates translation at two sites, AUG codon 1 and AUG codon 107, to form a full-length protein (419 aa) and a chPylRS C-terminal protein (313 aa) in equal amounts23,24. ESI-MS analysis of the latter confirmed translation initiation with M107 (Supplementary Fig. 8). Sequencing of the chPylRS variants 24B and 25C revealed deletion of a T residue (Δt293) from codon 98 or 99 in chpylS (Supplementary Note), resulting in a –1 frameshift causing chain termination at the UGA codon 103. Western blot analysis (Supplementary Fig. 7b) of variant 24B and 25C gene expression revealed production of split PylRS enzymes consisting of an N-terminal (102 aa) protein and a C-terminal (313 aa) protein. SfGFP expression (Supplementary Fig. 6) demonstrated that the evolved split chPylRS enzymes (24B and 25C) are more active than the ancestral chPylRS. However, the NTD is not active by itself (Supplementary Fig. 6c)19. These data are in line with the fact that the NTD is required for in vivo PylRS activity12 and that mutations in the NTD enhance pylS-dependent UAG 15 (ref. 25). Since the active site is located in the CTD14,26 one must assume that the communication of the presence of both domains occurs through the tRNA. Supplementary Figure 6c also shows the importance of mutations (S158N, G343D) endowing the 24B CTD with substantial in vivo activity, which the wild-type chPylRS CTD lacks19. This may be explained by increased tRNAPyl binding caused by the S158N mutation15 (Supplementary Fig. 9). The existence of a CTD that is catalytically active in vivo adds credence to the bioinformatic and proteomic discovery of isolated pylSc genes in diverse archaeal species1,27.</p><p>As the mutations in the evolved PylRS variants were mainly found in the NTD, we co-crystallized the mutant M. mazei N-terminal domain (32A NTD) with tRNAPyl and explored how the mutations affect PylRS contacts with tRNAPyl. The structure was determined at 2.8 Å resolution (Fig. 1c). It revealed that two of the PylRS mutations, which are located directly at the PylRS/tRNA interface, appear to weaken PylRS/tRNA contacts. Thus, in the wild-type complex (Fig. 1b) the K3 side chain forms two H-bonds with the T-loop of the tRNA molecule, whereas in the mutant complex the K3N mutation disrupts these H-bonds (Fig. 1c). Furthermore, the H62Y mutation disrupts another two H-bonds between the PylRS and T-loop moieties of tRNAPyl, although it establishes new and seemingly weaker contacts with a phosphate moiety of G21 and the base of A20. Collectively, this mutant structure suggested that the PANCE-evolved NTD mutations decrease the apparent affinity of the PylRS N-terminal domain for cognate tRNAPyl.</p><p>PylRS is less catalytically efficient than the canonical aaRSs (e.g., leucyl-tRNA synthetase, LeuRS). This is plausible, as PylRS needs to provide much less Pyl-tRNA (servicing ~50 codons per Methanosacina genome) compared to Leu-tRNA (for ~220,000 codons in E. coli). Additionally, altering the enzyme's Pyl binding site in order to recruit diverse ncAA substrates (e.g., Supplementary Fig. 5) leads to decreased kcat/KM for the cognate amino acid Pyl and increased kcat/KM for many ncAAs9,10,28. To better understand how mutations in the evolved PylRS variants improve specific PylRS activity, we performed kinetic analysis to measure reaction rates and PylRS affinity to tRNAPyl, Pyl and BocK (Table 1). For this purpose, we carried out aminoacylation assays29 using purified PylRS mutants 32A, 24B, and 25C and in vitro transcribed tRNAPyl. Our measurements demonstrated that the improvement of each evolved variant stems from two major factors: a marked increase in PylRS affinity for BocK and even greater increase in PylRS affinity for tRNAPyl (Table 1). Collectively, these changes resulted in up to 10-fold increased catalytic efficiency of PylRS, as measured by Kcat/Km ratio (Table 1).</p><p>For one of the PylRS variants, 32A, we also measured its ability to discriminate between Pyl and BocK (Table 1). We found that, compared to the wild-type chPylRS, the evolved PylRS mutant had a markedly lower affinity for Pyl, reflected in a ~500 fold increase of the KM value. At the same time, its affinity to BocK was higher than that of the parental protein, which is reflected in a ~5-fold decrease of KM for BocK (Table 1). These measurements showed that PANCE evolution substantially improved PylRS specificity to BocK. The evolution of PylRS variants with lower KM values for ncAAs may ease the challenge of in vivo toxicity by certain ncAAs30.</p><p>Next, we estimated the individual contribution of the mutated NTD to the overall increase of PylRS activity. For this purpose, we used the 32A-Nter PylRS variant (Supplementary Table 2) that has the catalytically active CTD of the wild-type protein sequence. Kinetic measurements revealed that 32A-Nter mutant has profoundly weakened apparent affinity to tRNAPyl (Table 1), consistent with the reduced contacts observed in the crystal structure (Fig. 1). This finding is notable, because it implies that the decreased apparent affinity of the mutated N-terminal domain to tRNAPyl is effectively counterbalanced by additional mutations in the C-terminal domain of the evolved PylRS variants. Indeed, 32A-Nter variant has ~6 times lower tRNAPyl affinity, while the 32A variant has ~10 higher tRNAPyl affinity compared to the parental enzyme (as measured by KM values); and the only difference between these two PylRS variants is the presence of three additional mutations (E119K in the NTD, and K258Q and Y349F in the C-terminal domain of 32A). Collectively, these kinetic observations indicate that PylRS improvement by PANCE is achieved in part by shifting the burden of tRNA recognition via weakening the N-terminus/tRNAPyl interaction and reinforcing tRNAPyl binding to the CTD. It is therefore not surprising that one of the evolved mutants, PylRS 24B, acquired a capacity to function in vivo in the absence of the N-terminal domain (Supplementary Fig. 6c).</p><p>While measuring the kinetic properties of 32A-Nter variant (Table 1), we could not escape noticing that mutations in the PylRS NTD influence the PylRS catalytic site. Thus, mutations of the NTD in the 32A-Nter variant decreased the KM value for BocK and increased it for Pyl. Taken together, this suggests a model wherein mutations in the N-terminal domain enable a more flexible protein structure, coupled with C-terminal domain alterations to position tRNAPyl into an orientation specifically conducive to BocK aminoacylation. This demonstrates that binding of tRNAPyl and amino acid are intrinsically interdependent, consistent with findings for other aminoacyl-tRNA synthetases31,32.</p><p>Analysis of previous crystal structures suggests that the mutations that we observed in the CTD of chPylRS affect direct contacts with both tRNAPyl and the amino acid substrate. Mutation Y349F, identified in PANCE variant 32A (Supplementary Table 2), was previously shown (as Y384F in M. mazei) to improve aminoacylation with BocK (and other ncAAs) by M. mazei PylRS26 likely through direct contacts with the amino acid14. Other mutations in the C-terminal domain appear to improve tRNA binding (Supplementary Fig. 9).</p><!><p>Here we report the crystal structure of the N-terminal domain of M. mazei PylRS captured in the complex with its cognate substrate, tRNAPyl. This structure provides mechanistic insights into the exceptionally high specificity of PylRS to tRNAPyl, and explains how changes in the PylRS N–terminal domain structure may be used to improve catalytic properties of this unique enzyme and hence facilitate genetic encoding of non-canonical amino acids. The key properties that make PylRS-tRNAPyl highly orthogonal are: (i) discrimination against canonical tRNAs based on the larger size of the variable arm, (ii) demonstration that PylRS:tRNA recognition is 'anticodon blind', (iii) the enzyme may 'surround' the tRNA as the C- and N-terminal domains of PylRS have the largest interface area of the known 20 aaRS:tRNA complexes, and (iv) opening up the structure (Fig. 1) lowers binding to tRNAPyl and leads to increased ncAA recognition albeit with lower catalytic activity9,10.</p><p>We also show that amino acyl-tRNA synthetases can be efficiently evolved into better enzymes by using a non-continuous PANCE approach. The results of the PANCE experiment are consistent with the separately conducted PylRS improvement by PACE19. In the PACE experiment, most of the PylRS mutations were also found in the PylRS NTD, and led to increased catalytic activity of PylRS19. Furthermore, two mutations (H62Y and T56P) were found at the very same sites, although, in the PANCE approach, PylRS variant 25C had a T56A mutation instead of the T56P mutation observed from PACE. Frameshift mutations were also observed in PACE lineages, resulting in PylRS enzymes split into two separate proteins. Separate evolutionary lineages consistently evolved N–terminal fragments containing 91–101 aa and a C–terminal fragment containing 313 aa, which was generated by translation initiation at M107. This variability in the length of the PylRS NTD reflects differing points of termination within the naturally variable linker region, which likely no longer plays a role in protein function following protein splitting. Curiously, this 'splitting' of the two PylRS domains into separate proteins appears to mirror the natural expression pattern of pylSc and pylSn genes found in bacteria11,12 and some archaeal lineages1. Comparing chPylRS mutations across different PANCE and PACE-derived lineages19, the genotypic diversity produced following the same selection shows that many evolutionary trajectories can improve PylRS activity.</p><p>Since its development, PACE has provided a powerful tool for directed protein evolution. Here we show that the PANCE approach can produce comparable results within a rapid timeframe and without the need for continuous flow machinery, simplifying adoption of this successful protein engineering approach. Nonetheless, it is important to note that while the experimental apparatus required for PACE may require additional effort to set up, continuous flow experiments confer several advantages, such as a shorter experiment duration, greater constancy of selective pressure and population size throughout evolution33, and facilitation of deeper study of the population dynamics of phage evolution, as many variables (such as population size, mutation rate, selection stringency) can be systematically altered to assess effects on adaptive outcomes34. By contrast, PANCE evolution entails constant flux between stationary and active growth phases, complicating such analyses. Consequently, contrasting mutations between PANCE and PACE observed in this study may reflect adaptation to these distinct growth conditions.</p><p>In summary, we anticipate that this study may facilitate rational PylRS engineering to help resolve future challenges in expanding the chemistry of living systems.</p><!><p>The DNA fragment encoding the initial 101 residues of PylRS from M. mazei (MmPylRS NTD) was cloned into the Nde I and Xho I sites of modified pET28b vector in which the thrombin site and extra residues upstream of the N-terminal His tag were deleted. The plasmid was transformed into E. coli BL21 (DE3). Cells were grown in LB medium containing 25 μg/ml kanamycin at 37°C until the A600 reached 0.6. The culture was then induced by the addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 100 μM and shifted to 25°C for approximately 16 h before harvesting. The cells were harvested and resuspended in buffer A [50 mM Tris-HCl (pH 8.5), 1200 mM NaCl, 5 mM MgCl2, 10% glycerol, 1 mM 2-mercaptoethanol] with 0.5 mg/ml lysozyme, 0.1 mg/ml DNase. After sonication, the His6-tagged protein was purified by immobilized metal-ion affinity chromatography using a Ni-NTA (Qiagen). The protein bound to the column was washed with buffer A containing 15 mM imidazole and eluted by buffer B [20 mM Tris-HCl (pH 8.5), 300 mM NaCl, 5 mM MgCl2, 10% glycerol, 1 mM 2-mercapto-ethanol] containing 250mM imidazole. Eluted proteins were loaded onto a HiTrap Heparin HP column (GE Healthcare) and eluted with a gradient of 300–1000 mM NaCl in buffer B. Finally, the protein was loaded onto HiLoad 16/60 Superdex 200 pg (GE Healthcare) equilibrated with buffer C [20 mM Tris-HCl (pH 8.5), 200 mM NaCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT]. The protein was concentrated using an Amicon Ultra 10,000 MWCO (Millipore), flash-frozen and stored at −80°C.</p><p>Trial and error studies had shown that addition of Zn2+ led to increased solubility of the NTD. (This was later clarified by finding a Zn2+ ion bound near H24 in the MmPylRS NTD•tRNAPyl structure). For selenomethionine labeled protein preparation, cells were grown in M9 minimal medium supplemented with 20 μM ZnCl2 at 37°C until the A600 reached 0.4. Then, 60 mg/l L-selenomethionine, 100 mg/l each of L-Lys, L-Phe and L-Thr and 50 mg/l each of L-Ile, L–Leu and L-Val were added to the medium. When the A600 reached 0.7, the culture was induced by the addition of IPTG to a final concentration of 100 μM and shifted to 25°C for approximately 16 h before harvesting. MmPylRS NTD with D2N, K3N, T56P and H62Y mutations (32A NTD) was prepared in the same way as was WT MmPylRS NTD.</p><!><p>As is customary with PylRS, tRNAPyl transcripts were used in biochemical experiments. Thus, M. mazei tRNAPyl was transcribed using T7 RNA polymerase as described previously35. Transcribed tRNAs were purified by a HiTrap DEAE FF column (GE Healthcare) as previously described36. Pooled tRNAs were precipitated with isopropanol and dissolved in buffer D [20 mM Hepes-NaOH (pH 7.5), 10 mM MgCl2].</p><!><p>MmPylRS NTD or 32A NTD were mixed with tRNAPyl in a molar ratio of 1:1.2 in buffer E [20 mM Hepes-NaOH (pH 7.5), 50 mM NaCl,10 mM MgCl2, 5% glycerol, 1 mM DTT]. The resulting mixture was concentrated to A260 of 200 by ultrafiltration. Crystallization experiments were performed with the sitting-drop vapor diffusion method at 19°C. Crystals of the MmPylRS NTD•tRNAPyl complex were obtained in a reservoir solution containing 0.1 M Hepes-NaOH (pH 7.5), 0.2 M MgCl2, and 15% PEG 3350. Crystals of the selenomethionine labeled MmPylRS NTD•tRNAPyl were obtained in a reservoir solution containing 0.1 M Hepes-NaOH (pH 6.9), 0.2 M MgCl2, and 21% PEG 3350. Crystals of 32A NTD•tRNAPyl were obtained in the buffer containing 0.1 M Bis-Tris (pH6.7), 0.2 M MgCl2 and 19% PEG3350. Crystals were cryo-protected with 30% xylitol in the reservoir solution. The diffraction data sets were collected at 100 K on beamline 24ID-C and 24ID-E of Advanced Photon Source. Collected data were indexed, integrated, scaled and merged using XDS37.</p><!><p>The structure of MmPylRS NTD•tRNAPyl complex was determined by single wavelength anomalous diffraction method. Selenium sites were identified and used for phasing by Phenix AutoSol38 and initial model was build using Phenix AutoBuild39. This model was used for structural analysis of the native MmPylRS NTD•tRNAPyl complex. After several cycles of refinement with the program phenix.refine40, autoBUSTER41, fitting of tRNA with NAFIT42 and manual fitting with Coot43, the Rwork- and Rfree- factors were converged to 21.6% and 24.2%, respectively (Supplementary Table 3). For the structure determination of 32A NTD•tRNAPyl, after rigid body refinement with the structure of MmPylRS NTD•tRNAPyl complex using phenix.refine, the structure was rebuilt and modified manually using Coot. Molecules of tRNA were rebuilt and fitted by NAFIT. Then, the structure was further refined using phenix.refine. The Rwork- and Rfree- factors were converged to 20.6% and 24.9%, respectively (Supplementary Table 3).</p><p>We also prepared and solved a co-crystal structure of the 120 aa N-terminal fragment. The structure of this complex was the same as that of MmPylRS NTD•tRNAPyl; in both cases the residues after position 87 were disordered.</p><!><p>The chPylRS variants were cloned into the pTech plasmid using insertion primers that incorporate the N-terminal c-Myc sequence (MEQKLISEEDL-) and the C-terminal 6xHis sequence (-GSHHHHHH). BL21 star (DE3) cells (Thermo Fisher Scientific) transformed with the appropriate pTech plasmids were grown in LB media (United States Biologicals) supplemented with 25 μg/mL chloramphenicol. For each variant, a saturated overnight culture was prepared from a single colony, and a 1:100 dilution of culture was made into 5 mL of fresh LB media containing chloramphenicol. The starter culture grew at 37°C while shaking at 230 rpm until the cell density reached A600 = 0.3. The starter culture was then used to inoculate a 1 L culture of LB media containing chloramphenicol, which continued to incubate while shaking for an additional 16 h. Cells were harvested by centrifugation at 5,000g for 10 min at 4°C, and cell pellets were resuspended in lysis buffer [20 mM Tris (pH 7.4), 300 mM NaCl, 10 mM imidazole, and EDTA-free protease inhibitor cocktail (Roche)]. The cells were lysed by sonication on ice, and the crude extract was centrifuged at 15,000g for 15 min at 4°C. Lysates were loaded onto columns containing 2 mL of HisPur Ni-NTA resin (Thermo Fisher Scientific) that had been pre-washed with two bed-volumes of equilibration buffer. The resin was washed with 10 bed-volumes of wash buffer [20 mM Tris (pH 7.4), 25 mM imidazole, 300 mM NaCl] and protein was then eluted in 3 mL of elution buffer [20 mM Tris (pH 7.4), 250 mM imidazole, 300 mM NaCl]. The purified protein was dialyzed against 20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol. Purified protein was stored in 20% glycerol at −80 °C until analysis.</p><!><p>Cell lysates (30 μL) of expressed protein were combined with 25 μL of XT Sample Buffer (Bio-Rad), 5 μL of 2-mercaptoethanol, and 40 μL water. The samples were heated at 70°C for 10 min and 7.5 μL of prepared sample was loaded per well of a Bolt Bis-Tris Plus Gel (Thermo Fisher Scientific). Precision Plus Protein Dual Color Standard (4 μL) Bio-Rad was used as the reference ladder. The loaded gel was run at 200V for 22 min in 1x Bolt MES SDS running buffer (Thermo Fisher Scientific). The gel was transferred to a PVDF membrane using the iBlot 2 Gel Transfer Device (Thermo Fisher Scientific). The membrane was blocked for 1 h at room temperature in 50% Odyssey blocking buffer (PBS) (Li-Cor) and was then soaked 4 times for 5 min in PBS containing 0.1% Tween-20 (PBST). The blocked membrane was soaked with primary antibodies [rabbit anti-6xHis (1:1,000 dilution) (Abcam, ab9108) and mouse anti-c-Myc (1:7,000 dilution) (Sigma-Aldrich, M4439)] in 50% Odyssey buffer (PBS) containing 0.2% Tween-20 for 4 h at room temperature. The membrane was washed 4 times in PBST, and then soaked for 1 h in the dark at room temperature with secondary antibodies [donkey anti-mouse 800CW (1:20,000 dilution) (Li-Cor) and goat anti-rabbit 680RD (1:20,000 dilution) (Li-Cor)] in Odyssey buffer containing 0.01% SDS, 0.2% Tween-20. The membrane was washed 4 times in PBST and finally rinsed with PBS. The membrane was scanned using an Odyssey Imaging System (Li-Cor).</p><!><p>Purified protein samples were diluted to 10 μM in dialysis buffer lacking reducing agent or glycerol prior to analysis on an Agilent 6220 ESI-TOF mass spectrometer equipped with an Agilent 1260 HPLC. Separation and desalting was performed on an Agilent PLRP-S Column (1,000A, 4.6 x 50 mm, 5 μm). Mobile the phase A was 0.1% formic acid in water and mobile phase B was acetonitrile with 0.1% formic acid. A constant flow rate of 0.250 mL/min was used. Ten μL of the protein solution was injected and washed on the column for the first 3 min at 5% B, diverting non-retained materials to waste. The protein was then eluted using a linear gradient from 5% B to 100% B over 7 min. The mobile phase composition was maintained at 100% B for 5 min and then returned to 5% B over 1 min. The column was then re-equilibrated at 5%B for the next 4 min. Data was analyzed using Agilent MassHunter Qualitative Analysis software (B.06.00, Build 6.0.633.0 with Bioconfirm). The charge state distribution for the protein produced by electrospray ionization was deconvoluted to neutral charge state using Bioconfirm's implementation of MaxEnt algorithm, giving a measurement of average molecular weight. The average molecular weight of the proteins were predicted using ExPASy Compute pI/Mw tool (http://web.expasy.org/compute_pi/).</p><!><p>A pTECH plasmid containing the AARS of interest and a pBAD plasmid containing the sfGFP of interest were cotransformed into chemically competent TOP10 cells (Thermo Fisher Scientific). The transformed cells recovered in SOC (New England Biolabs) for 1 h while shaking at 37°C and were then plated and grown overnight at 37°C on LB agar containing 100 μg/mL carbenicillin and 25 μg/mL chloramphenicol. Single colonies were used to inoculate 3 mL of LB media (United States Biologicals) containing antibiotics and were grown overnight at 37 °C while shaking at 230 rpm. The saturated overnight cultures were diluted 100-fold in a 96-well deep well plate using 500 μL of LB media containing the required antibiotic. The plate was shaken at 37 °C for 3 h at 230 rpm and an additional 0.5 mL of LB was added containing antibiotics and additional components to provide each well with a final concentration of 1 mM BocK where denoted and 1.5 mM arabinose to induce expression of sfGFP. The cultures incubated with shaking at 37°C for an additional 16 hr after induction of sfGFP, and 150 μL of each culture was transferred to a 96-well black wall, clear bottom plate (Costar). The A600 and fluorescence (excitation = 485 nm; emission = 510 nm; bandwidth of excitation and emission = 5 nm) readings from each well were taken using an Infinite M1000 Pro microplate reader (Tecan). Background A600 and background fluorescence measurements were taken on wells containing LB media only. The background-subtracted fluorescence value from each well was divided by the background-subtracted A600 value of the same well to provide the fluorescence value normalized to cell density. All variants were assayed in biological quadruplicate, and error bars represent the standard deviation of the independent measurements.</p><!><p>The aminoacylation of tRNAPyl variants was carried out at 37°C in the buffer containing 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 10 mM amino acids, 100 nM PylRS variants, 24 μM unlabeled tRNAPyl, and 3.6 μM 32P-labeled tRNAPyl with a total volume of 25 μL. Various concentrations of BocK (0.1–12.8 mM), Pyl44 (5–500 μM for chPylRS and 0.1–10mM for variant 32A), and tRNA (0.5–16 μM) were used to determine KM values for corresponding substrates. A 2 μL aliquot was taken out from each of the reaction mixtures at the time points of 5 min, 20 min and 30 min, and the reactions were immediately quenched by adding 3 μL quenching solution [0.66 μg/μL nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)]. The nuclease P1 mixtures were then incubated at room temperature for 30 min and 1 μL aliquots were spotted on PEI-cellulose plates (Merck) and developed in the running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots for AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and visualized and quantified by phosphorimaging using a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of aminoacylated tRNA to total tRNA was determined to monitor reaction progress.</p><!><p>In kind with PACE evolution, an initial linkage between activity of M13 phage gIII protein (encoded within an E. coli cell) and activity of the gene to be evolved (encoded inside M13 phage) must first be established, as previously described16. This linkage makes phage growth dependent on activity of the gene of interest (for chPylRS evolution, see Supplementary Fig. 4). A series of stringency conditions must then be established, such that the initial activity of the gene of interest enables growth during low-stringency conditions, while improved activity is required for growth under higher stringency conditions. For this study, the initial activity of chPylRS was sufficient to mediate read-through of gIII containing single UAG codon (gIIIP29am, see Supplementary Table 4), however additional activity was required to enable phage propagation with two or more UAG codons in gIII.</p><p>Prior to beginning PANCE adaptation, host E. coli culture is first grown to A600 = 0.3–0.5. This culture must contain a low-stringency AP which permits SP propagation, a plasmid mediating increased mutagenesis (such as such as MP622), and is to be grown in 2xYT media with 20 mM glucose, 5 mM magnesium, and appropriate antibiotics. As plasmid MP6 is induced by arabinose, glucose supplementation is essential to reduce undesired mutagenesis prior to phage infection. E. coli cultures can be grown as a large batch in advance of phage infection and stored at 4°C, however storage is recommended for a maximum of 3–5 days as culture containing MP6 exhibits reduced infectivity following prolonged storage.</p><p>To begin PANCE adaptation, SP containing the gene to be evolved are first outgrown to a high titer (≥ 106 plaque forming units (PFUs)) in the absence of selection (e.g., using permissive strain S1059 containing WT gIII). The first selection growth is initiated by transferring an aliquot of infected culture at a volume of 5–200 μL containing a minimum of 5 x 106 PFUs to a flask containing 50 mL of E. coli culture in a 125 mL baffled flask (aliquoted from larger batch culture as described above). If reaching this population size requires more than 200 μL, the previous phage growth is to be repeated. Arabinose (5 mM) is added at this time to induce mutagenesis, as well as any other supplements cogent to the selection experiment (such as BocK, used in this study prepared at 100 mM concentration in 1N NaOH). Infected cultures are subsequently grown for 8–12 hr at 37°C with shaking at 225 rpm. Subsequently, a 1 mL aliquot is then stored at 4°C, and phage titers are measured using permissive host S1059. After phage growth is confirmed, an aliquot containing 5 x 106 PFUs is used to inoculate a subsequent culture, and this process is iteratively repeated until the desired phenotype is evolved.</p><p>Every 1–3 transfers, phage will be tested for growth under higher-stringency conditions by inoculating into an additional E. coli culture previously established to be non-permissive of ancestral SP phage growth. In the context of this experiment, E. coli containing plasmids pDB038a and pDB038b, encoding gIIIP29am/E84am and gIIIP29am/E84am/Y183am (respectively), served as higher stringency growth conditions, as they required read-through of an increasing number of stop codons (Online Methods and Supplementary Table 4). Each PANCE growth cycle is to be conducted at the highest stringency condition permissive to phage growth.</p><p>Following multiple PANCE growth cycles at the highest desired levels of stringency, additional high-stringency cycles are conducted in the absence of a mutagenic plasmid to allow for loss of neutral or deleterious mutations within the population. Individual phage plaques are isolated and sequenced to identify evolved mutations. Additional characterization of evolved protein can then be performed to better describe a biochemical basis for evolved phenotypes (see Table 1 and Supplementary Fig. 6 for examples from this work).</p><!><p>E. coli strain S103045 containing plasmid MP622 and the desired accessory plasmid (either pDB038, pDB038a, or pDB038b, see Supplementary Table 4) was initially inoculated from a plate into a large volume (~600 mL) of 2xYT media containing 100 μg/mL spectinomycin, 35 μg/mL chloramphenicol, 20 mM glucose, and 5 mM magnesium chloride in a 1L baffled flask. Cells were grown at 37°C with shaking at 225 rpm until reaching A600 = 0.3 – 0.5, at which point the culture was stored at 4°C for 3–5 days.</p><p>Phage growth was then begun by first removing a 50 mL aliquot of cell culture from the larger flask, and transferring it to a sterile 125 mL baffled flask. This aliquot was then supplemented with 5 mM arabinose and 1 mM BocK, followed by transfection with a minimum inoculum of 5 x 106 PFUs of selection phage taken from the previous growth in the PANCE cycle (Prior to beginning the first phage growth in the PANCE cycle, phage were grown in strain S1030 containing pJC175e which supplies WT gIII, termed strain S105946, to reach the required population size). Transfected cells were then grown at 37°C with shaking at 225 rpm for 8–12 hr. A 1 mL aliquot of phage was removed from the flask and was used to inoculate the subsequent phage growth in the PANCE cycle. Phage samples were subsequently stored at 4°C.</p><p>For each transfection, a standard volume of 5 μL of infected E. coli cells was used. To ensure a minimum inoculum size of 5 x 106 PFUs of phage was reached during each transfection, a phage titer was performed for each sample grown using permissive strain S1059 as the host strain. For samples with a concentration below 106 PFU/μl, phage growth was repeated, and inoculation volume was increased to a maximum of 0.2 mL to reach a total inoculum size 5 x 106 PFU. If reaching this population size required more than 0.2 mL, the previous phage growth was also repeated. BocK stock was prepared at 100 mM concentration in 1N NaOH.</p><!><p>Growth with mutagenesis plasmid rapidly evolves the desired phenotype. Following 40 transfers across three independent lines with plasmid pDB038 in the absence of a mutagenesis plasmid, phage were unable to grow on pDB038a. Following 8–12 transfers on pDB038 in the presence of MP6, phage were able to grow with pDB038a (Fig. 3b). Propagation in the presence of selection (plasmid pDB038b) and in the absence of mutagenesis plasmid MP6 resulted in the loss of neutral and deleterious mutations from the population. Selection without mutagenesis plasmid MP6 was conducted until sequenced populations showed convergence. In line A, samples 27A – 32A were grown without MP6; these are the last 6 transfers. In line B, samples 23B–24B were grown without MP6; these are the last two transfers. In line C, sample 25C was grown without MP6; this is the last single transfer.</p><!><p>Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5UD5 and 5V6X. Selection plasmids used in this study will be available through Addgene. Other materials are available upon reasonable request from the corresponding author.</p>

PubMed Author Manuscript

Solution-Phase Synthesis of the Chalcogenide Perovskite Barium Zirconium Sulfide as Colloidal Nanomaterials

Chalcogenide perovskites such as BaZrS3 have promising optoelectronic properties. Methods to produce these materials at low temperatures, especially in the solution phase, are currently scarce. We describe a solution-phase synthesis of colloidal nanoparticles of BaZrS3 using reactive metal amide precursors. The nanomaterials are crystallographically and spectroscopically characterized.The search for efficient and low-cost materials for thin-film photovoltaics has in recent years been dominated by a focus on hybrid lead halide perovskites due to their low cost, facile processing, and high efficiency-exceeding 29% when combined with silicon in a tandem device. 1 However, concerns about the stability, toxicity, and potential environmental effects of these lead-based materials have already begun to drive substantial research efforts towards the development of related materials with higher stability and lower toxicity. 2-4 Among the many proposed materials, chalcogenide perovskites and related materials with general formula ABQ3 (A = Ca, Ba, Sr; B = Zr, Hf, Ti; Q = S, Se) show particularly strong promise based on their optoelectronic properties and excellent stability. [5][6][7] Of these materials, BaZrS3 has drawn the most attention because of its distorted perovskite structure and useful optical properties (Figure 1A). In particular, at ~1.8 eV its bandgap is higher than the ideal for a single-junction solar cell but competitive to replace perovskites in tandem applications; moreover, the bandgap could

solution-phase_synthesis_of_the_chalcogenide_perovskite_barium_zirconium_sulfide_as_colloidal_nanoma

<p>be lowered closer to the ideal by alloying or the use of related Ruddlesden-Popper phases. 6,[8][9][10] Despite the theorized potential of BaZrS3, development and testing of it in thin-film devices has been largely hindered by the lack of low-temperature methods to deposit it as a thin film. 6 The first bulk syntheses of BaZrS3 required temperatures near or above 1000 o C. 11,12 Further tuning of the chemistry and stoichiometry eventually lowered this to 450 o C. 13,14 Initial efforts to generate BaZrS3 thin films have relied on sulfurization of oxide films or co-sputtering approaches, but high temperatures (>900 o C) were usually needed to complete the reaction and achieve crystalline materials. [15][16][17] Recently, epitaxial film growth has been achieved at lower temperatures (>700 o C) using pulsed laser deposition. 18 Most promisingly, film growth and crystallization at 600 o C has been achieved using a sputtering/sulfurization approach. 19 Many of these techniques also require more complex and expensive equipment compared to the solution-phase growth and processing that is possible with the hybrid lead halide perovskites.</p><p>In cases where direct solution-phase growth of a thin film is not feasible, an alternative approach is to use a colloidal suspension of nanocrystals as a precursor ink. 20,21 For this reason, among others, there has been some interest in the preparation of BaZrS3 as colloidal nanomaterials. There has been one report of the successful preparation of colloidal BaZrS3 nanoparticles by grinding bulk BaZrS3 to a fine powder and treating it with appropriate solvents/ligands to extract a population of small (40-60 nm) colloidal particles, which were successfully processed into thin-film devices. 22 However, methods for the direct solution-phase synthesis of BaZrS3 nanomaterials are still lacking.</p><p>In this communication, we report the synthesis of BaZrS3 as colloidal nanoparticles using reactive metal amide precursors in oleylamine solution, with N,N'-diethylthiourea as the sulfur source, using a procedure adapted from that we previously reported for the synthesis of BaTiS3 nanomaterials. 23 The reaction was successful at temperatures ranging from 365 o C to as low as 275 o C. However, nanomaterials synthesized at the lower temperatures showed structural distortions and a more pronounced platelet-like morphology as compared to those synthesized at the upper end of the temperature range.</p><p>Briefly, in a typical synthesis, Ba[N(TMS)2]2(THF)2, 24 Zr[N(CH3)2]4, and N,N'diethylthiourea are combined in a 1:2:60 mole ratio in rigorously dried oleylamine, at a concentration of 0.08 M in Ba 2+ (Figure 1B). The reaction is carried out in a Schlenk reaction tube under inert gas using a set-up similar to that we previously reported for the heat-up synthesis of BaTiS3. 25 The reaction mixture is heated to the desired reaction temperature (e.g., 365 o C) and maintained at this temperature for 30 minutes before being allowed to cool to room temperature. During heating, the reaction mixture takes on a deep red-brown color and remains homogeneous in appearance. Following precipitation and washing of the nanomaterials from the reaction solution using anhydrous chloroform and ethanol, BaZrS3 nanoparticles are isolated as an orange-red powder.</p><p>We found that a large excess of the sulfur precursor and a high concentration in solution were both important for the success of the reaction; otherwise, impurity phases were commonly observed. Additionally, the use of the readily soluble and reactive metal amide precursors greatly facilitated the successful production of crystalline BaZrS3. Attempts to synthesize this material using simple chloride and acetate salts of Ba 2+ and Zr 4+ have thus far been unsuccessful in our hands, as have attempts to use alternative sulfur precursors including CS2 and (Me3Si)2S; in these cases, binary phases such as BaS and ZrS2 are frequently observed, or no detectable crystalline phase is observed at all. Although the synthesis itself must be carried out under rigorously anhydrous conditions due to the reactive nature of the precursors, the resulting nanomaterials are quite stable to air and moisture (vide infra). However, the initial nanoparticle purification must also be carried out under air-and water-free conditions in order to avoid contamination of the sample with amorphous oxide byproducts (e.g. ZrO2).</p><p>Figure 2 shows characterization data for a sample of nanoparticles obtained at the highest readily accessible temperature-approximately 365 o C, corresponding to vigorously refluxing oleylamine. Figure 2a compares the powder diffraction pattern of the nanoparticles to the predicted pattern based on the reported orthorhombic distorted perovskite structure of bulk BaZrS3; the diffraction peak positions are reasonably well-matched, with the expected sizerelated broadening. 26 TEM imaging shows nano-sized particles which appear to have a platelike morphology and tend to be highly aggregated, making precise size distribution measurements challenging. Typically, the particles are non-uniform and polydisperse with the majority of particles falling within a lateral size range from approximately 10 to 40 nm. Lattice at lower temperatures showed some apparent structural changes (Figure 4); we refer to these material as LT-BaZrS3. We note that this result was also sometimes observed in nanoparticle samples synthesized at 365 o C for reasons that are not currently clear. Powder X-ray diffraction analysis of these nanomaterials showed evidence of deviations from the reported bulk structure, as illustrated in Figure 4A. Key differences include the presence of additional diffracted intensity around 2θ = 34 o and 22.5 o , along with small shifts of the other major diffraction peaks towards higher angles and the appearance of a broad shoulder on the lowangle side of the peak near 44.5 o . The Ruddlesden-Popper Ba3Zr2S7 and Ba2ZrS4 phases do not appear to be a better match to the experimental data (Figure 4A). 27,28 It is difficult to completely rule out that the samples could actually be a mixture of related phases, although they do appear uniform by TEM (Figure 4B-C). TEM imaging (Figure 4B-C) of LT-BaZrS3 shows nanoplatelet-like particles. Elemental analysis (EDX) data is similar to that measured for HT-BaZrS3, although the samples tend to have a higher barium/zirconium ratio. Given the platelet-like morphology of the particles, this discrepancy could be partly accounted for if the lateral surface planes are Ba/S-rich. However, we cannot conclusively determine if this is the case.</p><p>To better understand the origin of the apparent structural distortions, and to further confirm the identity of the material as a distorted perovskite BaZrS3, preliminary structural refinement was carried out using pair distribution function (PDF) analysis of synchrotron-based X-ray scattering data (see Supporting Information for details and fit parameters). The resulting data and fits are shown in Figure 5. Data from the high-temperature particles (HT-BaZrS3) are shown in Figure 5a, overlaid with a fit to the bulk BaZrS3 structure. During the fitting, refinement of the lattice parameters and isotropic atomic displacement parameters was allowed while other structural parameters were fixed to those of the reported bulk structure.</p><p>Overall the PDF data appears consistent with a Pnma distorted perovskite BaZrS3 structure. comparison, attempted fits of this PDF data to the reported structures of Ba3Zr2S7 and Ba2ZrS4 are shown in Figure 4C and by visual inspection are less successful than the fit to BaZrS3; these fits also have higher Rw values (see SI). Therefore, the PDF data suggests that these nanoparticles may still possess the perovskite-like structure of bulk BaZrS3 at least on the local level. The exact nature of the structural distortions suggested by PXRD and PDF analysis, and their relationship to the particle morphology and synthesis temperature, is not yet known; investigations into this question are ongoing and will be elaborated in future reports.</p><p>Figure 6A shows an extinction spectrum for a colloidal solution of the HT-BaZrS3 nanoparticles. The spectrum shows a shallow sloping onset around 750 nm and a steeper slope commencing around 600 nm. A broad shoulder is detectable at 460 nm, and an additional peak at 300 nm. The absorption onset is in line with those reported in the literature for bulk BaZrS3, and is also reflected by the reddish-orange color of the materials (Figure 6B-C). 6 The exact value of the intrinsic band gap for BaZrS3 is not well-established, with reports ranging at least from 1.75 eV to 1.94 eV. 17,29 Unfortunately, we were unable to detect significant luminescence from our materials; it has been noted that the luminescence of BaZrS3 nanomaterials can be highly dependent on surface treatment, so further optimization of the surface termination could elicit emission in the future. Finally, we tested the stability of the nanoparticles to exposure to ambient atmosphere and to direct immersion in water, to determine if the excellent stability observed for bulk BaZrS3 might hold true on the nanoscale (Figure 7). For a sample of HT-BaZrS3, the PXRD pattern did not change significantly over the course of 9 weeks of exposure to ambient atmosphere. Upon immersion in water, a small unidentified impurity peak was observed in the sample after 30 minutes. Therefore, although the stability of these nanomaterials against water is higher than that of lead halide perovskite nanocrystals or BaTiS3 nanocrystals, 25 they may be less resilient than bulk BaZrS3.</p><p>In conclusion, we have demonstrated that colloidal suspensions of BaZrS3 nanomaterials can be obtained using a low-temperature, solution-phase process. The approaches reported here may be generalizable to other related materials.</p>

ChemRxiv

Biotinylated Bioluminescent Probe for Long Lasting Targeted in Vivo Imaging of Xenografted Brain Tumors in Mice

Bioluminescence is a useful tool for imaging of cancer in in vivo animal models that endogenously express luciferase, an enzyme that requires a substrate for visual readout. Current bioluminescence imaging, using commonly available luciferin substrates, only lasts a short time (15\xe2\x80\x9320 min). To avoid repeated administration of luciferase substrate during cancer detection and surgery, a long lasting bioluminescence imaging substrate or system is needed. A novel water-soluble biotinylated luciferase probe, B-YL (1), was synthesized. A receptor-targeted complex of B-YL with streptavidin (SA) together with a biotinylated epidermal growth factor short peptide (B-EGF) (SA/B-YL/B-EGF = 1:3:1, molar ratio) was then prepared to demonstrate selective targeting. The complex was incubated with brain cancer cell lines overexpressing the EGF receptor (EGFR) and transfected with the luciferase gene. Results show that the complex specifically detects cancer cells by bioluminescence. The complex was further used to image xenograft brain tumors transfected with a luciferase gene in mice. The complex detects the tumor immediately, and bioluminescence lasts for 5 days. Thus, the complex generates a long lasting bioluminescence for cancer detection in mice. The complex with selective targeting may be used in noninvasive cancer diagnosis and accurate surgery in cancer treatment in clinics in the future.

biotinylated_bioluminescent_probe_for_long_lasting_targeted_in_vivo_imaging_of_xenografted_brain_tum

INTRODUCTION<!>RESULTS AND DISCUSSION<!>CONCLUSIONS<!>General Methods<!>Poly(ethylene glycol)23-diazide (8)<!>Amino-poly(ethylene glycol)23-azide (9)<!>N-(Azido-poly(ethylene glycol)23)-hexahydro-2-oxo-(3aS,4S,6aR)-1H-thieno[3,4-d]imidazole-4-pentanamide (5)<!>N\xe2\x80\xb2-Propargyl N\xe2\x80\xb3-(2-Cyano-6-benzothiazolyl)-glycinamide (4)<!>N-(5-(N\xe2\x80\xb3-(2-Cyano-6-benzothiazolyl)-glycinamido-methyl)-1H-1,2,3-triazolyl-poly(ethylene glycol)23)-hexahydro-2-oxo- (3aS,4S,6aR)-1H-thieno[3,4-d]imidazole-4-pentanamide (6)<!>6-(3-N-{N-[5-(Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl)-1-oxopentyl]-,[3aS-(3a\xce\xb1,4\xce\xb2,6a\xce\xb1)]}-aminoethyl-poly(ethylene glycol)21-oxyethyl-1H-1,2,3-triazol-5-yl-methylamino-oxomethylamino)-2-benzothiazolyl]-4,5-dihydro-,(4S)-4-thiazole-carboxylic Acid (1)<!>Measurement of Bioluminescence Spectrum of Probe B-YL (1)<!>Preparation of a Complex of Streptavidin with B-YL and Free Biotin (B/SA/B-YL, 1:3:1 Molar Ratio)<!>Preparation of a Complex of Streptavidin with B-YL and EGF Peptide (EGF-B/SA/B-YL, 1:3:1 Molar Ratio)<!>In Vitro Bioluminescence Imaging<!>In Vivo Bioluminescence Imaging<!>Statistical Analysis<!>

<p>Imaging has become one of most important techniques in identifying and monitoring cancer in noninvasive diagnosis as well as in accurate surgical resection during treatment.1–6 First, it is crucial to detect tumor cells at an early stage of development.7 The most commonly used methods for detecting cancer in patients include magnetic resonance imaging (MRI), computerized tomography (CT), and positron-emission tomography (PET). Second, cancer treatments can include surgical resection, chemotherapy, and radiation.8,9 Surgery is one of the most effective ways to remove tumors and avoid metastatic disease spread. Surgery cures approximately 45% of all cancer patients with solid tumors. To be considered successful, a surgeon must remove the entire tumor and any lymph nodes or satellite nodules containing tumor cells. Partial removal of the solid tumor and incomplete removal of all the tumor cells decrease a patient's survival rate by 5-fold. Therefore, it is important to map the solid tumor accurately using imaging before surgery and double check for any residual tumor cells during and after surgery.</p><p>Recently, fluorescence imaging has been suggested for use in both cancer detection and resection; however, it is a light-dependent method.5 This strategy uses an external light source to excite exogenously added fluorescence agents and is not very versatile because many biological molecules present in the body have significant absorption of wavelengths at both visible and infrared regions of the light spectrum. Further, many wavelengths generated from the external excitation light source cannot penetrate tissues to reach the imaging fluorescent molecules, especially when they are in solid tumors. When the fluorescent molecules are not excited, no light is emitted for detection of the cancer. To overcome this problem, luminescence imaging is being developed to emit light from within the solid tumor. The process involves an enzyme, such as luciferase, which catalyzes the oxidation of a substrate, luciferin, to generate a bioluminescent signal, which is measurable by photon emission.10 Light is generated without application of an external excitation light source. The enzymatically generated photons are able to travel through solid tumors during the detection process.7</p><p>Although humans and animal models of cancer do not have naturally occurring bioluminescent genes, such as luciferase, the genes or proteins can be introduced for imaging purposes. For instance, bacteria encoded with a luciferase gene can accumulate in C6 glioma tumors in a mouse model. The expressed luciferase is then used for cancer detection in vivo.11 Furthermore, mammalian cells genetically modified with a luciferase gene can be delivered directly into tumors in live animals for tumor detection using bioluminescence.7,12,13</p><p>In addition, tumor bioluminescence can be a useful tool during surgery as shown in an animal model.8 Bioluminescence was able to precisely detect tumors both preoperatively and intraoperatively. For the enzyme to produce bioluminescence within the tumor, d-luciferin or other small molecule substrates, such as coelenterazine, vargulin, and 6-aminoluciferin derivatives, must be administered.10,14–22 However, bioluminescence generated from these substrates lasts for a very short time, only 15 to 20 min per administration.10 This is not practical in a clinical setting as the surgery to remove a brain tumor takes 3– 5 h. Checking and rechecking for tumor remnants by bioluminescence would require multiple new administrations of substrate. The additional application steps would lengthen the surgical time and potentially complicate the surgery and reduce the success rate of the surgery. Therefore, a new substrate with longer lasting bioluminescence signal is needed.</p><p>To overcome current limitations in bioluminescence imaging of cancers for detection and surgery, here, we report the development of a novel platform system for detection of brain tumors in mice in vivo (Scheme 1). First, we synthesized a novel biotin containing bioluminescent probe, B-YL (1), which acts as a substrate for luciferase. The probe possesses an aminoluciferin unit as a bioluminescent reporter, a poly-(ethylene glycol) (PEG-1000) link for improving cell penetrating ability, and a biotin tail for binding to streptavidin.19,23,24 Then, we constructed a complex, which contains streptavidin (SA), the bioluminescent probe B-YL, and a biotinylated epidermal growth factor short peptide (B-EGF) (SA/B-YL/B-EGF = 1/3/1, molar ratio), to target the complex. The EGF peptide binds to the EGF receptor, a biomarker overexpressed in 30–50% of high-grade gliomas. We then applied the complex to detect implanted brain tumor cells encoded with the luciferase gene by bioluminescence in vitro and in vivo.23</p><!><p>The synthesis of substrate B-YL is outlined in Scheme 2. Synthesis began with compound 2.15 Compound 2 was amidated with propargylamine (3) in the presence of 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC) and 1-hydroxy-benzotriazole (HOBt), resulting in compound 4 (88% yield). The latter was reacted with biotinylated azidoPEG (5) by click chemistry in the presence of Cu(CH3CN)2PF6 and tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), affording compound 6 (74% yield).25 Finally, compound 6 was reacted with d-cysteine in the presence of K2CO3 at pH 8.5 to produce probe B-YL (1) in 67% yield. The details of the synthetic procedures for the B-YL are described in the Methods.</p><p>The azidoPEG (5) is also a new and useful synthetic intermediate. The synthesis of compound 5 is also shown in Scheme 3.</p><p>After synthesis, the biotinylated probe, B-YL, was evaluated in vitro with a commercially available firefly luciferase. B-YL displayed bioluminescence with a maximum emission at 590 nm and was oxidized by commercial luciferase to emit bioluminescence photons (Figure 1).</p><p>The mechanism for generating bioluminescence is shown in Scheme 4. Luciferase, for example, from a firefly, generates light from a luciferin-based substrate in a multistep process. First, the substrate is adenylated by Mg-ATP to form luciferyl adenylate and pyrophosphate. Luciferyl adenylate is then oxidized by oxygen to form a dioxetanone ring. Decarboxylation forms an excited state oxyluciferin, which tautomerizes between the keto–enol forms. The reaction emits light as the oxyluciferin returns to the ground state.10</p><p>The B-YL substrate was then incubated with the brain cancer cell line U87-luc, which was derived from the parental brain cancer cell line U87 after stable transfection with a luciferase gene. As shown in Figure 2a, B-YL applied to cancer cells without luciferase, parental U87 cells, did not reveal bioluminescence activity regardless of the number of cells. However, as shown in Figure 2b, when there were as few as 7500 U87-luc cells or more in a well, bioluminescence signal was detected by using B-YL. B-YL clearly adsorbed across the cell membrane and was oxidized by luciferase. Therefore, B-YL can be used for the detection of the cancer cells by bioluminescence.</p><p>Using classical avidin–biotin complex (ABC) formation, complexes were made with a streptavidin core, which lacks any carbohydrate modification and has a near-neutral pH, and biotinylated ligands in the presence or absence of free biotin (B). The complex EGF-B/SA/B-YL (SA/B-YL/B-EGF = 1/3/1, molar ratio) is a targeting complex, which can be used as an active imaging agent for the detection of brain cancer cells.18 Complex EGF-B/SA/B-YL possesses targeting functionality because it has an EGF short peptide (12 amino acids). The complex B/SA/B-YL (SA/B-YL/B = 1/3/1, molar ratio) is a control complex with no targeting ability (no conjugated EGF short peptide).</p><p>The EGF-B/SA/B-YL complex was then tested for the ability to target and image brain cancer cell lines that overexpress the biomarker EGFR using bioluminescence in vitro. U87 (gray hatch bars) and U87-luc (black bars) cells were treated with free B-YL (150 µg/mL), untargeted B/SA/B-YL complex (150 µg/mL), or EGFR-targeted EGF-B/SA/B-YL complex (50–150 µg/mL). Bioluminescence was detected immediately. As shown in Figure 3, increasing concentrations of EGF-B/SA/B-YL complex actively accumulated into both U87 and U87-luc cells due to the presence of the EGF short peptide. However, only cells with luciferase were able to convert the EGF-B/SA/B-YL complex into detectable photons. Bioluminescence intensity depends directly on the amount of administered complex. Signal intensity increased linearly with increasing concentrations of the EGF-B/SA/B-YL complex. Cells without luciferase (U87) did not oxidize the EGF-B/SA/B-YL complex and release photons.</p><p>Untargeted B/SA/B-YL passively accumulated within the brain cancer cells. Luciferase activity for untargeted B/SA/B-YL was comparable to that of adsorbed free B-YL at the same concentration in U87-luc cells. The bioluminescence signal intensity from the targeted EGF-B/SA/B-YL complex is 2.5-fold higher than that of the untargeted B/SA/B-YL complex, suggesting that the complex EGF-B/SA/B-YL could image cancer cells U87-luc much better than complexed B/SA/B-YL. Neither of the complexes (B/SA/B-YL or EGF-B/SA/B-YL) nor free B-YL were converted to bioluminescence photons by U87 cancer cells because they do not have luciferase activity.</p><p>The targeted EGF-B/SA/B-YL complex or untargeted B/ SA/B-YL complex was then administered to mice (N = 5 per group) with subcutaneously implanted xenograft brain tumors derived from U87-luc cancer cells (right flank) or U87 cancer cells (left flank) and evaluated for bioluminescence activity.23 In Figure 4a, targeted EGF-B/SA/B-YL revealed increasing bioluminescence in the right flank tumor region (U87-luc) of a mouse from 0.3 to 6 h. The signal was strong 6 h after injection of the complex. In contrast, administration of the untargeted B/SA/B-YL resulted in a weak bioluminescence signal after 0.3 h. The signal was completely gone after 4 h. The results suggest that the EGF-B/SA/B-YL complex is a suitable imaging agent for detection of luciferase activity within tumors.</p><p>Furthermore, in Figure 4b, longitudinal imaging of mice injected with either targeted EGF-B/SA/B-YL complex (solid line) or untargeted B/SA/B-YL complex (dashed line), revealed that the signal from untargeted B/SA/B-YL complex peaked at 0.02 h after injection, suggesting that the B/SA/B-YL complex had an even shorter retention time than that reported for d-luciferin in mice (20–30 min). Both the B/SA/B-YL complex and d-luciferin do not have targeting function. Bioluminescence signal intensities were then normalized across the treatment groups (N = 5 for each group) at 0, 0.02, 6, and 24 h (Figure 4c).</p><p>When the EGF-B/SA/B-YL complex was used, the maximum bioluminescence intensity was achieved at 24 h, suggesting the retention time for the EGF-B/SA/B-YL complex was very long. When the B/SA/B-YL complex was used, the maximum bioluminescence intensity was achieved at 0.02 h (12 min); there is 1200-fold increase in terms of retention time of bioluminescence from EGF-B/SA/B-YL. Furthermore, it took approximately 3 h to accumulate EGF-B/SA/B-YL within the tumor, EGF-B/SA/B-YL can be retained, and bioluminescence will last 24 h. The signal of bioluminescence finally disappeared after the sixth day, suggesting that the bioluminescence from EGF-B/SA/B-YL complex had very long retention time. It could be used to image cancer cells long-term in vivo. Current studies are underway to evaluate the ability of the biotinylated bioluminescent probe to cross the BBB in orthotopic brain tumors in mice and spontaneously formed gliomas in dogs.</p><!><p>In conclusion, a novel biotinylated probe, B-YL, has been synthesized. B-YL possesses a biotin unit for binding to streptavidin, a PEG linker for improving cell penetration, and a luciferin unit for bioluminescence triggered by luciferase. After synthesis, the B-YL was tested and found to be a substrate of firefly luciferase. B-YL was then used to form complexes with streptavidin together with biotinylated EGF short peptide to create targeted and untargeted complexes. The EGF-B/SA/B-YL complex produced stronger bioluminescent signal in brain cancer cell lines with an encoded luciferase gene (U87-luc). By using the EGF-B/SA/B-YL complex, bioluminescence signal in xenograft brain tumors in mice was also detected. More importantly, the bioluminescence in the xenografted mouse lasted several days, suggesting that the targeted complex could be a long lasting bioluminescent agent for brain cancer imaging in vivo. To the best of our knowledge, this is the first long lasting bioluminescent imaging agent, which can last as long as 5 days in vivo. This imaging agent could find applications in cancer detection and surgery in the brain using bioluminescence, when tumors are removed and the surgery lasts several hours.</p><!><p>All chemicals used were purchased from commercial sources and used without further purification. Firefly luciferase was purchased (Sigma, MO). Flash chromatography was performed with SiliaFlash P60 silica (70–230 mesh; SiliCycle, Canada) and monitored by thin layer chromatography (TLC) with silica gel matrix plates (pore size 60 Å; Sigma-Aldrich, MO). The 1H spectra were recorded on a 400 MHz Bruker Nanobay-400 (9.4 T) instrument (Bruker, MA) with methanol-d4, DMSO-d6, and CDCl3. The chemical shifts of protons are given in ppm relative to the signal of tetramethylsilane (TMS) as the internal standard. The purification of compound 1 was carried out using a high performance liquid chromatography (HPLC) instrument (Dionex Ultimate 3000; Thermo Scientific, MA) as described in more detail below in the preparation and purification of probe 1. Luminescence spectroscopy measurement was performed using a SynergyMx (BioTek, VT). High-resolution mass spectrometry (HRMS) spectra were performed in the Mass Spectrometry Service Center of UC Riverside, CA. A Waters GCT Premier mass spectrometer (Waters, MA) was utilized with electron ionization (EI) and chemical ionization (CI) capabilities. Positive or negative ion mode was performed. Accurate mass measurements for these ionization modes were carried out as outlined.26</p><!><p>PEG-1000 (20 g, 0.02 mol) was dehydrated by refluxing with toluene (100 mL), which was distilled out after reflux. After cooling, the PEG-1000 was dissolved in THF (150 mL). To the solution were added mesyl chloride (4.88 mL) in one portion and triethylamine (9.16 mL) in tetrahydrofuran (THF) (80 mL) dropwise over 30 min at 0 °C. The mixture was stirred for 1 h at 0 °C and 3 h at room temperature, affording PEG dimesylate solution in THF.27 A small amount of solution was extracted with dichloromethane, washed with water and dried over Na2SO4. After concentration, the residue was used for characterization. 1H NMR (400 MHz, CDCl3, ppm) δ 3.11 (s, J = 5.2 Hz, 4 H), 3.65 (s, 85 H).</p><p>To the solution of PEG dimesylate in THF were added sodium bicarbonate (5%, 44 mL) and sodium azide (5.2 g). The resultant mixture was concentrated to 250 mL, stirred at room temperature overnight, and refluxed for 5 h. After cooling, the mixture was extracted with dicloromethane (3 × 100 mL). The resultant organic layer was dried over MgSO4 and concentrated to give PEG diazide (20.7 g, 98%). The product was used for the next step without further purification. 1H NMR (400 MHz, CDCl3, ppm) δ 3.40 (t, 6 H), 3.67 (s, 81 H), 3.78–3.80 (m, 4 H), 4.40–4.41 (m, 4 H). Mass calculated for C44H88N6O21+ (M + H+) 1037, found 1032.</p><!><p>To the PEG diazide (8, 10.7 g, 10.2 mmol) solution in ethyl acetate (EtOAc, 60 mL) were added HCl (1 N, 12 mL) and triphenylphosphine (2.67 g, 10.2 mmol). The mixture was stirred for 12 h at room temperature. Water (12 mL) was added into the reaction mixture, and the mixture was extracted with EtOAc (2 × 50 mL) to remove unreacted PEG diazide. Solid potassium hydroxide (9.28 g) was added at 0 °C. The mixture was then extracted with CH2Cl2 (3 × 50 mL). The organic layer was washed with brine, dried over Na2SO4, and concentrated to 9.8 g of amino containing PEG compounds (azido PEG monoamine and PEG diamine) in 94% yield.2 1H NMR (400 MHz, CD3OD, ppm) δ 2.80–2.82 (m, 2 H), 3.39–3.42 (m, 2 H), 3.55 (m, 2 H), 3.63 (s, 86 H). Mass calculated for C44H90N4O21+ (M + H+) 1011, found 1012.</p><!><p>To the above amino containing PEG compounds 9 (9.8 g) in DMF (70 mL) and NEt3 (2.76 mL) was added biotin NHS (3.93 g, 11.48 mmol). After stirring overnight at room temperature, the reaction mixture was concentrated, dissolved in CH2Cl2, centrifuged to a clear solution, and purified with column chromatography (silica gel, dichloromethane–dichloromethane/ acetone/methanol (2:7:1)–dichloromethane/methanol (9:1)) to yield product 5 (67%).28 1H NMR (400 MHz, CDCl3, ppm) δ 1.45–1.49 (m, 2 H), 1.70–1.75 (m, 4 H), 2.28 (t, J = 7.9 Hz, 2 H), 2.72–2.79 (m, 1 H), 2.92 (dd, J = 12.8, 4.8 Hz, 1 H), 3.17–3.18 (m, 1 H), 3.41 (t, J = 5.0 Hz, 2 H), 3.59 (t, J = 5.0 Hz, 2 H), 3.65–3.75 (m, 75 H), 4.34–4.37 (m, 1 H), 4.53–4.56 (m, 1 H), 6.21 (s, 1 H), 6.76 (s, 1 H). Mass calculated for C54H104N6O23SNa+ (n = 22, M + Na+) 1259.677, found 1259.681.</p><!><p>To a solution of N-(2-cyano-6-benzothiazolyl)-glycine (2, 0.57 g, 2.6 mol) in tetrahydrofuran (100 mL) and acetonitrile (70 mL) were added propargyl amine (3, 0.33 mL, 5.2 mmol), 1-ethyl-3-(3- (dimethylamino)propyl)carbodiimide (EDC, 0.996 g, 5.2 mmol), and 1-hydroxybenzotriazole (HOBt, 0.8 g). The mixture was stirred for overnight, concentrated in vacuo, dissolved in dichloromethane (50 mL), washed with saturated sodium bicarbonate (50 mL), and dried over magnesium sulfate. After concentration, residue was purified by column chromatography (silica gel, hexane, ethyl acetate, then acetone) to yield 0.59 g (88%) of product 4.3 1H NMR (400 MHz, CD3COCD3, ppm) δ 2.65 (t, J = 2.4 Hz, 1 H), 3.97 (d, J = 5.6 Hz, 2 H), 4.05 (dd, J = 5.6, 2.5 Hz, 2 H), 6.30 (s, 1 H), 7.17 (m, 1 H), 7.19 (s, 1 H), 7.79 (s, 1 H), 7.96 (d, J = 8.6 Hz, 1 H). Mass calculated for C13H11N4OS+ (M + H+) 271.065, found 271.064.</p><!><p>To a solution of 4 (0.185 g, 0.72 mmol) and N-(azido-poly(ethylene glycol)23)-hexahydro-2-oxo-(3aS,4S,6aR)-1H-thieno [3,4-d]imidazole-4-pentanamide (5, 0.45 g, 0.36 mmol) in DMF (4.0 mL) were added tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 3.3 mg) and Cu(CH3CN)2PF6 (29.8 mg) under N2 stream at room temperature. The resultant mixture was stirred at 40 °C overnight. After evaporation of solvent in high vacuum, the residue was purified with column chromatography (silica gel, dichloromethane–dichloromethane/ methanol (88/12 (v/v))) to yield 0.402 g (74%) of product 6.25 1H NMR (400 MHz, CD3OD, ppm) δ 1.47–1.48 (m, 2 H), 1.63– 1.74 (m, 4 H), 2.27 (t, J = 7.5 Hz, 2 H), 2.75 (d, J = 12.8 Hz, 1 H), 2.98 (dd, J = 12.8, 2.5 Hz, 1 H), 3.22–3.24 (m, 1 H), 3.37–3.39 (m, 2 H), 3.60–3.65 (m, 82 H), 3.86 (d, J = 5.0 Hz, 2 H), 3.96 (s, 2 H), 4.31–4.35 (m, 1 H), 4.51–4.60 (m, 5 H), 7.08–7.10 (m, 2 H), 7.92–7.94 (m, 2 H), 8.02 (s, 1 H). Mass calculated for C63H107N10O22S2 (n = 18, M + H+) 1419.700, found 1419.700.</p><!><p>To a solution of compound 6 (15.07 mg, 0.9 mmol) in methanol (6 mL), was added a solution of d-cysteine (9.31 mg) in water (1.8 mL). The mixture was titrated with potassium carbonate (0.05 M) to pH 8. The mixture was then stirred for 10 min at room temperature. After concentration with N2 stream, the residue was purified with high performance liquid chromatography (HPLC) using a reverse phase column (Luna 5 µ C18 column, 4.6 µ, 250 mm; Phenomenex, CA)29 with a 20–100% acetonitrile gradient at a flow rate of 5 mL/min and the detector set at 260 nm, affording probe 1 (10.1 mg, 67% yield).15 1H NMR (400 MHz, CD3OD, ppm) δ 1.46–1.47 (m, 2 H), 1.65–1.69 (m, 4 H), 2.24 (m, 2 H), 2.74 (d, J = 12.4 Hz, 1 H), 2.93–2.97 (m, 1 H), 3.22 (m, 1 H), 3.33–3.66 (m, 84 H), 3.82 (s, 2 H), 3.91 (s, 2 H), 4.10–4.12 (m, 1 H), 4.33 (s, 1 H), 4.52 (s, 4 H), 6.90–6.99 (m, 2 H), 7.76–7.60 (m, 2 H). Ultraviolet maximum absorption was at 365 nm and maximum fluorescence emission was at 515 nm in Leibovitz's L-15 medium (pH 7.8). Mass calculated for C66H112N11O24S3 (n = 18, M + 1H+) 1538.704, found 1538.706.</p><!><p>In a well of 96 quartz plate (500 µL), HEPES buffer (30 mM) solution (400 µL, pH = 7.7) containing MgSO4 (5.0 mM), ATP (2.6 mM), DTT (3.5 mM), CoA (1.5 mM), and commercially available firefly luciferase (40 µg/mL) were mixed. The probe B-YL was then added and mixed to a final concentration (32 µM). Bioluminescence spectrum of B-YL was then taken immediately with a Synergy Mx (BioTek, VT) over the wavelength range 520–650 nm.</p><!><p>Streptavidin (189 µM, stock solution A) was prepared by dissolving streptavidin solid (10.0 mg) in PBS buffer (1.0 mL, pH 7.4). Biotin was prepared in dimethyl sulfoxide (DMSO) (1.0 mM). Biotin (1.0 mM, 0.189 mL) and B-YL (6.5 mM, 0.0852 mL) were added to DMSO (0.20 mL), resulting in solution B after mixing. Solution A was then mixed with solution B evenly. The resulting mixture was incubated at room temperature for 30 min. The mixture was then transferred into a membrane filter cassette (20 kDa cutoff) and dialyzed in PBS buffer (1000 mL) overnight. The B/SA/B-YL solution was removed from the cassette. Concentration was measured by UV spectroscopy (76.4 µM, 353 µg/mL).</p><!><p>Biotinylated EGF short peptide (B-Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile-NH2) was prepared in dimethyl sulfoxide (DMSO) (1.0 mM). Biotinylated EGF short peptide (1.0 mM, 0.189 mL) and B-YL (6.5 mM, 0.0852 mL) were added to DMSO (0.20 mL), resulting in a solution B after mixing evenly. Solution A was then mixed with B evenly. The resulting mixture was incubated at room temperature for 30 min. The mixture was then transferred into a membrane filter (cassette, 20 kDa cutoff) and dialyzed in PBS buffer (1000 mL) overnight. The EGF-B/SA/BYL solution was drawn out of the membrane filter. Concentration was measured by UV spectroscopy (111 µM, 512 µg/mL).</p><!><p>For the in vitro luciferase assay, cells were plated in triplicate on black walled 24-well plates with increasing cell numbers (0–60000 cells) of either U87 not expressing luciferase or with luciferase. Cells were grown overnight with regular growth medium. After 24 h, the regular medium was replaced with 100 µg/mL B-YL Leibovitz's L-15 medium with MgCl2 (5 mM). Bioluminescence images were taken immediately after adding the substrate into the cells using an IVIS 200 In vivo Imaging System (PerkinElmer, MA).</p><!><p>U87 cells were implanted subcutaneously on the left (without luciferase) and right flanks (with luciferase) of athymic nude mice as per IUCAC approved protocols. Prior to in vivo imaging, the mice were anesthetized with isoflurane. Targeted EGF-B/SA/B-YL (N = 5) or untargeted B/SA/B-YL (N = 5) solution was then injected intraperitoneally (150 µg/kg body weight). The mice were imaged using an IVIS Spectrum. Mice were imaged every hour for 8 h and then every 24 h thereafter. Relative luminescence was quantitated by creating a region of interest (ROI) over the tumor and expressed as photons/(s·cm2). To standardize the data, a ROI was drawn around a white light image of each tumor. Light emission was quantified from the same surface area (ROI) for each tumor. Corresponding gray scale photographs and color luminescence images were superimposed and analyzed using Living Image analysis software version 3.1 (Caliper Life Sciences, CA).30</p><!><p>All data is expressed as mean ± SD. All data analysis was performed using GraphPad Prism software version 6 (La Jolla, CA) unless specified. Multiple variables were analyzed via analysis of variance techniques; a p value <0.05 was considered statistically significant.</p><!><p> Author Contributions </p><p>The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.</p><p>The authors declare no competing financial interest.</p>

PubMed Author Manuscript

Delivery and Tracking of Quantum Dot Peptide Bioconjugates in an Intact Developing Avian Brain

Luminescent semiconductor ~9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap-(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-\xce\xb2-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

delivery_and_tracking_of_quantum_dot_peptide_bioconjugates_in_an_intact_developing_avian_brain

<!>Synthesis of QD-Peptide Bioconjugates<!>Initial Comparison of PEG and CL4 Coated QDs<!>Evidence for Migratory Neuroblast Tracks<!>Widespread Distribution of QD-CL4-JB577 in the Developing Central Nervous System at E8<!>Changing Pattern of QD Distribution with Developmental Time<!>QDs in the Choroid Plexus<!>Neural Differentiation after QD Incorporation<!>Quantum Dots<!>Peptides<!>Quantum Dot-Peptide Bioconjugate Microinjection into the Embryonic Spinal Cord<!>Histology<!>mRNA in Situ Hybridization<!>Microscopy and Image Analysis<!>Simulating the QD Ligand and Peptide Structures

<p>A major obstacle to the treatment of neurological diseases with small molecules or peptides/proteins is the delivery to individual cells in the central nervous system. For small molecules, the overall charge of the molecule and a mechanism to prevent sequestration in the endosome are thought to be critical factors. For example, positively charged dopamine does not cross the blood-brain barrier, whereas its zwitterionic precursor (3,4-dihydroxyphenylalanine or L-DOPA) readily crosses the barrier and is taken up by neurons to relieve some of the symptoms of Parkinson's disease.1 For larger peptides or proteins, a specific targeting sequence has been employed with limited success, for example, the 29 amino acid RVG sequence from the rabies surface coat protein2,3 or a synthetic peptide based on the positively charged TAT peptide.4,5 As an initial step toward designing a vehicle that could deliver biologically active chemicals into the brain, we previously showed that JB577, a pharmacological chaperone (an inhibitor of palmitoyl:protein thioesterase based on palmitoylated-K-Ras4A C-terminal sequence required for membrane association) could uniquely promote both membrane penetration and endosomal escape.6,7 This chaperone lipopeptide contained a positively charged lysine-rich region and a noncleavable palmitic acid tail. We then appended the peptide tail with a polyproline sequence and a glycine spacer together with a 6 amino acid polyhistidine domain which allowed it to self-assemble by binding to Zn on the surface of luminescent CdSe/ZnS semiconductor quantum dots (QDs). The intense QD fluorescent signal along with peptide presentation in a localized high avidity format allowed us to evaluate peptide functional properties in a variety of cell lines and a hippocampal brain slice model.6,8,9 A series of Förster resonance energy transfer studies confirmed that the peptide stayed associated with the QD for at least 72 h, and this study confirms that QDs coated with specific types of chemical coatings are stable nanoparticles that can be utilized to understand many of the mechanistic processes and intricacies surrounding the burgeoning field of nanoparticle-mediated drug delivery both in vitro and in vivo.10</p><p>QDs have the advantage that they can be inherently chemically synthesized with a discrete and narrow emission profile by adjusting their physical dimensions, and their spectral properties allow them to fluoresce intensely and with extended longevity.7,9,11 Furthermore, the surface physiochemical properties and character of the QD can be engineered to specifically target a particular type of cell or tissue. It has been previously shown that coating the QDs with negatively charged carboxyl groups increased leukocyte adherence and migration whereas positively charged amino groups and neutral PEG did not.12 Other studies have focused on further modification of the QD surface with active targeting moieties, for example, an RGD sequence ligand specifically targeted QDs to brain tumors,13 whereas an in vivo injection of uncoated (neutral) QDs into the hippocampus of live mice mainly targeted the QDs to microglia.14 These and other similar results confirm that the coating of QDs can affect its destination following injection. More importantly, multifunctional QDs have been demonstrated to deliver an active druglike cargo of siRNA to knockdown EGFRvIII receptors in human glioma cells,15 suggesting that QDs can be designed to deliver cargo to a specified target cell type. However, there is still limited data reagarding their fate in an intact normal brain and no conclusive studies conducted on the fate of chemically modified QDs injected into a developing brain.</p><p>We have previously reported that CdSe/ZnS core/shell QDs coated with a dihydrolipoic acid (DHLA)-based zwitterionic compact ligand were able to specifically target QDs to neurons, with minimal uptake by glial cells or microglial cells, in actively myelinating and physiologically intact rat hippocampal slices.9 The combination of small size (≤10 nm hard diameter), uniquely intense spectral properties, the ability to synthesize different compact zwitterionic ligand coatings, and the ability to spontaneously conjugate to JB577 peptide enabled us to follow their fate after injection into embryonic chick brain. We have achieved the successful introduction of such QDs by a novel microinjection procedure at an early embryonic stage of development and can demonstrate the extensive brain distribution in a developing central nervous system without overtly affecting brain development, despite the rapid increase in size and complexity of the brain during this period.</p><p>The developing chick embryonic brain presents numerous advantages for assessing the ability of QDs to deliver a chemical cargo such as JB577 (and/or other bioactive peptides) to all parts of a developing nervous system. While rats and mice have often been used to study embryonic neuronal and glial cell differentiation and migration,16,17 this requires the use of timed pregnant mice, and large mouse colonies in order to get enough embryos. Furthermore, although methods for electroporation and viral infection have been established for both mouse and chick, the in ovo method is much simpler than the in utero method. The chick model also allows for larger sample sizes, ease of handling, and the ability to harvest brains at precise developmental intervals. The chick model is absent of confounding maternal or placental influences and upon hatching, has a comparable developmental maturity to the human brain by birth.18 We chose to microinject the QDs at embryonic day 4 (E4), because at this age the chick brain consists of a neuroepithelium that differentiates in waves to eventually generate the neurons, astrocytes, and oligodendrocytes that form functional centers in a schedule consistent with that observed in mammals. The chick is therefore a model whose embryonic development is well-studied and documented, and the anatomy of the brain is well-defined in particular for the laminar structure of the optic tectum in the midbrain.19,20 We have not previously observed any gross toxicity with the CdSe/ZnS QDs in cells or actively myelinating neonatal rat hippocampal slices,9 and our experience with interventions in the developing chick embryo in vivo suggested that injecting the QDs at E4 would cause minimal trauma and injury to the embryo and not result in major experimental embryo death. We therefore utilized the chick as a system in which to follow the distribution of peptide-functionalized QDs to a developing brain without adversely affecting critical brain structures or their function.</p><!><p>We started with 625 nm emitting CdSe/ZnS core/shell QDs with a quantum yield in PBS of ~50%. These bright red emitting QDs provided for easy distinction and separation of QD fluorescence from naturally occurring autofluorescence in the dense chicken embryonic brain tissue. We initially began testing delivery with QDs surface functionalized and made hydrophilic with DHLA derivatives displaying either a long, neutral PEG moiety or a far more compact zwitterionic functional group (CL4: see Figure 1 for ligand structures and schematics of the QD-peptide conjugates21,22). The PEG mediated colloidal stability across a broad pH range via its ~15 ethylene oxide repeats, while the CL4 exploited its zwitterionic nature to accomplish the same goal while significantly reducing the QDs hydrodynamic size. Both ligands are also known for their nonfouling and nonbinding nature and were superior to a range of other coats tested.</p><p>The relatively large surface area of these ~9.5 nm diameter 625 nm emitting QDs (266 ± 25 nm2 with a radius of 4.6± 0.4 nm) provided for the optimal conjugation of a 25:1 ratio of peptide:QD or as required.8,23 For the controlled display of peptides on the QD surface, we relied on the metal affinity driven coordination of polyhistidine sequences, (His)n, with the ZnS outer shell of the QD.8–2126 This rapid, high-affinity cooperative interaction only required mixing of the requisite amounts of peptide per QD and in many cases provided for control over the ratio of peptide displayed per QD along with the subsequent orientation.24 The peptide tested was a chemical chaperone (GDap[palmitoyl]VKIKK, based on the ras-4A palmitoylation site) designed to promote the refolding of misfolded protein (palmitoyl:protein thioesterase) when delivered to cells. This lipopeptide is part of the cell penetrating Palm-1 lipopeptide referred to here as JB5778 and was chosen for its unique ability to effect both cellular delivery and endosomal escape of various nanoparticles in a nontoxic manner.8 The modular nature of the peptide sequence, W•G•Dap(Palmitoyl)•VKIKK•P9•GG•H6 with a peptide bond "•" separating functional modules, provided several desired functions to the peptide. The final peptide includes Trp (W) for monitoring peptide purification via UV–vis absorption; Gly2 as flexible hinges and Dap(Palmitoyl) and VKIKK (based on the K-Ras4A C-terminal sequence utilized for membrane association and penetration) to jointly provide for endosomal release in a manner that is still not fully understood. (Pro)9 acts as a rigid linker that displaces the peptide N-terminus away from the QD surface and prevents it from folding back on the QD (in the case of PEGylated ligands). The (His)6 binds the Zn molecules on the QD surface and thereby mediates long-term peptide attachment to the QD8. Similar QDs have been reported to be stable in monkey brain for at least 90 days.27</p><!><p>Given the challenging nature of the environment in which the QD-peptide conjugates would be placed, we initially sought to determine the optimal ligand coating for these experiments. Embryos at E4 were injected in the spinal cord canal with either CL4-coated or PEG-coated QDs assembled with a ratio of 25 JB577 per QD following the protocol described in the Methods. These embryos were then harvested and sectioned at embryonic stage E6. The distribution of both the CL4-coated and PEG-coated QDs were then visually analyzed in longitudinal sections (40 μm) from comparable regions along the spinal column (see Figure 2). Clusters of fluorescence appeared more frequently with PEG-QDs as compared to CL4-QDs although the reasons for this observation are not understood at the present time. One possibility is that the extended length of the PEG surface on the QD could result in the screening or obscuring of a key portion of the JB577 needed for cellular uptake or tissue binding in this particular context. The more compact CL4 surface allows such interactions to occur. Additionally zwitterionic groups are commonly found in biological systems and are one of the primary mechanisms by which proteins remain both soluble and noninterfering. The CL4-QD peptide conjugates were better tolerated in this environment.25 Supporting this latter notion, Muro et al. reported that QDs coated with a structurally similar zwitterionic DHLA-sulfobetaine group remained far more dispersed and were far better tolerated in both HeLa cells and Xenopus laevis embryos than QDs capped with micelles or polymers.26 Aggregation of QDs in vivo is believed to increase the risk of cell-toxicity.27 Therefore, in terms of coupling and stability in vivo and minimizing any potential toxicity issues, the CL4-coated QDs were utilized throughout the rest of the study, and in conjunction with the JB577 peptide. These are referred to hereafter as "QD-CL4-JB577".</p><!><p>Using just the QD-CL4-JB577 conjugates, we continued on with our experimental format and observed clear and extensive clonal dispersion of the QDs, and the representative example provided in Figure 3 shows them visualized in the E8 midbrain. The QD-CL4-JB577 conjugates always appeared to be distributed along several parallel "tracks" extending radially outward from the ventricular zone. This distribution most likely indicates that several neural stem cells located in the ventricular surface28 at the time of injection had taken up massive amounts of QDs and distributed them to their progeny. At the time of injection, E4, most of the neural stem cells are expected to express SOX2,29,30 maintain a radial glial morphology, and generate neuroblasts.31 Following this, during the subsequent 2 days, the newly generated neuroblasts should migrate radially to their specific layer location along the radial glial tracks in the midbrain.31 Thus, the observed columnar distribution of cells at E8 (Figure 3) could represent neuroblasts migrating radially and carrying the QDs with them. Furthermore, a layer of heavily QD labeled cells can be distinguished which corresponds to the densely packed neuronal nuclei layer confirming their intracellular localization (bracket in Figure 3B). Although beyond the present scope of this study, the evidence of cell migration in many areas suggests that the QD-CL4-JB577 could be used to study migratory patterns in the developing nervous system and how this could potentially be used to deliver peptides and proteins to all regions of the brain.</p><!><p>We observed maximum brain labeling around E8 at which time QDs were distributed extensively and uniformly throughout the entire brain, forebrain, midbrain, and hindbrain, forming distinct patterns of labeling, as observed from longitudinal sections of the brain (see Figure 4). Although the QDs tend to densely populate some regions over others (especially ventricular areas), confocal microscopy confirmed that QDs generally labeled the entire brain at E8 from forebrain to hindbrain. Also, aggregation of QDs in the ventricular walls was occasionally evident (Figure 4). Confocal microscopy further confirmed that the fluorescent particles detected were not surface artifacts but were inside the cells.9 This widespread distribution of the QDs was encouraging from the point of view of eventually treating genetic disease where each cell bears the lethal mutation.</p><p>As evident from the distribution of the QDs in midbrain at E8, the layer of heavily QD-labeled cells (Figure 4, bracket) most likely corresponds to the neuronal layer which formed from neural stem cells located at the ventricular zone at the time of QD delivery (E4). These observations confirm the significant initial intracellular uptake of QDs at the time of their delivery. Since QDs were evident in the ventricles for a few days after the initial delivery, it is probable that incorporation of QDs into the tissue proceeds for several days. This would also help explain why the QD emission decreases from E8 onward. The extensive labeling by QDs transported along the spinal cord and throughout the developing brain to all types of cells and the developing brain from E6 to E15, and even a small amount of labeling at E19 compares favorably with the localized distribution of viral constructs following injection into brain.28 While the QDs were numerous and uniform at E8, the continual differentiation of cells and increase in brain size32 was associated with the formation of complex structures absent from the early developmental stages such as the choroid plexus. These became evident by E11, and became intensely labeled from E11 to E15. After E15, the level of QD labeling declined steeply so that QDs were mostly undetectable at E21.</p><!><p>In order to track the labeling pattern in brain as development progresses, embryos injected in the spinal canal with QD at E4 were sacrificed at E8, E11, E15, E19, and E21 (hatching) days (3–4 embryos per age) and processed for fluorescent analysis. A representative section from the different developmental ages is depicted in Figure 5 in order to appreciate the overall labeling pattern. A view of a random but representative areas (box) with both DAPI staining (middle panel) and QDs alone (lower panel) showed no abnormalities attributable to the QDs. Although they only represent a small region of the brain, the lower panels are accurate indicators of the frequency of fluorescent QDs (roughly corresponding to the amount of QDs in a given cross-sectional area) at each stage of development. Using ImageJ, Z-stack analysis of E15 brain sections selected at random showed that approximately 50% of detectable QDs were intracellular (inside cells) (data not shown). Comparison of the fluorescence intensities and frequencies among each stage of development revealed the following: (i) Peak fluorescence and labeling occurred at E8. (ii) A steep decline in fluorescence and labeling was observed after E15. (iii) Very little fluorescence was detected at E21. Although this was not strictly a quantitative study, we concluded that overall QD labeling was maximum at E8, declined slightly from E8 to E15, and then steeply declined to almost no detectable fluorescence at E21(hatching). Interestingly, some ventricular content can still be observed until E15. Previous studies have shown that the size of the chick brain increases 8-fold from E4 to E6, 10-fold from E6 to E8, and a further 2–3-fold to hatching. Overall, there is a 150-fold increase in size during this time frame so considerable dilution of the dots is expected. However, we believe that some of the dramatic decrease in overall fluorescence is connected to the development of the choroid plexus.</p><!><p>At E11, we observed that the labeling pattern dramatically changed (Figure 6A), and that it coincided with the extensive labeling of the choroid plexus (E11, Figure 6B), a structure that develops between E8 and E15 in the chick brain.33,34 The enrichment of QDs in the choroid plexus was also visible at E15 (Figure 6C). This clearly suggested that the microinjection of QDs had no visible or functional negative impact on the assembly of this complex structure. QDs were injected at E4 when the brain is just a simple neuroepithelium that contains only neural stem cells which are differentiating into neurons. As development progresses, the neural stem cells give rise to astrocyte and oligodendrocyte precursors and eventually to specialized glial cells such as ependymal cells. After migration and terminal differentiation the cells ultimately organize into collective systems with distinct structures and functions. The choroid plexus (CP) is one such structure that can be distinguished from E11 onward and from this study seems to play an important role in the later distribution of QDs. Thus, it was surprising that despite the >100-fold increase in tissue size from E6 to E15, in all experiments we still observed intense staining of the choroid plexus at E15 (Figure 5, upper panel). This intense labeling was transient since it declined rapidly to E18 until it was barely detectable at birth (E21) (Figure 5). The choroid plexus develops on the anterior ventricular roof from a sagitally oriented fold and some posteriorly located transverse folds.33 It is a collection of modified ependymal cells and capillaries that form a system of producing and distributing cerebrospinal fluid (CSF.) These ependymal cells are glial cells whose apical surface is surrounded by cilia that push around the CSF, similar to the brush-border interface of microvilli in the intestine. Furthermore, they produce CSF and transfer the fluid through tight junctions and so effectively secrete the QDs. By E11, round cells start to appear on the entire plexus (Figure 6A). The QD-CL4-JB577 injected at E4 were stable enough to extensively label the developing choroid plexus 11 days later, and both the E11 and E15 samples clearly showed the expected folds of the CP (see Figure 6B and C). Concurrently, QD labeling was more concentrated in the CP compared to the other regions of the brain. While E8 samples showed uniform distribution of QDs, E11 and E15 samples displayed a shift in fluorescence toward the CP and less fluorescence in other regions. Ultimately, at birth (E21), fluorescence was barely detectable (Figure 5).</p><!><p>Since the QD were injected in a critical period of neural differentiation and expansion of neural precursors, it was important to verify that the progression into different neural cell types was not affected by their presence or incorporation. To this end we compared the expression patterns of different known markers of neural stem cells and their differentiated progeny between E8 brains from embryos injected at E4 with either QD-CL4-JB577 or control vehicle (5% DMSO). mRNA in situ hybridization was then used to show that the expression pattern of the transcription factor SOX2, proteolipid protein (PLP), class III-tubulin beta (TUBB3), and brain lipid binding protein (BLBP) was normal in E8 and E15 midbrain and hindbrain (Figure 7). The timing and pattern of expression of these markers is extremely conserved and quickly shifts as the cells differentiate, making them ideal indicators of developmental progression.</p><p>SOX2 mRNA is strongly expressed in proliferating neural precursors and differentiation induces downregulation of SOX2, although expression is subsequently transiently upregulated in some subpopulations of mature neurons.30 At E8 and E15, we detected expression of SOX2 mRNA associated with the ventricular zone precursors (neural stem cells) and a specific layer of mature neurons in the midbrain in sections from both QD and vehicle injected embryos (Figure 7). The absence of any detectable change in the pattern of expression gives further support to the benign nature of the QDs and JB577 used in this study.</p><p>Proteolipid protein (PLP) mRNA is expressed in oligodendrocyte progenitors at E8 on cells located in the ventral midline.35 The expression pattern of PLP was as expected and showed no differences between QD-CL4-JB577 and 5% DMSO- injected embryos. Additionally, the PLP expression pattern was also analyzed later on in development (E15) during the expansion of oligodendrocyte precursors in chicks with similar patterns of expression noted for both QD and 5% DMSO-injected brains (Figure 7), further suggesting the lack of overt or gross toxicity originating from the QDs used in this study. We chose to compare the expression of the brain-specific member of the lipid-binding protein family (BLBP) since its expression is spatially and temporally correlated with neural differentiation in many parts of the CNS, including in the postnatal cerebellum, embryonic spinal cord, and cerebral cortex. BLBP is transiently expressed in radial glia in the embryonic ventricular zone36 and it is required for the establishment of the radial glial fiber system necessary for the migration of immature neurons to establish cortical layers.37 Thus, our finding that expression of BLBP is normal in E8 hindbrain radial glial is strong evidence that chick brain development was unaffected by QD injection. After neuronal migration, the BLBP positive precursors migrate away from the ventricle and initiate the astrocytic phase of the differentiation process. By E15, this process is well underway in the chick midbrain and expression of BLBP in QDs and vehicle injected brain was the same (Figure 7). We also analyzed the expression of GLAST (solute carrier family 1 (glial high affinity glutamate transporter) member 3) at E15. GLAST belongs to an astroglial glutamate transporter family which maintain physiological extracellular glutamate concentrations and are expressed by astrocyte precursors.38,39 Their expression correlates with the status of glial differentiation/maturation, making them a good measure of normal brain developmental progress. Again GLAST expression was unaffected by the QDs (Figure 7). Lastly, we present evidence that beta-tubulin-III (TUBB3), which is expressed in postmitotic neurons, was also normally expressed in the midbrain and hindbrain regions at E8 and E15 and was unaffected by QD incorporation (Figure 7). In particular, in E15 midbrain the expected number of neuronal layers along with their distribution and thickness was observed in the QD-injected brain indicating that the neuronal cell proliferation over generations and migration is occurring normally in these brains. We have previously used similar in situ hybridization assays to demonstrate increases in expression of astrocyte differentiation genes GFAP (glial fibrillary acidic protein), GLAST, and GS (glutamine synthase) in the nanomelic chick which lacks aggrecan (a proteoglycan with a major role in glial cell maturation) and in chick injury models.18,35 Thus, the absence of any change in this expression is good evidence of lack of toxicity of the QDs and the chicks appeared normal at hatching.</p><p>There have been many attempts to deliver QDs to cells and tissues by either passive delivery, relying on surface functionalization and charge,6–8 active delivery, such as electroporation, or facilitated delivery, where, for example, a cell-penetrating peptide and the cargo are conjugated to the QD.9,40,41 However, relatively few studies have followed the fate of QDs in a living, intact brain.15,42 We therefore designed a novel microinjection system to deliver a precise amount of the synthetic lipopeptide JB5776–8 into the spinal cord of a developing chick embryo at E4 and showed that the QD-complex was initially targeted to neurostem cells and the neuroblasts present in the brain at time of the injection. Distribution was even and widespread, with very little sign of QD aggregation or chemical instability in tissues. Because of the high intrinsic fluorescence of the QDs used, we were able to follow the fate of the QDs in the chick brain through hatching (17 days later at E21), even though its volume increased >150-fold.32</p><p>Successful microinjection of QDs at E4 resulted in extensive labeling at E6, and replacing the choice of a neutral PEG coating with a zwitterionic compact ligand, "CL4" (Figure 1) gave improved distribution of label at both E6 and E8. Therefore, for all the subsequent studies described, we used QDs coated with CL4 to which JB577 was attached via the His6-linkage. The uniform labeling of QDs along the spinal tract not only demonstrates the broad perfusion of QDs into the administered region but also the ability of the QDs to migrate a significant distance from the site of injection along the spinal cord canal and into the ventricular system of the brain, penetrating the surrounding nervous system tissue in the process. Whereas our previous study explored the cell-type specific targeting of different types of QDs in specified cell types,9 the present study explored the widespread-distribution of QDs in the central nervous system in a model system as it developed. At the time of delivery of the QD (E4), the neural plate has already closed, a neural tube has formed along the whole longitudinal axis of the embryo, and the neuroepithelium cells have gone through several cell divisions. At this stage, most of the cells are migrating neuroblasts or neural stem cells of radial glial morphology, which remain associated with the ventricular zone.17,19,28,31 We conclude that significant amounts of QDs are taken up by these cells and then distributed to their progeny as they migrate radially, carrying their QDs with them.</p><p>Evidence that normal milestones were achieved in E8- and E15-brains which received QDs was provided by a series of in situ hybridization studies. TUBB3 expression was high and well-distributed in the expected patterns, indicating normal generation and migration of neurons. The transcription factor SOX-229,30 showed a normal pattern of expression as well indicating that neural stem cells distribution and survival were unaffected by the QDs. Glial cell markers PLP, BLBP, and GLAST18,35–38 appeared at the expected time and in normal distribution patterns, suggesting that developmental progression to the gliogenic phase of brain development was proceeding normally. We conclude that there was no evidence of growth retardation or acceleration of growth or differentiation. The chickens developed normally and hatched from their eggs on schedule where they displayed normal pecking and feeding behavior identical to control animals.</p><p>We have previously demonstrated that QD-CL4-JB577 particles target the perinuclear region of neuronal cell bodies in the CA3 cell layer of hippocampal slice cultures with no toxicity or morphological changes (up to 72 h in culture). A similar lack of any overt toxicity was observed in the developing chick brain with the same QDs. Both stability and toxicity of the QDs had been an issue in previous studies in which QDs/nanoparticles were administered intravenously in monkeys and mice and in both instances there was diminished overall fluorescence in the long term in in vivo systems.13,27 We similarly observed a time-dependent decrease in overall fluorescence in our study which greatly accelerated after E11. At E19, only a few QDs were visible in the brain and were scattered throughout the section in a seemingly sporadic rather than the uniform manner observed at E8-E15. However, the structure of the brain appeared completely normal. Between E15 and E19, the choroid plexus was no longer intensely labeled by QDs, and overall fluorescence was greatly reduced. The QDs were most likely transported out of the brain after the embryo developed its functioning choroid plexus. The structure likely recognized the QD/CL4s/QD-CL4-JB577 as foreign particles, and removed QDs from circulation in the brain. Previous studies22 suggest stability in vivo up to 90 days so they are unlikely to have ceased to be emissive due to formation of surface defects or traps or to be degraded sufficiently to release Cd2+ ions to the intracellular and intercellular environments. One additional factor is that a significant dilution of the QDs occurs because the brain increases in volume some 150-fold and after the development of the choroid plexus there is accelerated removal of QDs via the glymphatic system.43 We conclude that a combination of these explanations is the most likely.</p><p>The main advantage of chemically synthesizing QDs with negatively charged coats (CL4) and a cell-penetrating peptide (JB577) is that they can deliver additional cargo beyond the 25 peptides/QD. Should the Cd prove to be toxic in future studies, we can replace them with less toxic QD materials such as those with InP cores.7,10,11 Interestingly, reports focusing on the toxicity issue are somewhat inconsistent in terms of answering major issues. Mice injected with amphiphilic PEG-coated QDs at 20 pmol/g animal weight were viable until necropsy at 133 days. However, injection of maltodextrin coated CdSe QDs into the air sac of early stage E1–2 chickens was reported to be extremely cytotoxic and embryotoxic.44 We believe this difference from our experience with embryonic chicks to be primarily a technical issue, either the quality of the nanoparticles or the time of injection (E1–2 compared with E4 in our study) or the use of airsac absorption. There was certainly no indication of any intrinsic toxicity of QDs as we prepare them. Moreover, we note that the Cd in those core-only QDs had direct access to the biological environment and were not protected by an overcoating shell as our materials are. Another report of toxicity occurred when a synthetic peptide (APWHLSSQYSRT) was conjugated to CdSe/ZnS QDs and targeted to Rhesus monkey embryonic stem cells. This study used a small S-CH2COOH QD coating, which is known to undergo pH dependent aggregation,7 something that is not an issue for the DHLA-based zwitterionic coatings used in this and our previous studies.21 More in agreement with our findings is a recent long-term study in Rhesus monkeys which showed the QDs to be intact in the body for up to 90 days and showing no toxicity after 3 years.27</p><p>The primary aim of this study was to show that we could deliver a QD-peptide functionalized construct to an intact normally developing nervous system and observe the distribution of the QDs with their surface cargo to all brain regions. This has been achieved, and we have supplied ample evidence that our QD constructs produced minimal observable gross or overt toxicity in chicken embryos. We suggest that the previously observed QD toxicity is very much dependent on the conditions of their use, the composition of the QD core and whether it is protected by an overcoating layer, the chemicals used to coat the QDs, the conjugated peptides attached to the QDs, and the technique used to administer the QDs. We conclude that, by carefully choosing the chemistry of the core and coating, QDs have the potential to be safe for long-term research applications such as demonstrating that JB577 can function as a chemical chaperone in the CNS.</p><p>A major barrier to applying chemistry to treating neurodegenerative disease with QDs is the blood-brain barrier. This study does not address this question directly since we injected directly into the spinal cord, but we believe that the uniform distribution of the QD label in the brain suggests that JB577 is functioning as a brain barrier cell-penetrating peptide. Previously, nanoparticle (NP) iron chelators have been used with limited success to treat Alzheimer's disease and other neurologic disorders while PEGylated polymeric NPs have been tried therapeutically in prion diseases but with limited success.45 Other strategies to target the brain with NPs have taken advantage of the interaction with specific receptor-mediated transport such as the transferrin receptor (TFR). TFR binding antibodies were attached to cell penetrating peptides, and melanotransferrin was incorporated into chimeric constructs but all have met with limited success. Polysorbate 8038 and antitransferrin receptor monoclonal antibodies (MAbs) have been tested extensively but a lot of research is still needed before they can be considered for treatment of human diseases. The hexapeptide dalargin, tubocurarine, and the lipid-soluble P-glycoprotein substrates loperamide-45 and doxorubicin have all been coupled to polysorbate 80-coated nanoparticles to little avail,46 so we conclude that QDs with the appropriate chemical modifications (QD-CL4-JB577) have enormous potential to overcome the failures of traditional therapeutics, which often fail because of poor water solubility or a lack of target specificity.42,46</p><p>In summary, we conclude that QDs offer the exciting potential of uniformly delivering small molecule therapeutics or peptides (or proteins) to the developing brain in a model system together with the equally important ability to directly track chemically synthesized, biologically active compounds and see if they are actually delivered to the correct cells and subcellular compartments. This is especially critical if, for example, QDs were to be used to deliver pharmacological chaperones to promote folding of misfolded proteins in the ER47–49 in inherited diseases resulting from a single missense mutation in a gene. In our example, the drug is a small molecule inhibitor of the enzyme palmitoyl:protein thioesterase48,49 (the DAP1 structure incorporated into JB577) which functions as a chaperone. By coupling to QDs, we can follow its fate in detail. To date, there is only one chemical chaperone (Migalastat) which is believed to function as a chaperone in certain patients with α-galactosidase deficiency (Fabry disease),47 but this is clearly a rapidly expanding area of small molecule chemical therapeutics in the CNS with great potential to benefit from the unique properties that nanoparticles such as QDs can provide.</p><!><p>CdSe/ZnS core/shell QDs50 with an emission maxima centered at 625 nm were from Invitrogen Life Technologies (Carlsbad, CA) and were made hydrophilic by exchanging the native surface with DHLA (dihydrolipoic acid) appended to either polyethylene glycol (PEG, Mw ~ 750/~15 ethylene oxide repeats) terminating in a methoxy group or the zwitterionic compact ligand CL4 as described previously.21,22 These are referred to as DHLA-PEG or DHLA-CL4 ligands/QDs.</p><!><p>The palmitoylated peptide (JB577, WGDap(Palmitoyl)-VKIKKP9GGH6, N-terminal acetylated/C-terminal amidated) sequence used was based on that of Ras-4A(GC(Palmitoyl) VKIKK)51 where "Palmitoyl" corresponds to a C16:0 palmitate group that is covalently attached to a nonhydrolyzable diaminopropionic acid (Dap) functionality synthesized into the peptide backbone. All peptides were synthesized using Boc-solid phase peptide synthesis,6,8,52 purified by HPLC, and characterized by electrospray ionization mass spectrometry. All peptide sequences are written in the conventional amino-to-carboxy terminus orientation.</p><!><p>QD-Palm-1 bioconjugates were diluted from a stock solution of preformed peptide-QD complexes (1 μM QD assembled with a ratio of 25 JB577 peptides per QD-CL4 in 5% DMSO in PBS) into complete growth medium to a final QD concentration of 50–100 nM for 1 h. Fertilized White Leghorn chicken (Gallus gallus) eggs were obtained from Sunnyside Hatchery (Beaver Dam, WI) and incubated at 37.9 °C and 90% relative humidity in a Midwest incubator. At embryonic day 4 (E4) (stage 21), chicken embryos were made directly accessible by cutting a window in the eggshell and carefully removing the extra-embryonic membranes covering the embryo, using forceps and spring scissors. Injection of a mixture of QD-CL4/PEG-JB577 and methyl green (0.25 mg/mL) into the central canal of the spinal cord was performed with a glass capillary needle. Control embryos were injected with vehicle solution (5% DMSO, 0.25 mg/mL methyl green in PBS). The maximum injection volume (2–4 μL) was achieved when the dye injected into the ventricle by this new procedure had reached the brain vesicle as visualized by the distribution of the methyl green colored solution. After retraction of the capillary, the embryo was hydrated with sterile PBS, the window in the eggshell was resealed with 3 M vinyl tape, and the embryos allowed to incubate for the specified times.18 As with all similar studies, including those designed to electroporate DNA constructs at E4,53,54 there was an average of 50% mortality in injected chick embryos from damaging the allantoic and vitelline extra-embryonic vessels during the procedure. Typically we injected 18 embryos with QD-peptide conjugates and dye and compared this to 18 injected with dye alone over the time period studied. There was no difference in mortality between QD-injected and control animals. A total of 180 eggs over a period of 2 years were microinjected for these studies. We observed no size or gross physical differences between control and QD-injected chick embryos during these experiments. All age embryos were euthanized by decapitation prior to dissection of the brain in agreement with recommendations of the Institute for Laboratory Animal Research, the NIH intramural recommendations for rodent neonates, and the AVMA Panel on Euthanasia.</p><!><p>Eggs were removed from the incubator after their allotted incubation period (E6, E8, E11, E15, E19, E21), embryos harvested, and the brains dissected and fixed in 4% paraformaldehyde in PBS (PFA).25–27 E11 embryos and older were euthanized by decapitation prior to dissection of the brain in agreement with recommendations of ILAR, the NIH intramural recommendations for rodent neonates, and the AVMA Panel on Euthanasia. Brains and spinal cords dissected from fixed bodies or heads were cryoprotected by incubation in 20% sucrose, 10% formalin in PBS solution for 24 h, embedded in 10% gelatin, 20% sucrose blocks and cut into 40 μm sections using a SM 2000R Leica microtome. Sections were mounted on Superfrost Plus glass slides (Fisherbrand, Pittsburgh,PA). Nuclei were counterstained with DAPI.</p><!><p>To prepare the digoxigenin (DIG)-labeled RNA probes used for in situ hybridizations, cDNA fragments from the PLP (proteolipid protein), TUBB3 (class III-β-tubulin), and BLBP (brain lipid binding protein) genes were prepared as previously described.18,35 cDNA fragments for SOX2 (SRY-related HMG-box gene 2) (nucleotides 402–948 of GenBank: D50603.1) was obtained by PCR from E8 chick brain cDNA using specific primers based on GenBank sequences. Riboprobes incorporating DIG-labeled nucleotides were synthesized from linearized plasmid templates with SP6 or T7 polymerase (Roche). Slices of 40 μm were prepared for mRNA in situ hybridization as described previously.18,35,55 After hybridization, DIG-labeled RNA duplexes were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche). Alkaline phosphatase activity was then detected using 5-bromo-4-chloro-3′-indolyphosphate p-toluidine (BCIP) and nitro-blue tetrazolium (NBT) substrates (Roche) as described previously.18,35,55</p><!><p>The intracellular distribution of QDs was assessed from fluorescent images obtained with a Leica SP2 spectral confocal microscope in the Integrated Light Microscopy Core Facility of The University of Chicago Cancer Research Center. Complete brain images were reconstructed as montages from individual images (obtained with a Zeiss AxioCam Mrtm) through manual tiling (<20 images) or automatic tiling (20–100 images). ImageJ software was used for image analysis.</p><!><p>The hard core–shell structure of the 625 nm emitting QD is a sphere of diameter ~10 nm based on TEM analysis. The DHLA-PEG ligand is a shell extending ~30 Å from the QD surface, and the DHLA-CL4 ligand is a shell extending 16.2 Å from the QD surface. The DHLA-CL4 extension used is based on previous results along with energy minimization21 and suggests that the ligand only extends to the portion of the peptide corresponding to the Gly2-Pro9 interface. It is important to note that these ligands may actually be even more compacted than depicted here (Figure 1). The palmitoylated peptide (JB577) is shown as a ball and stick structure oriented onto the QD surface by the terminal hexahistidine (His6) motif. These structural models were created using the same methods as described previously8 and only suggest one of many different possible configurations for each.</p>

PubMed Author Manuscript

Excited state dynamics for visible-light sensitization of a photochromic benzil-subsituted phenoxyl-imidazolyl radical complex

Visible-light sensitized photoswitches have been paid particular attention in the fields of life sciences and materials science because long-wavelength light reduces photodegradation, transmits deep inside of matters, and achieves the selective excitation in condensed systems. Among various photoswitch molecules, the phenoxyl-imidazolyl radical complex (PIC) is a recently developed thermally reversible photochromic molecule whose thermal back reaction can be tuned from tens of nanoseconds to tens of seconds by rational design of the molecular structure. While the wide range of tunability of the switching speed of PIC opened up various potential applications, no photosensitivity to visible light limits its applications. In this study, we synthesized a visible-light sensitized PIC derivative conjugated with a benzil unit. Femtosecond transient absorption spectroscopy revealed that the benzil unit acts as a singlet photosensitizer for PIC by the Dexter-type energy transfer. Visible-light sensitized photochromic reactions of PIC are important for expanding the versatility of potential applications to life sciences and materials science.

excited_state_dynamics_for_visible-light_sensitization_of_a_photochromic_benzil-subsituted_phenoxyl-

Introduction<!>Steady-state absorption spectra<!>Nanosecond-to-microsecond transient absorption spectra<!>Femtosecond-to-nanosecond transient absorption spectra<!>Effect of triplet-triplet energy transfer<!>Experimental Synthetic procedures<!>4'-Hydroxy-[1,1'-biphenyl]-2-carbaldehyde (1)<!>1-(4-(2-(4'-Hydroxy<!>Benzil-PIC<!>Steady-state measurements<!>Nanosecond transient absorption measurements<!>Femtosecond transient absorption measurements

<p>Photochromism, which is defined as the reversible transformation of a chemical species between two structural isomers by light, has been extensively studied over decades [1][2][3][4]. Recently, visible-light sensitized photochromic materials have been paid particular attention in the fields of life sciences and materials science because long-wavelength light reduces photodegradation, transmits deep inside of matters, and achieves the selective excitation in condensed systems [5][6][7][8][9][10][11][12]. General strategies for the sensitization of the photochromic reactions to visible light are to extend the π-conjugation and to utilize photosensitizers. Especially, triplet photosensitizers, which form the triplet state of a molecule by the triplet-triplet energy transfer, have been frequently used in photoresists, photodynamic therapy, and photocatalysts because the lowest triplet excited (T 1 ) state can be formed by light whose energy is smaller than that of the optically active transition [13][14][15][16]. However, photochromic reactions of some systems do not proceed via the T 1 state. For example, it was reported that the photochromic reaction of hexaarylbiimidazole (HABI), which is a well-known radicaldissociation-type photochromic molecule [17][18][19][20], is not sensitized by triplet photosensitizers [21][22][23]. On the other hand, it was reported that singlet photosensitizers effectively sensitize the photochromic reaction of HABI to the visible light [21,23]. While the S 0 -S 1 transition of HABI is located at the visiblelight region, the transition is optically forbidden. Therefore, the photochromic reaction of HABI without singlet photosensitizers occurs via the S 0 -S n transition, which is located at the UV region. On the other hand, singlet photosensitizers efficiently transfer the visible-light energy to the optically inactive S 1 state of HABI, and thus the photochromic reaction of HABI proceeds with visible light. The phenoxyl-imidazolyl radical complex (PIC, Scheme 1) is one of the recently developed rate-tunable T-type photochromic compounds which reversibly generate an imidazolyl radical and a phenoxyl radical (biradical form) in a molecule upon UV light irradiation [24]. The great advantage of PIC is the tunability of the thermal back reaction from tens of nanoseconds to tens of seconds by simple and rational molecular design [25]. The wide ranges of thermal back reactions of photoswitches expand the potential applications of photochromic materials such as to dynamic holographic display [26][27][28], switchable fluorescent markers [29][30][31], and anticounterfeit inks. However, PIC is photosensitive only in the UV region, which limits the application fields. It was reported that the S 0 -S 1 transition of PIC is optically forbidden and is located at the visible-light region as similar to that of HABI [32]. It is expected that the photochromic reaction of PIC occurs via the optically forbidden S 1 state as similar to other radical dissociation-type photochromic molecules such as HABI and pentaarylbiimidazole (PABI) [33][34][35]. Therefore, if we could substitute a singlet photosensitizer unit to PIC, the visible-light sensitivity could be achieved by singlet-singlet energy transfer. The visible-light sensitization of PIC expands the versatility of the rate-tunable photoswitches of PIC systems.</p><p>In this study, we synthesized a novel PIC derivative conjugated with a visible-light photosensitizer (Benzil-PIC, Scheme 1) and investigated the excited state dynamics. We used a benzil framework as a photosensitizer unit because aryl ketones have been widely used as visible-light photosensitizers [36]. While most of aryl ketones were used as triplet photosensitizers, the benzil unit in the present study acts as a singlet photosensitizer. The detail of the sensitization processes was investigated by wide ranges of time-resolved spectroscopies.</p><!><p>The synthetic procedure of Benzil-PIC is described in the Experimental part. Benzil-PIC has two structural isomers (isomer A and isomer B) as shown in Scheme 1. These isomers were separated by high-performance liquid chromatography (HPLC), and each isomer was characterized by steady-state absorption spectra and time-dependent density functional theory (TDDFT) calculations as shown below. Figure 1 shows the steady-state absorption spectra of the two isomers of Benzil-PIC and PIC in benzene at 298 K. While the absorption of PIC appears only at wavelength shorter than 350 nm, those of the two isomers of Benzil-PIC are extended to the visible-light region. The simulated absorption spectra by TDDFT calculations (MPW1PW91/6-31+G(d,p)// M05-2X/6-31+G(d,p) level of the theory) are also shown as the vertical lines in Figure 1. The simulated absorption spectra well explain the experimental absorption spectra of the two isomers. Therefore, the absorption spectra of isomers A and B were assigned as shown in Figure 1. The absorption band at 357 nm of isomer A is assigned to the electronic transition from the molecular orbital distributed around the triphenylimidazole unit (highest occupied molecular orbital: HOMO) to that around the benzil unit (the second lowest unoccupied molecular orbital: LUMO+1) (Figure S14, Supporting Information File 1). On the other hand, the absorption band at 375 nm of isomer B is assigned to the electronic transition from the molecular orbital distributed around the triphenylimidazole unit (HOMO) to that around the benzil unit and the phenoxyl unit (mainly the lowest unoccupied molecular orbital: LUMO and LUMO+1, Figure S15, Supporting Information File 1). While the HOMOs of isomer A and isomer B are very similar, the LUMO and LUMO+1 of isomer B are more delocalized than the LUMO+1 of isomer A, suggesting that the LUMO and LUMO+1 levels of isomer B are lower than those of isomer A. This would be the most plausible reason why isomer B has an absorption band at the longer wavelength than isomer A.</p><p>PIC generates the biradical species upon UV-light irradiation and shows the broad transient absorption spectrum over the visible-to near infrared-light regions. The half-life of the thermal back reaction of the biradical in benzene is 250 ns (the lifetime is 360 ns) at 298 K. To investigate the difference in the photochromic properties between two isomers of Benzil-PIC, we measured the absorption spectra and nanosecond-tomicrosecond transient absorption dynamics of isomer A in benzene upon repeated irradiation of 355 nm nanosecond laser pulses (355 nm, 7 mJ pulse −1 , Figure S8a, Supporting Information File 1). The absorption band at 357 nm of isomer A gradually decreases upon irradiation of the nanosecond laser pulses and the absorption edge alternatively shifts to the longer wavelength. It indicates that the irradiation of the UV pulse induces the photochromic reactions (breaking of the C-N bond) and interconverts between isomer A and isomer B. The system reaches the photostationary state (PPS) within 696 shots of the laser pulses. The ratio of isomer A and isomer B is estimated to be 22:78 by the curve fitting of the absorption spectrum at the PPS with those of pure isomer A and isomer B (Figure S9, Supporting Information File 1). Figure S8b (Supporting Information File 1) shows the nanosecond-to-microsecond transient absorption dynamics of isomer A probed at 650 nm under repeated irradiation with the 355 nm nanosecond laser pulses at 298 K. While the transient absorption dynamics of isomer A accumulated by 8 shots are slightly fluctuated most probably because of the low signal-to-noise ratio, the decay kinetics do not change by repeated irradiation with UV-light pulses. It shows that both isomers generate the same biradical form by UV-light irradiation as shown in Scheme 1, indicating that the excited state dynamics of the two isomers of Benzil-PIC after the bond breaking are identical. Therefore, the mixture solution of the two isomers was used for further time-resolved spectroscopic measurements.</p><!><p>To investigate the photochromic properties of Benzil-PIC, the nanosecond-to-microsecond transient absorption measurements were conducted by the randomly interleaved pulse train (RIPT) method [37]. Figure 2a shows the transient absorption spectra of Benzil-PIC in benzene (2.9 × 10 −4 M) under argon atmosphere at room temperature excited with a 355 nm picosecond laser pulse (pulse duration = 25 ps, intensity = 30 μJ pulse −1 ). At 0.5 ns after the excitation, two broad transient absorption bands are observed at 660 and <450 nm. The spectral shape is more or less similar to that of the biradical form of PIC [24], indicating Benzil-PIC generates the biradical by 355 nm light irradiation. The transient absorption spectra gradually decay with a time scale of hundreds of nanoseconds and another absorption band at 580 nm remains after 900 ns. The transient absorption dynamics at 590 nm was fitted with a biexponential decay function and the lifetimes are estimated to be 260 and 820 ns (Figure 2c). On the other hand, while the transient absorption spectra of Benzil-PIC in benzene under air show the same transient absorption spectrum as under argon at 0.5 ns, the transient absorption band at 580 nm is not observed in the time scale of microseconds. The transient absorption dynamics at 590 nm can be fitted with a single exponential decay function and the lifetime is 220 ns (Figure 2d), which is almost identical to that of the fast decay component under argon atmosphere. Because the transient absorption spectrum at 0.5 ns is similar to that of PIC and because the fast decay component does not depend on the molecular oxygen, the fast and slow decay components can be assigned to the biradical form generated by the C-N bond breaking and the T 1 state of Benzil-PIC, respectively. It is worth mentioning that the T 1 state of Benzil-PIC would be formed by some portions of the S 1 of the benzil unit where the energy transfer did not occur to the PIC unit (discussed below).</p><!><p>To investigate the sensitization process by the benzil unit of Benzil-PIC in detail, we performed femtosecond transient absorption measurements using a 400 nm excitation pulse. The instrumental response function is ≈170 fs. It is noted that the excitation wavelength for femtosecond transient absorption spectroscopy (400 nm) is slightly different from that for nanosecond transient absorption spectroscopy (355 nm). The difference may affect the ratio of isomer A and isomer B at the photostationary state (PSS) and initial relaxation kinetics at subpicosecond time scales. Benzil was used for a reference sample. Figure 3a shows the time evolution of the transient absorption spectra of benzil in benzene (6.8 × 10 −2 M). At 0.3 ps after the excitation, a transient absorption band is observed at 546 nm. The transient absorption band continuously shifts to 531 nm and a shoulder is observed at 500 nm. It was reported that the spectral shift of the transient absorption spectra of benzil at the sub-picosecond time scale was assigned to the structural change from the skewed structure to the planar structure [38]. Solvent and vibrational relaxations would also take place in this time scale. After the rapid spectral shift, the transient absorption spectra are preserved until 100 ps. This signal can be assigned to the excited state absorption from the lowest vibrational level of the S 1 state. The transient absorption band at 531 nm gradually decreases with a time scale of nanoseconds and another transient absorption band appears at 485 nm. The transient absorption band at 485 nm was assigned to the T 1 state according to previous studies [39][40][41]. The quantum yield of the formation of the triplet excited state was reported as 92% [42], indicating that most of the S 1 state is converted to the T 1 state in benzil.</p><p>Figure 3b shows the transient absorption spectra of Benzil-PIC in benzene (2.2 × 10 −3 M) excited at 400 nm with a femtosecond laser pulse. The signal around 800 nm was omitted because it was perturbed by the second order diffraction of the excitation pulse around 400 nm. At 0.3 ps after the excitation, two transient absorption bands are observed at 520 and 563 nm, which are most probably assigned to the transient absorption of the benzil unit of Benzil-PIC. The absorption is slightly shifted to the red as compared to those of benzil probably due to the extended π-conjugation of the benzil unit connected to the PIC unit. The two peaks continuously shift to the shorter wavelength (503 and 543 nm, respectively) with a time scale of picoseconds as similar to that of benzil, which supports that these bands are originated from the benzil unit. In addition to the two bands, a broad absorption band over the visible-light region is also observed at 0.3 ps. Because the spectral band shape of this absorption band is similar to that observed in Figure 2, this absorption band is ascribable to the biradical form of PIC, which was directly excited at 400 nm and underwent the rapid radical formation in the sub-picosecond time range. The instantaneous formation of the biradical form under these excitation conditions suggests that a peak at ≈430 nm at 0.3 ps would be most probably assigned to the S 1 state of the PIC unit. In addition to this rapid appearance of the biradical form, the gradual increase of the absorption due to the biradical is observed in picoseconds to tens of picoseconds region, together with the decay of the S 1 state of the benzil unit. This slow process of the biradical formation indicates the energy transfer from the benzil unit to the PIC unit. The amplitude of the increased biradical form with a time scale of tens of picoseconds is larger than the instantaneously generated biradical form at the early time scale, indicating that the energy transfer process is dominant for the photochromic reaction of Benzil-PIC under the excitation with 400 nm. In the nanoseconds time region, the absorption around 580 nm slightly increases with a time scale of nanoseconds.</p><p>To elucidate the details of the reaction dynamics, we performed global analyses with singular value decomposition (SVD) with the Glotaran program (http://glotaran.org) [43]. We tentatively used the three-state sequential kinetic model for benzil (Equation 1) and the five-state sequential kinetic model for Benzil-PIC (Equation 2) convolved with Gaussian pulse. The detail of the SVD analyses are shown in Supporting Information File 1.</p><p>(1)</p><p>(2)</p><p>The evolution associated spectra (EAS) thus obtained indicate the resolved transient absorption spectra into each component of the kinetic models. Because the time window of our measurements was limited to 2 ns, it was difficult to determine the time constant of nanosecond time scale exactly. Therefore, the lifetimes of the intersystem crossing (ISC) of benzil and the benzil unit of Benzil-PIC were fixed to a reported value of benzil (2.5 ns) [44]. The lifetime of the T 1 state of benzil was fixed to 2.0 μs according to the nanosecond-to-microsecond transient absorption spectroscopy. In the benzil system, time constants of three EAS are revealed to be 420 fs, 2.5 ns (fixed), and 2.0 μs (fixed), respectively (Figure 3c). Each EAS species (A to E in the Equation 1 and Equation 2) is denoted as EAS1 to EAS5 in the order of the time constants as shown in Figure 3c and Figure 3d. The fastest time constant of benzil reflects the structural change from the skewed structure to the planar structure and solvent and vibrational relaxations. However, it should be noted that the lifetime of 420 fs is the apparent lifetime because the conformational change from the skewed to the planar structure at sub-picosecond time scale induces the continuous spectral shift. Because the present SVD global analyses do not consider the continuous spectral shift, it is difficult to extract the exact time constant at the early stage of the transient absorption spectra. The EAS with time constants of 2.5 ns and 2.0 μs are safely assigned to the absorption spectra of the S 1 and the T 1 states, respectively, because of the similarity of the spectra to those reported previously [39,40].</p><p>In the Benzil-PIC system, the time constants of five EAS were obtained to be 160 fs, 1.4 ps, 38 ps, 2.5 ns (fixed), and 240 ns (fixed), respectively (Figure 3d). EAS1 has 4 peaks located at 430, 520, 582, ≈710 nm, respectively. The absorption bands at 430 and ≈710 nm are ascribable to the S 1 state of the PIC unit and the biradical generated instantaneously, respectively. It indicates that the biradical was also formed by the direct excitation of the PIC unit with 400 nm light. The spectral evolution from EAS1 (160 fs, grey line in Figure 3d) to EAS2 (1.4 ps, red line in Figure 3d) shows the C-N bond cleavage of the PIC unit and the spectral shift due to the benzil unit (from 582 nm to 556 nm). In PABI, which is a similar photochromic molecule to PIC, it was reported that the C-N bond fission occurs with the time constant of 140 fs and the broad absorption assigned to the biradical form was formed with a time constant of ≈2 ps [45]. The similarity of the time constant of the bond breaking to that of EAS1 supports that the C-N bond is cleaved by the direct excitation of the PIC unit. The spectral evolution from EAS2 (1.4 ps) to EAS3 (38 ps, green in Figure 3d) shows the continuous spectral shift due to the benzil unit and the increase in the absorption due to the biradical form (660 nm). Because the continuous spectral shift due to the benzil unit is still observed in EAS2 (1.4 ps), it is suggested that the structural change of the benzil unit of Benzil-PIC is somehow slightly decelerated as compared to that of benzil (420 fs). However, it should be mentioned that it was difficult to resolve the structural change of the benzil unit and the formation process of the PIC unit by the present SVD analysis.</p><p>The spectral evolution from EAS3 (38 ps) to EAS4 (2.5 ns, fixed, blue line in Figure 3d) shows the decay of the S 1 state of the benzil unit and the alternative increase in the biradical form of the PIC unit. This result clearly shows that the energy of the S 1 state of the benzil unit is used for the photochromic reaction of the PIC unit. It is important to note that the S 0 -S 1 transition energy of PIC, which is optically forbidden, was reported to be 2.8 eV (≈440 nm) [32]. These results suggest that the energy transfer occurs from the S 1 state of the benzil unit to the ground state of the PIC unit with the time constant of 38 ps. Since the bond-breaking process from the S 1 state of the PIC unit would be much faster than this time scale (hundreds of femtoseconds), the time constant of 38 ps reflects the singlet-singlet energy transfer process from the benzil unit to the PIC unit. It should be noted that the fluorescence quantum yield of benzil was quite low (<0.001) [43] and the PIC unit has no absorption in the emission wavelength of the benzil. Accordingly, the effective energy transfer by the Förster mechanism is not plausible. The energy transfer of the 38 ps time constant is probably due to the Dexter mechanism at weak or very weak coupling regimes owing to the overlap of the wave functions of the benzil and the PIC units in the excited state.</p><p>The spectral evolution from EAS4 (2.5 ns, fixed) to EAS5 (240 ns, fixed, purple line in Figure 3d) shows the increase in the absorption around 580 nm. Although both lifetimes of EAS4 and EAS5 are longer than the measured time window (≈2 ns), the spectral difference around 580 nm at 10-100 ps and that at nanosecond time scales enable to resolve these spectra. The increased absorption band is similar to the transient absorption band assigned to the T 1 state of Benzil-PIC (Figure 2a). It indicates that the spectral evolution over nanosecond time scale is ascribable to ISC of the benzil unit. It should be noted, however, that this slow rise of the T 1 state of the benzil unit by ISC indicates that some portions of the benzil unit do not undergo the effective energy transfer to the PIC unit because the S 1 state of the benzil was deactivated with the time constant of 38 ps. Although the clear mechanism is not yet elucidated at the present stage of the investigation, the reason for the two relaxation pathways (energy transfer and ISC) from the S 1 state of the benzil unit of Benzil-PIC might be due to the difference in the mutual orientation of benzil and PIC units including the structural isomers (isomer A and isomer B). As was discussed above, the energy transfer is due to the overlap of the wave function of the both units, of which mechanism might be sensitive to the difference in the mutual orientation.</p><!><p>Ultrafast spectroscopy revealed that the benzil unit acts as a singlet photosensitizer for Benzil-PIC by the Dexter-type energy transfer. It was reported that benzil was often used as a triplet photosensitizer because the quantum yield for the T 1 state formation is 92% [42]. To investigate the possibility for the triplet-triplet energy transfer process in Benzil-PIC, we performed two experiments. Firstly, we measured the phosphorescence spectra of benzil and PIC in EPA (diethyl ether/isopentane/ethanol 5:5:2) at low temperature to estimate the energy levels of the T 1 states of benzil and PIC. In the conventional emission measurement setups at low temperature, both fluorescence and phosphorescence are observed upon irradiation of excitation light. To extract the phosphorescence spectra, the excitation light (continuous wave laser, 355 nm, 1 mW) was chopped at 1 Hz and the afterglow emission under blocking the beam was accumulated as the phosphorescence spectra. Figure 4 shows the phosphorescence spectra of benzil in EPA at 77 and 100 K. While the phosphorescence spectrum of benzil at 77 K is broad and observed at 500 nm, that at 100 K becomes sharper and the peak is shifted to 567 nm with a vibrational fine structure at 625 nm. The spectral shift with the increase in temperature is most probably due to the rigidity of the environment of molecules. At 77 K, it is expected that the solvent is too rigid for benzil to change the conformation in the excited state, namely, the conformation of benzil is fixed to the skewed conformation. On the other hand, it is expected that the increase in the temperature to 100 K softens the rigid matrix and allows the benzil to form the planar conformation at the T 1 state. The energy level of the T 1 state of benzil was estimated from the phosphorescence at 100 K because the T 1 state of benzil in solution forms the planar conformation. The energy level of the T 1 state was determined by an edge of the high energy side of the phosphorescence, where a tangent line crosses the x-axis. The energy level of the T 1 state of benzil is estimated to be 53 kcal mol −1 , which is consistent with a reported value (53.7 kcal mol −1 ) [38]. On the other hand, the phosphorescence of PIC was only observed at 77 K and the signal is very weak. Because the conformation of PIC is relatively rigid, we tentatively estimated the T 1 state energy level from the phosphorescence at 77 K. The T 1 state energy level of PIC is estimated to be 63 kcal mol −1 . It suggests that the T 1 state energy level of benzil is slightly lower than that of PIC. Moreover, the triplet photosensitization was examined by the microsecond transient absorption measurements of the mixture solution of benzil and PIC in benzene (3.7 × 10 −3 M and 2.8 × 10 −5 M for benzil and PIC, respectively). A 450 nm excitation pulse was used to selectively excite benzil. The transient absorption dynamics of the mixture solution of benzil and PIC probed at 500 nm is identical to that of benzil, which is assigned to the T 1 state (Figure S13, Supporting Information File 1). It indicates that the triplet-triplet energy transfer is negligible between the benzil and PIC units. The plausible reason for the negligible triplet-triplet energy transfer is the lower energy level of the T 1 state of the benzil unit than that of the PIC unit. While PIC absorbs light of wavelength only shorter than 350 nm, the introduction of the benzil unit extends the photosensitivity of the photochromic reaction to the visible-light region. When Benzil-PIC absorbs visible light, the conformation of the benzil unit, which is the skewed structure in the ground state, quickly changes to the planar structure with a time scale of picoseconds and the S 1 state of the benzil is formed. While the photochromic reaction partly proceeds via the direct excitation of the PIC unit, most of the photochromic reaction is induced via the Dexter-type singlet-singlet energy transfer from the benzil to the PIC units with the time constant of 38 ps. The triplet photosensitization does not occur in Benzil-PIC most probably because the triplet energy level of the PIC unit is higher than that of the benzil unit. The clarification of the visible-light sensitization mechanism of PIC is important for expanding the versatility of potential applications of PIC in life and materials sciences.</p><!><p>All reactions were monitored by thin-layer chromatography carried out on 0.2 mm E. Merck silica gel plates (60F-254). Column chromatography was performed on silica gel (Silica Gel 60N (spherical, neutral), 40-50 μm, Kanto Chemical Co., Inc.). 1 H NMR spectra were recorded at 400 MHz on a Bruker AVANCE III 400 NanoBay. DMSO-d 6 and CDCl 3 were used as deuterated solvents. Mass spectra (ESI-TOF-MS) were measured by using a Bruker micrOTOFII-AGA1. All reagents were purchased from TCI, Wako Co. Ltd., Aldrich Chemical Company, Inc. and Kanto Chemical Co., Inc., and were used without further purification.</p><p>The synthetic procedure of Benzil-PIC is shown in Scheme 2. The synthetic procedure is analogous to that of PIC [24].</p><!><p>Compound 1 was prepared according to a literature procedure [24].</p><!><p>4'-Hydroxy-[1,1'-biphenyl]-2-carbaldehyde (1, 0.088 g, 0.44 mmol), 1,4-bisbenzil (0.176 g, 0.51 mmol) and ammonium acetate (0.240 g, 3.12 mmol) were stirred at 110 °C in acetic acid (2.7 mL) for 6 h. The reaction mixture was cooled and neutralized by aqueous NH 3 . The precipitate was filtered and Scheme 2: Synthetic procedure of Benzil-PIC (analogous to synthesis of PIC in [24]).</p><p>washed with water. The crude product was purified by silica gel column chromatography (CH 2 Cl 2 /AcOEt 20:1 to 3:1), to give the desired product as a mixture of two structural isomers as a yellow solid, 0.0674 g (0.130 mmol, 29%). 1</p><!><p>A solution of potassium ferricyanide (0.968 g, 2.94 mmol) and KOH (0.741 g, 13.2 mmol) in water (3.3 mL) was added to a suspension of 2 (70 mg, 0.14 mmol) in benzene (7.3 mL). After stirring for 3 h at room temperature, the resultant mixture was then extracted with benzene and the organic extract was washed with water and brine. After removal of solvents, the crude product was purified by silica gel column chromatography (AcOEt/ hexane 2:3) to give the desired product as a yellow powder, 42 mg (0.081 mmol, 58%). Two structural isomers were separated by HPLC (eluent: CH 3 CN/H 2 O 7:3). 1</p><!><p>Steady-state absorption spectra were measured with an UV-3600 Plus (SHIMADZU) at room temperature with 1 cm quartz cuvette. Phosphorescence spectra were measured by home-build millisecond time-resolved emission spectrometer at 77 K with nitrogen cryostat (OptistatDN2, Oxford instruments). Briefly, the cooled samples in EPA (diethyl ether/isopentane/ ethanol 5:5:2) under argon atmosphere were excited with a 355-nm continuous wave (CW) laser (Genesis CX355 100SLM AO, Coherent) and the emission was detected by EMCCD (Newton DU920P-OE, Andor Technology). The excitation light was blocked with 1 Hz by an optical shutter (76992 and 6995, ORIEL) and the time evolution of the emission spectra was measured to separate the fluorescence and phosphorescence. The shutter was controlled by LabVIEW.</p><!><p>The laser flash photolysis experiments were carried out with a TSP-2000 time resolved spectrophotometer system (Unisoku Co., Ltd.). A 10 Hz Q-switched Nd:YAG laser (Continuum Minilite II) with the third harmonic at 355 nm (pulse width, 5 ns) was employed for the excitation light and the photodiode array was used for a detector. Transient absorption measurements on the nanosecond to microsecond time scale were conducted by the randomly interleaved pulse train (RIPT) method [37]. A picosecond laser, PL2210A (EKSPLA, 1 kHz, 25 ps, 30 μJ pulse −1 for 355 nm), and a supercontinuum (SC) radiation source (SC-450, Fianium, 20 MHz, pulse width: 50-100 ps depending on the wavelength, 450-2000 nm) were employed as the pump-pulse and probe sources, respectively. A 355 nm laser pulse was used to excite the samples. The measurements were performed in a benzene solution placed in a 2 mm quartz cell under stirring at room temperature. We used the mixture solution of isomer A and isomer B as was obtained by the synthesis and irradiated a 355 nm pulse laser during the measurements. By considering the duration of the measurements (usually it takes one hour) and the total photon numbers, the system probably reaches the PSS. The ratio of isomer A and isomer B at the PSS upon excitation with the 355 nm pulse is 22:78.</p><!><p>Transient absorption spectra in the visible-light region were measured using a home-built setup. The overall setup was driven by a Ti:Sapphire regenerative amplifier (Spitfire, Spectra-Physics, 802 nm, 1 W, 1 kHz, 100 fs) seeded by a Ti:Sapphire oscillator (Tsunami, Spectra-Physics, 802 nm, 820 mW, 80 MHz, 100 fs). The output of the amplifier was equally divided into two portions. The first one was frequencydoubled with a 50 μm β-barium borate (BBO) crystal, and the generated second harmonics was used for excitation of the sample. The second portion was introduced into a collinear optical parametric amplifier (OPA, TOPAS-Prime, Light Conversion) and converted into the infrared pulse at 1180 nm. This 1180 nm pulse was focused into a 2 mm CaF 2 plate after passing through a delay stage, so as to generate femtosecond white light continuum for the probe pulse. The probe pulse was divided into signal and reference pulses. The signal pulse was guided into the sample and then the both pulses were detected using a pair of multichannel photodiode array (PMA-10, Hamamatsu). The chirping of the white light continuum was evaluated by an optical Kerr effect of carbon tetrachloride and used for the corrections of the spectra. The FWHM of the cross correlation between the excitation and probe pulses was ca. 170 fs. The polarization of the excitation pulse was set to the magic angle with respect to that of the probe pulse. The typical excitation power was 100 nJ pulse −1 at the sample position. During the measure-ment, the sample solution was circulated with a home-made rotation cell with 1 mm optical length. Steady-state absorption spectra were recorded before and after the transient absorption measurement to examine photodegradation of the sample and no permanent change in absorbance was observed. We used the mixture solution of isomer A and isomer B as was obtained by the synthesis and irradiated a 400 nm pulse laser during the measurements. By considering the duration of the measurements (usually takes several hours), the system probably reaches the PSS. Under the irradiation of the 400 nm laser, the ratio of isomer A and isomer B at the PSS depends on each absorption coefficients and the efficiency for the bond cleavage. The absorption coefficients of isomer A and isomer B at 400 nm are 2.1 × 10 3 M −1 cm −1 and 4.1 × 10 3 M −1 cm −1 , respectively.</p>

Beilstein

Discovery of Key Physicochemical, Structural, and Spatial Properties of RNA-Targeted Bioactive Ligands

While myriad non-coding RNAs are known to be essential in cellular processes and misregulated in diseases, the development of RNA-targeted small molecule probes has met with limited success. To elucidate guiding principles for selective small molecule:RNA recognition, we analyzed cheminformatic and shape-based descriptors for 104 RNA-targeted ligands with demonstrated biological activity (RNA-targeted BIoactive ligaNd Database, R-BIND). We then compared R-BIND to both FDA-approved small molecule drugs and RNA ligands without reported bioactivity. Several striking trends emerged for bioactive RNA ligands, including: i) compliance to medicinal chemistry rules; ii) distinctive structural features; and iii) enrichment in \xe2\x80\x9crod-like\xe2\x80\x9d over other shapes. This work provides unique insights that directly facilitate the selection and synthesis of RNA-targeted libraries with the goal of efficiently identifying selective small molecule ligands for therapeutically relevant RNAs.

discovery_of_key_physicochemical,_structural,_and_spatial_properties_of_rna-targeted_bioactive_ligan

<p>The identification of regulatory RNAs, including those that may have therapeutic relevance, has exploded in recent years.[1] Indeed, functional non-coding RNAs have been implicated in diseases ranging from bacterial[2] and viral infections[3] to metastatic cancer.[4] At the same time, methods for the molecular characterization of these RNAs are more difficult and/or lacking compared to those for proteins.[5] Chemical probes, for example, have greatly progressed the study of many protein classes and related diseases[6] but have been challenging to develop for non-ribosomal RNAs. Slow progress in this field has been attributed, in part, to a lack of guiding principles for achieving specific small molecule:RNA interactions.[7]</p><p>Previous attempts to quantify distinct properties of RNA ligands have been notable but limited. For example, a cheminformatic analysis by Aboul-ela et al. in 2009 identified small differences between RNA-binding ligands and other libraries, including FDA-approved drugs.[7a, 8] In studies targeting r(CUG)-repeat sequences in 2015, Disney et al. tested a drug-like, RNA-biased library of bis-benzimidazoles and similar core structures and found that bioactive molecules had statistically significant differences in topological polar surface area (tPSA) as well as hydrogen bond acceptors (HBA) and donors (HBD).[9] Inspired by this previous work and the recent surge in discoveries of biologically active RNA-targeting ligands,[10] we proposed to more broadly analyze the key guiding principles for specific RNA-targeting. In this work, we report the discovery of distinct physicochemical, structural, and spatial properties of RNA-targeted bioactive ligands, which are expected to facilitate the rapid discovery of RNA-targeted chemical probes and subsequent evaluation of the therapeutic potential of non-ribosomal RNAs.</p><p>First, we gathered reports from reviews[10-11] and recent primary literature that described organic small molecule probes for non-ribosomal RNAs with evidence of both in vitro binding and target engagement in cell culture or animal models (SI-1). To focus on small organic molecules, peptides and oligonucleotides were omitted.[12] Aminoglycosides were excluded due to known promiscuous RNA-binding behavior.[10c, 11b, 13] Molecules satisfying the selection criteria and highlighted in the conclusion of each report were included in the RNA-targeted BIoactive ligaNd Database (R-BIND) (available as RBIND.xls). While compiling R-BIND, we identified two strategies utilized to target RNA in biological systems: monovalent small molecules (SM) and multivalent ligands (MV) (Figure 1). We generally classified MV ligands as those with the presence of an alkyl, aryl, or peptide-like linker between two or more binding moieties and a molecular weight greater than 500 amu (SI-1a).</p><p>To analyze the physicochemical properties of these and other ligands, we adapted a list of 20 cheminformatic parameters from Tan and co-workers (SI Table 2-1).[16] Before analysis, all molecules were unbiasedly adjusted to their major protomeric and tautomeric structures (pH = 7.4) using ChemAxon (SI 2). In our first analysis, we compared R-BIND (SM) and R-BIND (MV) sub-libraries to identify similarities and/or differences between the design strategies (SI Table 3-1, SI Figure 3-1). Since both the SM and MV ligands had non-normal parameter distributions, we used an independent two-group Mann-Whitney U test for all statistical comparisons. In line with the increased molecular weight of R-BIND (MV) compared to R-BIND (SM), many parameter distributions in the initial analysis displayed highly significant differences (P < 0.001). The exceptions were LogP (n-octanol/water partition coefficient) and LogD (n-octanol/water distribution coefficient), suggesting a common lipophilicity for RNA-binding and/or bioavailability, as well as ring complexity (SysRR). Next, the parameter values for each library member were scaled by molecular weight, and the Mann-Whitney U test was repeated (SI Table 3-2). Three parameters with previously observed differences were no longer statistically significant: HBA, oxygen atoms (O), and total charge (TC). Interestingly, a closer look at TC revealed that both ligand classes have approximately one positive charge for every 250-350 amu, suggesting an important but restricted role for ionic interactions in RNA recognition. Many of the significant differences between the two libraries can be attributed to the presence of alkyl or peptide-like linkers in R-BIND (MV), such as the dramatic increase in rotatable bonds (RotB). These analyses confirmed that, even when scaled by molecular weight, R-BIND (SM) and R-BIND (MV) represent two unique classes of RNA-binding ligands and thus distinct targeting strategies.</p><p>To identify distinctive properties of bioactive RNA ligands, we compared R-BIND to two additional libraries. First, we compiled all RNA-binding small molecules from the Nucleic Acid Ligand Database (NALDB)[17] and filtered the library analogously to R-BIND (SI-1). Small molecules present in R-BIND were removed from NALDB (SM) and (MV) to represent RNA-binding ligands without reported bioactivity (SI-1). In addition, the FDA-approved chemical entities list was downloaded from DrugBank[18] and filtered to accurately compare organic small molecules (SI-1). The FDA library represented both "drug-like" space and "protein-targeted" space as ~90% of FDA-approved drugs target proteins.[19]</p><p>As a preliminary comparison, principal component analysis (PCA) was used to reduce the dimensionality of the data and visualize the relative relationships between the libraries.[16] PCA defines orthogonal axes, i.e. principal components (PCs), that represent the maximum variance in the data in an unbiased manner, prior to defining which data points are in which groups. In this analysis, the first three PCs described 67% of the variance within the data (SI Table 4-1). Generally, PC 1 was heavily influenced by molecular weight and related parameters, PC 2 by solubility and polarity-related parameters, and PC 3 by differences in aromatic rings and sp3-hybridized carbons (SI Table 4-2). Most small molecules clustered near the origin while the larger and multivalent molecules extended along the positive PC 1 axis (Figure 2, SI Figure 4-2). In comparison, the NALDB (SM) and R-BIND (SM) occupied a similar region, though R-BIND (SM) occupied a smaller subset of FDA chemical space along PC 1 and PC 3. To assist in determining the overlap between the libraries, we performed nearest neighbor clustering analysis (SI Table 5-1 and 5-2). Using 95% of the variance (PC 1-10), notable overlap was observed between the SM libraries with 13% of R-BIND (SM) ligands and 19% of NALDB (SM) ligands falling within the other library's cluster. Further, 31% of R-BIND (SM) and 9% of the FDA library overlapped with the other library's cluster, suggesting that bioactive RNA-binding ligands can possess drug-like properties.</p><p>To determine the properties responsible for these similarities and differences, we again used a Mann-Whitney U test to compare the parameter distributions. For the comparison of R-BIND (SM) and FDA, we included only FDA molecules within the molecular weight range of R-BIND (SM) to account for the presence of larger and potentially multivalent molecules within the FDA library (SI Figure 3-2, SI Table 3-3). Interestingly, 4/7 medicinal chemistry properties (molecular weight (MW), HBA, RotB, tPSA) did not show significant differences (P > 0.05) while statistically significant increases were observed for water solubility (LogP, LogD) and HBD in R-BIND (SM). Among the molecular recognition properties, highly significant differences (P < 0.001) were observed with increased TC and a higher yet narrower range of accessible surface area (ASA). Further, there were statistically significant differences in the molecular complexity of the molecules, where R-BIND (SM) contained fewer sp3-hybridized carbons (Fsp3) and stereocenters (nStereo) compared to the FDA library. We cannot determine, however, the extent to which these differences can be attributed to reduced complexity in synthetic and commercial screening libraries, from which many R-BIND (SM) ligands were discovered.[20]</p><p>Strikingly, the differences in distribution of 8/9 structural parameters were found to be highly statistically significant between R-BIND (SM) and FDA libraries (Figure 3, SI Figure 3-2, SI Table 3-3). These differences included atom counts where concurrently higher N count and lower O count were observed for R-BIND (SM). While the differences in ring count were modest, the ring types were different, and R-BIND (SM) had more aromatic rings (ArRings) and heteroatom-containing rings (HetRings). Collectively, these observations signify that monovalent biologically active RNA-binding ligands can possess "drug-like" properties but that these properties have thus far been achieved through distinct chemical architectures relative to FDA-approved drugs.</p><p>To compare differences between RNA-binding ligands with and without reported bioactivity, the Mann Whitney U test was repeated to compare the distributions of R-BIND (SM) and NALDB (SM) (SI Table 3-5). All medicinal chemistry and molecular recognition properties were found to not have statistically significant differences with the exception of relative polar surface area (relPSA). In contrast, all of the structural parameter distribution differences were found to be statistically significant except SysRR and Fsp3 (Figure 3, SI Figure 4). Repeating the analysis using the same molecular weight restriction as the FDA library (140-590 amu) yielded similar statistical results (SI Table 3-6). These comparisons further our hypothesis that the structural composition of small molecules is crucial for the specific recognition of RNA in biological systems.</p><p>As shape is known to be a critical component of RNA:protein recognition,[21] we also analyzed the spatial properties of R-BIND (SM), FDA, and NALDB (SM) using calculated principal moments of inertia (PMI) (SI-6).[22] The normalized PMI coordinates are plotted on a triangular graph where the vertices represent prototypical ligand shapes: rod, disc, and sphere (Figure 4A). Upon visual inspection, the libraries populate distinct regions within the triangular plot (Figure 4A, SI Figure 6-1). These observations are supported by the library averages (SI Table 6-2) and cumulative fraction graphs (Figure 4B, SI Figure 7-1). The Kolmogorov-Smirnov (KS) statistical test and cell-based partitioning were used to quantitatively compare the library distributions (SI-6). The KS test confirmed that the shape distributions of R-BIND (SM) are statistically different from both the FDA and NALDB (SM) libraries, containing more rod-like and less disc- and sphere-like character (SI Table 6-3). Upon partitioning the libraries into four sub-triangles, 72% of R-BIND (SM) populated the "rod" triangle compared to 56% and 52% for the FDA and NALDB (SM) libraries, respectively (SI Table 6-4). Further partitioning into 16 sub-triangles showed that molecules in R-BIND (SM) largely populate the sub-triangles farthest from the sphere vertex (SI Table 6-5). While many libraries are enriched in rod-like molecules,[23] the differences of R-BIND (SM) compared to the FDA and NALDB (SM) libraries suggest that flat, rod-like small molecules may represent a particularly privileged shape for the recognition of RNA in biological systems.</p><p>In summary, we identified properties of biologically active RNA ligands that distinguish them from both RNA binders without reported bioactivity and from FDA-approved drugs that largely target proteins. Specifically, we identified two clear classes of ligands, the monovalent R-BIND (SM) and the multivalent R-BIND (MV), which differ in size and expected binding mode but have both demonstrated RNA target engagement in cell culture and/or animal models. Interestingly, R-BIND (SM) resembled the FDA library in most medicinal chemistry and molecular recognition properties but differed significantly in almost all structural parameters. Notable differences for R-BIND (SM) included a narrow range of positive charge relative to molecular weight, a concurrent increase in nitrogen count and decrease in oxygen count, and differences in ring type and number. The structural properties were also found to be statistically different between the RNA-binding libraries with and without reported bioactivity. Further, the first spatial analysis of R-BIND (SM) small molecules revealed a statistically significant increase in rod-like character of the library and, in particular, a lack of sphere-like shape when compared to other libraries. Together, these differences support the existence of a "privileged chemical space" for RNA targeting and suggest several important future directions, including the explicit testing of these guiding principles against several RNA targets and the expansion of the properties analyzed. Methods including machine-based learning[24] and multi-parameter optimization[25] will be explored to design and create libraries[26] for efficient RNA-focused screening. We expect that our analysis will serve as a stepping stone to facilitate the discovery of chemical probes for RNAs and ultimately facilitate the discovery of small molecule drugs with novel modes of action against humankind's most challenging diseases.</p>

PubMed Author Manuscript

Compound prioritization methods increase rates of chemical probe discovery in model organisms

SUMMARY Pre-selection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ~81,000 compounds in S. cerevisiae and identified ~7,500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. This data was used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ~7,500 growth-inhibitory molecules has been made commercially available and the computational model and filter used are provided.

compound_prioritization_methods_increase_rates_of_chemical_probe_discovery_in_model_organisms

INTRODUCTION<!>Small molecules that inhibit yeast growth increase the phenotypic hit-rate in other model organisms<!>Physicochemical properties of growth-inhibitory compounds<!>Application of a Na\xc3\xafve Bayes model allows prediction of yactives<!>Predictive approaches enrich for phenotype-inducing compounds in diverse organisms<!>Yactives are a rich source of lead probes<!>DISCUSSION<!>SIGNIFICIANCE<!>Reagents, strains and equipment<!>Screening chemical libraries on S. cerevisiae<!>Screening chemical libraries on E. coli, B. subtilis, S. pombe, C. albicans, C. neoformans, C. elegans and human cell line A549<!>HaploInsufficiency Profiling (HIP) Assay<!>Computational Analysis<!>Statistical Analysis

<p>Current methods for identifying lead chemical probes frequently rely on high-throughput screening against select targets of interest. This approach assumes that the in vitro high potency of small molecules will translate to low-dose efficacy in vivo. However, this is often not the case (Gleeson, et al., 2011). In contrast, in vivo model organism screening provides a direct measure of cellular potency, bypassing the bias of target pre-selection typically used in modern drug discovery. A growing number of academic labs are pursuing model organism screens to identify chemical probes for use as powerful molecular tools to probe biological function (Frearson and Collie, 2009). Chemical probes complement standard genetic approaches to elucidate gene function while offering distinct advantages. For example, when applied to a cell or whole organism, the effects induced by chemical probes are often rapid, reversible and tuneable (Morgan, et al., 2008; Oprea, et al., 2009; Workman and Collins, 2010). Moreover, chemical probes can often be transferred across model organisms, regardless of their genetic tractability (Specht and Shokat, 2002). One drawback of chemical and chemical-genetic screens is that the percentage of compounds that results in a desired phenotype is often small; for example, in a C. elegans study, only 2% of pharmacologically active compounds resulted in a phenotype (screened at 25μM) (Kwok, et al., 2006) and in a study using a hyperpermeable E. coli strain, only 3.5% of compounds (screened at 50μM) resulted in growth inhibition (Li, et al., 2004). These observations, combined with the fact that model organism screening can be both compound-intensive and time-consuming (Burns, et al., 2010; Wheeler and Brändli, 2009) places an emphasis on compound selection prior to screening in contrast to typical in vitro high-throughput screening campaigns (Agresti, et al., 2010; Lipinski and Hopkins, 2004) where the number of total hits is higher and compound consumption is lower. Such pre-screening compound selection strategies may include enriching for known active substructures against multiple targets ("privileged structures")(Klekota and Roth, 2008) and/or enriching for compounds most likely to accumulate in the organism of interest (Burns, et al., 2010).</p><p>The pre-selection strategy described here is aimed at increasing the discovery rate of lead chemical probes in model organisms by first identifying small molecules that inhibit yeast growth. Growth is a comprehensive phenotype, combining multiple effects on cellular physiology into a single quantitative metric (Botstein and Fink, 1988). Moreover, growth measurements can be made in a rapid, high-throughput and low cost manner (Paixao, et al., 2008; Proctor, et al., 2011). Here, we first screened ~81,000 commercially available synthetic compounds and identified ~7,500 compounds that inhibit growth of S. cerevisiae. It is noteworthy that yeast screens often require significantly higher doses (approximately 5-10x) compared to typical mammalian cell culture screens or in vitro assays (Figure S1 and (Blackburn and Avery, 2003; Buurman, et al., 2005; Ericson, et al., 2008; Kwak, et al., 2011)). While our initial yeast screening concentrations are relatively high (maximum 200μM), this high dose does not sacrifice specificity (Blackburn and Avery, 2003; Botet, et al., 2007; Dias, et al., 2010; Dorer, et al., 2005; Ericson, et al., 2008; Giaever, et al., 2004; Khozoie, et al., 2009; Kwak, et al., 2011; Muren, et al., 2001). Several biological factors also contribute to yeast's ability to resist chemical perturbation, including the physical barrier of the yeast cell wall (Dielbandhoesing, et al., 1998) and a dynamic defense known as the pleiotropic drug response (PDR). The PDR is comprised of efflux pumps that reduce the intracellular dose of a broad spectrum of diverse small molecules (Ernst, et al., 2010; Kolaczkowski, et al., 1998; Rogers, et al., 2001).</p><p>Once we had identified the ~7,500 yeast bioactives or "yactives", we then tested the set on a diverse set of model organisms for bioactivity. We found that the yactives significantly increased phenotypic hit-rates compared to randomly selected compounds. Using the physicochemical properties of the yactives, we designed a two-property compound filter based on a simple modification of the Lipinski's rule-of-five (Lipinski, et al., 1997) and in addition, built a Naïve Bayes model to identify substructures present in yactives. We demonstrate both empirically and in silico (using publicly available datasets) that application of the two-property filter and the Naïve Bayes model result in an enrichment for phenotype-inducing compounds in diverse model organisms. Finally, we address the question of whether growth inhibitory compounds have the potential to become specific chemical probe leads by testing twenty of the most potent growth-inhibitory compounds in vivo against all ~1100 essential yeast proteins using our well-validated HaploInsufficiency Profiling (HIP) assay (Baetz, et al., 2004; Giaever, et al., 2004; Giaever, et al., 1999; Lain, et al., 2008; Lum, et al., 2004; Xu, et al., 2007). Several of these compounds exhibit specific genome-wide profiles, identifying candidates for the most likely protein target(s). We pursued two of the most promising target candidates; one supporting lanosterol synthase (Erg7 in yeast, mammalian homolog LSS) as the primary target, and the second fatty acid desaturase (Ole1 in yeast, mammalian homolog SCD) as the potential target. We confirmed these two targets genetically and in independent secondary assays. Taken together, our results demonstrate that pre-selection and prioritization of compound libraries increase the likelihood of identifying specific chemical probe leads for model organisms while decreasing overall costs. To disseminate these tools, the yactives have been made available through Chembridge, Inc. and we provide a prioritized list of compounds generated by applying our model to all commercially available small molecules (Irwin and Shoichet, 2005) on our supplementary website (http://chemogenomics.med.utoronto.ca/supplemental/bioactive/).</p><!><p>To identify small molecules that decrease yeast fitness or growth, we screened 81,320 commercially available synthetic compounds (Table S1) and identified 7,476 small molecules that inhibit wild-type S. cerevisiae growth by at least 30% (IC30)(Experimental Procedures). We next asked if this set of yeast bioactive compounds, or yactives, were enriched for molecules that induce a phenotype when tested across a diverse set of model organisms, spanning substantial evolutionary distance (Figure 1A). Subsets of the 7,476 yactives were screened against our panel of model organisms (as well as a human cell line) and the results compared to those obtained from screening random compounds (Figure 1B, Table S2). Yactives significantly enriched for compounds that inhibited growth (IC50 or greater) in human A549 non-small cell lung carcinoma cells, S. pombe, C. neoformans, E. coli, B. subtilis, and C. albicans. The increase in phenotypic hit-rate was independent of evolutionary distance. Notably, in the model metazoan C. elegans (where hit-rate was determined by visual inspection) the yactives increased the discovery rate 6.6x over random compounds (Figure 1B).</p><!><p>Because the majority of the 81,320 synthetic compounds screened adhere to Lipinski's rule-of-five (intended to define chemical properties that reflect oral bioavailability (Lipinski et al. 1997)) we asked if a simple modification of these rules could be used as a yactive filter. Two of four physicochemical properties that comprise Lipinski's rule-of-five were significantly different (p<1×10−15) in yactive compounds versus inactive compounds (Table S3). First, Lipinski's rule states compounds should have a calculated octanol-water partition coefficient LogP ≤5. In contrast, yactive compounds are more lipophilic (mean LogP = 4.0) than inactive compounds (mean LogP =3.1) (Figure 2A). This observation suggests that they are more likely to be passively transported into the cell as their solubility in a lipid rich-environment would be expected to contribute to cell permeability (Al-Awqati, 1999; Gamo, et al., 2010; Hacker, et al., 2009). Second, Lipinski's rule includes compounds that have ≤10 hydrogen acceptors, while yactives are best described using a limit of ≤6 hydrogen acceptors (Figure 2B). This decreased number of hydrogen acceptors also reflects the likelihood that such compounds can be passively transported across the cell membrane (Muegge, 2003). The increased hit-rate achieved by applying a 2-property filter based on these observations (compounds pass if they have a LogP≥2 and hydrogen acceptors ≤6) (12.7% compared to 9.2%, p-value of 10−15, Table S2) pre-purchase, would have reduced the number of compounds screened from 81,320 to 53,480 (a 30% cost savings) while still identifying 91% of the original 7,475 yactives, demonstrating that even such a modest increase in hit-rate can result in substantial cost savings.</p><!><p>Encouraged by the increased hit-rate resulting from our two-property modification of Lipinski's rule-of-five, we built a Naïve Bayes model to better enable prediction of yactives. Specifically, in this model, substructures in active compounds are weighted higher than those found in inactive compounds, resulting in a prioritized list of compounds for screening. The Naive Bayes model was built using the data from our original 81,320 compounds as a training set. ECFP_4 topological fingerprints (Rogers, 2005) was selected to represent substructures because it outperformed three other representational methods (Figure 3A). Five-fold cross validation was used, with 4/5ths of the original screening data used as the training set, and the remaining 1/5 used to test the model's performance. This procedure was repeated five times, and the model's performance reported as the average over the iterations. At a cutoff of the top 10% of ranked compounds, the ECFP_4 model resulted in an enrichment factor of ~4.5, defined as ~4.5-fold the number of yactives compared to a random set of compounds (Figure 3A).</p><p>To address potential overestimation bias from using the same library for model building and testing, we assessed the performance of the model on an independent chemical library. Because this library has different structural property distributions than the training set, it better represents real world performance. To generate this dataset, the Spectrum Library of ~2,000 compounds was screened against S. cerevisiae to identify yactives followed by application of the Naive Bayes model. The top 10% of compounds ranked by the model showed an enrichment factor of ~3.5 for the yactives (Figure 3B). Extending these tests, we applied our model to publically available chemical screening data. Specifically, the model was applied to the data from 29 yeast assays available from PubChem (Wang, et al., 2010); 23 of the assays were designed to identify modulators of yeast growth (in mutant backgrounds or in wild-type strains), and six relied on readouts other than growth inhibition (see Table S4 for assay details, Table S5 for 2-property filter results, Table S6 for Naïve Bayes model results). Our model performed well across nearly all of these diverse assays, achieving a median enrichment factor 1.85 in the top 10% of compounds ranked (Figure 3C).</p><!><p>We next asked how well the Naïve Bayes model and the 2-property filter enriched for phenotype-inducing compounds when applied to our data for other model organisms (S. pombe, B. subtilis, E. coli, and C. elegans). Comparison of the performance of three approaches (yactives/Naïve Bayes/2-property filter) revealed that empirical screening of the yactives gave the best performance (median enrichment factor 5.95), the Naïve Bayes model performed nearly as well (median enrichment factor 4.30), while the 2-property filter performed appreciably lower (median enrichment factor 1.64) (Figure 4A, see also Tables S2 and S7). To avoid overestimating the level of performance of the Naïve Bayes model due the model being tested on the same library as the training set, we tested the performance using results from nine publically available small molecule screens performed in four organisms (E. coli, C. elegans, C. reinhardtii, and D. rerio) from PubChem (Wang, et al., 2010). As was the case in the yeast assays, the Naïve Bayes model performed best (median enrichment factor 2.10), while the 2-property filter exhibited only modest improvement (enrichment factor 1.28) (Figure 4B, see also Tables S8 and S9 for individual results). The increase in enrichment factors observed across such diverse model organisms (see also Figure 4A and Figure 4B) demonstrates that these approaches are broadly generalizable across a very wide range of model organisms and are therefore valuable methods for compound selection and prioritization.</p><!><p>To be useful as a chemical probe, a compound should act in a specific manner to inhibit a protein or cellular activity. We therefore tested the twenty most potent yactives using our well-validated genome-wide HaploInsufficiency Profiling (HIP) assay (Baetz, et al., 2004; Giaever, et al., 2004; Giaever, et al., 1999; Lain, et al., 2008; Lum, et al., 2004; Xu, et al., 2007) to identify candidate protein targets. The HIP assay allows an unbiased, in vivo quantitative measure of the relative drug sensitivity of all ~1100 essential yeast proteins in a single assay, and results in a list of candidate protein targets ranked in order of compound sensitivity. The profiles of the 20 yactives revealed that 13 of the 20 tested exhibited a degree of specificity for an essential protein or protein(s) in the HIP profile while the remaining 7 compounds did not (Figure S2). We chose the two compounds that exhibited the highest degree of specificity for detailed follow-up studies. Our data suggest these compounds target Erg7, lanosterol synthase, and Ole1, fatty acid desaturase, respectively.</p><p>The HIP profile of Chembridge 95809153 (ERG7.153, Figure 5A) supports Erg7 as the most likely target. ERG7 encodes lanosterol synthase, an essential protein involved in ergosterol biosynthesis (Lees, et al., 1995), a pathway exhibiting structural and functional conservation with the biosynthesis of cholesterol in human. Erg7 performs an essential step in ergosterol biosynthesis and holds promise as an antifungal target based on the success of antifungal agents that target other steps of this pathway (Jolidon, et al., 1990; Voyron, et al., 2010). In addition, the human homolog of Erg7 (LSS, lanosterol synthase BLASTP e-value 5e-148) has potential therapeutic relevance as a cholesterol-lowering agent (Charlton-Menys and Durrington, 2007). Two supporting studies demonstrated that compounds sharing structural similarity to ERG7.153 inhibit lanosterol synthase (Figure S3A). One of these compounds was demonstrated to inhibit lanosterol synthase (Erg7) in C. albicans (Buurman, et al., 2005), while the other was shown to inhibit the human lanosterol synthase, LSS (Fouchet, et al., 2008) (Figure S3A). To genetically confirm that ERG7.153 inhibits Erg7, we tested the individual erg7 heterozygous deletion for the expected compound hypersensitivity to the wild type (Figure S3B). Because ERG7.153 was not available for resupply, we carried out further testing with a close analog, CB 83425298 (ERG7.298), which induced similar hypersensitivity in the S. cerevisiae erg7Δ heterozygous deletion strain (Figure 5B). Analogous growth assays of an erg7Δ heterozygous deletion mutant and a conditional promoter shut-off allele in the human fungal pathogen C. albicans also exhibited hypersensitivity to compound, providing several lines of gene-dose support for Erg7 as the drug target of ERG7.153 and ERG7.298 (Figure 5B). Two additional heterozygous deletion strains, neo1Δ and pik1Δ, encoding a putative aminophospholipid translocase (flippase) (Paulusma and Oude Elferink, 2005) and a phosphatidylinositol 4-kinase (Flanagan, et al., 1993), respectively, are also sensitive to ERG7.153. Both of these genes have been previously classified as multi-drug resistant (MDR) (Hillenmeyer, et al., 2008). In this study, neo1Δ heterozygous deletion strain was sensitive in 14 out of 20 profiles (70%) in this study and the pik1Δ heterozygous deletion strain sensitive to 7 of 20 (35%) compounds tested.</p><p>To independently test if Erg7 is the target of ERG7.298, we analyzed the lipid metabolites from cells grown in the presence of this inhibitor (Figure 5C) by mass spectrometry. As predicted for a bona fide Erg7 inhibitor, the substrate of Erg7 (oxidosqualene) showed significant accumulation in the presence of inhibitor compared to vehicle alone. As a positive control, the level of oxidosqualene was measured in cells treated with cerivastatin which inhibits HMG-CoA reductase, the rate-limiting step of the ergosterol and cholesterol biosynthetic pathways (Endo, 1988). As expected, cells treated with cerivastatin did not accumulate oxidosqualene. Relative measurements of ergosterol, the end product of the ergosterol biosynthetic pathway, showed depletion in cells treated with the Erg7 inhibitor and with cerivastatin. Finally, we were able to partially rescue the growth defect caused by cerivastatin and ERG7.298 by adding ergosterol to the growth medium. Although yeast do not typically incorporate exogenously supplied lipids, we used a S. cerevisiae strain carrying the upc2-1 mutation (Li and Prinz, 2004) (Figure 5D, Figure S3C) which allows cells to take up exogenously supplied sterols under aerobic growth conditions (Crowley, et al., 1998).</p><p>A second HIP profile supports Ole1 as a likely protein target (Figure 6A) of a novel compound. Ole1 encodes the yeast delta(9) fatty acid desaturase, which converts stearic acid to oleic acid and which has previously been proposed as a potential antifungal target (Hu, et al., 2007). Furthermore, the human homolog of Ole1, SCD (stearoyl-CoA desaturase, BLASTP e-value 3e-52), has attracted interest for its potential modulation for the treatment of diabetes (Lenhard, 2011; Ntambi, et al., 2002). Two other heterozygous deletion strains unrelated to fatty acid desaturase inhibition, (lsg1Δ and rpb8Δ) are sensitive to this compound. Given that both of these genes encode ribosomal components which frequently come up as sensitive in diverse chemical screens (unpublished data), they were not further pursued in this study. We used a S. cerevisiae ole1 DAmP loss-of-function allele (Schuldiner, et al., 2005; Yan, et al., 2008) to confirm compound hypersensitivity. Hypersensitivity was also seen with an ole1 conditional promoter shut-off allele in C. albicans, further supporting Ole1 as the target of OLE1.041(CB 11119041)(Xu, et al., 2009) (Figure 6B). This compound was also effective in vitro, inhibiting the enzymatic activity of Ole1 in S. cerevisiae, C. albicans and human HepG2 cells (Figure 6C). Finally, we found that two C. elegans mutants with reduced stearoyl-CoA desaturase activity (fat-5;fat-7 and fat-5;fat-6) (Brock, et al., 2006) are hypersensitive to OLE1.041 (Figure 6D). These two examples highlight the ability of the HIP assay to identify additional effects that can be monitored during compound optimization.</p><!><p>Compounds that inhibit yeast growth are more likely to induce phenotypes in other model organisms. Modeling the properties of the subset of drug-like compounds that inhibit yeast growth allows prioritization of compounds for model organism screening, reducing screening costs and increasing efficiency. Over time, as the research community accumulates compound screening data, these models can be refined to be both organism and phenotype-specific resulting in increasing the predictability and accuracy. As an important first step, we have demonstrated that compounds that inhibit yeast growth are more likely to induce phenotypes of interest in other model organisms and in mammalian cell culture assays. In order to address whether the inhibitory compounds we identified could act in a specific manner, we followed up on two compounds that looked particularly promising based on their genome-wide profile of drug sensitivity. The mechanism of action of these compounds (ERG7.298, OLE1.041) was shown to be consistent with inhibition of their presumed molecular targets in both genetic and cellular biochemical assays. Both ERG7 and OLE1 are highly conserved with human LSS and SCD, and represent recognized targets of medical relevance. ERG7 and LSS inhibitors have clinical relevance as potential antifungal and anti-cholesterol lowering agents, respectively, while the human homolog of OLE1, SCD, may represent a potential target for diabetes treatment.</p><p>The identification of two compounds that act with a high degree of specificity in a relatively short experimental time frame underscores the benefits of prioritizing compounds. While no filtering or prioritization method can trump an exhaustive screening campaign and perfectly predict all compounds of interest, our results clearly indicate that pre-selection methods, when applied across diverse assays and organisms, can identify and prioritize those compounds most likely to induce a phenotype. The advantage of using such compounds as starting points for chemical probe discovery in model organisms is that a wide variety of genetic tools in different organisms can be used to validate the mode of action, as well as to identify off-target effects. To provide a publically accessible resource, we applied our 2-property filter and our Naïve Bayes model to compounds available in 1) the NIH Molecular Screening program (Austin, et al., 2004) and 2) the Zinc catalogue of approximately 14 million purchasable compounds (Irwin and Shoichet, 2005). These results are available on our website, http://chemogenomics.med.utoronto.ca/supplemental/bioactive/.</p><p>Finally, a primary goal of this work was to encourage compound suppliers to provide libraries directed at model organism screening to the research community. Towards this end, ChemBridge, Inc. has agreed to make our ~7,500 yactive compounds available for purchase as a pre-plated compound set. This library should prove a valuable resource for chemical screening labs working to develop chemical probes using model organisms.</p><!><p>We have presented three approaches, based on yeast growth inhibition, to guide compound selection to reduce the costs associated with model organism screening programs. First, we have demonstrated that compounds that inhibit yeast growth enriches for compounds that induce a variety of phenotypes in diverse model organisms, and these compounds, with further optimization, may yield specific chemical probes. The first approach is then to simply screen compounds for those that inhibit yeast growth. A second approach is to prioritize compounds based on those that pass the two-property filter described here. This approach, depending on the model organism, can decrease costs by ~25%, and is straightforward to implement. The third approach is to purchase compounds based on their likelihood to result in a desired phenotype by applying our Naïve Bayes model. This approach can also dramatically reduce costs. Newly generated screening data can be used to rebuild the model described here in the context of the model organism of interest to increase performance. This iterative approach is key as no model will perform optimally in all applications. Finally, as an experimental resource the yactive compounds are available as a pre-plated collection from ChemBridge, and a list of 14M purchasable compounds scored by our Naïve Bayes model and 2-property filter is available for download from our website (http://chemogenomics.med.utoronto.ca/supplemental/bioactive/).</p><!><p>The chemical libraries screened were obtained from ChemDiv (Divers, San Diego, CA, USA) and Chembridge (NOVACore and DIVERSet, San Diego, CA, USA) in a 96-well format at 10mM in DMSO. The Spectrum library (Microsource, Gaylordsville, CT, USA) of 2000 compounds was supplied at 2.5mM in DMSO and was a gift from D. Desveaux and D. Guttman (University of Toronto).</p><p>E. coli strain BW25113 (Datsenko and Wanner, 2000) (lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) was a gift from Andrew Emili (University of Toronto), S. pombe strain TK1/972 (h−) was a gift from Charlie Boone (University of Toronto) and B. subtilis strain 168 1A700 (trpC2) was a gift from Alex ter Beek and Gertien Smits (University of Amsterdam). C. albicans heterozygous deletion (Xu, et al., 2007) and conditional shut-off or GRACE mutants (Roemer, et al., 2003) in the SC5314 background were a gift from Terry Roemer (Merck-Frosst Canada Ltd). C. neoformans strain H99 was a gift from Joseph Heitman (Duke University). A549 human lung cancer cells (ATCC number: CCL-185) and HepG2 cells (ATCC number HB-8065) were obtained from American Type Culture Collection (ATCC, Rockville MD, USA). S. cerevisiae WPY361 (MATa upc2-1 ura3-1 his3-11,-15 leu2-3,-112 trp1-1) was a gift from William Prinz (NIH, Bethesda, MD, USA) (Li and Prinz, 2004). The BX110 fat-7(wa36);fat-5(tm420)V double, the BX160 fat-6(tm331)IV;fat-5(tm420)V double, and the wild-type (N2) worm strains were obtained from the C. elegans Genetics Center (U. Minnesota) maintained at 20°C using standard techniques (Lewis, 1995).</p><p>Growth assays were performed in clear, flat bottom 48-, 96- and 384-well microplates (Greiner) sealed with adhesive plate seals (Cat. No. AB-0580, ABgene) using a custom developed platform incorporating microplate readers GENios, Infinite, and Safire2 (Tecan-US, Durham NC, USA), the Packard Multiprobe II four-probe liquid-handling system (PerkinElmer, Waltham MA, USA). Genome-wide assays were analyzed on Genflex_Tag_16K_dev microarrays (Item No. 511331, Affymetrix, Santa Clara CA, USA) using GeneChip Fluidics Station 450 and GeneChip Scanner 3000 (Affymetrix). For protocol detail see (Pierce, et al., 2007).</p><!><p>Our wild-type yeast strain, BY4743 (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0), was grown in YPD medium at 30°C. 20mM HEPES pH 7.0 was added to the YPD medium as indicated in the supplementary files available online. Cells were diluted from a fresh overnight culture to 0.0625 OD595 in a final volume of 100μL for 96-well and 30μL for 384-well plates. Compounds were added to the culture using a 2μL or 600nL pin tool (V&P Scientific, San Diego, CA, USA) for 96-well or 384-well microplates respectively, to dilute the compounds 50 times to a final DMSO concentration of 2%. The ChemDiv Diverse, NOVACore SAR and NOVACore DIVERSet libraries were screened at 200μM final concentration, and the Spectrum library was screened at 50μM final concentration. Yeast growth was monitored for up to 24 hours by measuring the OD595 every 15 minutes as described (Giaever, et al., 2004; Lee, et al., 2005). The majority of the compounds screened were soluble at 200uM, with less than 8% compounds having a starting optical density that was significantly different from the DMSO controls indicating either a colored compound or a solubility issue.</p><p>The fitness of BY4743 in compound was expressed as the ratio of the average generation time (AvgG) (AvgG reference/AvgG compound) where the reference condition was grown on the same plate in 2% DMSO. Average generation time is calculated by (time to 5 generations/5)(Lee, et al., 2005). A compound was scored as active when the ratio AvgG was 0.7 or less, corresponding to an IC30 or greater. Automatic flagging of actives was confirmed by visual inspection of the data.</p><!><p>Compounds scored as growth inhibitory were transferred to a new 96-well microplate, the hit plate. Hit plates and random naïve plates were screened at 200μM final concentration on E. coli strain BW25113 grown in Luria Broth (LB), B. subtilis strain 1A700 grown in Nutrient Broth (NB), S. pombe strain TK1 grown in YES medium, S. cerevisiae strain BY4743, C. neoformans strain H99 and C. albicans strain HIS3 grown in YPD medium. All media was buffered with 20mM HEPES pH 7 and growth temperature was 37°C for bacteria and 30°C for yeast.</p><p>A compound was considered active in S. pombe, C. albicans, C. neoformans, E. coli or B. subtilis if the area under the growth curve after 20 hours of growth was 50% of that compared to the DMSO control (ratio (compound/control) < 0.5). Compounds that showed a high ratio, defined as greater than 1.2 were excluded. We found the area under the growth curve to be a more robust method for measuring growth rate in the other organisms than the ratio AvgG. A value of 0.5 gives a similar hit-rate as a ratio AvgG of 0.7 in S. cerevisiae.</p><p>Phenotypic screening of C. elegans was performed as reported previously (Kwok, et al., 2006). In brief, molecules were screened in duplicate in 24-well format at 25μM concentration. Two L4 stage N2 animals were deposited per well on agar and the progeny were visually assessed for phenotype, including slow growth, egg laying abnormalities and embryonic lethality, using an MZ12 dissection microscope (Leica Microsystems GmbH, Wetzlar, Germany).</p><p>A549 human lung cancer cells were maintained in Dulbecco's Modified Eagle medium (Wisent, St-Bruno, QC, Canada) supplemented with 10% fetal bovine serum (Wisent) and 100 U/ml penicillin/streptomycin (Wisent) in a humidified incubator with 5% CO2 at 37°C. The cells were seeded in 96-well plates with a density of 2200 cells per well and treated for 48 hours with 50μM compound in 0.5% DMSO. Cell survival was measured using the Sulforhodamine B (SRB) colorimeteric assay (Vichai and Kirtikara, 2006) and readout using a SpectraMax Plus384 (Molecular Devices, Sunnyvale, CA, USA) with the following modification, the cells were stained with 50μL of 0.4% SRB. Actives for the A549 cells were defined as compounds causing ≤ 50% viability after 48 hours of growth in the presence of the compound.</p><!><p>By serial dilution of 16 hit plates, each containing ~86 active compounds, 79 compounds were found to completely inhibit the growth of wild-type S. cerevisiae at a four-fold dilution (50μM). 20 diverse compounds were selected for testing in the HIP assay. The molecular weights of these 20 compounds were verified by liquid chromatography and mass spectrometry to confirm their structure (see Supplementary Methods).</p><p>The HIP assay was performed as previously described (Pierce, et al., 2007). Two biological replicates were generated for each compound condition. A significant hit is defined as a gene with a log2 ratio greater than 1 of the intensity of the DMSO control intensity/drug treatment intensity in both replicates. All raw and ratio data files are available on the supplementary website. All HIP profiles are shown in Figure S2. In addition, the microarray data is available on ArrayExpress (http://www.ebi.ac.uk/microarray-as/ae/).</p><!><p>All chemical analysis and Naïve Bayes model building was performed using the cheminformatic package in Pipeline Pilot version 6.1 (Scitegic Inc. Accelyrs, San Diego, CA USA). Marvin version 5.4.1(ChemAxon, http://www.chemaxon.com) was used for drawing and displaying chemical structures.</p><p>Pipeline Pilot was used to standardize the representation of all compounds studied including removing inorganic compounds, salts and duplicates. All data used for the model building is available on our supplementary website. Three methods were tested to represent molecules for the Naïve Bayes model using five-fold cross validation: 1) A vector constructed from physical properties for each compound (LogP, Molecular Weight, Molecular Polar Surface Area, Molecular Solvent Accessible Area, the number of Hydrogen acceptors and donors, the number of rotatable bonds, the number of rings, and the number of aromatic rings). 2) A vector based on MDL Public Keys (Durant, et al., 2002) where the presence or absence of specific substructures is recorded. 3) A vector using the Extended Connectivity Fingerprints (Rogers, 2005) method where the compound is represented by overlapping fragments of a diameter of up to 2/4 bond lengths (ECFP_2/ECFP_4). The enrichment factor was used as a measure of accuracy. This is calculated by ranking the library of compounds to be tested by the model score. Next, for different thresholds the number of observed actives was compared with the number of actives expected by random selection.</p><p>Tanimoto coefficient, also known at the Jaccard Coefficient (Rogers and Tanimoto, 1960), was used to calculate the similarity between two compounds and calculated based on the number of features in common between the compounds divided by the total number of features present.</p><p>LogP values were calculated using the Ghose and Crippen algorithm (Viswanadhan, et al., 1989).</p><!><p>The significance of the effect of the 2-property and Lipinski filters was calculated using the hypergeometric test, termed "phyper" function, in R. To test if the distribution of LogP and the number of hydrogen acceptors for active and inactive compounds was significantly different, a two-sample Kolmogorov-Smirnov test (Durbin, 1973) was implemented using R.</p>

PubMed Author Manuscript

Enantioselective Total Synthesis of Decytospolide A and Decytospolide B Using an Achmatowicz Reaction

Enantioselective syntheses of decytospolide A and decytospolide B are described here. The current synthesis highlights an Achmatowicz rearrangement of an optically active furanyl alcohol followed by reduction of the resulting dihydropyranone hemiacetal with BF3\xe2\x80\xa2OEt2 and Et3SiH to provide the saturated tetrahydropyranyl alcohol directly. This reduction was investigated with a variety of other Lewis acids. The synthesis also features Noyori asymmetric transfer hydrogenation and Friedel-Craft acylation. Overall, the synthesis provides ready access to the natural products and may be useful in the preparation of bioactive derivatives.

enantioselective_total_synthesis_of_decytospolide_a_and_decytospolide_b_using_an_achmatowicz_reactio

Introduction<!>Results and discussion<!>Conclusions<!>Experimental Section<!>(R)-1-(Furan-2-yl)butan-2-yl acetate (12):<!>(R)-1-(5-Hexanoylfuran-2-yl)butan-2-yl acetate (13):<!>(R)-1-(5-((R)-1-Hydroxyhexyl)furan-2-yl)butan-2-yl acetate (10):<!>(R)-1-((2R,5S,6R)-5-Hydroxy-6-pentyltetrahydro-2H-pyran-2-yl)butan-2-yl acetate (8):<!>1-((2R,5S,6R)-5-((tert-Butyldimethylsilyl)oxy)-6-pentyltetrahydro-2H-pyran-2-yl)butan-2-one (15):<!>Decytospolide A (6):<!>Decytospolide B (7):<!>Methyl 2-(5-hexanoylfuran-2-yl)acetate (18):<!>Methyl (R)-2-(5-(1-hydroxyhexyl)furan-2-yl)acetate (19):<!>Methyl 2-((2R,5S,6R)-5-hydroxy-6-pentyltetrahydro-2H-pyran-2-yl)acetate (20):<!>2-((2R,5S,6R)-5-Hydroxy-6-pentyltetrahydro-2H-pyran-2-yl)-N-methoxy-N-methylacetamide (21):<!>Decytospolide A (6) via Weinreb amide 21:<!>Decytospolide B (7) via Weinreb amide 21:

<p>Functionalized tetrahydropyrans are important structural features present in many bioactive natural products.1,2 Over the years, several methods have been developed for the synthesis of substituted tetrahydropyran rings.3,4,5 However, there are limitations with respect to readily available starting materials, stereo- and regiochemical issues and lack of potential for incorporation of multiple substitutions within the tetrahydropyran ring system. Additionally, many of these transformations rely on transition metals such as a Pd-catalyzed decarboxylative allylation and an indium catalysed Prins cyclization.6,7 The Achmatowicz reaction, an oxidative ring enlargement of a furanyl alcohol, has been developed into a very practical reaction with immense potential.8 In recent years, the Achmatowicz reaction has been utilized in the synthesis of a variety of natural products.10,11 We have utilized this reaction in the synthesis of a number of bioactive natural products containing functionalized tetrahydropyran rings.10,12,13 In particular, the Achmatowicz reaction of furanyl alcohol 1 (Figure 1) provides dihydropyranone hemiacetal 2 which upon reduction, typically with triethylsilane in the presence of trifluoroacetic acid (TFA) provided a variety of 2,6-disubstituted dihydropyranone derivatives 3.10,11 Such enones have been utilized in the synthesis of bioactive natural products, including the potent anticancer agent, herboxidiene 4.10,13</p><p>Thus far, dihydropyranone hemiacetal reduction provides access to a range of enones under a variety of reaction conditions.10,11 However, the potential for reduction of an Achmatowicz reaction product enone hemiacetal to a saturated tetrahydropyran derivative and further reduction to the alcohol functionality has been scarcely explored. For further development of the Achmatowicz reaction as well as its application, we sought to synthesize 2,6-disubstituted tetrahydropyranyl alcohols as exemplified by alcohol 5 using a silane in the presence of a Lewis acid. There have been limited studies for this transformation.14 This transformation would provide easy access to natural products containing substituted tetrahydropyran rings with three contiguous chiral centers. Such functionalized tetrahydropyran rings are imbedded in a variety of bioactive molecules, including decytospolides A (6) and B (7) and their derivatives.</p><p>The decytospolides contain three asymmetric centers surrounding a central tetrahydropyran ring flanked by two alkyl chains. Both natural products were recently isolated by Zhang and co-workers from the endophytic fungus, Cytospora sp. No ZW02, from Ilex canariensis, an evergreen shrub from the Canary Islands.15 The chemical structure of both decytospolides was determined by extensive NMR studies and HRMS analysis. The absolute configuration was established through Mosher ester analysis.15,16 Decytospolide B exhibited moderate cytotoxicity in A549 and QGY cancer cell lines with IC50 values of 14.8 and 46.8 μg/mL, respectively. Since decytospolide A did not show appreciable cytoxicity, the acyl group in decytospolide B may be responsible for its moderate anticancer activity. There is potential for further improvement through modification of this acyl group. Several syntheses of these natural products have been reported.6,7,17–19 We, however, planned to assemble the functionalized tetrahydropyran ring using the Achmatowicz reaction as the key step. Herein, we report our synthesis of decytospolides A and B using the Achmatowicz, Noyori reduction, and Friedel-Craft reactions as the key steps. We particularly sought to synthesize 2,5,6-trisubstituted tetrahydropyran derivatives in a highly stereoselective manner in optically active form in a one-pot operation directly from the Achmatowicz product, the dihydropyranone hemiacetal.</p><!><p>Our synthetic strategies to decytospolides A and B is shown in Scheme 1. We planned to synthesize trisubstituted tetrahydropyran alcohol 8 in an optically active manner by reduction of dihydropyran hemiacetal 9 which would be obtained directly from furanyl alcohol 10 using an Achmatowicz rearrangement. We particularly planned to synthesize alcohol 8 stereoselectively from dihydropyran hemiacetal 9.10,11 Furan derivative 10 would be synthesized from furan 11 by a Friedel-Crafts acylation followed by asymmetric reduction.20</p><p>The synthesis of furanyl alcohol 10 in optically active form is shown in Scheme 2. Deprotonation of furan 11 with n-BuLi in THF at 0 °C to 23 °C followed by addition of commercially available (R)-butylene oxide furnished the corresponding alcohol through epoxide opening. The resulting alcohol was acetylated with acetic anhydride in the presence of Et3N and DMAP to afford furan derivative 12 in 84% yield over two steps. We specifically planned to install the C3-acetoxy group with defined stereochemistry to avoid forming a mixture of diastereomers and provide easy access to C3 stereo-defined derivatives for biological evaluation Furan derivative 12 underwent a Friedel-Craft acylation by reaction with hexanoyl chloride in the presence of SnCl4 at 0 °C for 30 min to provide ketone derivative 13 in 75% yield.21 For enantioselective reduction of the ketone, we planned an asymmetric transfer hydrogenation reaction developed by Noyori and co-workers.22,23 Therefore, reaction of 13 with a catalytic (1 mol%) amount of Noyori's catalyst, (R,R) RuCl(mesitylene)-Ts-DPEN, in the presence of Et3N and formic acid in CH2Cl2 at 55 °C for 12 h, furnished alcohol 10 in 93% yield. The asymmetric reduction proceeded with high diastereoselectivity as alcohol 10 was isolated as the single product by1H- and13C-NMR analysis (diastereoselectivity > 20:1).</p><p>The synthesis of the decytospolides is shown in Scheme 3. Initially, the Achmatowicz reaction of 10 was carried out with a catalytic amount of VO(acac)2 andtBuOOH; however, the reaction was sluggish.24,25 Achmatowicz reaction with oxone in the presence of NaHCO3 in a mixture (4:1) of THF and water at 23 °C for 30 min smoothly converted 10 to the corresponding dihydropyranone hemiacetal (9).26 The resulting hemiacetal was initially subjected to reduction with Et3SiH and TFA at −45 °C for 3 h. This resulted in a mixture of dihydropyranone 14 and a small amount of alcohol 8. Further optimization of the reaction with an excess of BF3•OEt2 at −45 °C for 3 h provided alcohol 8 as the exclusive product in 47% yield over 2-steps. The stereochemistry at C8 was determined by1H NMR coupling with the proton at C9, which has two J values (2.4 and 9 Hz). Having J = 9 Hz indicates a trans relationship between the protons at C8 and C9 and since the stereocenter at C9 was set by the Noyori reduction, the stereochemistry at C8 was assigned based on that. We then, investigated silane reduction of the dihydropyranone hemiacetal 9 in the presence of a number of other Lewis acids. The results are showin in Table 1. As can be seen, the use of BF3•OEt2 and SnCl4 as the Lewis acids provided alcohol 8 exclusively in 56% and 82% yield, respectively (entries 3 and 4). Reductions with Lewis acids Sc(OTf)3 and Cu(OTf)2 yielded only trace amounts of dihydropyranone 14, while using TiCl4 as the Lewis acid gave 14% of 14.</p><p>To complete the synthesis of the decytospolides, alcohol 8 was protected as a TBS-ether with TBSOTf in the presence of 2,6-lutidine in CH2Cl2 at 0 °C to 23 °C for 12 h. The acetate was hydrolyzed with K2CO3 in MeOH at 23 °C to provide the corresponding alcohol. Oxidation of the resulting alcohol with Dess-Martin periodinane in the presence of NaHCO3 in CH2Cl2 furnished ketone 15 in 89% yield over 3-steps. Removal of the silyl ether was carried out with tetrabutylammonium fluoride (TBAF) in THF at 23 °C for 12 h to provide decytospolide A (6) {[α]D23+18.4 (c 0.45, CHCl3)} in quantitative yield. Treatment of decytospolide A (6) with acetic anhydride in the presence of pyridine and DMAP in CH2Cl2 at 23 °C for 1.5 h furnished decytospolide B (7) {[α]D23+28.4 (c 0.75, CHCl3)} in quantitative yield. The1H- and13C-NMR spectra of synthetic decytospolides are in complete agreement with the spectra reported for the natural decytospolide A {[α]D20+6.1 (c 0.08, CHCl3)} and decytospolide B {[α]D20+26.6 (c 0.02, CHCl3)}.14</p><p>We also investigated an alternative route to the decytospolides in an effort to synthesize the saturated tetrahydropyranol directly following the Achmatowicz reaction. As shown in Scheme 4, commercially available furural, was converted to furanyl methyl acetate 17 by a one-carbon homologation using a Jocic reaction as developed by Snowden and co-workers.27,28 Methyl ester 17 is also commercially available. Methyl ester 17 was reacted with hexanoyl chloride in the presence of SnCl4 as described above to provide furanyl-ketone 18 in 57% yield. Reduction of ketone 18 using Noyori's catalyst RuCl(mesitylene)[(R,R)-Ts-DPEN] (1 mol%) in the presence of Et3N and formic acid at 50 °C for 12 h afforded optically active furanyl alcohol 19 in 83% yield and 99% ee determined by chiral HPLC analysis (Please see experimental section for details).20,21 Achmatowicz reaction of 19 with oxone at 23 °C for 30 min furnished the corresponding dihydropyranone hemiacetal which was subjected to reduction with excess Et3SiH in the presence of BF3•OEt2 at −40 °C for 16 h to provide saturated tetrahydropyranol derivative 20 as a single product by1H-NMR analysis. Presumably, the reduction of enone first provided the ketone which was reduced by Lewis acid chelation followed by axial delivery of hydride.</p><p>Tetrahydropyranol derivative 20 was readily converted to the decytospolides as shown in Scheme 5. Reaction of methyl ester 20 with NH(OMe)Me•HCl in the presence of i-PrMgCl in THF at −30 °C for 5 h provided Weinreb amide derivative 21. Treatment of Weinreb amide 21 with EtMgBr in THF at 0 °C to 23 °C for 5 h afforded decytospolide A, 6 in 97% yield {[(α)D23+7.8 (c, 1.33, CHCl3)]}. Acylation of 6 with acetic anhydride in the presence of pyridine and DMAP furnished decytospolide B, 7 {[α]D23+22.8 (c, 1.96, CHCl3)} in 86% yield. The1H-NMR and13C-NMR spectra of these synthetic decytospolides are in complete agreement with reported spectra for the natural products.14</p><!><p>In summary, we have accomplished an enantioselective total syntheses of decytospolides A and B. The synthesis features an Achmatowicz rearrangement of an optically active furanyl alcohol which was obtained conveniently by use of the Friedel-Craft reaction followed by a Noyori asymmetric transfer hydrogenation reaction as the key steps. The synthesis highlights a highly stereoselective reduction of the Achmatowicz product, a dihydropyranone hemiacetal to the saturated tetrahydropyranol derivative using BF3•OEt2 and Et3SiH. Reduction presumably proceeds through Lewis Acid chelation followed by delivery of an axial hydride. The current work will provide access to structural variants of these natural products for further studies. Further studies and applications are in progress in our laboratories.</p><!><p>Chemicals and reagents were purchased from commercial suppliers and used without further purification. Anhydrous solvents were obtained as follows: dichloromethane and toluene from calcium hydride, diethyl ether and tetrahydrofuran from sodium/benzophenone, and methanol from activated magnesium. All other solvents were reagent grade. All moisture-sensitive reactions were either carried out in flame- or oven-dried (120 °C) glassware under an argon atmosphere. TLC analysis was conducted using glass-backed thin-layer silica gel chromatography plates (60 Å, 250 μm thickness, F254 indicator). Column chromatography was performed using Silicycle 230–400 mesh, 60 Å pore diameter silica gel.1H and13C NMR spectra were recorded on either Bruker ARX400, Bruker DRX-500, Bruker AV500HD, or Bruker Avance-III-800 spectrometers. Chemical shift (δ values) are reported in parts per million and are referenced to the residual solvent signal (CDCl31H singlet = 7.26,13C triplet = 77.16). Characteristic splitting patterns due to spin-spin coupling are identified as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sep = septet, m = multiplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, td = triplet of doublets, dq = doublet of quartets, brs = broad singlet, app = apparent. All coupling constants are measured in hertz (Hz). Optical rotations were recorded by a Perkin Elmer 341 polarimeter. IR spectra were recorded on a Perkin Elmer Spectrum Two FT-IR Spectrometer. LRMS and HRMS spectra were recorded at the Purdue University Department of Chemistry Mass Spectrometry Center. HPLC data was obtained on an Agilent 1290 Infinity II.</p><!><p>To furan (2.02 mL, 27.74 mmol) dissolved in THF (23 mL) at 0 °C was added n-BuLi (15.6 mL, 24.97 mmol) dropwise upon which a bright yellow color developed. After stirring for 1 h at this temperature, (R)-(+)-butylene oxide (1.21 mL, 13.87 mmol) was added and the reaction was slowly warmed to room temperature. After stirring for 12 h, the deep red solution was quenched with satd. NH4Cl, extracted with EtOAc, washed with brine and dried over Na2SO4. Purification by column chromatography (10% EtOAc/Hexanes) gave 1.69 g (87% yield) of the resulting furan alcohol as a yellow oil.</p><p>To (R)-furan alcohol (868 mg, 6.19 mmol) dissolved in CH2Cl2 (21 mL) at 0 °C was added acetic anhydride (1.2 mL, 12.4 mmol), Et3N (1.3 mL, 9.3 mmol) and a few crystals of DMAP. The reaction was allowed to warm to room temperature. After 6 h, the reaction was quenched with satd. NaHCO3, extracted with CH2Cl2, washed with brine and dried over Na2SO4. Purification by column chromatography (5% to 10% EtOAc/Hexanes) afforded 1.1 g (97% yield) of acetate 12 as a clear oil [α]D20 +19.6 (c 0.73, CHCl3);1H NMR (500 MHz, CDCl3) δ : 7.31 (dd, J = 1.9, 0.9 Hz, 1H), 6.28 (dd, J = 3.2, 1.9 Hz, 1H), 6.05 (dd, J = 3.2, 0.9 Hz, 1H), 5.04 (m, 1H), 2.87 (d, J = 6.3 Hz, 2H), 2.02 (s, 3H), 1.65–1.55 (m, 2H), 0.92 (t, J = 7.5 Hz, 3H);13C NMR (125 MHz, CDCl3) δ : 170.7, 151.9, 141.5, 110.4, 107.0, 73.8, 32.5, 26.7, 21.3, 9.7; FT-IR (neat) νmax = 2965, 2925, 2852, 1738, 1507, 1461, 1436, 1376, 1239, 1012, 739 cm−1</p><!><p>Hexanoic acid (604 mg, 5.20 mmol) in an excess of thionyl chloride was refluxed overnight. The thionyl chloride was removed by distillation, strictly keeping the system under argon. The remaining hexanoyl chloride was used immediately for the subsequent reaction. To hexanoyl chloride (699 mg, 5.2 mmol) in CH2Cl2 (15 mL) at 0 °C was added SnCl4 (8.7 mL, 8.7 mmol, 1 M in CH2Cl2) dropwise. After stirring for 1 h at this temperature, acetate 12 (789 mg, 4.33 mmol) dissolved in CH2Cl2 (5 mL) was added to the reaction mixture via cannula and remained stirring at 0 °C. After 30 min, the red-brown solution was quenched with ice, extracted with CH2Cl2, washed with brine and dried over Na2SO4. Purification by column chromatography (10% to 20% EtOAc/Hexanes) gave 909 mg (75% yield) of furan derivative 13 as a clear oil; [α]D20 +16.2 (c 0.86, CHCl3);1H NMR (500 MHz, CDCl3) δ : 7.07 (d, J = 3.5 Hz, 1H), 6.21 (d, J = 3.5 Hz, 1H), 5.07 (quint, J = 6.4 Hz, 1H), 2.99–2.91 (m, 2H), 2.74 (t, J = 7.9 Hz, 2H), 2.02 (s, 3H), 1.72–1.66 (m, 2H), 1.65–1.59 (m, 2H), 1.34–1.31 (m, 4H), 0.92 (t, J = 7.4 Hz, 3H), 0.89 (t, J = 6.5 Hz, 3H);13C NMR (125 MHz, CDCl3) δ : 189.4, 170.6, 157.0, 152.1, 118.3, 109.9, 73.2, 38.4, 32.8, 31.6, 26.9, 24.3, 22.6, 21.2, 14.0, 9.7; FT-IR (neat) νmax = 2961, 2931, 2866, 1741, 1674, 1588, 1516, 1374, 1238, 1023 cm−1</p><!><p>To furan derivative 13 (280 mg, 1 mmol) in CH2Cl2 (5 mL) was sequentially added Et3N (1.0 mL, 7.5 mmol), formic acid (0.28 mL, 7.5 mmol) and Noyori catalyst, (R,R) RuCl(mesitylene)-Ts-DPEN (6.2 mg, 0.01 mmol), and the reaction was set to reflux at 55 °C. After refluxing for 12 h, the orange solution was diluted with water, extracted with CH2Cl2, washed with brine and dried over Na2SO4. Purification by column chromatography (10% to 20% EtOAc/Hexanes) provided 261 mg (93% yield) of alcohol 10 as a clear oil. This alcohol was obtained as a single diastereomer by1H NMR (ratio >20:1) [α]D20 +13.5 (c 0.84, CHCl3);1H NMR (500 MHz, CDCl3) δ : 6.10 (d, J = 3.1 Hz, 1H), 5.97 (d, J = 3.1 Hz, 1H), 5.04 (m, 1H), 4.59 (q, J = 6.8 Hz, 1H), 2.88–2.80 (m, 2H), 2.01 (s, 3H), 1.92 (d, J = 5.2 Hz, 1H), 1.83–1.79 (m, 2H), 1.65–1.55 (m, 2H), 1.42 (m, 1H), 1.31–1.30 (m, 5H), 0.93–0.86 (m, 6H);13C NMR (125 MHz, CDCl3) δ : 170.7, 155.9, 151.3, 107.6, 106.7, 73.8, 68.0, 35.6, 32.6, 31.7, 26.8, 25.4, 22.7, 21.3, 14.2, 9.7; FT-IR (neat) νmax = 3447, 2958, 2933, 2859, 1739, 1559, 1464, 1433, 1373, 1242, 1021, 964, 792 cm−1; LRMS-ESI (+) m/z 305.1 [M+Na]+.</p><!><p>To furanyl alcohol 10 (317 mg, 1.12 mmol) dissolved in THF (8 mL) and H2O (2 mL) at 0 °C was added KBr (6.7 mg, 0.06 mmol), NaHCO3 (47 mg, 0.56 mmol) and oxone (826 mg, 1.34 mmol) after which a light yellow color developed. After stirring at 0 °C for 30 min, the reaction was quenched with saturated throughout NaHCO3, extracted with EtOAc, washed with brine and dried over Na2SO4. The resulting crude hemiacetal 9 was used directly for the subsequent reaction.</p><p>To crude hemiacetal dissolved in CH2Cl2 (10 mL) at –45 °C was added Et3SiH (0.89 mL, 5.6 mmol) and TFA (1.3 mL, 16.8 mmol) dropwise upon which a yellow color developed. The reaction stirred at this temperature for 3 h, then was allowed to warm to room temperature. After stirring for 30 min at 23 °C, the reaction was cooled to 0 °C and additional Et3SiH (0.895 mL, 5.6 mmol) was added followed by BF3∙OEt2 (0.415 mL, 3.36 mmol). After stirring for 30 min at 0 °C, the reaction was quenched slowly with satd. NaHCO3 until the effervescence ceased. The reaction was extracted with CH2Cl2, washed with brine and dried over Na2SO4. Purification by column chromatography (10% to 30% EtOAc/Hexanes) provided 151 mg (47% yield over two steps) of tetrahydropyran derivative 8 as a clear oil. It was obtained as a single diastereomer by1H NMR analysis (>20:1) [α]D20 +2.7 (c 0.49, CHCl3);1H NMR (500 MHz, CDCl3) δ : 5.01 (m, 1H), 3.30–3.22 (m, 2H), 2.95 (td, J = 9.0, 2.4 Hz, 1H), 2.06 (m, 1H), 2.03 (s, 3H), 1.79 (m, 1H), 1.65–1.53 (m, 6H), 1.43–1.24 (m, 9H), 0.90–0.85 (m, 6H);13C NMR (125 MHz, CDCl3) δ : 170.7, 82.2, 74.1, 72.9, 70.9, 40.0, 33.3, 32.2, 32.1, 32.0, 31.8, 27.8, 25.0, 22.7, 21.3, 14.2, 9.5; FT-IR (neat) νmax = 3451, 2929, 2859, 1739, 1718, 1461, 1436, 1373, 1242, 1080, 1056, 1024, 954 cm−1; LRMS-ESI (+) m/z 287.1 [M+H]+.</p><!><p>To tetrahydropyran derivative 8 (141 mg, 0.49 mmol) dissolved in CH2Cl2 (5 mL) at 0 °C was added 2,6-lutidine (0.23 mL, 1.97 mmol) and TBSOTf (0.34 mL, 1.48 mmol) and the reaction was warmed to 23 °C. After stirring for 12 h, the reaction was quenched with satd. NaHCO3, extracted with CH2Cl2, washed with brine and dried over Na2SO4. Purification by column chromatography (5% to 10% EtOAc/Hexanes) gave 200 mg (quantitative) of the resulting silyl ether as a clear oil.</p><p>To the silyl ether (186 mg, 0.46 mmol) in MeOH (3 mL) at 0 °C was added K2CO3 (6.4 mg, 0.05 mmol) upon which a yellow color developed. The reaction was warmed to room temperature. After 12 h, the reaction was diluted with H2O and EtOAc, extracted with EtOAc, washed with brine and dried over Na2SO4. Purification by column chromatography (5% to 20% EtOAc/Hexanes) gave 155 mg (93% yield) of the resulting alcohol as a clear oil.</p><p>To the above alcohol (144 mg, 0.40 mmol) dissolved in CH2Cl2 (4 mL) at 0 °C was added NaHCO3 (202 mg, 2.41 mmol) followed by DMP (341 mg, 0.8 mmol) and the reaction was warmed to room temperature. After 12 h, the reaction was quenched with a 1:1 mixture of satd. sodium thiosulfate and saturated NaHCO3. The mixture was extracted with CH2Cl2, washed with brine and dried over Na2SO4. Purification by column chromatography (5% to 10% EtOAc/Hexanes) gave 138 mg (96% yield) of ketone 15 as a clear oil [α]D23 +32.0 (c 0.58, CHCl3);1H NMR (500 MHz, CDCl3) δ : 3.71 (m, 1H), 3.21 (m, 1H), 3.02 (m, 1H), 2.62 (dd, J = 14.9, 8.2 Hz, 1H), 2.53–2.41 (m, 2H), 2.36 (dd, J = 14.9, 4.7 Hz, 1H), 1.94 (m, 1H), 1.78–1.69 (m, 2H), 1.52–1.22 (m, 9H), 1.03 (t, J = 7.3 Hz, 3H), 0.86 (s, 12H), 0.04 (s, 6H);13C NMR (125 MHz, CDCl3) δ : 210.3, 82.5, 74.3, 71.4, 48.7, 37.2, 33.6, 32.2, 31.9, 31.5, 25.9, 25.1, 22.7, 18.1, 14.2, 7.6, –3.9, –4.6; FT-IR (neat) νmax = 2954, 2929, 2855, 1721, 1464, 1376, 1253, 1105, 887, 837, 774 cm−1; LRMS-ESI (+) m/z 357.3 [M+H]+.</p><!><p>To ketone 15 (126 mg, 0.35 mmol) dissolved in THF (4 mL) at 0 °C was added TBAF (0.71 mL, 0.71 mmol, 1 M in THF) and the reaction was warmed to 23 °C. After 12 h, the reaction was concentrated and purification by column chromatography (30% to 50% EtOAc/Hexanes) provided 82 mg (96% yield) of decytospolide A (6) as a clear oil [α]D23 +18.4 (c 0.45, CHCl3);1H NMR (500 MHz, CDCl3) δ : 3.72 (m, 1H), 3.24 (td, J = 9.8, 4.5 Hz, 1H), 3.01 (m, 1H), 2.64 (dd, J = 15.0, 8.1 Hz, 1H), 2.53–2.41 (m, 2H), 2.37 (dd, J = 15.0, 4.8 Hz, 1H), 2.06 (m, 1H), 1.81–1.69 (m, 2H), 1.54 (brs, 1H), 1.47–1.24 (m, 9H), 1.03 (t, J = 7.3 Hz, 3H), 0.86 (t, J = 6.7 Hz, 3H);13C NMR (125 MHz, CDCl3) δ : 210.2, 82.3, 74.2, 70.6, 48.5, 37.2, 33.1, 32.1, 31.9, 31.4, 25.1, 22.7, 14.2, 7.6; FT-IR (neat) νmax = 3447, 2929, 2859, 1714, 1457, 1376, 1077 cm−1; LRMS-ESI (+) m/z 243.1 [M+H]+; HRMS-ESI (+) m/z calc'd for C14H27O3 [M+H]+: 243.1955, found 243.1958.</p><!><p>To decytospolide A (6) (73 mg, 0.30 mmol) dissolved in CH2Cl2 (3 mL) at 0 °C was added pyridine (73 μL, 0.9 mmol), acetic anhydride (85 μL, 0.9 mmol) and a few crystals of DMAP and the reaction was warmed to 23 °C. After 1.5 h, the reaction was diluted with H2O, extracted with CH2Cl2, washed with brine and dried over Na2SO4. Purification by column chromatography (10% to 20% EtOAc/Hexanes) provided 77 mg (89% yield) of decytospolide B (7) as a clear oil [α]D20 +28.1 (c 0.75, CHCl3);1H NMR (500 MHz, CDCl3) δ : 4.43 (td, J = 10.0, 4.6 Hz, 1H), 3.75 (m, 1H), 3.21 (td, J = 9.1, 2.4 Hz, 1H), 2.66 (dd, J = 15.2, 8.0 Hz, 1H), 2.52–2.40 (m, 2H), 2.36 (dd, J = 15.3, 4.9 Hz, 1H), 2.12 (m, 1H), 2.02 (s, 3H), 1.73 (m, 1H), 1.53–1.35 (m, 4H), 1.29–1.20 (m, 6H), 1.02 (t, J = 7.3 Hz, 3H), 0.85 ( t, J = 6.9 Hz, 3H);13C NMR (125 MHz, CDCl3) δ : 209.9, 170.4, 79.4, 74.3, 72.2, 48.3, 37.3, 32.0, 31.8, 30.9, 29.5, 24.9, 22.7, 21.3, 14.1, 7.6; FT-IR (neat) νmax = 2933, 2859, 1739, 1714, 1457, 1373, 1235, 1080, 1042 cm−1; LRMS-ESI (+) m/z 285.1 [M+H]+; HRMS-ESI (+) m/z calc'd for C16H28O4Na [M+Na]+: 307.1880, found 307.1884.</p><!><p>Hexanoic acid (94 μL, 0.75 mmol) was dissolved in thionyl chloride (6 mL) and refluxed for 3 h. The excess thionyl chloride was distilled off to give the resulting hexanoyl chloride as a dark yellow oil. It was then dissolved in CH2Cl2 (6 mL) and cooled to 0oC. SnCl4 (1.13 mL, 1 M in CH2Cl2, 1.13 mmol) was then added slowly dropwise and the resulting solution stirred at 0oC for 45 min. Methyl ester 17 (105.3 mg, 0.75 mmol) was dissolved in CH2Cl2 (2 mL) and the resulting solution was added slowly to the reaction over 10 min. The reaction was then stirred at 0oC for 45 min before being quenched with ice. The biphasic mixture was separated and the aqueous layer extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography (10 to 20% EtOAc/Hexanes) to give ketone 18 (102 mg, 57%) as yellow oil.1H NMR (400 MHz, CDCl3) δ : 7.12 (d, J = 3.5 Hz, 1H), 6.41 (d, J = 3.5 Hz, 1H), 3.77 (s, 2H), 3.74 (s, 3H), 2.84 – 2.66 (m, 2H), 1.70 (t, J = 7.4 Hz, 2H), 1.34 (h, J = 3.6 Hz, 4H), 0.90 (td, J = 7.1, 5.9, 3.4 Hz, 3H);13C NMR (101 MHz, CDCl3) δ : 189.33, 168.68, 152.16, 129.03 118.12, 110.76, 77.24, 76.92, 76.60, 52.38, 38.23, 34.01, 31.39, 24.06, 22.34, 13.82; ESI-API MS: [M+H] = 239.1; HRMS-ESI (+) m/z calc'd for C13H10O4 [M+H]+: 239.1280, found 239.1282.</p><!><p>Ketone 18 (60.5 mg, 0.25 mmol) was dissolved in CH2Cl2 (7 mL) and Et3N (0.5 mL, 3.81 mmol) was added followed by HCO2H (143 μL, 3.81 mmol). RuCl[(R,R)-TsDPEN](mesitylene) (3.9 mg, 0.006 mmol) was dissolved in CH2Cl2 (0.5 mL) and then added to the reaction. The reaction was then set to stir at 50oC for 12 h before being quenched with H2O. It was then extracted with CH2Cl2 and the combined organic layers washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography (10% to 30% EtOAc/Hexanes) to give optically active alcohol 19 (50.3 mg, 83%) as a clear oil.1H-NMR (400 MHz, CDCl3) δ 6.15 (d, J = 0.9 Hz, 2H), 4.62 (t, J = 6.8 Hz, 1H), 3.71 (s, 3H), 3.66 (s, 2H), 1.98 (s, 1H), 1.88 – 1.74 (m, 2H), 1.43 (tdd, J = 10.0, 8.1, 7.5, 4.1 Hz, 1H), 1.36 – 1.22 (m, 5H), 0.88 (q, J = 4.9, 4.1 Hz, 3H);13C NMR (101 MHz, CDCl3) δ 169.76, 156.47, 146.83, 108.51, 106.61, 67.66, 52.15, 35.30, 33.84, 31.48, 25.11, 22.44, 13.90; ESI-API MS: [M+Na] = 263.1; HRMS-ESI (+) m/z calc'd for C13H20O4Na [M+H]+: 263.1254, found 263.1256; 99% ee, determined by HPLC using Chiralpak IA3 and gradient of 0–10% isopropanol/hexanes (tmajor = 25.7 min, tminor =24.8 min)</p><!><p>Furanyl alcohol 19 (16.9 mg, 0.07 mmol) was dissolved in THF (2 mL) and H2O (0.5 mL) and cooled to 0oC. KBr (0.4 mg, 0.003 mmol), NaHCO3 (2.9 mg, 0.03 mmol) and oxone (51.9 mg, 0.08 mmol) were then added and the reaction slowly warmed to 23 °C. After 3 h, it was quenched with saturated NaHCO3 and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was used immediately for the next reaction.</p><p>The crude oil was dissolved in CH2Cl2 (3 mL) and Et3SiH (112 μL, 0.7 mmol) was added. The reaction was then cooled to −40oC and BF3•OEt2 (52 μL, 0.42 mmol) was added slowly dropwise. The reaction was then stirred at −40oC for 16 h before being warmed to 23 °C and stirred for an additional 1 h. It was then quenched with saturated NH4Cl and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography (20% to 30% EtOAc/Hexanes) give tetrahydropyran derivative 20 (10.0 mg, 58%) as a clear oil.1H NMR (400 MHz, CDCl3) δ : 3.80 – 3.69 (m, 1H), 3.67 (s, 3H), 3.27 (ddd, J = 10.4, 8.9, 4.6 Hz, 1H), 3.04 (td, J = 9.0, 2.4 Hz, 1H), 2.53 (dd, J = 14.9, 8.0 Hz, 1H), 2.40 (dd, J = 14.9, 5.4 Hz, 1H), 2.13 – 2.01 (m, 1H), 1.85 – 1.72 (m, 2H), 1.54 – 1.29 (m, 3H), 1.32 – 1.22 (m, 6H), 0.87 (t, J = 6.8 Hz, 3H);13C NMR (101 MHz, CDCl3) δ 171.67, 82.12, 73.83, 70.39, 51.50, 40.77, 32.75, 31.74, 31.65, 30.84, 24.85, 22.56, 13.95; ESI-API MS: [M+H] = 245.1, [M+Na] = 267.1; HRMS-ESI (+) m/z calc'd for C13H25O4 [M+H]+: 245.1747, found 245.1750.</p><!><p>To a solution of NH(OMe)Me•HCl (16.0 mg, 0.16 mmol) in dry THF (3 mL) and cooled to −30oC, i-PrMgCl (450 μL, 0.45 mmol) was added slowly dropwise and the reaction stirred at −30oC for 1 h. Methyl ester 20 (10 mg, 0.04 mmol) was dissolved in dry THF and then added slowly, dropwise to the reaction which was stirred at −30oC for an additional 4 h before being quenched with saturated NH4Cl. It was extracted with EtOAc and the combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography (50% to 80% EtOAc/Hexanes) to give Weinreb amide 21 (10.4 mg, 93%) as a clear oil.1H NMR (800 MHz, CDCl3) δ 3.85 – 3.80 (m, 1H), 3.71 (d, J = 2.2 Hz, 3H), 3.32 – 3.27 (m, 1H), 3.20 (s, 3H), 3.09 (ddd, J = 8.9, 6.5, 2.5 Hz, 1H), 2.85 (d, J = 13.4 Hz, 1H), 2.43 (dd, J = 15.1, 6.3 Hz, 1H), 2.13 – 2.08 (m, 1H), 1.90 – 1.79 (m, 2H), 1.55 – 1.44 (m, 2H), 1.46 – 1.39 (m, 1H), 1.41 – 1.31 (m, 1H), 1.33 – 1.27 (m, 5H), 0.90 (td, J = 7.0, 2.3 Hz, 3H);13C NMR (201 MHz, CDCl3) δ 172.04, 82.24, 74.14, 70.58, 61.33, 37.95, 33.02, 32.03, 31.90, 31.25, 29.71, 25.01, 22.66, 14.07; ESI-API MS: [M+H] = 274.0, [M+Na] = 296.1.</p><!><p>Weinreb amide 21 (10.4 mg, 0.038 mmol) was dissolved in dry THF (2 mL) and cooled to 0oC. EtMgBr in THF solution (305 μL, 0.3 mmol) was added slowly dropwise and the reaction slowly warmed to 23 °C. It was stirred for 5 h, then quenched with saturated NH4Cl and extracted with EtOAc. The combined organic layers were then washed with brine and dried over Na2SO4. The crude residue was purified by column chromatography (20% to 50% EtOAc/Hexanes) to give decytospolide A, 6 (8.9 mg, 97%) as a clear oil.1H NMR (400 MHz, CDCl3) δ 3.73 (dddd, J = 10.2, 8.0, 4.8, 2.0 Hz, 1H), 3.25 (ddd, J = 10.6, 9.0, 4.6 Hz, 1H), 3.02 (td, J = 8.9, 2.5 Hz, 1H), 2.65 (dd, J = 15.0, 8.1 Hz, 1H), 2.58 – 2.29 (m, 3H), 2.07 (ddd, J = 12.0, 5.5, 2.8 Hz, 1H), 1.85 – 1.70 (m, 2H), 1.53 – 1.41 (m, 1H), 1.46 (s, 2H), 1.44 – 1.33 (m, 1H), 1.37 – 1.24 (m, 5H), 1.03 (t, J = 7.3 Hz, 3H), 0.93 – 0.83 (m, 3H);13C NMR (101 MHz, CDCl3) δ 209.96, 82.04, 74.00, 70.42, 48.26, 36.98, 32.83, 31.84, 31.70, 31.16, 24.88, 22.51, 13.92, 7.41; ESI-API MS: [M+H] = 243.1, [M+Na] = 265.1; HRMS-ESI (+) m/z calc'd for C14H27O3 [M+H]+: 243.1955, found 243.1958.</p><!><p>Above synthetic decytospolide A (9.7 mg, 0.04 mmol) was dissolved in CH2Cl2 (3 mL) and cooled to 0oC. Pyridine (10 μL, 0.12 mmol), Ac2O (4 μL, 0.04 mmol) and a few crystals of DMAP were added and the reaction warmed to 23 °C. The reaction was stirred for 3 h before being quenched with water. It was extracted with CH2Cl2 and the combined organic layers washed with brine and dried over Na2SO4. The crude residue was purified by column chromatography (10 to 30% EtOAc/Hexanes) to give 7 (9.8 mg, 86%) as a clear oil.1H NMR (400 MHz, CDCl3) δ 4.44 (td, J = 10.0, 4.7 Hz, 1H), 3.76 (ddd, J = 10.9, 7.8, 4.9 Hz, 1H), 3.22 (td, J = 9.0, 2.6 Hz, 1H), 2.67 (dd, J = 15.2, 8.0 Hz, 1H), 2.58 – 2.33 (m, 3H), 2.13 (ddd, J = 11.3, 5.6, 3.3 Hz, 1H), 2.03 (s, 3H), 1.79 – 1.70 (m, 1H), 1.58 – 1.36 (m, 3H), 1.40 – 1.17 (m, 7H), 1.03 (t, J = 7.3 Hz, 3H), 0.86 (t, J = 6.9 Hz, 3H);13C NMR (101 MHz, CDCl3) δ 209.77, 170.22, 79.21, 74.09, 71.95, 48.09, 37.10, 31.82, 31.60, 30.70, 29.24, 24.71, 22.48, 21.10, 13.89, 7.40; ESI-API MS: [M+H] = 285.1, [M+Na] = 307.2; HRMS-ESI (+) m/z calc'd for C16H28O4Na [M+Na]+: 307.1880, found 307.1884.</p>

PubMed Author Manuscript

Evaluation of range-separated hybrid density functionals for the prediction of vibrational frequencies, infrared intensities, and Raman activities\xe2\x80\xa0

We present an assessment of different density functionals, with emphasis on range-separated hybrids, for the prediction of fundamental and harmonic vibrational frequencies, infrared intensities, and Raman activities. Additionally, we discuss the basis set convergence of vibrational properties of H2O with long-range corrected hybrids. Our results show that B3LYP is the best functional for predicting vibrational frequencies (both fundamental and harmonic); the screened-PBE hybrid (HSE) density functional works best for infrared intensities, and the long-range corrected PBE (LC-\xcf\x89PBE), M06-HF, and M06-L density functionals are almost as good as MP2 for predicting Raman activities. We show the predicted Raman spectrum of adenine as an example of a medium-size molecule where a DFT/Sadlej pVTZ calculation is affordable and compare our results against the experimental spectrum.

evaluation_of_range-separated_hybrid_density_functionals_for_the_prediction_of_vibrational_frequenci

I. Introduction<!>II. Computational details<!>A Estimation of vibrational frequencies<!>B Assessment of infrared intensities<!>C Assessment of Raman activities<!>D Basis set convergence of vibrational properties<!>E Raman spectrum of adenine<!>IV. Conclusions<!>

<p>Kohn–Sham density functional theory (DFT)1 has become an efficient computational chemistry tool for the accurate prediction of a variety of molecular properties. Common local, semilocal, and hybrid density functional approximations (DFAs) are, however, unsuccessful for several applications including, but not limited to, polarizability of long chains2 and charge-transfer and Rydberg excitations.3 Many of the failures of common DFAs can be traced back to the self-interaction error (SIE) they include.4 One-electron self-interaction error (1e–SIE) can be defined as inexactness for one-electron systems. (The complementary concept of many-electron self-interaction error has recently been emphasized.5,6) An important manifestation of 1e–SIE is the incorrect long-range behavior of the exchange–correlation (XC) potential. In molecules, the XC potential of semi-local functionals decays exponentially along with the density, while the asymptotic form of the exact potential is −1/r.7,8</p><p>In recent years, it has been suggested3,9–13 to introduce range separation into the exchange component of the XC potential as a means of recovering the exact −1/r asymptote. This can be implemented by splitting the Coulomb operator into short-range (SR) and long-range (LR) components by a scheme like (1)1r12=erfc(ωr12)r12︸SR+erf(ωr12)r12︸LR, where r12 is the interelectronic distance and ω is a parameter defining the extent of the short and long ranges. Admixing exact (Hartree–Fock-type, HF) exchange into SR and LR separately gives a general expression for a hybrid functional with range separation13 (2)Exc=aExSR-HF(ω)+(1−a)ExSR-DFA(ω)+bExLR-HF(ω)+(1−b)ExLR-DFA(ω)+EcDFA.</p><p>In screened hybrids (such as HSE14,15), the coefficient b = 0, while in long-range corrected hybrids (such as LC-ωPBE13,16), a = 0 and b = 1. The importance of middle-range exchange has recently been assessed.17,18 Long-range correction with 100% LR-HF exchange (b = 1) does not work properly for all common DFAs previously available. Yanai and coworkers19 proposed a "Coulomb-attenuating method" where b ≠ 1. The most obvious consequence of not including 100% LR-HF exchange is that one loses the correct asymptotic behavior of the XC potential.</p><p>The success of density functional theory for vibrational spectroscopy has been well documented.20–22 Density functional approximations, especially hybrids, outperform HF and other simple wavefunction methods (such as MP2) for the prediction of fundamental frequencies.22 DFT has also been successful in quantitative assessments of infrared intensities and Raman activities.23,24</p><p>It has long been known that the accurate prediction of infrared and Raman intensities is difficult because of their dependence on dipole moment and polarizability derivatives. These properties sample the tail of the density and require the use of large, diffuse basis sets.25,26 Electronic correlation effects are also important. The long-range corrected functionals discussed above, which recover the correct asymptotic XC potential, should improve the description of the tail of the density compared to semilocal functionals. Indeed, long-range correction has been shown to improve properties such as polarizabilities of long chains27 and excitation energies.28 Thus we were prompted to examine the performance of long-range corrected density functionals for predicting infrared and Raman intensities.</p><p>Long-range exact exchange is formally and computationally problematic in metals and small-band-gap systems.29 If we were to study molecules on the surface of a metal, 30–32 we would like to be able to predict infrared intensities and Raman activities accurately enough without the inclusion of long-range HF exchange. Thus, we also explored the behavior of a screened hybrid density functional, HSE, for predicting these properties. HSE has been successful in describing the electronic structure of solids, significantly improving the band gaps predicted by local and semi-local DFAs.29</p><!><p>All calculations were performed using the development version of the GAUSSIAN33 suite of programs. Full geometry optimizations were carried out for all molecules enforcing the proper point group symmetry of the ground state. Calculations that broke symmetry, where the symmetric geometry was a transition state (TS) or a higher-order saddle point, were discarded from the data sets (see Tables). Tight convergence criteria were used for both the scf and the optimization procedure [Gaussian keywords scf = tight and opt = tight]. All calculations use at least a pruned (99 590) numerical integration grid (Gaussian keyword integral (grid = ultrafine)). This grid turned out to be insufficient for some calculations, particularly with the M06 meta-GGAs. These calculations yielded relatively large imaginary frequencies for molecular rotations. Accordingly, we used larger integration grids (974 or 70 × 140 = 9800 angular points) for several calculations with the M06 functionals, including all of the M06-HF and M06-L Raman activities. We also used these large grids for all Raman activity calculations using the Sadlej pVTZ basis set. A few other calculations72 required larger grids for proper geometry convergence. Otherwise, larger integration grids do not change the reported statistics (up to round-off errors).</p><p>Harmonic frequencies and dipole moment derivatives were computed analytically for HF and all DFAs. Derivatives of the polarizability tensor were computed analytically for HF and by numerical differentiation of the analytic dipole derivatives with respect to the electric field for all DFAs. Static Raman activities (zero-frequency, non-resonant) were computed in the double harmonic approximation, ignoring cubic and higher force constants and omitting second and higher order polarizability derivatives. Infrared intensities were computed analytically within the double harmonic approximation. We used the convention theory—experiment to report mean errors (MEs).</p><p>The DFT approaches employed here consisted of the Becke three-parameter functional34 with the Lee–Yang–Parr correlation functional35 (B3LYP), the Perdew–Burke–Ernzerhof exchange and correlation functional36,37 (PBE), the global hybrid version of the PBE functional38,39 (PBEh), the long-range corrected PBE functional13,16 (LC-ωPBE), the Heyd–Scuseria–Ernzerhof screened-PBE hybrid functional14,15, (HSE), the Hamprecht–Cohen–Tozer–Handy exchange and correlation functional40–42 (HCTH or HCTH/407), two members of the Minnesota 06 family of density functionals43–45 (M06-L and M06-HF), and the "Coulomb-attenuating method" version of B3LYP19 (CAM-B3LYP) density functional. The density functionals used in this work can be classified as semi-local (PBE, HCTH, and M06-L), global hybrids (B3LYP, PBEh, and M06-HF), and range-separated hybrids (HSE, LC-ωPBE, and CAM-B3LYP). HCTH, M06-L, and M06-HF have a large number of empirical parameters fitted to extensive data sets. Of particular interest to this study, LC-ωPBE includes 100% LR-HF exchange, CAM-B3LYP uses 65% LR-HF exchange, and M06-HF incorporates 100% HF exchange in the entire range.</p><p>Three basis sets were considered for the assessment of frequencies and intensities. 6–31G(d) is split-valence, double zeta augmented with one set of polarization functions on heavy atoms.46,47 It is commonly used for vibrational spectroscopy because it constitutes a good compromise between computational cost and accuracy for the prediction of vibrational frequencies. Moreover, it has been used to predict infrared intensities at the HF level which are qualitatively correct.48 Sadlej pVTZ49,50 (polarized valence triple zeta) is a medium size basis set designed to give accurate molecular polarizabilities.73 It has been shown in ref. 24 that it is also very accurate for the prediction of Raman activities (which depend on the derivative of the polarizability tensor) with several density functionals. Lastly, aug-cc-pVTZ51–53 (augmented correlation-consistent polarized valence triple zeta) is a large basis set used here to estimate the convergence of vibrational properties in the basis set limit. We investigated the basis set dependence of vibrational frequencies, infrared intensities, and Raman activities of H2O using Dunning's cc-pVnZ, aug-cc-pVnZ, and d-aug-cc-pVnZ series, with n = 2–5.54</p><!><p>It is well known that harmonic vibrational frequencies calculated from electronic structure methods generally overestimate fundamental frequencies.55 Fundamental frequencies are the experimentally measured frequencies associated with the transition from the ground to the first excited vibrational state. This overestimation can be traced back to the neglect of anharmonicity (neglecting cubic and higher force constants), the approximate treatment of electron correlation, and the use of a finite basis set. Several algorithms have been proposed for predicting experimental fundamental frequencies from computed harmonic frequencies. Of these, probably the most popular are uniform rescaling and dual rescaling (where different scaling factors are used for low and high-frequency modes). Herein we adopt uniform rescaling and estimate scaling factors suitable for the various density functionals considered with 6–31G(d), Sadlej pVTZ, and aug-cc-pVTZ.</p><p>We used the F1 set (122 molecules with a total of 1066 frequencies; molecules contain ≤ 4 heavy atoms (1st–2nd row), ≤ 10 total atoms) compiled by Pople et al.56 to assess the performance of the various density functionals and to estimate optimum uniform scaling factors. AlCl3, BF3, B2H6, and BH3CO were excluded from the set in DFT/Sadlej pVTZ calculations because there are no basis available for B and Al. Additionally, we excluded C4H2 (diacetylene) in calculations using the Sadlej pVTZ basis set because all methods predict it to have a slightly bent minimum. Merrick et al.22 have computed optimum scaling factors and estimated RMS errors for a large list of density functionals using Pople-type basis sets with the F1 set. Halls et al.57 have later developed scaling factors for HF, MP2, and various DFAs for use along with the Sadlej pVTZ basis set over a slightly reduced F1 set.</p><p>Optimum scaling factors are calculated in order to minimize the residuals given by (3)Δ=∑iN(λωitheor−ν˜iexpt)2, where ωitheor and ν˜iexpt are the ith theoretical harmonic frequency and the ith experimental fundamental frequency (in cm−1), and the index i runs over all the N vibrational modes considered. This leads to the following expression for the scale factor λ (4)λ=∑iNωitheorν˜iexpt∑iN(ωitheor)2.</p><p>The results of our assessment are shown in Table 1.</p><p>It should be noted that our results vary slightly from the results shown in ref. 22 for the 6–31G(d) basis set. In particular, the RMS errors reported herein are ca. 3 cm−1 larger than the ones previously reported. This discrepancy appears to arise from the way one matches the computed harmonic frequencies to the experimental fundamentals. In previous studies,16 the authors seem to have sorted all computed frequencies of a given molecule and compared them directly to the experimental fundamentals without considering the symmetry of modes. Here, we consider the symmetry of the mode in the matching algorithm: modes are required to have the previously assigned symmetry. Indeed, there are only few cases (for the largest molecules) in which symmetry makes a difference. Ignoring symmetry underestimates the error of a given method to accurately predict experimental fundamentals. This is why we discard calculations in which molecules break symmetry.</p><p>In general, the scaling factors for all functionals are closer to unity than those for HF. The scaling factor for LC-ωPBE (0.9479 with the aug-cc-pVTZ basis) is the lowest one of all DFAs. We also note that the scaling factors for the Sadlej pVTZ and aug-cc-pVTZ basis are about 0.01 larger than the ones for 6–31G(d). It is interesting to note that Sadlej pVTZ does not seem to be the best basis for estimating fundamental frequencies with density functionals; it gives root mean square errors that are ca. 8–10 cm−1 larger than those for aug-cc-pVTZ, whereas 6–31G(d), once scaled, gives root mean square errors that are only slightly larger than the corresponding ones for aug-cc-pVTZ. This observation does not hold for HF, whose RMS error computed with Sadlej pVTZ is even slightly smaller than the one from aug-cc-pVTZ computations. From the results shown in Table 1, the best functionals for predicting fundamental frequencies are B3LYP and its "Coulomb-attenuating method" version (CAM-B3LYP). Indeed, B3LYP/6-31G(2df,p) is used to estimate thermal corrections from computed frequencies in G3X theory.58</p><p>From comparison of unscaled RMS errors, it seems that inclusion of HF exchange in the long-range (CAM-B3LYP, LC-ωPBE, and M06-HF) tends to degrade the performance of the parent density functional for the prediction of fundamental frequencies.</p><p>Experimentally, there is the possibility of measuring not only the fundamental but also other transitions, especially for diatomic molecules. One can then fit these energies to a polynomial to estimate the harmonic frequencies. It is interesting to assess the behavior of different functionals for the prediction of these "experimental" harmonic frequencies. The errors in the estimation of harmonic frequencies are not due to the harmonic approximation, but result only from the use of finite basis sets, approximate treatment of electron correlation, and experimental uncertainties.</p><p>We have used two different sets to evaluate the performance of density functionals in predicting harmonic vibrational frequencies. The T82F set59 consists of 82 diatomic molecules, radicals, and cations; most experimental harmonic frequencies in this set were taken from the CRC Handbook of Chemistry and Physics and deemed reliable. The T32F (introduced here) is a combination of the 28 experimental harmonics used by Besler et al.60 and the 4 experimental harmonics used by Tew et al.;61 we feel very confident about the accuracy of the experimental values in this second set. A total of 11 molecules are included in the T32F set: CH4, HCN, C2H2, CO2, H2O, H2CO, NH3, HF, N2, CO, and F2. Be2 was omitted from the T82F set for HF, as it is unbound. A few molecules failed to converge and were omitted (see Table 2). Several molecules had to be excluded from the T82F set in DFT/Sadlej pVTZ calculations because there are no basis available for B and Al: B2, BH, BF, BCl, BN, BO, BS, Al2, AlH, AlCl, AlO, and AlS. The results of our assessment are shown in Table 2. HF significantly overestimates the harmonic frequencies. Hybrids incorporating a significant amount of HF exchange, such as M06-HF and LC-ωPBE, also overestimate harmonic frequencies, though not as much as HF does. LC-ωPBE performs better than its PBE parent in the T32F set, but not in the T82F set. Interestingly, PBE and HCTH underestimate harmonic frequencies whereas all other functionals tend to overestimate them. B3LYP gives the best results for the prediction of harmonic frequencies.</p><!><p>In a previous study, Halls and Schlegel23 concluded that the agreement of electronic structure methods with respect to QCISD for the prediction of infrared intensities follows the order: hybrid DFT > MP2 > local DFT ≈ gradient-corrected DFT > HF, with hybrid functionals offering an excellent performance. Galabov et al.62 have shown that inclusion of electron correlation (at the QCISD, CCSD or CCSD(T) levels of theory) and extended basis sets (such as aug-cc-pVTZ) are needed to achieve quantitative agreement for infrared intensities between theoretical predictions and experimentally determined values. It should be noted that experimental infrared intensities are difficult to measure because of overlapping bands, resonances, intensity sharing, etc., limiting the accuracy to around ±10%.</p><p>Halls and Schlegel23 compared the results of infrared intensities at the QCISD/6–311+G(3df,3pd) and CCSD(T)/TZ(2df,2pd) levels for a small number of molecules. The authors found good agreement between both theoretical methods, but both showed large discrepancies with respect to experimental values. Therefore, a large fraction of the difference may be attributed to the experimental uncertainties and the double harmonic approximation. We compare our calculated intensities against experiment and against high-level ab initio results already reported in the literature. DFT and ab initio results should ultimately converge to the same limit within the double harmonic approximation. Our test set is a combination of the sets used by Halls and Schlegel23 and Galabov et al.:62 HF, CO, H2O, HCN, C2H2, H2CO, NH3, C2H4, CH2F2, CH4 and SiH4. The results are shown in Table 3. We did not consider CO2 as part of our test set because there is a large disagreement between experimental and ab initio results: the experimental intensity of the σu+ mode is 548 km mol−1, while the theoretical intensities are 749, 634 and 708 km mo−1 at the CCSD/TZ(2df,2pd), CCSD(T)/TZ(2df,2pd), and QCISD/6–311+G(3df,3pd) levels, respectively.23,63</p><p>In general, all functionals show similar behavior and the results are much better than the predictions of HF. Taking aug-cc-pVTZ as a basis, HSE and PBEh are the best performers when comparing against experiment, whereas comparison against ab initio results yields B3LYP and CAM-B3LYP as the best performers. PBE and HCTH give results in greatest disagreement with respect to ab initio results. When comparing against experimentally determined intensities, the long-range corrected hybrids LC-ωPBE and CAM-B3LYP are among the worst performers, although the RMS error is only ca. 3 km mol−1 larger than that for the best performers. M06-HF gives even larger errors. It is evident that 6–31G(d) is an insufficient basis set for the prediction of infrared intensities, giving RMS errors much larger than either the Sadlej pVTZ or the aug-cc-pVTZ basis. Most methods show very similar behavior with the Sadlej pVTZ and aug-cc-pVTZ basis. There is also a reasonable agreement when comparing results against experiment or against high-level ab initio computations.</p><!><p>Halls and Schlegel24 have shown that DFAs are almost as good as MP2 for the prediction of Raman activities. They observed that the difference between HF and MP2 is not as large as for infrared intensities. In addition, the convergence with basis set is slower for Raman activities than for infrared intensities, in agreement with the observation that Raman activities require a better description of the tail of the density. The density near the nuclei is held tightly by the nuclear charges, whereas the tail of the density is more polarizable and thus becomes important in the prediction of the Raman response, which depends on the derivative of the polarizability. Our test set is the same as that used in ref. 24: N2, H2O, CH4, C2H2, C2H4, C2H6, CH2F2 and NH3. A total of 29 vibrational modes were considered (degenerate modes were considered only once). The results of our assessment are shown in Table 4.</p><p>As in the case of infrared intensities, the 6–31G(d) basis set is insufficient, with RMS errors close to 50 Å4 amu−1. As observed by Halls and Schlegel,24 all functionals show a similar performance with large basis sets. LC-ωPBE is among the best DFT performers with both the Sadlej pVTZ and the aug-cc-pVTZ basis sets, giving results comparable to those of MP2. Interestingly, all methods but LC-ωPBE tend to overestimate the Raman activities with the Sadlej basis set; in the case of the aug-cc-pVTZ basis, all methods performing well (as judged by the RMS) give a mean error close to zero. CAM-B3LYP also performs well, though not as well as LC-ωPBE, suggesting that 100% HF exchange in the long range improves the predicted Raman activities. However, M06-L, which does not include HF exchange, performs also very well in conjunction with the aug-cc-pVTZ basis.</p><!><p>Electronic structure methods that can be used to estimate molecular properties are characterized by an approximate treatment of electron correlation and an incomplete basis set, thus limiting the accuracy of the computation. It is important to study how different molecular properties converge with respect to the size of the basis set for a given theoretical method. Water (H2O) has previously been used to assess this convergence for ab initio methods.48,62,64 In Table 5, we present an assessment of the basis set convergence of the vibrational properties of water with the long-range corrected LC-ωPBE density functional using Dunning's basis set series. The same assessment performed with the PBE and PBEh functionals is included as part of the supporting information for this paper.</p><p>Regarding the computed harmonic frequencies, the two high frequency modes are almost converged with a triple-ζ quality basis set (cc-pVTZ), while the low-frequency a1 mode requires, at the triple-ζ level, inclusion of a set of diffuse functions (aug-cc-pVTZ). Typically, scaling factors are used to correct for both of these deficiencies in computed values. In fact, the use of scaling factors also allows one to use a lower quality basis set (the scaling factor will be slightly different and will include contributions from basis set effects). It is interesting to notice that, at the quintuple-ζ level, inclusion of diffuse functions has a marginal effect on the prediction of harmonic frequencies. This confirms that it is not needed to describe very accurately the tail of the wavefunction (or the density) to get accurate harmonic frequencies, which is not the case for infrared intensities or Raman activities.</p><p>Inclusion of diffuse functions is almost mandatory for an accurate estimation of Raman activities (observe, for instance, that the predicted values for the two a1 modes are far from converged even at the cc-pV5Z level). The aug-cc-pVTZ basis set seems to be acceptably close to the converged values, although inclusion of a second set of diffuse functions (d-aug-cc-pVTZ) may significantly improve the prediction for the low-frequency a1 mode.</p><p>For the prediction of infrared intensities, the aug-cc-pVTZ basis gives results very close to convergence, while working without diffuse functions is not recommended. It is interesting to note that d-aug-cc-pVDZ (that is, including two sets of diffuse functions with a double-ζ basis set) yields very similar results to those of aug-cc-pVTZ for both infrared intensities and Raman activities.</p><p>The best compromise between accuracy and computational efficiency seems to be the Sadlej pVTZ basis sets, which gives results very close to those of aug-cc-pVTZ while being only slightly more expensive than aug-cc-pVDZ and cheaper than the d-aug-cc-pVDZ basis. Comparison of the results shown in Table 5 and those reported in the ESI† shows that the convergence of vibrational properties with basis sets is rather similar for PBE, PBEh, and LC-ωPBE, an observation that was difficult to estimate a priori.</p><p>With respect to experimental results, a very good estimate of the absolute Raman activities can be obtained with LC-ωPBE as one approaches the basis set limit. Indeed, the converged values are much closer to experiment than those for infrared intensities (vide supra). However, we recall that absolute infrared intensities are challenging to measure experimentally, and that LC-ωPBE gives infrared intensities close to other density functionals.</p><!><p>We were interested in evaluating the predicting power of DFT/Sadlej pVTZ computations for the Raman spectrum of middle-size molecules. We chose adenine as our target molecule for this purpose.</p><p>There has been some controversy over whether adenine has a planar or non-planar equilibrium geometry.65 It is not our intention to solve this issue here. We note that LC-ωPBE/Sadlej pVTZ predicts a planar (Cs) minimum, while B3LYP/6–31G(d) and B3LYP/Sadlej pVTZ predict a minimum slightly distorted from planarity, in which the NH2 group retains its pyramidal configuration. The computed theoretical spectra at the B3LYP/6–31G(d), B3LYP/Sadlej pVTZ, and LC-ωPBE/Sadlej pVTZ levels in the 300–1700 cm−1 region are shown in Fig. 1, along with the experimental spectrum for comparison purposes. Fig. 1 plots the calculated relative Raman intensities against scaled frequencies. The Raman intensity of a vibrational mode p with frequency ωp is proportional to Rp/ωp, where Rp is the "Raman activity" calculated using Placzek's approximation and reported by a program such as GAUSSIAN. (Theoretical details of Placzek's approximation for Raman intensities may be found in the textbook by Barron.66) A 5 cm−1 Lorentzian broadening was added to each of the peaks. The frequencies and relative intensities of the peaks in adenine's experimental spectrum were obtained from the Spectral Database for Organic Compounds, SDBS, from the National Institute of Advanced Industrial Science and Technology (http://riodb01.ibase.aist.go.jp/sdbs/). These were measured from a powder sample using a 4880 Å source. The "experimental" spectrum in Fig. 1 is these tabulated frequencies and intensities, plotted with a Lorentzian broadening as a guide to the eye. This approach suffices for our purposes, as we did not attempt to calculate the Raman peak shapes.</p><p>There is a good overall agreement between the computed spectrum using LC-ωPBE and the experimental spectrum. The relative intensities of all low-frequency modes are underestimated, and the predicted frequencies for low-frequency modes are slightly overestimated while for high-frequency modes are underestimated. The feature at approximately 1100 cm−1 from the experimental spectrum describing an in-plane motion appears severely underestimated (in relative intensity) in all theoretical spectra. The theoretical spectra at the B3LYP level are also in good agreement with experiment. In general, the frequencies predicted with B3LYP are in better agreement with experiment than those from LC-ωPBE, consistent with the results from previous sections.</p><!><p>We have shown that long-range corrected hybrids, LC-ωPBE in particular, improve the accuracy of common DFAs for the prediction of Raman activities. The one-parameter LC-ωPBE gives frequencies and Raman activities approaching the highly parametrized "density16 functional for spectroscopy" (M06-HF).44 Previous studies showed that LC-ωPBE is also very good for thermochemical kinetics. The observation that HSE does not degrade results from PBEh for the prediction of vibrational properties is encouraging, as this functional is particularly suitable to be used in molecule–metal interfaces using periodic boundary conditions. Inclusion of long-range exchange tends to degrade a functional's performance for vibrational frequencies and infrared intensities. This is especially true for M06-HF. However, the CAM-B3LYP and LC-ωPBE functionals still appear to provide acceptable accuracy for these properties. As in previous investigations, B3LYP with an empirical scale factor provides very accurate predictions of experimental fundamental and harmonic vibra-tional frequencies. The LC-ωPBE/Sadlej pVTZ adenine Raman spectrum is in good agreement with the experimental one, further demonstrating that long-range corrected functionals can be routinely applied to predicting the vibrational spectra of medium-sized molecules.</p><!><p>Electronic supplementary information (ESI) available: Basis set convergence of vibrational properties of H2O with PBE and PBEh.</p>

PubMed Author Manuscript

Direct catalytic conversion of cellulose to liquid straight-chain alkanes

High yields of liquid straight-chain alkanes were obtained directly from cellulosic feedstock in a one-pot biphasic catalytic system. The catalytic reaction proceeds at elevated temperatures under hydrogen pressure in the presence of tungstosilicic acid, dissolved in the aqueous phase, and modified Ru/C, suspended in the organic phase. Tungstosilicic acid is primarily responsible for cellulose hydrolysis and dehydration steps, while the modified Ru/C selectively hydrogenates intermediates en route to the liquid alkanes. Under optimal conditions, microcrystalline cellulose is converted to 82% n-decane-soluble products, mainly n-hexane, within a few hours, with a minimum formation of gaseous and char products. The dominant route to the liquid alkanes proceeds via 5-hydroxymethylfurfural (HMF), whereas the more common pathway via sorbitol appears to be less efficient. High liquid alkane yields were possible through (i) selective conversion of cellulose to glucose and further to HMF by gradually heating the reactor, (ii) a proper hydrothermal modification of commercial Ru/C to tune its chemoselectivity to furan hydrogenation rather than glucose hydrogenation, and (iii) the use of a biphasic reaction system with optimal partitioning of the intermediates and catalytic reactions. The catalytic system is capable of converting subsequent batches of fresh cellulose, enabling accumulation of the liquid alkanes in the organic phase during subsequent runs. Its robustness is illustrated in the conversion of the raw (soft)wood sawdust. Broader contextA novel one-pot catalytic approach is presented that is able to directly transform cellulose into straight-chain alkanes (mainly n-hexane). The carbon-based yields are high (up to 82%) and the process completes in less than 6 hours at only 493 K. The so produced and thus bio-derived light naphta fraction is an ideal green feedstock for existing processes that produce aromatics, gasoline or olens. Considering the vast and cheap amounts of cellulosic residue and the absence of its pretreatment for this process, this catalytic one-pot approach seems highly promising en route to more sustainable chemicals and fuels.

direct_catalytic_conversion_of_cellulose_to_liquid_straight-chain_alkanes

Introduction<!>Experimental<!>Rationale<!>Tuning the hydrogenation properties of Ru/C<!>Exploring cellulose to liquid alkanes conversion with modied Ru/C<!>View Article Online<!>Reaction network study<!>Process robustness: converting real wood feedstock and catalyst and reuse<!>Conclusions

<p>Interest in lignocellulosic biomass as a renewable feedstock for fuels, chemicals and materials has increased tremendously in recent years. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] The high oxygen-to-carbon ratio of cellulosic biomass creates ample opportunities to produce chemicals and polymer building blocks with high chemical functionality, which cannot be produced as cheaply from fossil feedstock. [15][16][17] 5-hydroxymethylfurfural (HMF), 18 (vinyl) glycolic acid, 19,20 lactic acid 21,22 and levulinic acid 23,24 are four examples of such chemicals, for which synthesis directly from cellulose is under investigation. Targeting fuels with biomass feedstock primarily concentrates on depolymerization and defunctionalization strategies to produce molecules with high heating value like alkanes and aromatics. [25][26][27][28][29][30][31][32][33][34][35] As the value of a fuel per tonnage is usually low, but the targeted volumes are enormous, process and energy cost should be kept to an absolute minimum. There are elaborate examples in literature describing the production of new generation biofuels from sugars, sugar alcohols or other platform molecules such as HMF and levulinic acid, 3,10,25,[30][31][32][33][35][36][37][38][39][40][41] but research on the direct route from low cost cellulose to alkanes is still in its infancy. Although high temperature hydropyrolytic routes from biomass towards mixtures of gasoline and other compounds are promising, [42][43][44][45][46][47][48][49] there is room to improve the carbon efficiency to liquid alkanes. Due to its high natural abundance 50 and uniform chemical structure with repeating C 6 sugar units, cellulose should be the ideal precursor for selectively making C 6 alkanes (and thus light naphtha) as C-C bond breaking and forming are not required.</p><p>The main challenge is to selectively break C-O in presence of C-C bonds.</p><p>The prospect of renewable n-hexane directly made from cellulose, as outlined in Scheme 1, is exciting as this alkane has many uses such as technical solvent, 51 fuels and building block for chemicals. For use as a transportation fuel, viz. gasoline, nhexane needs to be isomerized to branched hexanes with higher motor octane numbers (MON) like 2,2-dimethylbutane (MON 95). 52 It is well known that, unlike C 7+ alkanes, light (C 4 , C 5 and C 6 ) alkanes can be selectively isomerized with minimal cracking (e.g., in the Hysomer Process of Shell), and mixed in with gasoline. 52,53 Since highly branched alkanes, possibly mixed with some ethers, constitute the environmentally most friendly gasoline, 52 bio-based isomerized light naphtha (with or without ethers) may be an interesting option to improve the renewability of gasoline in short term. Besides fuel and solvent use, n-hexane may also serve as ideal feedstock for bio-benzene production 54 and for bio-ethylene and propylene production via steam or catalytic cracking. 55 During submission of this manuscript, the group of Tomishige reported the rst selective one-pot conversion of cellulose to n-hexane using Ir-ReO x /SiO 2 and H-ZSM-5. 56 The reaction proceeds via the hydrolytic hydrogenation of cellulose to sorbitol, which is subsequently converted to n-hexane through consecutive hydrodeoxygenation cycles. Apart from this report, only multistep processes 57 and the conversion of cellobiose 36 (94.8% n-hexane yield) and methylcellulose 58 into nhexane (80% total yield) were demonstrated. The major obstacles for direct cellulose conversion are its poor solubility in conventional solvents and high chemical recalcitrance. 59 These issues necessitate severe reaction conditions in terms of acidity and/or temperature, which can lead to unwanted side reactions. This paper reports a direct, fast and selective conversion of cellulose into liquid straight-chain alkanes, mainly n-hexane, by tuning the hydrogenation selectivity of a commercial Ru catalyst in a biphasic liquid system. The surface modication steers the reaction via a novel pathway, forming liquid alkanes through intermediate HMF (see Scheme 2).</p><!><p>A typical modication of commercial 5 wt% Ru/C proceeded as follows: Ru/C (1 g), tungstosilicic acid (TSA) hydrate (0.25 g) and water (40 ml) were loaded into a 100 ml stainless steel batch reactor (Parr Instruments Co.). The reactor was ushed with N 2 and subsequently pressurized with 5 MPa H 2 . The mixture was stirred at 700 rpm and heated to 483 K at an average rate of 10 K min À1 and kept at this temperature for 1 h. The reactor was then cooled, depressurized and opened. The synthesized catalyst (htTSA(2)Ru/C) was ltered, thoroughly washed with distilled water and dried to constant weight.</p><p>In a typical catalytic experiment, microcrystalline Avicel PH-101 cellulose (2 g), TSA hydrate (5 g), htTSA(2)Ru/C (0.5 g), water (20 ml) and n-decane (20 ml) were loaded into a 100 ml stainless steel batch reactor (Parr Instruments Co.). The reactor was ushed with N 2 and subsequently pressurized with 5 MPa H 2 . The mixture was stirred at 700 rpm and heated to 493 K at an average rate of 12 K min À1 from room temperature to 423 K and further to 493 K at a xed rate of 0.5 K min À1 . The mixture was kept at 493 K for an additional 40 min. Aer reaction, the reactor was cooled, depressurized and opened. Samples were taken from both the water and n-decane phases and centrifuged before GC and TOC analysis. For determination of cellulose conversion and catalyst reuse experiments, centrifuged particles were added back to the reaction mixture. The reaction mixture was subsequently ltered, thoroughly washed and dried to constant weight.</p><p>Complete experimental procedures are provided in the ESI. †</p><!><p>From literature on n-hexane production from sugar and sugarderived feedstock, 31,32 and the recent one-pot approach from Tomishige and co-workers, 56 one may deduce one major pathway, which proceeds via a combination of various reactions including hydrolysis, hydrogenation, dehydration and hydrodeoxygenation (HDO), catalyzed by bifunctional acid/redox catalytic systems (Scheme 2). There is general agreement that the route involves the initial formation of sorbitol as key intermediate towards n-hexane. 32,36,60,61 However, we propose here an alternative pathway that runs through HMF. Deep HDO of HMF to n-hexane, e.g. by direct metal-catalyzed C-O hydrogenolysis or acid/metal-catalyzed dehydration/hydrogenation cycles, 60,62 has not been demonstrated experimentally.</p><p>The use of cellulose rather than sugar solutions signicantly complicates the balance of reaction rates required for selective n-hexane formation. As fast cellulose hydrolysis generally requires strong acidic conditions or high temperatures, sorbitol produced from glucose may undergo rapid dehydration to sorbitan and isosorbide. As a remarkable stability of isosorbide in the presence of acid/redox catalysts at high temperatures was encountered, 63 isosorbide formation may be a signicant hurdle for the low energy conversion of cellulose to n-hexane. Isosorbide formation can be prevented as long as glucose dehydration to HMF is kinetically favored over glucose hydrogenation to sorbitol. Additionally, subsequent and fast hydrogenation of HMF to e.g., 2,5-dihydroxymethylfuran (2,5-DHMF, Scheme 2) and 2,5-dihydroxymethyltetrahydrofuran (2,5-DHMTHF) should be promoted to avoid HMF degradation into levulinic acid and humin (char). From that point on, a series of HDO cycles of the furanic species should ensue. A recent example of Buntara et al., showing a selective conversion of 2,5-DHMTHF to 1,6-hexanediol, underscores the potential of an HMF route that ultimately leads to n-hexane. 64 The main challenge seems the integration of the acidic hydrolysis of cellulose with a selective hydro(deoxy)genation of acid-sensitive HMF in presence of glucose. We anticipate that these demands can be fullled by (i) compartmentalization of the acidity in an aqueous phase and redox activity in an organic phase and (ii) modication of the redox catalyst to increase its selectivity towards HMF hydrogenation instead of glucose hydrogenation (vide infra). The biphasic system is essential to extract acid-sensitive intermediates from the acidic aqueous phase into the organic phase, while the organic phase should be a favorable medium for hydrogenation and dehydration reactions due to a higher hydrogen solubility and more efficient dehydration in organic solvents, respectively. The important role of catalysis at water-oil interfaces in biomass conversion has been suggested in other work as well. 65,66 The benecial effect of a biphasic solvent system on the selective hydrogenation of HMF to 2,5-DHMTHF for instance has been investigated and conrmed by Alamillo et al. 67 and Yang et al. 68 Furthermore, glucose hydrogenation is suppressed in favor of HMF hydrogenation by the use of a hydrophobic hydrogenation catalyst, which predominantly resides in the organic phase. Since HMF traverses phase boundaries, the latter will result in selective hydrogenation of HMF, avoiding sorbitol formation.</p><!><p>Since Ru/C is commercially used to hydrogenate glucose to sorbitol, 69 it seems at rst sight an unlikely catalyst choice, but its selection here was primarily based on its high affinity for the organic phase (see ESI, Fig. S1 †) and its commercial relevance.</p><p>In order to suppress its glucose hydrogenation ability and favor HMF hydrogenation, Ru/C was modied. Modifying the chemoselectivity of metal redox catalysts is usually accomplished by adding promoters. 70 Below, we show that the hydrogenation selectivity of commercial Ru/C is drastically changed in favor of HMF hydrogenation by hydrothermal treatment (ht) in presence of tungstosilicic acid (TSA, H 4 SiW 12 O 40 ). The modication was carried out under H 2 pressure (5 MPa at room temperature) at 483 K for 1 h in water in presence of varying TSA concentrations. Despite the harsh treatment, we barely noticed Ru leaching during the hot water treatment in presence of TSA: elemental analysis of the ltrate demonstrated the presence of 2.5 ppm Ru, corresponding to 0.3 wt% of the initial Ru content.</p><p>The change in hydrogenation selectivity was evidenced in a kinetic study. A rst series of experiments with glucose was carried out in water in presence of unmodied Ru/C, ht-treated Ru/C and Ru/C ht-treated in a 2 and 135 mM TSA solution, denoted as Ru/C, htRu/C, htTSA(2)Ru/C and htTSA(135)Ru/C, respectively. The TSA loading on Ru/C aer drying, studied by gravimetric analysis, correlates to the TSA concentration in the pretreatment mixture (Table S1 †), in agreement with the strong adsorption of heteropoly acids on carbon supports. [71][72][73][74][75][76][77] The nal TSA modied catalysts htTSA(2)Ru/C and htTSA(135)Ru/C contain approximately 9 and 27 wt% TSA (on dry basis), respectively. The kinetic proles are presented in Fig. 1. As expected, glucose is selectively converted to sorbitol by each catalyst. The data show a signicant decrease in activity aer modication with TSA, and this decrease is more pronounced with the higher TSA loading. Comparison of the initial conversion rates of htTSA(2)Ru/C and htTSA(135)Ru/C versus pristine Ru/C showed a remarkable three-and six-fold activity loss, respectively, whereas a hydrothermal treatment in absence of TSA only shows a minor impact on the hydrogenation activity.</p><p>A similar set of kinetic experiments was carried out for the hydrogenation of HMF (Fig. 2). All reactions formed 2,5-DHMF as main product. Interestingly, the hydrothermal modication of Ru/C both with and without TSA results in a hydrogenation activity increase.</p><p>Although the fundamentals behind the selective modication of Ru/C with TSA are unclear, CO chemisorption (see ESI †) showed a decreased number of total active sites upon modication and this decrease correlates linearly with the initial glucose hydrogenation activity of the different catalysts (Fig. S2a †). Unless CO is selectively probing glucose adsorption sites, this observation is indicative of a structural change of Ru e.g. Ru sintering. Calculation of the turnover frequency (TOF, s À1 ) shows that modication with TSA has little impact on glucose hydrogenation, while there is a signicant increase in the TOF (s À1 ) (calculated as mol converted HMF per mol surface Ru per second) of HMF hydrogenation (Fig. S2b †). The modied Ru surface thus seems to benet the planar adsorption of HMF, with strongly adsorbed C]C and parallel-oriented C]O bonds, 62 likely on atomically smoother Ru surfaces of the sintered Ru. Investigation of the physicochemical properties of the TSA-modied Ru/C catalyst is ongoing.</p><!><p>The modied htTSA(135)Ru/C catalyst, with its altered hydrogenation selectivity, was used to explore the one-pot conversion of cellulose to liquid alkanes. A biphasic water/n-decane (50 : 50 vol%) solvent mixture was initially chosen. Microcrystalline cellulose was used and its conversion to liquid alkanes was initially tested with htTSA(135)Ru/C at temperatures ranging from 483 K to 503 K at 5 MPa H 2 pressure. The reaction uses an additional amount of water-soluble TSA catalyst to accelerate cellulose hydrolysis. Unlike most inorganic solids like alumina and silica/alumina, TSA is a strong Brønsted acid, and most importantly, it shows a high selectivity to glucose during cellulose hydrolysis. 63,75,[78][79][80][81][82] The catalytic results are summarized in Table 1. The table includes the main reaction products found: n-hexane, methylcyclopentane (MCP), n-pentane, 2,5-DMTHF, 1-hexanol and some hexitols (sorbitol, mannitol and their anhydrides like sorbitan and isosorbide).</p><p>A rst experiment, in presence of 135 mM of soluble TSA and htTSA(135)Ru/C (Table 1, entry 1), showed appreciable yields of n-decane-soluble products (42%, including 22% n-hexane yield), while only 6% hexitol yield was obtained in one hour at 483 K, reached by rapidly heating the reactor (see conditions in Table 1). This rst result validates the concept of directly converting cellulose to liquid alkanes in the biphasic liquid conditions using hydrothermally TSA-treated Ru/C. The 41% carbon decit indicates signicant losses in form of gaseous and insoluble polymeric products. To minimize these side reactions, a slower heating rate of 0.5 K min À1 instead of 5.5 K min À1 was applied from 423 K onward (Table 1, entry 2). This stepwise heating protocol resulted in a notable yield increase of n-decane-soluble products to 60%, including 34% n-hexane and a 78% yield of identied liquid phase products. Insignicant amounts of gaseous products were detected in this experiment (mainly methane, see ESI †).</p><p>TSA in the aqueous phase plays a key role in the conversion of cellulose to liquid alkanes. As expected, low yields of liquid phase products are observed in absence of soluble TSA (Table 1, entry 6), since the acid is responsible for cellulose hydrolysis. An increase of TSA concentration from 15 to 135 mM considerably enhances the total liquid alkane yield, mainly at the expense of oxygenates like DMTHF and 1-hexanol (Table 1, entries 2-5). This yield increase is in line with the strong dehydration property of TSA, required to efficiently carry out series of bifunctional HDO reactions. Reactions with only TSA and no hydrogenation catalyst should obviously be avoided, as it leads to pronounced char formation.</p><p>Interestingly, reactions at higher temperatures require less acid (Table 1, compare entries 3, 7 and 8). 83 For instance, by increasing the reaction temperature to 493 K, 71 mM TSA is sufficient to completely convert cellulose to 65% n-decanesoluble products of which more than half is n-hexane. The total product yield from both liquid phases accounts for 80% of the carbon balance. Based on the amount of TSA in the aqueous phase, and assuming cellulose hydrolysis, various acid-catalyzed rearrangements and dehydration steps to break C-O bonds en route to n-hexane, a catalytic turnover of about 12 can be estimated for each proton, showing a catalytic contribution of TSA.</p><p>The effect of modifying Ru/C with TSA, as predicted in the rationale of this contribution, is apparent from the experiments in Table 2. Unmodied Ru/C (Entry 8) led to signicantly less alkane formation, while the hexitol yield considerably increased (from about 9 to 29%). Main compound in the hexitol fraction is isosorbide (with 18%), followed by sorbitan, isoidide and isomannide. This difference in product distribution is in line with the well-known glucose hydrogenation ability of commercial Ru/C. Hydrothermal treatment in absence of TSA (htRu/C, entry 7) partly decreases the hexitol fraction, but this decrease is not as efficient as with the TSA-modied Ru/C catalysts. Entries 1 to 6 illustrate the catalytic results with different htTSA(x)Ru/C catalysts, where x represents the TSA concentration during hydrothermal pretreatment (ranging from 2 to 135 mM). The highest carbon efficiency and liquid alkane yield, viz. 90% and</p><!><p>60% respectively, were obtained with the lowest TSA modication (entry 6).</p><!><p>The previous data displayed a wealth of intermediates and endproducts, with the liquid alkanes being the desired ones in this work. To gain more insight into the reaction network, a systematic catalytic study was carried out by feeding the major reaction intermediates into the reactor under identical conditions. The data are collected in Table 3.</p><p>In contrast to previously reported pathways to alkanes, 32,60,84,85 sorbitol and isosorbide turned out to be fairly unreactive (see Table 3, entries 1-4): only 30% of sorbitol carbon (or 14% in fed-batch mode) and 19% of isosorbide carbon were converted into n-decane-soluble products. Sorbitol was mostly dehydrated to isosorbide (here also referred to as 'hexitol'). These observations conrm a kinetically less favorable route from cellulose to alkanes via sorbitol. In line with our hypothesis, it predicts that hydrogenation of glucose should be slow compared to its dehydration to HMF in order to avoid yield loss to hexitols and their anhydrides. Interestingly, modication of Ru/C with TSA fullls this particular role.</p><p>Selected key intermediates, which were analyzed in the previous experiments and are likely involved in the alternative HMF route, are glucose, fructose, HMF, 2,5-DMTHF, 2,5-hexanedione, 2,5-hexanediol, 1,2-hexanediol, 2-hexanol and 1hexanol. Scheme 3 collects these chemicals in a tentative reaction network. Reactions with these molecules are presented in Table 3 (entries 5-18). Before giving a detailed description of the data and a network analysis, it can already be concluded from the data that high yields of n-decane soluble products were attained from all the above mentioned molecules, validating the proposed n-hexane pathway via HMF. Conversion of glucose and fructose with htTSA(2)Ru/C in presence of 71 mM TSA yields an insignicant amount of hexitols (in line with Fig. 1), while the alkane yield is highest for glucose (Table 3, entries 5-8). The formation of a signicant amount of n-pentane is apparent. A fed-batch approach was used next to the batch reactions to imitate the gradual release of glucose from cellulose, as was done successfully in the direct conversion of glucose to ethylene glycol. 86,87 The product distribution indeed changed with reactor type, the batch reactor systematically leading to higher alkane yields, in agreement with the preferred high contact time to form the alkane end-products. Indeed, test reactions with n-hexane and n-decane showed negligible conversion (data not shown), while longer reaction times provides higher liquid alkane yields, as will be demonstrated below.</p><p>Study of the HMF conversion, although the key molecule in the new n-hexane pathway, was somewhat problematic in batch mode due to its high reactivity (Table 3, entry 10). A low content of n-decane soluble products was observed and large quantities of polymers (char) and degradation products like levulinic acid were noticed. To obtain high liquid alkane yield from cellulose, gradual formation of HMF and subsequently fast HMF hydrogenation is thus important. Fed batch conversion of HMF is more efficient, yielding 22% alkanes and 23% 2,5-DMTHF (Table 3, entry 9), which will be further converted into alkanes upon longer reaction time. Indeed, reaction with 2,5-DMTHF shows an almost quantitative conversion to n-hexane, in agreement with our proposed HMF pathway (Table 3, entry 11). Presence of acidity in the aqueous phase is essential for the latter reaction. A similar reaction without TSA in the aqueous phase, results into a low 2,5-DMTHF conversion and showed additional formation of 2-hexanol (Table 3, entry 12). The acidcatalyzed ether bond hydrolysis of 2,5-DMTHF, likely rst to 2,5hexanediol, followed by a dehydration/hydrogenation to 2-hexanol, is thus an essential step en route to n-hexane (Table 3, entry 13 and Scheme 3). A suitable amount of acidity in the aqueous phase is thus crucial not only to hydrolyze cellulose to glucose and to dehydrate glucose to HMF, but also to achieve fast ring-opening hydrolysis of 2,5-DMTHF to 2,5-hexanediol. 2,5-Hexanediol is indeed very selectively converted to n-hexane, as demonstrated in entry 13 of Table 3.</p><p>2,5-Hexanedione was also occasionally analyzed in the cellulose experiments, especially at low contact time (see later). As this dione is reported to result from an acid-catalyzed ring opening hydrolysis/HDO of furans like MHMF (2-methyl-5hydroxymethylfuran) and DMF (2,5-dimethylfuran), 88,89 such reaction happens when ring hydrogenation is slower than hydrolysis. Interestingly, the occurrence of the reaction imposes no decrease of the n-hexane selectivity as 2,5-hexanedione is almost quantitatively converted into n-hexane in reaction conditions (Table 3, see entry 14). One may conclude at this point that 2,5-hexanediol, either resulting from 2,5-DMTHF or A similar ring opening hydrolysis, followed by dehydration/ hydrogenation, occurs with 2,5-DHMF, mainly forming 1hydroxy-2,5-hexanedione. 67,88,89 Accordingly, this intermediate is prone to convert ultimately to 1-and 2-hexanol through a family of diols such as 1,2-hexanediol (see Scheme 3) in our harsher reaction conditions. To better understand the reactivity and reaction pathways of the primary alcohol, a series of catalytic experiments was carried out with 1-and 2-hexanol and 1,2hexanediol. The data are reported in Table 3, entries 16 to 18.</p><p>Whereas 2-hexanol nearly quantitatively converts to n-hexane, 1,2-hexanediol and 1-hexanol yield remarkably lower n-hexane amounts (40% and 37%, respectively). Surprising amounts of npentane (53% and 49%) were obtained instead. The n-pentane is thus formed via C-C splitting of a primary alcohol under the reaction conditions, likely proceeding through a sequential dehydrogenation/decarbonylation reaction mechanism on the modied Ru/C. 60 This reaction should form CO as by-product, which was indeed analyzed (as methane) in gas phase analysis in equimolar amounts with n-pentane (Fig. S12-S13 in ESI †). Since formation of 1-hexanol entities entails a signicant loss of carbon yield in the liquid alkane fraction, ring opening of 2,5-DHMF should be delayed in favor of C-O hydrogenolysis and ring-hydrogenation. Besides n-pentane and n-hexane, the liquid alkane fraction also contains signicant amounts of methylcyclopentane (MCP). The presumable formation route proceeds through the acid-catalysed Piancatelli rearrangement from 2,5-DHMF or MHMF, 90 but this suggestion needs further conrmation. The reaction has been reported in the formation of cyclopentanone from furfural in presence of NiCu based catalysts under reducing conditions in water. 91 Optimizing the cellulose to n-hexane reaction Knowledge of the reaction network indicates that a minimum amount of redox catalyst htTSA(2)Ru/C is necessary to achieve efficient conversion of cellulose to n-hexane (Table 4, entry 1-3). Otherwise, char formation from HMF and water solubles like diols and triols will form, decreasing the content of n-decane solubles. Interestingly, reducing the original amount of catalyst twofold did not result in a signicant change in total product yield, indicating that the hydrogenation activity in the biphasic system is still sufficient. A fourfold reduction of htTSA(2)Ru/C causes a drop in total carbon yield in the organic phase.</p><p>In previous experiments the reaction mixture was analysed at a xed reaction time, showing in some occasions signicant amounts of intermediates like 1-hexanol and 2,5-DMTHF in the sampling mixture (Table 4, entry 4). As these molecules ultimately lead to n-hexane and n-pentane according to the results of Table 3, prolongation of the contact time is an obvious option to further increase the liquid alkane yields (see for instance an HPLC analysis of aqueous phase at various reaction times, Fig. S9 †). Fig. 3 plots the product distribution and total cellulose conversion in function of time (with an indication of the reaction temperature at each time). The plotted data indeed conrm the increase in liquid alkane yield from cellulose with time. During the reactor heating stage between 423 and 453 K, the conversion of cellulose proceeds rapidly, reaching 40% and 90% at 30 and 60 minutes, respectively. During this interval, 2,5-DMTHF, glucose, some hexitols and their anhydrides and other water-solubles including HMF, 2,5-hexanedione and 1,2-hexanediol were formed (Fig. S9 †). The accumulation of glucose and low hexitol (sorbitol, sorbitan and isosorbide) yield are indicative of the reduced glucose hydrogenation activity of the TSA-modied Ru/C, in agreement with the data presented in Fig. 1. The inability of this catalyst to rapidly hydrogenate glucose opens up a fast cascade route, involving glucose to HMF Scheme 3 Proposed reaction pathways from cellulose to n-hexane and n-pentane through HMF with TSA and htTSA(2)Ru/C, partially based on Liu et al., 88 Alamillo et al., 67 Li et al. 60 and Yang et al. 92 Intermediates tested in this study are indicated in blue. The most selective reaction pathway from cellulose to n-hexane is indicated with bold arrows. HDO, hydrodeoxygenation; HG, hydrogenation; DH/ DC, dehydrogenation/decarbonylation. conversion, which is mainly hydrogenated to 2,5-DMTHF and some DMF was analyzed as well. The water phase contains a family of alcohols, mainly the most stable primary alcohols like 1-hexanol and 1,2-hexanediol, and also the secondary alcohols like 2-hexanol. Aer 1.5 h contact time, when the reactor temperature is in the range of 453-473 K, n-hexane is formed in expense of the reactive secondary alcohols through dehydration/hydrogenation cycles, 60 while the primary alcohols remain largely untouched. 2,5-DMTHF and 1-hexanol are abundantly present, while also MCP is mainly formed in this period. Conversion of 2,5-DMTHF and the alcohols continues with longer reaction times (and increasing reaction temperature up to 493 K) until they are almost completely converted. At this temperature, n-pentane is formed, while 1-hexanol is completely converted. Thus, aer a short reaction time of about 6 hours, high n-decane soluble product yields (about 82% based on carbon) and C 5 -C 6 alkane yields (up to 75%, including 52% n-hexane) were obtained. In these conditions, the catalytic turnover based on surface Ru atoms can be estimated at about 200, assuming the consumption of 7H 2 molecules for n-hexane production per glucose unit (and thus 7-metal catalyzed turnovers). The deciency in the mass balance is due to some insoluble products (gas and solid, about 7% total estimated yield), while 11% carbon is present in the water phase as hexitols and some oligomeric products. Note that the hexitols were already formed very early in the reaction (aer 30 min), but largely survived the reaction conditions, again proving the importance of the novel HMF route and differentiating the current biphasic system with the known pathways via sorbitol.</p><!><p>The direct conversion of sowood sawdust (from a local sawmill) to n-hexane was investigated in the aforementioned optimal reaction condition to assess the robustness of the catalytic system. Sowood was deliberately chosen here due to its high polyhexose content 93 (here: 58%). Apart from cutting, no other pretreatment of the wood sample was foreseen as to omit biomass pretreatment costs and energy. Irrespective of that, an appreciable C 5 -C 6 alkane yield of almost 60%, including approximately 40% n-hexane, was attained at full conversion of the polysaccharide component.</p><p>Besides the use of real lignocellulosic feedstock, multiple catalyst reuse is of vital importance to a heterogeneous process and thus two types of reuse strategies were put forward to test the resilience and durability of the TSA-modied catalyst. At rst, the catalyst was recovered from the reaction medium by View Article Online ltration, washed and re-suspended in a fresh reaction medium aer drying. The results of two such consecutive recycling runs are summarized in Fig. 4. Some loss of catalytic activity was noticed, which could originate from catalyst loss during ltration. The possibility of reusing both the heterogeneous htTSA(2) Ru/C catalyst and the soluble TSA co-catalyst in two successive runs was also investigated, by adding fresh cellulose to the batch reactor aer each run and starting a new reaction, while accumulating the products. The results of this recycling are presented in Table 4 in entries 4 to 6, with yields based on the total amount of cellulosic carbon added. The catalytic system is acceptably reusable, not withstanding the harsh reaction conditions and high concentrations of products potentially inhibiting active metal sites. A small decrease in alkane and hexitol yield and an increase in oxygenate yield was monitored.</p><!><p>This contribution has demonstrated the feasibility of a one-pot conversion of cellulose to alkanes. In contrast to recently reported hydroprocessing processes, this biphasic liquid approach at moderate temperatures mainly produces straightchain alkanes with n-hexane and n-pentane as major components. The process allows an easy recuperation of alkanes, oating on top of a separate water phase, while hydrogen selectivity is high as almost no gaseous products are formed. A thorough reaction network study showed the dominant pathway, which deviates from the currently accepted sorbitol-toalkane route. 36,56,60 Instead, the major pathway proceeded via hydrolysis of cellulose to glucose, followed by dehydration into HMF. The latter needs to be hydrogenated quickly and leads to 2,5-DHMF and subsequently, via ring hydrogenation/hydrogenolysis, into 2,5-DMTHF. This cyclic ether is selectively converted into n-hexane via consecutive ring-opening hydrolysis and dehydration/hydrogenation cycles. Contribution of C]O hydrogenation/hydrogenolysis of HMF to the methyl-furans, DMF and MHMF, followed by furan ring opening constitutes a productive parallel pathway to n-hexane through 2,5-hexanedione. The ring-opening of 2,5-DHMF on the other hand leads to the formation of linear primary alcohols such as 1hexanol, and this path leads to a mixture of n-hexane, n-pentane and methane. The fast hydrogenation of 2,5DHMF to 2,5-DMTHF or hydrogenolysis to DMF or MHMF is thus an essential step in the cellulose-to-n-hexane reaction. MCP is proposed to be the result of a rearrangement reaction of 2,5-DHMF, but requires further conrmation. The critical elements of the presented catalytic system are: (i) the use of a biphasic reaction solvent system -with redox activity in the organic phase and acidity in the aqueous phase -to partition reactive intermediates and to provide the best conditions for the different reactions to occur; (ii) controlled reactor heating to gradually release glucose and to form HMF in the right temperature zone to avoid their degradation; and (iii) modication of the hydrogenation selectivity of commercial Ru/ C to steer the reaction from glucose to HMF hydrogenation to avoid sorbitol formation.</p><p>The catalytic system proved appreciably reusable and was applicable on raw sowood sawdust (almost 40% n-hexane yield). Future improvement in n-hexane yield is envisioned through a more selective formation of 2,5-DMTHF (or HMMF and DMF) to circumvent the n-pentane production. Identication of the modifying role of TSA on Ru/C, optimization of the stability of the catalytic biphasic system and decreasing the carbon content in the water phase are several focus points for future research.</p>

Royal Society of Chemistry (RSC)

Synthesis and Cellular Studies of Polyamine Conjugates of a Mercaptomethyl-carboranylporphyrin1

Seven polyamine conjugates of a tri(p-carboranylmethylthio)tetrafluorophenylporphyrin were prepared in high yields by sequential substitution of the p-phenyl fluoride of tetrakis(pentafluorophenyl)porphyrin (TPPF), and investigated as boron delivery agents for boron neutron capture therapy (BNCT). The polyamines used were derivatives of the natural-occurring spermine with different lengths of the carbon chains, terminal primary amine groups and, in two of the conjugates, additional aminoethyl moieties. A tri(polyethylene glycol) conjugate was also synthesized for comparison purposes. The polyamine conjugates showed low dark cytotoxicity (IC50 > 400 \xce\xbcM) and low phototoxicity (IC50 > 40 \xce\xbcM at 1.5 J/cm2). All polyamine conjugates, with one exception, showed higher uptake into human glioma T98G cells (up to 12-fold) than the PEG conjugate, and localized preferentially in the cell ER, Golgi and the lysosomes. Our results show that spermine derivatives can serve as effective carriers of boronated porphyrins for the BNCT of tumors.

synthesis_and_cellular_studies_of_polyamine_conjugates_of_a_mercaptomethyl-carboranylporphyrin1

Introduction<!>1. Chemistry<!>5,10,15-Tri(p-carboranylmethylthiotetrafluorophenyl)-20-pentafluorophenylporphyrin (1)<!>Conjugate 2<!>Conjugate 3<!>Conjugate 4<!>Conjugate 5<!>Conjugate 6<!>Conjugate 7<!>Conjugate 8<!>Conjugate 9<!>2. Octanol-water partition coefficients<!>3. Cell Studies<!>3.1. Dark Cytotoxicity<!>3.2. Phototoxicity<!>3.3. Time-Dependent Cellular Uptake<!>3.4. Microscopy<!>1. Synthesis and characterization<!>Cytotoxicity<!>Time-dependent uptake<!>Subcellular Localization<!>Conclusions

<p>Boron-containing porphyrins are promising boron delivery agents for the boron neutron capture therapy (BNCT) of tumors, due to their ability for selective delivery of high amount of boron (> 20 μg/g) to target tissues with low dark toxicity, and their long retention times in tumors.1,2 BNCT is a binary cancer treatment that involves the irradiation of 10B-containing tumor cells with low energy thermal or epithermal neutrons to produce high linear energy transfer (high-LET) alpha and lithium-7 particles, and releasing 2.4 MeV of kinetic energy, according to the equation below:3–5</p><p>The 10B nucleus has a much higher neutron capture cross section (3838 barns, 1 barn = 10−24 cm2), than 12C (0.0034 barn), 1H (0.33 barn) and 14N (1.8 barns), which make up approximately 96% of tissues, thus minimizing damage to normal cells. Indeed, BNCT has the potential to be highly selective, able to destroy 10B-containing malignant cells in the presence of normal boron-free cells, due to the limited path range (5–9 μm) of the high-LET cytotoxic particles in tissues. The only two boron compounds currently in BNCT clinical trials in Japan, Finland and Sweden are BSH (sodium mercaptoundecahydro-closo-dodecaborate) and BPA (L-4-dihydroxy-borylphenylalanine), used either alone or in combination; although improved patient survival has been reported using these drugs,6–9 high doses of these agents, particularly BPA, are typically required to achieve a therapeutic response, and both BSH and BPA have only low selectivity for tumor cells and low retention times in tumors. On the other hand, boronated porphyrins have shown increase tumor selectivity and retention times compared with BSH and BPA. Furthermore, boron-containing porphyrins retain the fluorescence and photosensitizing properties characteristic of this type of macrocycle, allowing the use of photodynamic therapy (PDT) as adjuvant treatment for BNCT, and facilitating the detection of tissue-localized boron and treatment planning.2,10 PDT involves the activation of a tumor-localized photosensitizer with red light, producing reactive oxygen species, such as 1O2, that cause irreversible photodamage to malignant tissues.11,12 Two porphyrin derivatives, Photofrin and Visudyne, are FDA-approved for the PDT treatment of cancer of the lung, digestive tract, genitourinary tract, melanoma, Barrett's esophagus and, in the latter case, the wet form of age-related macular degeneration.13,14 On the other hand, BNCT has been investigated for tumors that are difficult to treat by PDT, such as glioblastoma multiforme, due in part to limited light penetration and/or insufficient tissue oxygenation. Due to the large amount of 10B in tumor (20–35 μg/g, depending on the boron microdistribution) required for effective BNCT, recent research has centered on the synthesis of boron-containing porphyrins of high boron content, containing stable boron clusters and a tumor-targeting moiety, such as a peptide.15,16 Among the boron clusters, the negatively charged closo-B12H122− anion, the carborane derivatives closo-CB11H12− and nido-C2B9H11−, and metallo-bis(dicarbollides) such as [3,3′-Co(1,2-C2B9H11)2] −, have been the most common clusters used for attachment to porphyrins because of their high boron content, known chemistry, amphiphilic properties, and their high photochemical, kinetic and hydrolytic stabilities. As a result, most of the boron-containing porphyrin derivatives reported to date are negatively charged,2 although positively charged boronated agents potentially target the most sensitive tumor cell organelles, e.g. mitochondria and nuclei, and important biomolecules such as DNA and RNA.17 Furthermore, the amount of 10B required for effective BNCT can be substantially reduced if it localizes near or within the cell nucleus.18 Herein we report the synthesis and investigation of a series of amphiphilic closo-carboranylmethylthioporphyrins containing fluoride and conjugated to various polyamines, which are protonated under physiologic conditions. Fluorinated porphyrins have been previously shown to have increased photodynamic activities compared with their non-fluorinated analogs,19,20 and could facilitate imaging (using 19F-NMR and/or 18F-PET in addition to porphyrin fluorescence) and treatment outcome.21 On the other hand, polyamines are known to be essential for cell growth and differentiation, and are found in high concentrations in rapidly proliferating tumor cells, due to an up-regulated polyamine transport system.22–24 As a consequence, polyamine conjugation to porphyrins1 and other drugs29–34 is an attractive strategy for increasing tumor selectivity, uptake and overall biological efficacy. Several boron-containing polyamines have been synthesized and shown to bind to DNA;35–41 although significant toxicity was observed for some of these polycationic molecules, new low cytotoxicity derivatives were synthesized but no additional biological investigations are yet reported. Therefore, conjugation of polyamines to a boron-containing porphyrin could potentially increase boron uptake into tumor cells, favor DNA binding and overall BNCT efficacy. In this paper seven polyamines were selected for conjugation to a pre-formed carboranylporphyrin, containing between 3 and 5 amine groups and 2, 3 or 4 carbon spacers between the nitrogens. The uptake and toxicity of the polyamine-porphyrin conjugates were evaluated in human glioma T98G cells and compared with those of a tripegylated analog.</p><!><p>Reactions that are sensitive were conducted under argon atmosphere in oven-dried glassware. Commercially available reagents and solvents (HPLC grade) were purchased from Sigma-Aldrich, Acros Organics and used without further purification. p-carborane was purchased from Katchem, Inc. Anhydrous methanol was prepared by distillation from magnesium turnings and was stored under nitrogen over 3 A0 molecular sieves. Anhydrous THF was prepared by distillation from sodium and benzophenone. Analytical thin-layer-chromatography (TLC) was performed on polyester backed TLC plates 254 (pre-coated, 200 μm, Sorbent Technologies). Silica gel 60 (70–230 mesh, Merk) used for column chromatography and silica gel TLC plates (0.2 mm thickness) were purchased from Sorbent Technologies. 1H NMR and 13C NMR spectra were obtained using a Bruker AV-4 400 MHz spectrometer; chemical shifts are expressed in ppm. 19F NMR spectra were obtained using a Bruker DPX-250 250 MHz spectrometer; chemical shifts are expressed in ppm. Electronic absorption spectra were measured on a Perkin Elmer Lambda 35 UV-vis spectrophotometer. Mass analysis was conducted at the LSU Mass Spectrometry Facility on a Bruker Omniflex MALDI-TOF mass spectrometer and exact masses were obtained from HRMS-ESI on an Applied Biosystems QSTAR XL. Melting points were measured on a Thomas hoover melting point apparatus. Reversed-phase HPLC was performed on a Waters system including a 2545 quaternary gradient module pump with a 2489 UV-vis detector and a fraction collector III. An analytical column (4.6X250 mm-XBridgeTM BED300 C18 5 μm) was used for the purification of all polyamine conjugates (2–8) and a stepwise gradient from 0 to 100% Buffer B in the first 10 min to 50% B and 50% C in next 10 min to 100% B in next 10 min. A stepwise gradient 10–90% Buffer B with Buffer A was used for the PEG conjugate 9. Buffer A (0.1% TFA, H2O), Buffer B (0.1% TFA, acetonitrile), Buffer C (0.1% TFA, acetone). All Boc-protected polyamines were synthesized as previously described.42 1-Mercaptomethyl-p-carborane was prepared from p-carborane, as described in literature.45</p><!><p>To a solution of TPPF (19.52 mg, 0.02 mmol) in 2 ml of dry DMF was added anhydrous K2CO3 (16.6 mg, 0.12 mmol), mercaptomethyl-p-carborane (15.2 mg, 0.08 mmol). The reaction mixture was stirred at room temperature for 48 h. The resulting solution was diluted with ethyl acetate (50 mL) and washed with brine (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, the solvents evaporated under reduced pressure and the resulting residue was dissolved in dichloromethane (10 mL). A saturated solution of Zn(OAc)2 in methanol (10 mL) was added and the resulting mixture stirred at room temperature for 24 h. The solvents were evaporated under reduced pressure and the resulting residue was purified by prep-TLC using chloroform/petroleum ether 1:1 to give 23.3 mg (30%) of desired zinc(II) porphyrin, mp > 3000C. UV-Vis (DMSO) λmax (ε/M−1 cm−1) 426 (444 400), 555 (43 800). 1H-NMR (CDCl3, 400 MHz): δ 8.93 (s, 8H, β-H), 3.45 (s, 6H, -SCH2), 2.19–3.33 (m, 33H, BH, CH). MS (MALDI-TOF) m/z 1550.789 [M], calcd for ZnC53H47F17N4B30S3 = 1550.494. The Zn(II) porphyrin (23.3 mg, 0.015 mmol) was dissolved in 2 mL of TFA/chloroform 1:1 and stirred at room temperature overnight. After removal of the solvents under vacuum, the title porphyrin was obtained in quantitative yield. mp > 3000C. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (468 900), 511 (45 900), 555 (18 700), 585 (11 200), 650 (8 300). 1H-NMR (CDCl3, 400 MHz): δ 9.02 (s, 2H, β-H), 8.90 (s, 6H, β-H), 3.46 (s, 6H, -SCH2), 1.78–2.96 (m, 33H, BH & CH), -2.87 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 147.82, 145.34, 143.62, 141.07, 138.89, 136.35, 120.85, 115.54, 104.64, 81.88, 59.23, 14.13. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.7 (m, 6F), −139.8 (m, 6F), −140.4 (d, J = 15.7 Hz, 2F), −156.2 (t, J = 19.0 Hz, 1F), −165.0 (d, J = 16.1 Hz, 2F). MS (MALDI-TOF) m/z 1485.672 [M], calcd for C53H49F17N4B30S3 = 1485.565.</p><!><p>To porphyrin 1 (14.9 mg, 0.01 mmol) were added (N1,N3,N6–tri-tert-butoxycarbonyl)-1,8-di-amino-3,6-diazaoctane42 (6.7 mg, 0.015 mmol) and NMP (2 mL)43,44 and the mixture was heated at 100 °C for 4 h. After cooling to room temperature, the solution was diluted with ethyl acetate (50 mL) and washed with brine (5 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, the solvents removed under vacuum and the resulting reddish brown residue purified by silica gel column chromatography using dichloromethane for elution, followed by dichloromethane/ethyl acetate 9:1. The Boc-protected conjugate was obtained (18.6 mg) in 96% yield, mp = 292–294 °C. UV-Vis (DMSO) λmax (ε/M−1 cm−1) 416 (401 700), 510 (28 400), 555 (22 000), 585 (11 000), 650 (10 500). 1H-NMR (CDCl3, 400 MHz): δ 9.06 (s, 2H, β-H), 8.91 (s, 6H, β-H), 3.33–3.53 (m, 16H, SCH2, NCH2), 2.72–2.79 (m, 2H, CH2), 1.89–2.71 (m, 33H, BH and CH), 1.58 (s, 9H, tBu), 1.52 (s, 9H, tBu), 1.46 (s, 9H, tBu), −2.86 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 155.92, 155.61, 147.54, 146.84, 143.23, 142.87, 137.89, 136.15, 119.85, 115.44, 103.84, 81.58, 79.78, 79.67, 78.54, 59.43 52.67, 49.96, 47.19, 46.35, 45.43, 40.34, 38.98, 30.78, 28.14, 14.23. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.8 (m, 6F), −139.9 (m, 6F), −144.2 (d, J = 14.8 Hz, 2F), −162.3 (d, J = 15.6 Hz, 2F). The Boc-protected conjugate was dissolved in TFA/dichloromethane 1:1 (2 mL) and stirred at room temperature for 6 h. After removal of the solvent under vacuum, the resulting residue was purified by HPLC to give conjugate 2 (14.7 mg) in 95% yield; mp > 3000C. HPLC tR = 27.01 min. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (591 400), 510 (52 600), 555 (21 900), 585 (11 400), 650 (10 000). 1H-NMR (CDCl3, 400 MHz): δ 8.75–9.75 (br s, 8H, β-H), 3.33–3.69 (m, 16H, SCH2 & CH2), 2.35–3.11 (m, 35H, CH2, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.6 (m, 6F), −139.8 (m, 6F), −144.2 (d, J = 15.4 Hz, 2F), −162.4 (d, J = 15.9 Hz, 2F). HRMS (ESI-TOF) m/z found 1612.7414 [M+H+], 806.3720 [M+2H+]2+, calcd for C59H64F16N8B30S3 = 1612.7008, [C59H64F16N8B30S3]2+ = 806.3504.</p><!><p>A similar procedure was used to that described above for conjugate 2, using porphyrin 1 (14.9 mg, 0.01 mmol), (N1,N3,N6,N9-tetra-tert-butoxycarbonyl)-1,11-di-amino-3,6,9-triazaundecane42 (8.85 mg, 0.015 mmol) and NMP (2 mL).43,44 The Boc-protected conjugate was obtained (20 mg) in 96% yield, mp = 292–295 °C. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (592 500), 510 (21 200), 555 (18 000), 585 (9 400), 650 (8 600); 1H-NMR (CDCl3, 400 MHz): δ 9.05 (s, 2H, β-H), 8.90 (s, 6H, β-H), 3.25–3.45 (m, 20H, SCH2, NCH2), 1.75–2.98 (m, 33H, CH2, BH, CH), 1.57 (s, 9H, tBu), 1.50 (s, 18H, tBu), 1.43 (s, 9H, tBu), −2.86 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 156.92, 156.16, 155.39, 147.64, 145.60, 143.30, 141.16, 138.06, 135.68, 120.86, 115.30, 103.94, 81.89, 80.69, 79.14, 77.24, 60.41, 59.22, 50.21, 47.45, 47.36, 45.42, 40.62, 39.47, 29.71, 28.43, 28.21, 24.83, 21.06, 14.21. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.9 (m, 6F), −139.8 (m, 6F), −144.3 (d, J = 15.3 Hz, 2F), −162.5 (d, J = 16.0 Hz, 2F). The Boc protected conjugate was deprotected using TFA in dichloromethane, as described above, and conjugate 3 was obtained (15.1 mg) in 95% yield after HPLC purification; mp > 300 °C; HPLC tR = 26.68 min. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (596 900), 510 (54 500), 555 (24 400), 585 (12 500), 650 (10 900). 1H-NMR (CDCl3, 400 MHz): δ 8.88–9.60 (br s, 8H, β-H), 3.33–3.72 (m, 20H, SCH2, CH2), 2.22–3.35 (m, 35H, CH2, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.9 (m, 6F), −139.9 (m, 6F), −144.4 (d, J = 15.1 Hz, 2F), −162.3 (d, J = 16.4 Hz, 2F). HRMS (ESI-TOF) m/z found 1656.8000 [M+H+], 828.4016 [M+2H]2+, calcd for [C61H68F16N9B30S3] = 1656.7418, [C61H68F16N9B30S3]2+ = 828.3709.</p><!><p>A similar procedure was used to that described above for conjugate 2, using porphyrin 1 (14.9 mg, 0.01 mmol), (N1,N3,N6,N9,N12-penta-tert-butoxycarbonyl)-1,14-di-amino-3,6,9,12-tetraazatetradecane42 (11.0 mg, 0.015 mmol) and NMP (2 mL).43,44 The Boc-protected conjugate was obtained (21.4 mg) in 96% yield, mp = 293–2960C; UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (456 000), 510 (24 000), 555 (21 000), 585 (12 500), 650 (10 300). 1H-NMR (CDCl3, 400 MHz): δ 9.06 (s, 2H, β-H), 8.91 (s, 6H, β-H), 3.22–3.56 (m, 24H, SCH2, NCH2), 2.72–2.79 (m, 2H, CH2), 1.8–3.15 (m, 35H, CH2, BH, CH), 1.58 (s, 9H, tBu), 1.48–1.51 (m, 27H, tBu), 1.43 (s, 9H, tBu), −2.86 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 156.99, 156.14, 155.33, 147.64, 145.49, 145.16, 143.53, 138.09, 137.90, 121.06, 120.88, 115.31, 103.94, 81.90, 80.95, 80.63, 80.23, 80.04, 79.12, 60.41, 59.23, 47.39, 45.43, 39.45, 38.76, 37.65, 28.91, 21.06, 14.21. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.8 (m, 6F), −139.6 (m, 6F), −144.0 (d, J = 15.4 Hz, 2F), −162.2 (d, J = 16.1 Hz, 2F). The Boc protected conjugate was deprotected using TFA in dichloromethane, as described above, and conjugate 4 was obtained (15.5 mg) in 95% yield after HPLC purification, mp > 3000C; HPLC tR = 26.14 min. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (552 300), 510 (45 900), 555 (18 400), 585 (14 100), 650 (5 700). 1H-NMR (CDCl3, 400 MHz): δ 8.78–9.61 (br s, 8H, β-H), 3.25–3.87 (m, 24H, SCH2, CH2), 2.09–3.18 (m, 35H, CH2, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.4 (m, 6F), −139.7 (m, 6F), −144.3 (d, J = 15.8 Hz, 2F), −162.5 (d, J = 16.3 Hz, 2F). HRMS (ESI-TOF) m/z found 1698.8939 [M+H+], 849.9461 [M+2H+]2+, 566.9658 [M+3H+]3+; calcd for C63H72F16N10B30S3 = 1698.7639, [C63H72F16N10B30S3]2+ = 849.3819, [C63H72F16N10B30S3]3+ = 566.2546.</p><!><p>A similar procedure was used to that described above for conjugate 2, using porphyrin 1 (14.9 mg, 0.01 mmol), (N1,N3,N7-tri-tert-butoxycarbonyl)-1,9-di-amino-3,7-diazanonane42 (6.7 mg, 0.015 mmol) and NMP (2 mL).43,44 The Boc-protected conjugate was obtained (18.7 mg) in 96% yield, mp = 280–283 °C. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (512 300), 510 (35 900), 555 (16 700), 585 (12 100), 650 (9 000). 1H-NMR (CDCl3, 400 MHz): δ 9.06 (s, 2H, β-H), 8.91 (s, 6H, β-H), 3.47 (s, 6H, SCH2), 3.3–3.45 (m, 10H, NCH2), 1.73–3.09 (m, 37H, CH2, BH, CH), 1.58 (s, 9H, tBu), 1.51 (s, 9H, tBu), 1.45 (s, 9H, tBu), −2.86 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 156.07, 147.63, 145.30, 143.01, 135.67, 130.73, 120.86, 115.29, 103.94, 81.88, 80.63, 80.12, 79.28, 60.41, 59.22, 47.08, 45.59, 40.62, 39.62, 28.06, 14.21. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.9 (m, 6F), −139.4 (m, 6F), −144.6 (d, J = 15.2 Hz, 2F), −162.6 (d, J = 15.8 Hz, 2F). The Boc protected conjugate was deprotected using TFA in dichloromethane, as described above, and conjugate 5 was obtained (14.8 mg) in 95% yield after HPLC purification; mp = 292–294 °C. HPLC tR = 26.80 min; UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (482 600), 510 (41 800), 555 (17 696), 585 (10 863), 650 (6 763). 1H-NMR (CDCl3, 400 MHz): δ 8.90–9.70 (br s, 8H, β-H), 3.60 (s, 6H, SCH2), 3.33–3.53 (m, 10H, CH2), 2.24–3.11 (m, 37H, CH2, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.4 (m, 6F), −139.6 (m, 6F), −144.8 (d, J = 15.4 Hz, 2F), −162.1 (d, J = 15.9 Hz, 2F). HRMS (ESI-TOF) m/z found 1626.7602 [M+H+], 813.8820 [M+2H+]2+, calcd for C60H66F16N8B30S3 = 1626.7262, [C60H66F16N8B30S3]2+ = 813.3631.</p><!><p>A similar procedure was used to that described above for conjugate 2, using porphyrin 1 (14.9 mg, 0.01 mmol), (N1,N4,N8-tri-tert-butoxycarbonyl)-1,11-di-amino-4,8-diazaundecane42 (7.34 mg, 0.015 mmol) and NMP (2 mL).43,44 The Boc-protected conjugate was obtained (19.0 mg) in 96% yield, mp = 270–2740C; UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (496 100), 510 (39 600), 555 (18 600), 585 (10 800), 650 (9 300). 1H-NMR (CDCl3, 400 MHz): δ 9.04 (s, 2H, β-H), 8.91 (s, 6H, β-H), 3.47 (s, 6H, SCH2), 3.14–3.33 (m, 10H, NCH2), 1.71–3.05 (m, 41H, CH2, BH, CH), 1.56 (s, 9H, tBu), 1.51 (s, 9H, tBu), 1.45 (s, 9H, tBu), −2.85 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 156.05, 147.63, 145.15, 142.43, 141.09, 135.65, 130.95, 121.05, 115.30, 103.94, 81.89, 80.05, 79.88, 60.41, 59.22, 44.99, 40.62, 38.78, 36.54, 31.89, 31.76, 30.78, 21.06, 14.21. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.3 (m, 6F), −139.7 (m, 6F), −144.5 (d, J = 15.4 Hz, 2F), −162.3 (d, J = 15.9 Hz, 2F). The Boc protected conjugate was deprotected using TFA in dichloromethane, as described above, and conjugate 6 was obtained (15.0 mg) in 95% yield after HPLC purification, mp = 284–287 °C. HPLC tR = 26.35 min. UV-vis (DMSO) λmax (ε/M−1 cm−1) 417 (453 300), 510 (37 500), 555 (14 600), 585 (11 000), 650 (9 700). 1H-NMR (CDCl3, 400 MHz): δ 8.81–9.82 (br s, 8H, β-H), 3.33–3.83 (m, 16H, SCH2, NCH2), 2.06–3.29 (m, 41H, CH2, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.6 (m, 6F), −139.4 (m, 6F), −144.4 (d, J = 15.6 Hz, 2F), −162.3 (d, J = 16.0 Hz, 2F). HRMS (ESI-TOF) m/z found 1654.7848 [M+H+], 827.8940 [M+2H+]2+, calcd for [C62H70F16N8B30S3] = 1654.7577, [C62H70F16N8B30S3]2+ = 827.3788.</p><!><p>A similar procedure was used to that described above for conjugate 2, using porphyrin 1 (14.9 mg, 0.01 mmol), (N1,N4,N9-tri-tert-butoxycarbonyl)-1,12-di-amino-4,9-diazadodecane42 (7.5 mg, 0.015 mmol) and NMP (2 mL).43,44 The Boc-protected conjugate was obtained (19.1 mg) in 96% yield, mp = 276–278 °C. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (490 800), 510 (39 600), 555 (18 600), 585 (11 900), 650 (9 900). 1H-NMR (CDCl3, 400 MHz): δ 9.04 (s, 2H, β-H), 8.91 (s, 6H, β-H), 3.47 (s, 6H, SCH2), 3.33–3.43 (m, 10H, CH2), 1.57–3.13 (m, 43H, CH2, BH, CH), 1.55 (s, 9H, tBu), 1.50 (s, 9H, tBu), 1.45 (s, 9H, tBu), −2.86 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 156.10, 147.77, 145.64, 143.30, 139.15, 137.01, 121.05, 115.30, 103.94, 81.89, 79.90, 77.28, 59.22, 49.42, 46.82, 40.62, 30.68, 29.72, 29.58, 17.66. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.4 (m, 6F), −139.4 (m, 6F), −144.5 (d, J = 15.3 Hz, 2F), −162.1 (d, J = 16.2 Hz, 2F). The Boc protected conjugate was deprotected using TFA in dichloromethane, as described above, and conjugate 7 was obtained (15.2 mg) in 95% yield after HPLC purification; mp = 284–287 °C. HPLC tR = 26.17 min. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (491 875), 510 (40 656), 555 (15 991), 585 (10 700), 650 (6 500). 1H-NMR (CDCl3, 400 MHz): δ 8.90–9.24 (br s, 8H, β-H), 3.61 (s, 6H, SCH2), 1.89–3.47 (m, 53H, NCH2, CH2, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.5 (m, 6F), −139.8 (m, 6F), −144.4 (d, J = 15.4 Hz, 2F), −162.2 (d, J = 15.9 Hz, 2F). HRMS (ESI-TOF) m/z found 1668.8031 [M+H+], 834.9022 [M+2H+]2+, calcd for [C63H72F16N8B30S3] = 1668.7734, [C63H72F16N8B30S3]2+ = 834.3867.</p><!><p>A similar procedure was used to that described above for conjugate 2, using porphyrin 1 (14.9 mg, 0.01 mmol), (N1,N4,N7-tri-tert-butoxycarbonyl)-1,10-di-amino-4,7-diazaoctane42 (7.1 mg, 0.015 mmol) and NMP (2 mL).43,44 The Boc-protected conjugate was obtained (18.8 mg) in 96% yield, mp = 289–292 °C. UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (480 800), 510 (41 500), 555 (18 900), 585 (11 600), 650 (8 900). 1H-NMR (CDCl3, 400 MHz): δ 9.03 (s, 2H, β-H), 8.90 (s, 6H, β-H), 3.33–3.68 (m, 14H, SCH2, NCH2), 1.6–3.1 (m, 41H, CH2, BH, CH), 1.56 (s, 9H, tBu), 1.52 (s, 9H, tBu), 1.45 (s, 9H, tBu), −2.86 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 156.10, 155.89, 147.63, 145.15, 143.65, 140.89, 137.78, 121.04, 115.29, 103.94, 81.87, 80.19, 79.87, 77.89, 60.40, 59.22, 45.76, 45.34, 44.95, 40.62, 36.78, 36.54, 31.56, 28.96, 28.65, 28.50, 21.05, 14.20. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.4 (m, 6F), −139.8 (m, 6F), −144.2 (d, J = 15.5 Hz, 2F), −162.3 (d, J = 16.1 Hz, 2F). The Boc protected conjugate was deprotected using TFA in dichloromethane, as described above, and conjugate 8 was obtained (15.0 mg) in 95% yield after HPLC purification; mp = 295–298 °C. HPLC tR = 26.77 min. UV-vis (DMSO) λmax (ε/M−1 cm−1) 417 (438 225), 510 (40 593), 555 (19 538), 585 (12 560), 650 (9 800). 1H-NMR (CDCl3, 400 MHz): δ 8.78–9.24 (br s, 8H, β-H), 3.60(s, 6H, SCH2), 3.33–3.53 (m, 8H, NCH2), 2.13–3.29 (m, 41H, CH2, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.6 (m, 6F), −139.6 (m, 6F), −144.4 (d, J = 15.6 Hz, 2F), −162.4 (d, J = 16.1 Hz, 2F). HRMS (ESI-TOF) m/z found 1640.7763 [M+H+], 820.8882 [M+2H+]2+, calcd for [C61H67F16N8B30S3] = 1640.7323, [C61H67F16N8B30S3] = 820.3661.</p><!><p>A similar procedure was used to that described above for conjugate 2, using porphyrin 1 (14.9 mg, 0.01 mmol), tert-butyl-12-amino-4,7,10-trioxadodecanoate (4.1 mg, 0.015 mmol) and NMP (2 mL). 43,44 The Boc-protected conjugate was obtained (16.8 mg) in 95% yield, mp = 289–292 °C; UV-vis (DMSO) λmax (ε/M−1 cm−1) 416 (460 800), 510 (38 450), 555 (16 700), 585 (10 200), 650 (8 100). 1H-NMR (CDCl3, 400 MHz): δ 9.02 (s, 2H, β-H), 8.90 (s, 6H, β-H), 3.89 (s, 4H, CH2), 3.68–3.82 (m, 12H, CH2), 1.67–3.10 (m, 33H, BH, CH), 1.43 (s, 9H, tBu), −2.88 (s, 2H, NH). 13C-NMR (CDCl3, 100 MHz): δ 171.23, 149.78, 149.51, 145.42, 145.28, 143.77, 131.17, 125.50, 120.45, 120.07, 119.23, 118.86, 114.49, 101.13, 100.60, 80.16, 79.93, 53.45, 46.56, 35.85, 27.41. 19F-NMR (acetone-d6, 233.3 MHz): δ −135.4 (m, 6F), −139.6 (m, 6F), −144.8 (d, J = 15.5 Hz, 2F), −162.3 (d, J = 16.1 Hz, 2F). HRMS (MALDI-TOF) m/z found 1747.822 [M]+, calcd for C66H75F16N5B30S3 O5 [M]+ = 1747.746. The Boc protected conjugate was deprotected using TFA in dichloromethane, as described above, and conjugate 9 was obtained (15.4 mg) in 95% yield after HPLC purification; mp = 295–298 °C; HPLC tR = 51.21 min. UV-vis (DMSO) λmax (ε/M−1 cm−1) 417 (430 200), 510 (36 700), 555 (18 500), 585 (10 500), 650 (8 800). 1H-NMR (CDCl3, 400 MHz): δ 8.88–9.32 (br s, 8H, β-H), 3.92 (s, 4H, CH2), 3.68–3.82 (m, 18H, CH2, SCH2), 2.01–3.31 (m, 33H, BH, CH). 19F-NMR (acetone-d6, 233.3 MHz): δ −135.5 (m, 6F), −139.8 (m, 6F), −144.6 (d, J = 15.6 Hz, 2F), −162.4 (d, J = 16.2 Hz, 2F). HRMS (MALDI-TOF) m/z found 1691.758 [M]+, calcd for C62H67F16N5B30S3O5 [M]+ = 1691.684.</p><!><p>The partition coefficients (log P) were measured at room temperature by adding 0.3 mL of a porphyrin stock solution in DMSO (0.333 mM) to a 4 mL volumetric tube containing 2.0 mL of HEPES buffer (50 μM, pH 7.4), followed by addition of 2.0 mL of 1-octanol.46 After vortexing for 5 min, the phases were separated by centrifugation. An aliquot of 0.3 mL from each layer was diluted with 2 mL of methanol and the absorbance was read on a Perkin Elmer Lambda 35 UV-Vis spectrophotometer with 10 mm path length quartz cuvettes.</p><!><p>All tissue culture medium and reagents were purchased from Invitrogen (Carlsbad, CA). Human glioma T98G cells were purchased from ATCC and cultured in ATCC-formulated Eagle's Minimum Essential Medium containing 10% FBS and 1% antibiotic (Penicillin Streptomycin). The cells were split twice weekly to maintain a sub-confluent stock. All compound solutions were filter-sterilized using a 0.22 μm syringe filter.</p><!><p>10,000 T98G cells were plated per well in a Costar 96 well plate and allowed to grow 36 h. Porphyrin stock solutions (32 mM) were prepared in DMSO and then diluted into final working concentrations (25, 50, 100, 200, 400 μM). The cells were exposed to increasing concentrations of porphyrin up to 400 μM and incubated overnight. The loading medium was removed and the cells washed with 100 μL PBS. Then medium containing Cell Titer Blue (Promega) 120 μL was added as per manufacturer's instructions. After incubating for 4 h the cytotoxicity was then measured by reading the fluorescence at 520/584 nm using a BMG FLUOstar plate reader. The signal was normalized to 100% viable (untreated) cells and 0% viable (treated with 0.2% saponin from Sigma) cells.</p><!><p>The T98G cells were prepared as described above for the dark cytotoxicity assay and treated with porphyrin concentrations of 0, 6.25, 12.5, 25, 50, and 100 μM. After compound loading, the medium was removed and replaced with medium containing 50 mM HEPES pH 7.4. The cells were exposed to a NewPort light system with 175 W halogen lamp for 20 min, filtered through a water filter to provide approximated 1.5 J/cm2 light dose. The cells were kept cool by placing the culture on a 50C Echotherm chilling/heating plate (Torrey Pines Scientific, Inc.). The cells were returned to the incubator overnight and assayed for viability as described above for the dark cytotoxicity experiment and adding medium containing Cell Titer Blue to determine the toxicity of the compounds.</p><!><p>The T98G cells were prepared as described above for the dark cytotoxicity assay. The cells were exposed to 10 μM of each conjugate for 0, 1, 2, 4, 8, and 24 h. At the end of the incubation time the loading medium was removed and the cells were washed with 200 μL PBS. The cells were solubilized upon addition of 100 μL of 0.25% Triton X-100 (Calbiochem) in PBS. To determine the porphyrin concentration, fluorescence emission was read at 415/650 nm (excitation/emission) using a BMG FLUOstar plate reader. The cell numbers were quantified using the CyQuant cell proliferation assay (Invitrogen) as per the manufacturer's instructions, and the uptake was expressed in terms of nM compound per cell.</p><!><p>The HEp2 cells were incubated in a glass bottom 6-well plate (MatTek) and allowed to grow for 48 h. The cells were then exposed to 10 μM of each porphyrin conjugate for 6 h. Organelle tracers were obtained from In- vitrogen and used at the following concentrations: LysoSensor Green 50 nm, MitoTracker Green 250 nm, ER Tracker Blue/white 100 nm, and BODIPY FL C5 Ceramide 1 mm. The organelle tracers were diluted in medium and the cells were incubated concurrently with porphyrin conjugate and tracers for 30 min before washing 3 times with PBS and microscopy. Images were acquired using a Leica DM RXA2 upright microscope with 40 X NA 0.8dip objective lens and DAPI, GFP and Texas Red filter cubes (Chroma Technologies).</p><!><p>TPPF is a commercially available porphyrin that can easily be synthesized in multi-gram scale using a published procedure,47 and functionalized via nucleophilic substitution of the p-fluoro phenyl groups.48–50 The boron cluster used for attachment to TPPF was the 1,12-dicarba-closo-dodecaborane, also designated p-carborane, rather than the most common 1,2-dicarba-closo-dodecaborane or o-carborane analog, because of the lower reactivity and higher stability of p-carboranes toward base deboronation and degradation.10,51 The reaction of TPPF with 1-mercaptomethyl-p-carborane45 in the presence of K2CO3 in DMF at room temperature gave a mixture of p-carboranyl-containing porphyrins that were very difficult to separate. The highest yields of the tri-substituted porphyrin 1 were obtained when 4 equiv. of 1-mercaptomethyl-p-carborane were used relative to TPPF. To facilitate purification, Zn(II) was inserted by reaction with Zn(II) acetate in methanol, and after purification by preparative TLC the zinc metal was quantitatively removed using TFA in chloroform. Tri(mercaptomethyl-p-carboranyltetrafluorophenyl)pentafluorophenylporphyrin 1 was obtained in 30% overall yield.</p><p>The p-fluoro phenyl group of porphyrin 1 underwent nucleophilic substitution with the primary amino group of Boc-protected polyamines42 and commercially available tert-butyl-12-amino-4,7,10-trioxadodecanoate,43,44 as shown in Scheme 1. Deprotection of the Boc and tert-butyl protecting groups using TFA in dichloromethane, gave conjugates 2–9 in 91% overall yields, after reversed-phase HPLC purification. This methodology affords the targeted polyamine-porphyrin conjugates in higher yields than those normally obtained via solid-phase coupling and other methodologies used to conjugate polyamines to porphyrins.25–27 The polyamines chosen for conjugation to porphyrin 1 contain between 2 and 4 secondary amine groups and terminal primary amine groups, with various lengths of the intermediate carbon chains. Conjugate 7 contains a spermine group, which is a naturally-occurring polyamine with a 3-4-3 carbon backbone; all other polyamines are derivatives of spermine with different lengths of the carbon chains, of the 2-2-2 (2), 2-3-2 (5), 3-2-3 (8) or 3-3-3 (6) types, and conjugates 3 and 4 contain one or two additional aminoethyl moieties, respectively. It has been observed that slight changes in the chemical structure of polyamines can induce large changes in their biological efficacy, and that spermine-type compounds with a 3-3-3 or a 3-4-3 carbon skeleton are particularly effective anti-tumor agents.29 The PEG-conjugate 9 was prepared for comparison purposes, since a PEG group normally increases the solubility and cellular uptake of porphyrin macrocycles,52 and it is often used as a spacer in cell-targeted porphyrin-peptide and – antibody conjugates.</p><p>All porphyrin conjugates were structurally characterized by NMR, MS, and UV-Vis spectroscopy. The partition coefficient (log P) values between 1-octanol and HEPES buffer (pH 7.4) were obtained using the shaking-flak method46 and are shown in Table 1. The hydrophobic character for the conjugates follows the order 7 < 4 ~ 3 < 6 < 5 < 8 < 2 ~ 9, depending on the number of amine groups and the carbon skeleton of the polyamine. The most hydrophobic polyamine conjugate was found to be 2 with a log P value similar to that of the PEG conjugate 9, whereas 7 bearing a spermine group was the least hydrophobic of this series.</p><!><p>The concentration-dependent dark and phototoxicity of all conjugates 2 – 9 were investigated in T98G cells and the results are shown in Figures 1 and 2, respectively. All conjugates showed low cytotoxicity in the dark, with determined IC50 (50% inhibition of cell proliferation based on dose-response curves) > 250 μM; of all conjugates, the PEG-porphyrin 9 showed the highest dark toxicity with determined IC50 = 296 μM. Upon exposure to 1.5 J/cm2, the spermine derivatives 7, 8, 6 and 5 were found to be the most toxic with IC50 = 40, 41, 64 and 87 μM, respectively. Compound toxicity is a key limiting factor that can prevent potential new boronated drugs from becoming practically useful in a clinical setting because of the high boron concentration requirement in BNCT. Significant toxicities (IC50 < 25 μM in F98 rat glioma cells) were reported for o-carborane-containing derivatives of spermidine and spermine, and in particular for the terminally (rather than internally) N-substituted derivatives.35 In contrast all our conjugates showed very low cytotoxicity in human glioma T98G cells, maybe as a result from attachment of the carborane clusters to the porphyrin macrocycle rather than directly to the polyamine chain, and the use of closo-1,12- (para) rather than closo-1,2- (ortho) dicarbadodecaboranes; in the o-carborane clusters the boron atoms bound to both carbons are highly susceptible to nucleophilic attack by amine groups, producing the corresponding negatively charged nido-1,2-dicarbaundecaboranes, therefore changing the overall charge and lipophilicity of the conjugates.51</p><!><p>The time-dependent uptake of porphyrin conjugates was evaluated at a concentration of 10 μM over a time period of 24 h and the results obtained are shown in Figure 3. The extent of cellular uptake followed the order 7 > 6 > 3 ~ 4 > 5 > 8 > 2 ~ 9, generally increasing with the hydrophilicity of the conjugates. All polyamine conjugates with the exception of 2 showed better plasma membrane permeability and uptake into T98G cells compared with the PEG conjugate 9; the compound taken up the most by cells at all time points investigated was spermine conjugate 7, about 12 times more than 9. This result might be due to active transport via the polyamine transporter system, particularly for conjugates 7 and 6 containing a 3-4-3 and 3-3-3 carbon backbone, and for 3 and 4 due to their larger number of nitrogens; such derivatives have been found in structure-activity studies to accumulate in cells via the polyamine transport system,29,53 although recent studies found no effects in the cellular uptake of polyamine-substituted phthalocyanines upon addition of spermidine or α-difluoromethylornithine.54 Nevertheless, the protonation of polyamines under physiological conditions (the pKa of the amine groups are in the range 7–10)55 likely favors interactions with membrane-containing phosphate groups inducing higher cellular uptake compared with the PEG conjugate. The plasma membrane of tumor cells usually contains higher net negative charge compared with normal cells, due to the over-expression of polysialic acid residues.56 In the polyamine-porphyrin conjugates the cationic polyamine groups likely facilitate binding to the negatively charged tumor cell plasma membranes, while the hydrophobic nature of the fluorinated porphyrin and carborane moieties additionally favor penetration of the conjugate through the lipid membrane. Our results suggest that polyamines, in particular derivatives of spermine, can serve as carriers of boronated porphyrins for BNCT, enhancing boron transport across plasma membranes and delivery into tumor cells. Furthermore, a polyamine group might be more effective than a PEG as a linker, in the synthesis of porphyrin-peptide conjugates.16</p><!><p>Fluorescence microscopy was used to examine the intracellular localization of all conjugates in live cells. Human HEp2 rather than T98G cells were used in these studies because they adhere and spread nicely on glass cover slips, thus facilitating the imaging process. The organelle specific fluorescent probes ERTracker Blue/White (ER), MitoTracker Green (mitochondria), BODIPY-FL Ceramide (Golgi), and LysoSensor Green (lysosomes) were used in overlay experiments, as shown in Figures 4–10. Figure S9 of the Supporting Information shows the corresponding images for the PEG-conjugate 9. All conjugates were found to preferentially localize in the cell ER, as seen by the purple color in Figures 4d-10d. In addition, the polyamine conjugate 2 and the PEG conjugate 9 were also found to localize in mitochondria. We have previously observed that a porphyrin containing one PEG group localized subcellularly in the ER and mitochondria.52 Minor sites of localization for the polyamine conjugates were the Golgi and the cell lysosomes. Both negatively and positively charged carboranylporphyrins have been observed to localize mainly in the cell lysosomes, probably as a result of an endocytic mechanism of uptake.10,57 However, we believe that the presence of the polyamine and PEG groups favors localization of conjugates 2–9 in the ER, an important target in PDT58 and probably also in BNCT. Although the cell nuclei were apparently not targeted by the polyamine conjugates, the delivery of boron to their vicinity, localized in the ER, Golgi and lysosomes, might enhance the biological efficacy of these agents.</p><!><p>A series of fluorinated porphyrin-polyamine conjugates containing para-carborane clusters were synthesized in high yields via nucleophilic substitution of the p-phenyl fluorides of TPPF, and investigated as boron carriers for BNCT. para-Carborane clusters were used rather than the most common ortho-carboranes, due to their higher stability in the presence of nucleophilic amine groups. The hydrophobic character of the conjugates was investigated by determining the logarithm of their partition coefficient (log P) between 1-octanol and buffered water (pH = 7.4). The most hydrophilic spermine-porphyrin conjugate 7 (log P = 1.06) accumulated the most within human glioma T98G cells of all conjugates studied, about 12-fold more than a pegylated-porphyrin derivative. This might be due to the target of the polyamine transport system, in spite of the bulky carboranylporphyrin, and/or to favorable interactions between the cationic polyamine chain and the negatively charged plasma membranes, and the overall lipophilic character of the conjugates. All polyamine conjugates showed very low dark toxicities (IC50 > 400 μM, lower than the PEG conjugate with determined IC50 = 296 μM), a critical feature for potential boron delivery agents because of the very high boron concentration requirement in BNCT (20–35 μg/g). In addition all polyamine-porphyrin conjugates showed relatively low phototoxicity, the most phototoxic were the spermine derivatives 5, 6, 7 and 8 with determined IC50 = 87, 64, 40 and 41 μM, respectively, at 1.5 J/cm2, further indicating their usefulness as boron carriers for BNCT. In contrast to most currently known carboranylporphyrins, the main intracellular sites of localization for all conjugates were the ER; in addition, the polyamine conjugates were also observed in the Golgi and lysosomes. Among the polyamines, the spermine derivatives containing a 3-4-3 or 3-3-3 carbon skeleton are the most efficient at increasing cellular uptake and therefore are the most promising as boron delivery agent for BNCT.</p>

PubMed Author Manuscript

Hydronium Ions Accompanying Buried Acidic Residues Lead to High Apparent Dielectric Constants in the Interior of Proteins

Internal ionizable groups are known to play important roles in protein functions. A mystery that has attracted decades of extensive experimental and theoretical studies is the apparent dielectric constants experienced by buried ionizable groups, which are much higher than values expected for protein interiors. Many interpretations have been proposed, such as water penetration, conformational relaxation, local unfolding, protein intrinsic backbone fluctuations, etc. However, these interpretations conflict with many experimental observations. The virtual mixture of multiple states (VMMS) simulation method developed in our lab provides a direct approach for studying the equilibrium of multiple chemical states and can monitor pKa values along simulation trajectories. Through VMMS simulations of staphylococcal nuclease (SNase) variants with internal Asp or Glu residues, we discovered that cations were attracted to buried deprotonated acidic groups and the presence of the nearby cations were essential to reproduce experimentally measured pKa values. This finding, combined with structural analysis and validation simulations, suggests that the proton released from a deprotonation process stays near the deprotonated group inside proteins, possibly in the form of a hydronium ion. The existence of a proton near a buried charge has many implications in our understanding of protein functions.

hydronium_ions_accompanying_buried_acidic_residues_lead_to_high_apparent_dielectric_constants_in_the

INTRODUCTION<!>SNase Variants.<!>Equal-Molar VMMS Simulations.<!>VMMS Simulations Show Low Dielectric Constants inside Many SNase Variants.<!>Conformational Relaxation and Water Penetration Are Not Enough To Account for the High Apparent Dielectric Constants.<!>Discovery of Nearby Cations and Their Correlation with High Apparent Dielectric Constants.<!>Nearby Proton Hypothesis.<!>Nearby Proton Hypothesis is Supported by VMMS Simulations with Added Hydronium Ions.<!>Nearby Protons are Energetically Favored for Buried Acidic Groups.<!>With a Nearby Hydronium Ion, Buried Ionizable Sidechains Show Better Agreement with X-ray Structures.<!>CONCLUSIONS

<p>Buried ionizable groups are important for biomolecular functions, such as catalysis,1 redox reactions,2,3 proton transport,4,5 and proton-coupled-electron-transfer reactions.6 During biomolecular function cycles, ionizable groups that experience microenvironment changes may adopt different protonation states.</p><p>A major mystery that remains after decades of studies is the high apparent dielectric constants experienced by buried ionizable groups. pKa is a property that measures the tendency of deprotonation. When an ionizable group transfers from aqueous solution to the hydrophobic environment inside proteins, its pKa shifts. The pKa shift reflects the difference in dielectric behaviors between aqueous solution and the environment of the protein interior and can be converted to the apparent dielectric constant. The pKa values of many ionizable groups in proteins have been measured via NMR spectroscopy,7,8 from which it has been determined that the apparent dielectric constants around internal ionizable groups can be as high as 10–20, which is much higher than 2–4 expected inside proteins.9</p><p>Over the decades, to explain the high apparent dielectric constants, many experimental and theoretical studies have been performed. Among many labs studying buried ionizable groups, Garcia-Moreno's lab has systematically engineered a series of staphylococcal nuclease (SNase) variants with ionizable residue mutations at various positions.10–15 These variants provide systematic targets for studies on the properties of the internal environment of proteins.</p><p>Many interpretations for the high apparent dielectric constants around buried ionizable groups have been proposed. Water penetration is thought to be a major factor,16–21 even though some crystal structures do not show water molecules around ionizable groups and long-lived water penetration is not observed in certain variants.22 Another interpretation is conformational relaxation and local unfolding.13,17,18,22–25 However, many studies found no detectable conformational changes upon charging buried ionizable groups.10,26–29 A high apparent dielectric constant has also been suggested as being a property resulting from the intrinsic backbone fluctuations originating from its structural architecture.11 In addition, some studies show that the structural responses involving ionization of buried ionizable groups are in a time scale beyond microseconds,30,31 implying that the mystery of the high apparent dielectric constants is difficult to address with nanosecond-scale molecular dynamics simulations.</p><p>Many computational methods for structure-based calculation of pKa values have been developed to examine pKa's molecular determinants.9,32–40 The large energy barriers between different charge states when simulating with explicit solvent makes sequential sampling of all charge states highly inefficient. The virtual mixture of multiple states (VMMS) method41 was designed to directly simulate chemical state equilibrium and can sample different charge states efficiently in explicit solvent. VMMS simulations produce instantaneous pKa values along simulation trajectories and are convenient for deciphering molecular determinants behind pKa shifts. This method has been applied to calculate the pKa values of SNase variants with lysine mutations, and the results agree with experimental measurements reasonably well.42 Water penetration was found to be the major reason for the high apparent dielectric constants around buried lysine residues.</p><p>In this work, we performed VMMS simulations for SNase variants with buried acidic residues, Asp and Glu. Our simulation results show that water penetration and conformation relaxation are not enough to account for the high apparent dielectric constants. Surprisingly, we found that cations floated into the buried charges and brought pKa close to experiment values. This finding, combined with additional chemical, energetic, structural, and simulation analysis, led us to believe that nearby protons, possibly in the form of hydronium ions, are a main contributor to the high apparent dielectric constants around buried acidic residues.</p><!><p>The initial conformations of the 10 SNase variants, listed in Table I, were taken from PDB database when available. For the Asp mutation variants that did not have crystal structures, we built their initial conformations from the PDB structures of their corresponding Glu mutation variants. All variants contain a SNase protein, a thymidine-3′,5′-diphosphate ligand, and a calcium ion. The VMMS systems were constructed by dissolving the initial conformations into a box of TIP3P water together with several ions to produce a neutral system in a cubic box of 46.655 × 46.655 × 46.655 Å3. The number of water molecules and ions in the deprotonated states are listed in Table I. The protonated state has one sodium ion replaced with a water molecule. In the simulations with a hydronium ion, the deprotonated state was created from the protonated state by replacing a water near the acidic group with a hydronium ion.</p><p>The deprotonated state has a dummy hydrogen so that the SNase variants in both states have the same number of atoms. The dummy hydrogen atom is identical to a hydrogen atom except that it has no charge. The atomic charges of the ionizable residues can be found in ref 41.</p><p>The reference deprotonation free energies (ΔGHDref) of Asp and Glu, which are needed to calculate the pKa values of these residues in the SNase variants, were obtained with model compounds. The model compounds consist of an ionizable residue, Asp or Glu, with an acetyl group (ACE) at the N-terminal and a methyl amide group (NME) at the C-terminal: ACE-Asp-NME or ACE-Glu-NME. The reference systems were built by dissolving the model compounds into a cubic TIP3P43 water box, including several chloride and sodium ions. The box side was 31.1 Å.</p><!><p>An equal-molar VMMS system contains two subsystems, one for the protonated state and one for the deprotonated state. At every time step, interaction forces at each subsystem are calculated independently. The solvent forces at each state are used to drive the motion of the solvent atoms. The solute forces from both states are averaged (50% from each state) to produce combined forces that drive the motion of the solute atoms. Therefore, solvent atoms in different subsystems experience different forces and sample their conformations differently, whereas solute atoms in all subsystems experience the same combined forces and sample their conformational space the same way. At every specific interval (10 fs in simulations presented here), the energy changes of the solute in transition from the current state to another state were calculated to evaluate the quantities for free-energy calculation. More details can be found in our previous work.141,42</p><p>All simulations presented here were performed with a modified version 39 of CHARMM44,45 with the VMMS method implemented. The all-atom CHARMM36 force field46 was used for energy calculation. All simulations were performed in a constant volume and a constant temperature of 300 K using the self-guided Langevin dynamics via generalized Langevin equation method47 with a local averaging time tL = 0.2 ps, a guiding factor λ =1, and a friction constant ξ = 10/ps. A time step of 1 fs was used, and the SHAKE algorithm48 was employed to fix hydrogen connecting bond lengths.</p><!><p>Table I lists the 10 SNase variants simulated in this work. Their pKa values have been measured experimentally and can be compared with our simulation results. The pKa profiles of these variants during our VMMS simulations are shown in Figure 1 (black curves). Not surprisingly, we see large discrepancies between the calculated pKa values and the experimental results (dashed lines) for many variants. As can be seen in Figure 1, V66D, L25D, L38D, I92D, and I92E's pKa values fluctuate between 15 and 40, much higher than their experimental values ranging between 5 and 10. These large discrepancies indicate that the apparent dielectric constants in the simulations are significantly lower than those observed in experiments. In other words, the high apparent dielectric constants observed in experiment are not captured in these simulation systems.</p><!><p>The conformational changes and numbers of water molecules around the ionizable groups are shown in Figure 2. The root-mean-square deviations (rmsd) of the backbones from X-ray structures are all under 1.5 Å, indicating conformational changes are minimal, especially for the local structures around the ionizable groups. There are water penetrations in both the protonated and deprotonated states for all variants, and the deprotonated states have more water molecules than the protonated states. The large discrepancies in pKa values indicate that the water penetration and the conformational relaxation observed in these simulations cannot account for the high apparent dielectric constants. Although water penetration can increase the apparent dielectric constant, without significant local conformational change, the few water molecules that can be accommodated around the ionizable groups are not enough to increase the apparent dielectric constants to experimental values.</p><p>Force field inaccuracy could be a reason for the inability to reproduce the high apparent dielectric constants at buried ionizable groups. However, more accurate force fields, such as polarizable force fields, will not make too much difference inside proteins where the hydrophobic microenvironment provides little polarizability to increase the apparent dielectric constant.</p><p>Examining the simulation trajectories, we found that the large fluctuations in pKa values shown in Figure 1 are due to changes in the number of penetrated water molecules in both the protonated and deprotonated states. The standard deviations of the penetrated water molecule numbers are shown in Figure 2 as error bars.</p><!><p>Although large discrepancies in pKa values are seen for many variants, there are several cases where the pKa values from the simulations are close to the experimental values. As shown in Figure 1, the pKa value of V66E is around 20 at the beginning, but approaches the experimental value of 9.1 at 42 ns and remains there until 55 ns. For L25E, its pKa fluctuates between 15 and 25 until 33 ns, when its pKa drops below the experimental value of 7.5. I72D shows a pKa around 12 for about 3.2 ns before dropping to values below the experimental value of 7.6. I72E's pKa is very close to the experimental value of 7.3, but frequently drops to around 2. A drop in pKa values corresponds to an increase in the apparent dielectric constant. These simulation trajectories with pKa approaching experimental values provide us opportunities to examine atomic details behind the high apparent dielectric constants.</p><p>Examining the conformations where pKa values are low, we found one common phenomenon: there is always a sodium ion near the deprotonated acidic groups. Figure 3 shows the conformations of these variants with low pKa values where a sodium ion can be seen near the deprotonated acidic group. The correlation between presence of a nearby cation and the low pKa values indicates that a nearby sodium ion can bring down the pKa value. For V66E, L25E, and I72D, the lower pKa values resulting from the nearby sodium ions agree more with their experimental results. For I72E, on the other hand, the higher pKa values without a nearby sodium ion are more consistent with the experimental result. Examining the conformations reveals that the sidechain of E72 extends into the solvent and becomes unburied. These results tell us that a deprotonated acidic group is likely to have a nearby sodium ion when buried inside proteins, but not when unburied. This is understandable because for unburied charge groups, a nearby sodium ion can easily diffuse into bulk solvent. Therefore, the presence of a nearby sodium ion may be a reason for the high apparent dielectric constants. It is common to see that naturally buried ionizable residues interact with metal ions, such as in the crystal structures of ATPase (PDB code: 1SU4) and cytochrome C oxidase (PDB code: 5DJQ) where Ca2+ ions are buried and interact with charged Asp or Glu residues.</p><!><p>Because sodium ions are the only available cations in these simulation systems, sodium ions are found near deprotonated acidic groups do not mean that only sodium ions can lead to the high apparent dielectric constants. Cations other than sodium ions would have a similar effect.</p><p>Examining both the protonated and deprotonated states of these simulation systems, we find that sodium ions exist near the acidic groups only in the deprotonated state. This is understandable because a deprotonated group has charge–charge interactions with cations, whereas a protonated group is neutral and does not have charge–charge interactions. Even though metal ions are often found in protein structures, there is no metal ion near the buried acidic groups in the crystal structures of these proteins. A proton is a natural product of the deprotonation process and is possibly hydrated to form a hydronium (1)AH+H2O⇔A−+H3O+ Therefore, the nearby cation is mostly likely a proton, possibly in the form of a hydronium, instead of a metal ion. On the basis of this rationale, we propose a hypothesis that a deprotonated acidic group can be stabilized by a nearby proton, possibly in the form of a hydronium ion, when buried in an apolar environment.</p><p>Why we do not see protons near acidic groups in crystal structures of these SNase variants? First, most of the X-ray structures are obtained under conditions, in which the acidic groups are in a protonated state. A protonated acidic group is neutral and has no charge–charge interactions with protons, agreeing with what was seen in our VMMS simulations. Second, protons cannot be seen with X-rays. X-rays are scattered by electrons, and hydrogen is the smallest of atoms with just one electron, making it very difficult to see. Electron-bare protons are completely invisible. Third, hydronium ions could be mistaken as water molecules due to the invisibility of protons. Even though protons cannot be seen by X-ray crystallography, the short distance between a water oxygen atom and its hydrogen-bonding acceptor implies possible hydronium ions.50,51 An existing example of a buried ionizable residue interacting with water molecules is Glu-109 in chymosin, with one crystal water molecule and a serine hydroxyl group within hydrogen-bonding distance.51 The buried water molecule has three oxygen atoms within 2.9 Å, with the shortest of these contacts, 2.5–2.6 Å, being one of the carboxyl oxygens of the buried glutamate. The geometric features prompted the suggestion that this water molecule might be a hydronium ion (H3O+). Alternatively, neutrons are scattered by atomic nuclei and can be used to visualize hydrogen atoms and protons, which can distinguish hydronium ions from water molecules. Using neutron crystallography, hydronium ions have been successfully observed in some protein structures.52–56 Through quantum mechanics/molecular mechanics calculations, Ikeda et al. have shown that isolated hydronium ion can stably exist in the interior of proteins when hydrogen bonded with acidic residues, such as aspartic and glutamic acids.57</p><p>In the X-ray crystal structures of some SNase variants, we do see some crystal water molecules that are very close to the ionizable groups. For example, as shown in Figure 4, in the structure of Δ+PHS T62K/V66E at pH = 9 (PDB code: 5KYI), water A332 is 2.67 Å from E66 atom OE2, and in the structure of the same variant at pH = 7 (PDB code: 5KYL), water A305 is only 2.41 Å from E66 atom OE1. Because a typical hydrogen bond length is around 3 Å, a short distance around 2.5 Å would suggest the existence of a hydronium ion.</p><!><p>To verify this hypothesis, we performed VMMS simulations with a hydronium ion added near the deprotonated acidic groups. The pKa profiles of these VMMS simulations are also shown in Figure 1 with the red curves. Clearly, it can be seen that with the nearby hydronium ions, all pKa values from the VMMS simulations approach experimental values. Large fluctuations in the pKa values are due to water penetrations in both protonated and deprotonated states. These results clearly demonstrate that the nearby hydronium ion can account for the high apparent dielectric constants. Without the nearby hydronium ions or other cations, pKa values obtained from our VMMS simulations are significantly higher than the experimental results, as indicated by the black curves were also observed in Figure 1. Similar results in simulations with the AMBER force field58 and we believe the nearby proton hypothesis is a generalizable concept.</p><!><p>The proton produced from a deprotonation process can either stay near the deprotonated group or difluse into the bulk solvent. For the nearby proton hypothesis to stand, a proton needs to energetically favor the nearby placement. To calculate the interaction energies of hydronium ions in bulk solvent, we performed simulations of the model compounds, ACE–Asp–NME and ACE–Glu–NME, with a hydronium ion added. Figure 5 shows the distances between a hydronium ion and the ionizable groups of the model compounds during the VMMS simulations. As can be seen from Figure 5, the hydronium ions frequently move away from the deprotonated ionizable groups of the model compounds. These distance changes correspond to transitions between the complexing (nearby) state (r ~ 2.5 Å) and the noncomplexing state (r > 6 Å). The interaction energies of both states are calculated and are listed in Table I. The hydronium interaction energy in bulk water is the interaction energy of the noncomplexing state, which are −116.61 ± 0.14 and −116.50 ± 0.15 kcal/mol, for the Asp and Glu model compound systems, respectively.</p><p>In the simulations of the variants, the hydronium ions stay near the deprotonated ionizable groups throughout the 100 ns simulations for all 10 variants. As shown in Table I, the hydronium interactions in all variants are stronger than interactions in bulk water. With the exceptions of V66D, V66E, and I92D, the hydronium interactions are even stronger than those of the complexing state of the model compounds. These strong interactions force the hydronium ions to stay near the buried acidic groups.</p><p>The nearby proton hypothesis does not apply to nonburied acidic groups. For ionizable residues on protein surfaces, the proton released from deprotonation would diffuse into bulk solvent, as that occurs in the model compound simulations shown in Figure 5. The model compounds, ACE-Asp-NME and ACE-Glu-NME, resemble residues on a protein surface. Therefore, there is not much difference if a hydronium ion is included for a surface ionizable group. As can be seen from Figure 5, the hydronium ions frequently move away from the deprotonated ionizable groups of the model compounds and under physiologic conditions (pH ~ 7) where the proton concentration is low, the hydronium ions will be mainly in the noncomplexing state.</p><p>The movement of cations toward the buried charge groups in the VMMS simulations of V66E, L25E, I72D, and I72E implies that the nearby placements of cations are low free-energy states. For the other variants, the simulations may be too short to see the move-in events. These move-in processes themselves indicate that cations prefer to stay near the buried charges. The interaction energies, shown in Table I, provide a quantitative support of the nearby proton hypothesis.</p><p>A nearby hydronium ion not only has stronger interactions with its surroundings, including with protein, penetrated water, and the deprotonated acidic group, but can also significantly reduce the deprotonation free energy of the buried acidic groups, which in turn lead to the high apparent dielectric constants. This is the energetic basis for a hydronium ion to favor a nearby placement. In other words, the proton produced from deprotonation prefers to stay nearby where it has a lower potential energy, which stabilizes the deprotonated state.</p><!><p>As a further support of the existence of protons near buried deprotonated acidic groups, we examined the conformations of these variants in the simulations with and without hydronium ions. We found that the buried ionizable residues kept a similar conformation to the X-ray structures when hydronium ions were added, whereas without the hydronium ions, the ionizable residues changed their conformation so that they could reach solvent. Figure 6 shows the conformations of L25E and I72E obtained from the simulations with and without a nearby hydronium ion. With nearby hydronium ions, the conformations of L25E and I72E are very similar to their X-ray structures, 3EVQ and 3ERO, respectively. In the X-ray structures, I72E is half buried and I25E is fully buried. Without a nearby hydronium ion, the carboxyl groups turn toward the solvent and become unburied, which are clearly different from the X-ray structures where the carboxyl groups point away from the solvent. For L25E, to allow the ionizable group to reach solvent, the β-sheet must break up to allow the E25 sidechain to pass through, further distorting the protein structure. In other words, without a nearby hydronium, the X-ray structure of L25E is not stable.</p><p>The nearby hydronium ions neutralize the deprotonated groups, allowing them to be stably buried inside proteins. This could be the reason why proteins often show little conformational change with buried charges. For acidic residues inside proteins, their deprotonation may proceed in two steps. First, the proton goes to a nearby water molecule to form a hydronium ion. Second, the proton transfers from a hydronium ion to bulk solvent. At the first stage, the overall buried group is neutral and the protein structure remains stable. At the second stage, the overall buried group is charged and could cause large-scale conformational relaxation and local unfolding. NMR measurement very likely reports the deprotonation at the first stage, where the nearby proton produces a high apparent dielectric constant without noticeable conformational relaxation or local unfolding.</p><p>Our simulation study is distinct from other simulation studies of the SNase variants due to the explicit treatment of the solvent environment. Because of the large energy barrier involving the reorientation of water molecules between different charge states, most of pKa related simulations were performed with implicit solvation models. Implicit solvation models do not have molecular details, like the protons nearby, and cannot correctly describe the microenvironment necessary for pKa determination. For example, Liu et al.59 performed pH replica exchange molecular dynamics simulations using the generalized Born solvation model to study SNase variants with internal Lys, Glu, or Asp residues. With implicit solvation models, conformational change is the only way to shift pKa to match experiment measurement. Not only pKa values in the buried and exposed states are needed, the equilibrium constants between the buried and exposed states are also introduced as additional parameters to fit experimental pKa values. The lack of details in implicit solvation models leads them to conclude that a coupled-ionization-conformational equilibrium is required to understand the properties of interior ionizable residues. The pKas of introduced buried residues in staphylococcal nuclease were also analyzed with the multi-conformation continuum electrostatic program.60 The results depend on parameters, such as the protein dielectric constant (εprot). An εprot of 8–10 and a Lennard-Jones scaling of 0.25 produce the best match with experimental values.</p><p>The idea that the proton from deprotonation can linger near the charged sidechains has been mentioned by many studies.61–63 The nearby proton concept played a role to explain proton pumping mechanisms in bacteriorhodopsin.64 Hydronium ions have been shown to have an important role in proton channels to transfer protons.50 These examples illustrate that the existence of hydronium ions near deprotonated acidic groups inside proteins could be a generalizable concept and has many implications in understanding protein functions.</p><p>This simulation study was performed with a highly stable form of SNase known as Δ+PHS. The stability of these variants may play a role to accommodate nearby hydronium ions without noticeable conformational relaxation or local unfolding. For other less stable proteins, deprotonation of buried acidic groups may result in conformational relaxation or local unfolding and let hydronium ions diffuse into the bulk solvent. Therefore, the nearby hydronium ions may only exist in stable proteins like Δ+PHS and bacteriorhodopsin.</p><!><p>We applied the VMMS method to simulate SNase variants with Asp or Glu mutations. As expected, large discrepancies between the calculated and experimental pKa values were observed for many variants. Of the 10 variants simulated, some variants showed pKa values very close to experimental results, which provide clues to the molecular determinants behind the high apparent dielectric constants. Conformational analysis reveals the presence of cations near the deprotonated acidic groups when the pKa approaches experimental values. This finding relates the high apparent dielectric constants to nearby cations. On the basis of available X-ray crystallography structures and deprotonation chemistry, we proposed a nearby proton hypothesis that the proton produced in a deprotonation process remains nearby to have stronger interactions with and stabilize the deprotonated group in an apolar environment. On the basis of this hypothesis, we set up VMMS simulations with a hydronium ion placed near the deprotonated group. The pKa values produced in these simulations correlate excellently with experimental observations. Energy analysis shows that it is energetically favorable for the hydronium ion to stay near the buried deprotonated group. Although other mechanisms, such as water penetration and conformational relaxation, play important roles, protons near buried deprotonated acidic groups should be a natural way to elevate apparent dielectric constants without significant conformational relaxation or local unfolding.</p>

PubMed Author Manuscript

Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration

We describe an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of protein targets in a microfluidic device. The key weaknesses of aptamer as a binding agent (weak binding strength/fast target dissociation) were counteracted by continuous injection of fresh sample while band-broadening phenomena were minimized due to self-focusing effects. With 30 minutes of continuous signal enhancement, we can detect 4.4 pM of human immunoglobulin E (IgE) and 9 pM of human immunodifficiency virus 1 reverse transcriptase (HIV-1 RT), which is among the lowest limit of detection (LOD) reported. IgE was detected in serum sample with LOD of 39 pM due to nonspecific interactions between aptamers and serum proteins. The method presented in this paper also has broad applicability to improve sensitivities of various other mobility shift assays.

continuous_signal_enhancement_for_sensitive_aptamer_affinity_probe_electrophoresis_assay_using_elect

<!>Principle of the assay<!>Reagents and Chemicals<!>Microchip Fabrication<!>Microchip Operation<!>Measurement Instrument and Image Analysis<!>Optimization of assay<!>Detection of IgE and HIV-1 RT from buffer<!>Detection of IgE from serum sample<!>CONCLUSION

<p>For decades, antibody based immunoassays have been the method of choice for disease diagnosis that require highly specific and sensitive recognition elements. However, aptamers are recently emerging as an increasingly popular alternative to antibodies as affinity probes. Aptamers are single stranded oligonucleotides that have undergone multiple rounds of in-vitro selection to bind specifically to various molecular targets1, 2, rivaling antibodies in terms of sensitivity and selectivity. Furthermore, they excel antibodies in other aspects such as low cost production by chemical synthesis, ability to survive harsh conditions, and labeling simplicity that ensures batch-to-batch uniformity3. Recently, high affinity aptamers have been generated for >800 human proteins that are potential biomarkers4. These are promising signs that aptamer biosensors will find widespread applications.</p><p>Several groups have demonstrated that capillary electrophoresis (CE) using aptamers as affinity probes can be used to detect specific target proteins such as IgE5–7, thrombin5, 8, ricin9, and HIV-1 reverse transcriptase (HIV-1 RT)6, 10, 11. Unlike heterogeneous immunoassay methods such as Enzyme-Linked Immunosorbent Assay (ELISA) that require several hours and multiple washing steps, the homogeneous CE assay is performed in one step with only a short incubation time (≤ 30minutes). Due to the ease of use and short assay time, CE methods are highly attractive for developing point-of-care biosensor platforms. However, CE assays are generally less sensitive than ELISA due to the ability of enzymes in ELISA to continuously convert a substrate to visible product over time. Furthermore, band dispersion and complex dissociation when using lower affinity (high Kd) aptamers as affinity probe in CE limits their applicability to detect low abundance biomolecules that could be important biomarkers.</p><p>Herein, we report an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of low abundance biomarkers in a microfluidic device. This scheme features three simultaneous processes: 1) continuous injection, 2) focusing, and 3) separation of the free aptamers and aptamer-protein complexes. One of the significant disadvantages of aptamer affinity probe CE is that complex may dissociate during long migration times, leading to weak or even absence of signal12. Decreasing the time spent on column, either by applying very high electric fields or utilization of hydrodynamic flow was often necessary to achieve reliable detection of the aptamer-protein complex12. In this new scheme, we counteract dissociation of the aptamer-protein complex by continuous injection and accumulation of fresh sample from the inlet reservoir. Band broadening phenomena commonly encountered in CE are also minimized due to the self-focusing effect. When a continuous flux of sample from the equilibrium mixture in the reservoir is subjected to simultaneous focusing and separation the signal-to-noise ratio increases with time. A good signal enhancement scheme is the key to highly sensitive assays such as ELISA. The major contribution of this paper is the use of electrokinetic concentration to realize a continuous signal enhancement scheme applicable to homogeneous mobility-shift assay.</p><p>Various schemes that combine sample concentration and CE analysis have been reported previously, including sample sweeping8, preconcentration using a size-exclusion membrane7, transient isotachophoresis (t-ITP)13, and temperature gradient focusing (TGF)14. In the first two cases, preconcentration and separation are carried out sequentially, thus band broadening during separation reduces the sensitivity enhancement. The t-ITP method results in very high sensitivity improvement, but imposes certain restrictions on the sample and running buffer and concentration factor is limited by injected plug volume. Use of photomultiplier tubes (PMT) in conjunction with Laser Induced Fluorescence (LIF) further improves the sensitivity of the first three assays. In the TGF example, high concentration factors are obtained as sample is focused continuously throughout the 7.5min experiment. However, special temperature sensitive buffer is needed and higher limit of detection (LOD) is expected since detection is based on monitoring a small decrease in the large free aptamer peak. In all these examples, high voltages of ≥ 1kV are required.</p><p>Our group has previously reported on nanofluidic electrokinetic concentration devices that can continuously collect negatively charged molecules in a given sample into a much smaller volume, thereby increasing local concentration significantly15. This electrokinetic concentration effect has been used to enhance protein binding kinetics to surface-bound antibodies16, increase the sensitivity of homogeneous enzyme assays17, 18, as well as improve the sensitivity of ELISA by more than an order of magnitude19. However, simultaneous concentration and separation of biomolecules based on mobilities have not been demonstrated due to the predicted low separation resolutions at high electric field gradients in these devices. The assay described in this paper exploits the fact that low molecular weight aptamers with high charge density undergo a significant mobility shift upon binding to a larger target protein with small net charge6, 7. Therefore, bound and unbound aptamers could be baseline-separated even under high electric field gradient conditions. Moreover, binding of the aptamers to the proteins makes the complex negatively charged6, which facilitates the electrokinetic concentration of neutral or basic proteins in buffers at physiological pH.</p><p>In this paper, we demonstrate electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assays for two different disease biomarkers, namely human Immunoglobulin E (IgE) and Human Immunodificiency Virus 1 Reverse Transcriptase (HIV-1 RT). IgE is the least abundant class of antibodies produced in human, and plays an important role in generating allergic response as well as defending against parasites20, 21. Some recent studies have suggested the use of serum IgE as a predictive biomarker for diseases such as asthma and peanut allergy21, 22. On the other hand, HIV-1 RT is a key diagnostic and therapeutic target of HIV-123, 24. Many aptamer based sensor have been used to detect IgE with different LOD, these include methods based on fluorescence enhancement (57 pM) 25, 26, carbon nanotube field effect transistors (250 pM)27, surface plasmon resonance (18.5 pM)28, CE (46 pM)5–7 and aptamer microarray using labeled IgE (10 pM)29, 30. Meanwhile, for detection of HIV-1 RT, the methods reported are predominantly based on CE (100 pM)6, 10, 11, temperature gradient focusing (84 pM), transient isotachophoresis (<1pM) and CE followed by PCR (30 fM). It is worth noting that the LOD reported is dependent on detection instruments and the affinity of the particular aptamers, and that coupling separation with amplification step often leads to dramatic increase in sensitivity.</p><p>Using our platform, we obtained LOD of 4.4 pM and 9 pM for human IgE and HIV-RT respectively in simple buffer after 30 minutes preconcentration, compared to LOD of 46 pM5 and 100 pM6 obtained with conventional CE methods. These are the lowest assay LOD reported in the literature for aptamer affinity probe capillary electrophoresis in spite of the inferior detector used for our assays (arc lamp and CCD) versus LIF and PMT for CE. To demonstrate the applicability of this assay to complex sample analysis, we performed the assay in 10-fold diluted donkey serum. Initial experiments showed significant nonspecific interaction between DNA aptamers and serum proteins. However, we found that addition of nonspecific and nonfluorescent oligonucleotides largely suppresses the matrix interference, thus enabling us to detect IgE in 10% donkey serum with a LOD of 39 pM.</p><!><p>Figure 1 shows the key operation of the poly(dimethylsiloxane) (PDMS) microfluidic electrokinetic concentration chip. Under the voltage configuration shown in Figure 1a, ion depletion zones are created in the sample channels at the vicinity of the ion selective membrane due to concentration polarization phenomena. The conductivity gradient at the boundary of the ion depletion zone gives rise to a stable electric field gradient that can effectively focus negatively charged biomolecules at separate locations where electrophoretic velocity balances bulk flow velocity as illustrated in Figure 1b. Free aptamers, which have very high electrophoretic mobilities due to the highly negative-charged backbone of the oligonucleotide, are concentrated at the low electric field region. On the other hand, the aptamer-protein complex has a lower mobility due to its larger mass; therefore it concentrates nearer to the cation selective membrane where the electric field is higher. Conventional aptamer affinity probe CE operate in a nonequilibrium condition, since there are no targets in the run buffer that allow for rebinding of aptamers that have dissociated from their initial target during separation. A unique advantage of this platform is that the free target protein molecules are able to travel downstream beyond the concentrated aptamer-protein complex band (an even higher electric field is needed to stop the low mobility free protein). Therefore, aptamers that have dissociated from their target in the complex band can quickly rebind with free proteins in the run buffer and regenerate the complex, akin to the Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (ECEEM)31 where a plug of equilibrium aptamer-target mixture is injected and separated in a capillary prefilled with target. This is an important advantage which allows even aptamers with relatively high Kd's to be used in this platform with good sensitivity. Using this device, we realized a multiplexed microfluidic platform where homogeneous aptamer affinity probe electrophoresis assays can be performed with low voltages (30V) and gravitation-induced flow without the need of periphery equipments (syringe pumps, temperature blocks) or multiple buffers.</p><!><p>Unless stated otherwise, all chemicals used in the experiments were purchased from Sigma (St. Louis, MO). Human myeloma IgE was purchased from Athens Research and Technology, Inc. (Athens, GA). Recombinant HIV-1 reverse transcriptase (HIV-1 RT) was purchased from Worthington Biochemical Corporation (Lakewood, NJ). Oligonucleotides were synthesized and fluorescently labeled by Integrated DNA Technologies, Inc. (Coralville, IA). IgE –binding aptamer (5′-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3′) was labeled with 6-carboxyfluorescein (FAM) during synthesis at the 5′ end using ethylene glycol linker5. HIV-1 RT-binding aptamer (5′-AT CCG CCT GAT TAG CGA TAC TCA GAA GGA TAA ACT GTC CAG AAC TTG GA-3′) was labeled directly at the 5′ end with FAM6. Nonfluorescent nonspecific oligonucleotides (5′-TGG TCT TGT GTG GCT GTG GCT ATG TCT GAT CTT AAT CCA CGA AGT CAC C-3′)6 were also obtained from the same source. Donkey serum was purchased from Innovative Research (Novi, MI). All solutions were made with deionized water (18.2MΩ) by Fluid Solutions (Lowell, MA).</p><!><p>The microchip was fabricated using poly(dimethysiloxane) PDMS (Sylgard 184, Dow Corning Inc., Midland, MI) irreversibly bonded on a glass slide. Microchannels were molded in PDMS by replica molding technique16. To obtain the positive master mold, the desired design was photolithographically patterned onto a silicon wafer using positive photoresist. Next, the wafer was etched to a depth of 6 μm via a reactive ion etching (RIE) process. The silicon master was further treated with trichlorosilane (T2492, UCT Specialties, Bristol, PA) in a vacuum desiccator overnight to prevent adhesion to PDMS.</p><p>We fabricated the ion-selective nanoporous structures by using the microflow patterning technique to obtain a thin strip of Nafion film on a standard glass slide17, 18. A 50 μm deep and 200 μm wide PDMS microchannel was used to define the flow path of the Nafion solution (20 wt% solution in lower aliphatic alcohol/H2O mix, Sigma Aldrich, St. Louis, MO). The PDMS chip with microchannels was irreversibly bonded on top of the glass slide by standard plasma bonding.</p><p>Figure 1c showed the top view of the actual PDMS device used in the experiments. 0.1–10 μL pipette tips (USA Scientific, Ocala, FL) were cut at the tip end with a razor blade and inserted into the punched PDMS holes to act as fluid reservoirs. There were five separate inlets connecting to one outlet, allowing five samples to be preconcentrated simultaneously. Two side channels flanked the inlet channels to provide symmetrical electrical ground. The ion-selective nanojunction was fabricated at the center of the device to concentrate sample molecules by electrokinetic trapping when voltages are applied. The channels were filled with dyed solution for visualization purpose.</p><!><p>Before the experiment, the PDMS device channels were passivated with 1% BSA for 10 minutes to reduce nonspecific binding of the sample to channel walls. After that, the channels were flushed with DI water 3 times and filled with buffer solution (10mM Tris-HCl, pH 7.4) until the samples were ready to be loaded. Sample was prepared by mixing 5 nM of fluorescently labeled aptamer with different concentrations of analyte in buffer solution (10mM Tris-HCl, 1mM MgCl2, 200μg/mL BSA, pH 7.4 (IgE)/pH 8(HIV-1 RT)).</p><p>After 30 minutes incubation at room temperature, 30 μL of sample was loaded into each of the five inlet reservoirs and drawn into the microchannel by applying a brief suction at the outlet reservoir. The liquid height difference between the inlet reservoir and the empty outlet reservoir caused a well-controlled gravitational flow of sample solution from inlet to outlet, without any need for external pump.</p><p>Electrodes were inserted into the inlet and buffer reservoirs on the chip and connected to a power supply (Stanford Research Systems, Sunnyvale, CA). To initiate the concentration-enhanced affinity probe electrophoresis assay, we applied 30 V at the inlet reservoirs while grounding the side channels. An ionic concentration gradient was induced near the ion-selective membrane by concentration polarization effect. Meanwhile, charged sample molecules are continuously separated and stacked at the location where its electrophoretic velocity equals the bulk flow velocity. Within the experimental duration of 30 minutes, the fluorescent intensity of the stacked molecules increases linearly with time while background noise remained constant, resulting in a high signal-to-noise ratio. To study the reproducibility of the assay, we repeated the experiment in the same device after removing the contents in the inlet reservoirps and replacing them with new samples.</p><!><p>An inverted epifluorescence microscope IX 71 (Olympus, Center Valley, PA) equipped with a cooled CCD camera (SensiCam, Cooke Corp., Romulus, MI) was used for fluorescence imaging. A mechanical shutter which only opens for 100 ms every 5 s when images are taken was used to prevent photobleaching of the fluorescent molecules. The images were analyzed using the NIH ImageJ software. Flat-field correction was performed by dividing a reference image of the device taken before each experiment. Concentrations of bound and unbound aptamers were assumed to be directly proportional to the focused peak height as demonstrated in previous work14. Complex peak heights are normalized by the sum of complex peak height and free aptamer peak height. Dose response curves were fitted using a four-parameter logistic model. Origin 7 software (OriginLab Corp., Northampton, MA) was used for curve fitting.</p><!><p>We first determine the optimal conditions that promote stable aptamer-protein complex formation in free solution. The presence of divalent cations such as Mg2+ has been reported to be necessary for certain aptamer-protein complex formation32. Without Mg2+, no aptamer-IgE complex is formed while the aptamer-HIVRT complex band is only weakly fluorescent. Addition of 1mM of MgCl2 greatly improves the interaction between the species. We have also found that complex stability is a sensitive function of buffer pH. Best results were obtained in 10mM Tris-HCl buffer at pH 7.4 and pH 8.0 for IgE and HIV-1 RT assays respectively.</p><p>In initial experiments, aptamer and target proteins (IgE and HIV-1 RT) were simultaneously concentrated and separated in bare PDMS-glass devices. We observed no complex bands until high concentrations (> 10nM) of target proteins are added. Precoating the microchannels with 1% BSA for 10 minutes enabled us to clearly visualize the complex band corresponding to 750 pM of IgE (Figure 2a), suggesting that prevention of nonspecific adsorption of proteins to the microchannel surface is important to increase sensitivity. Precoating microchannel with 5% BSA did not lead to additional improvement in sensitivity. Interestingly, as shown in Figure 2, adding BSA into the sample increases sensitivity of the assay. Adding 50 μg/mL of BSA led to a clear complex band corresponding to 75 pM of IgE, while addition of 100 μg/mL of BSA to the sample enabled detection of 7.5 pM of IgE. No further sensitivity improvement was obtained when more than 200 μg/mL of BSA was added to the sample. Similar trends were observed with the HIV-1 RT assays. This observation is thought to be due to BSA stabilizing the aptamer-protein complex33. Based on previous reports, it has been suggested that the presence of BSA in solution helps maintain the correct aptamer and target protein conformation for optinal binding33. Presence of BSA in the solvent could also maintain the ratio of hydrophobic and hydrophilic regions on the target protein, thus preventing it from denaturation34. Both the IgE and HIV-1 RT specific aptamers did not interact with BSA, as the negative controls containing BSA but no target proteins did not form a visible complex band. In all our subsequent experiments, the microchannel surfaces were passivated with 1% BSA for 10 minutes and 200 μg/mL of BSA were added to the samples to obtain the best sensitivites.</p><p>We also observed that the separation distance between the bound and free aptamers increased with the addition of high concentrations of BSA into the sample. We believe that this is due to an isotachophoresis-like effect where preconcentration of an intermediate mobility species (BSA) results in a broadening electric-field plateau that separates the bound and unbound aptamer bands35. This suggests a method whereby the separation resolution between two species can be independently tuned by adding a spacer molecule with intermediate mobility in the sample.</p><!><p>We first demonstrate electrokinetic concentration-enhanced affinity probe electrophoresis assay of IgE using a specific aptamer for this protein. Upon binding to IgE, the mobility of a free aptamer (−2.81×10−4 cm2 V−1 s−1) is expected to shift to −0.58×10−4 cm2 V−1 s−1 6. Figure 3a,b shows the representative results for electrokinetic concentration-enhanced affinity probe electrophoresis assay for human IgE using anti- IgE aptamer as affinity probe. Experiments were performed in optimized buffer conditions (10mM Tris-HCl, pH 7.4, 1mM MgCl2, 200 μg/mL BSA) with constant aptamer concentration (5nM) and varying concentrations of human IgE protein (5pM to 75nM). During the 30 minutes experiment, the fluorescent intensity of the bands increased linearly with time and achieved concentration factors of >1000. The aptamer and complex bands were well-resolved (resolution~3.9). The position of the aptamer-protein band was also remarkably stable; moving less than 200 μm after it reached an equilibrium position at around 2 minutes. Slight variations in the band locations are due to differences in gravitation-induced flow, but the ratiometric assay results are relatively insensitive to the exact band locations. As expected, with increasing target protein concentration, the free aptamer peak decreased and the complex peak increased. Figure 3d shows the aptmer-protein complex peak due to 4.92 pM of IgE. For comparison, no complex band was observed in the negative control experiment.</p><p>The full dynamic range of the assay is shown in Figure 3e. The dose response curve was fitted using the four-parameter logistic model. The average and standard deviation of the zero dose response is calculated by performing two separate experiments where the sample contains no IgE, and taking the peak ratios as described in the methods section. Figure 3f shows a linear relationship in the log-log plot obtained at low IgE concentrations (5 pM – 7 nM). The LOD for IgE, calculated to be the analyte concentration needed to produce a signal three standard deviations above the zero dose response, is 4.4 pM. This is the lowest LOD reported to date for detection of IgE using aptamers. Interestingly, the apparent Kd (1.85 nM) is found to be more than an order of magnitude lower than the reported dissociation constant for this aptamer (64 nM)5. One possible explanation for this binding enhancement is that aptamer-protein rebinding events in the electrokinetic concentration zone more than offset the effect of dissociation during the experiment. Given that aptamers generally have higher Kd compared to antibodies, this scheme could significantly increase the sensitivity and utility of aptamer based assay.</p><p>Similar experiments were performed using Human Immunodeficiency Virus 1 Reverse Transcriptase (HIV-1 RT) and an aptamer against this protein to demonstrate that this method is general and can be applied to multiple analytes. Upon binding to IgE, the mobility of a free aptamer (−2.81×10−4 cm2 V−1 s−1) is expected to shift to −0.50×10−4 cm2 V−1 s−1 5, 6. The results (Figure 4) showed a LOD of 9 pM, which is also among the lowest LOD reported to date for this aptamer-protein pair.</p><!><p>To demonstrate the applicability of this assay to complex sample analysis, we performed the IgE assay in a buffer that consists of diluted donkey serum. Initial experiments showed significant interactions of serum components with the aptamers. Figure 5 showed a representative IgE assay performed in 1% donkey serum. We observed fluorescent precipitation in the sample solution. Moreover, there is formation of an extra fluorescent band in between the free aptamer and aptamer-protein band. Interestingly, the extra band is well defined and baseline-separated from the other two bands. This suggests that the aptamer is interacting with a particular species in serum, such as DNA binding proteins found in mammalian serum36. Although this interference did not interfere with the formation and determination of the free aptamer and the aptamer-protein complex (they are all baseline separated), the fluorescent precipitation in the sample solution caused large spikes in the electrophoregram.</p><p>It has been reported that serum matrix interference on aptamer affinity probe capillary electrophoresis can be suppressed by addition of nonspecific oligonucleotides that bind competitively to the interfering serum proteins6. We found that addition of 10 μM of a nonspecific and nonfluorescent 49-mer oligonucleotide6 eliminated the extra band and fluorescent precipitation in a sample solution containing 10% donkey serum.</p><p>Figure 6 shows the experimental results for IgE assay in 10% donkey serum after 1 minute preconcentration, with addition of 10 μM of nonspecific oligonucleotide to suppress matrix interference. Due to the high total protein concentration in the sample, preconcentration leads to a rapid broadening of electric field plateau between the free aptamer and the aptamer-protein complex as discussed in the previous section. We can only perform experiments for 2 minutes before the separation distance exceeds the microscope field of view. Due to the shorter preconcentration time, there is less sensitivity enhancement. We obtained a LOD of 39 pM for IgE assay in 10% donkey serum.</p><p>The LOD using this scheme is ultimately restricted by the specificity, rather than the affinity (Kd) of the aptamers against the target protein. Experiments in the serum sample showed that two bands were observed even in the case of the negative control (Figure 6b), which indicated that nonspecific binding was not completely eliminated by addition of nonspecific oligonucleotides. On the other hand, experiments in simple buffer showed that signal-to-noise ratio increased with time. We can obtain better sensitivities at the expense of longer assay time. The key advantage of this technique is a continuous influx of sample that counteracts the effects of dissociation and a self-focusing ability that minimizes band dispersion, so even aptamers with relatively high dissociation constant can be used in this assay.</p><!><p>In conclusion, this paper demonstrates the use of electrokinetic concentration to realize a continuous signal amplification scheme that increases the sensitivity of homogeneous mobility shift assay. Aptamers are attractive alternatives to antibodies for point-of-care diagnostic purposes due to their stability, low cost, and homogeneity. Our aptamer based affinity probe electrophoresis assay in a lab-on-chip device could detect 4.4 pM and 9 pM of IgE and HIV-RT in simple buffers, and detect 39 pM of IgE in 10% serum sample. These are among the lowest LOD obtained for aptamer affinity probe capillary electrophoresis experiments. Furthermore, this method has an advantage over many other assays since it is rapid, uses low voltage, consumes very little sample, can be multiplexed, and is very user-friendly (no multiple processing steps required).</p><p>Miniaturized capillary electrophoresis devices are one of the first microfluidic systems that gained popular acceptance, and remains a mainstay in lab-on-chip platforms. The method presented in this paper has broad applicability to improve the sensitivity of various capillary electrophoresis assays, such as those involving protein-protein interactions and enzymatic reactions. We plan to pursue these topics and apply them to clinically relevant samples in the future.</p>

PubMed Author Manuscript

Structural basis for targeting T:T mismatch with triaminotriazine-acridine conjugate induces a U-shaped head-to-head four-way junction in CTG repeat DNA

The potent DNA-binding compound triaminotriazine-acridine conjugate (Z1) functions by targeting T:T mismatches in CTG trinucleotide repeats (TNR) that are responsible for causing neurological diseases such as myotonic dystrophy type 1 (DM1), but its binding mechanism remains unclear. We solved a crystal structure of Z1 in complex with DNA containing three consecutive CTG repeats with three T:T mismatches. Crystallographic studies revealed that direct intercalation of two Z1 molecules at both ends of the CTG repeat induces thymine base flipping and DNA backbone deformation to form a four-way junction. The core of the complex unexpectedly adopts a U-shaped head-to-head topology to form a crossover of each chain at the junction site. The crossover junction is held together by two stacked G:C pairs at the central core that rotate with respect to each other in an X-shape to form two non-planar minor groove aligned G\xc2\xb7C\xc2\xb7G\xc2\xb7C tetrads. Two stacked G:C pairs on both sides of the center core are involved in the formation of pseudo-continuous duplex DNA. Four metal-mediated base pairs are observed between the N7 atoms of G and CoII, an interaction that strongly preserves the central junction site. Beyond revealing a new type of ligand-induced, four-way junction, these observations enhance our understanding of the specific supramolecular chemistry of Z1 that is essential for the formation of a non-canonical DNA superstructure. The structural features described here serve as a foundation for the design of new sequence-specific ligands targeting mismatches in the repeat-associated structures.

structural_basis_for_targeting_t:t_mismatch_with_triaminotriazine-acridine_conjugate_induces_a_u-sha

INTRODUCTION<!>Binding of Z1 forces deformation of the T:T mismatch DNA.<!>Structural detail of central crossover junction.<!>Structural details of the Z1 binding sites.<!>Comparison with the Z1\xe2\x80\x93DNA complex with central Watson-Crick-Franklin base pair instead of T:T mismatch.<!>DISCUSSION<!>Drugs and oligonucleotides.<!>Melting temperature.<!>Job plot analysis.<!>Crystallization.<!>X-ray data collection, phasing, and structure refinement.

<p>Errors during DNA replication cause misincorporation of nucleotide bases in the DNA sequence leading to mispairing or mismatches between the bases.1–2 These mismatches are ubiquitous, and their insertion into the genome often have various physiological and pathological implications.3–4 The abnormal expansion of trinucleotide repeats (TNRs) often form non-canonical structures containing mismatches sandwiched between Watson-Crick-Franklin base pairs.5 Deficiency in the repair systems may lead to elevated rates of mismatches resulting in genetic instabilities, giving rise to neurological defects.6 Small molecules that recognize mismatches can be used as probes for the recognition mismatches to detect TNR related diseases that arise from defective DNA repair mechanisms.7</p><p>Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG trinucleotide repeats in the 3'-untranslated region of myotonic dystrophy protein kinase gene.8–9 The number of expanded CTG repeats correlates well with disease inheritability, age-of-onset and severity.10 Therefore, the CTG repeat length is often considered as a disease diagnostic marker and thus used for disease onset prediction.11–12 Acridine-based small molecules have been known since the 19th century to perform various therapeutic functions including acting as anti-parasitic, antiviral, and antibacterial agents.13 Moreover, acridine-based therapeutics were investigated for their antitumor activity and ability to bind to topoisomerase II.14 Triaminotriazine-acridine conjugate (Z1) (Figure 1a) was first developed using structure-based design to selectively recognize both d(CTG) and r(CUG) sites for the treatment of DM1.15 Z1 exhibited strong binding affinity to CTG repeat sequences, inhibiting transcription by binding to the mismatches in CTG repeats. The triaminotriazine unit was proposed to recognize and form a base triplet with T:T or to flip out one of the pyrimidine rings forming a Watson-Crick-Franklin-like base-pair with thymine while stacking on the acridine.16 However, the structural basis for the recognition of Z1 ligands to CTG repeats remains unclear.</p><p>In the present study, the structure of Z1 bound to a palindromic duplex containing three consecutive CTG repeats has been determined. The DNA duplex is intercepted by two Z1s at both ends that induce significant structural distortions resulting in a new type of four-way junction featuring a double U-shaped conformation that assembles in a head-to-head manner. We also solved the crystal structure of Z1-d(TTCTGCTGCTGAA/TTCTGCAGCTGAA) to investigate the structural basis behind this interaction with a non-consecutive CTG motif. The unexpected features of the crystal structures with T:T mismatch specificity clearly demonstrates the ability of Z1 to induce a non-canonical DNA superstructure resembling a four-way junction. The structure allows the interactions between the Z1 and CTG repeat-associated DNA to be elucidated with atomic-level detail. These findings further enhance our knowledge about the adoption of higher-order non-canonical DNA conformations that depend on the nature of the interacting ligand and its selective supramolecular interactions.</p><!><p>To understand the structural basis behind the preference of Z1 for CTG DNA, we solved the crystal structure of a Z1 in complex with the palindromic DNA sequence, d[(5-BrU)TCTGCTGCTGAA], denoted Z1-TTT, at 1.55 Å resolution (Figure 1b and S1). The asymmetric unit shows two perpendicular antiparallel duplexes with four DNA strands, labeled as chain A, chain B, chain C and chain D (Figure 1c). The detailed analysis of complex structure reveals that the intercalation of Z1 to the end of the CTG repeat duplex induces thymine (T4) base flipping that allows for DNA backbone deformation of the central 5'-GCTGC. The backbone bends over 90o and forms a non-canonical four-way junction. These findings are also consistent with the Job-type titration plot of Z1 binding to DNA which showed a maximum at c.a. 0.67 (mole fraction of Z1), identifying the 2:1 stoichiometry of the complex in solution (Figure S2).</p><p>The core of the complex adopts a U-shaped head-to-head topology to form a crossover of each chain at the junction G5-C6-T7-G8-C9 penta-sequence (Figure 1d). The U-shaped kink is associated with an abrupt change in backbone torsion angle α by c.a. 140° between bases T4 and G5 in each chain thereby forming an unusual structure upon Z1 binding (Table S1). The central crossover core is formed by two stacked G:C pairs (denoted as GC-I, and GC-II) between 5'-G5pC6 of A and C chains and the C6pG5– 5' of B and D chains. The adjacent G8pC9 bases of chains A and C are hydrogen bonded with C9pG8 from the B and D chains, respectively, to form two additional stacked G:C pairs (denoted as GC-III and GC-IV) on both sides (Figure 1e). In addition, the central stacked G5:C6 pairs from A, C and B, D chains are sandwiched between the T7 of each chain to form stable and continuous stacking interactions. These stacking interactions are similar to a previously reported structure where quinoxaline ring of echinomycin stabilized the continuous CpG base pairs in DNA containing a T:T mismatch.17 The thymine (T4) is present on the head of the U-turn with a χ angle in the range of −121° to −134° adopts an anti-conformation. The two backbone dihedral parameters α and ζ between T4 and G5 residue steps undergo drastic transitions (−167° to 67° for α and 52° to −95° for ζ, respectively) (Table S2). This causes T4 to be flipped in the opposite orientation of the DNA backbone with a higher degree of distortion generating an anti-conformation. Interestingly, the flipped out thymine shows no disordered electron density map and is found to be stabilized by the stacking interactions of symmetry-related 3'- terminal adenine (A13) residue in the crystal packing with an e-motif as observed previously.18</p><p>Furthermore, two arms of U-shaped duplex stack on each other to form a pseudo-continuous right-handed duplex DNA with G8 and C9 of strands A and C chains base-paired with C9 and G8 of strands B and D, respectively. Z1 intercalates through the minor groove side into the 5'-C3pT4 step (Figure 2). The binding of Z1 causes remarkable distortion in the pseudo duplex including the widening of the major (22.5 Å) and minor grooves (19.6 Å) at the central 3'-GpT step compared to the groove widths of canonical duplexes. This forces the backbone to unwind to generate additional space. The 5' thymine (T4) is flipped out of the helix upon Z1 binding and the complementary thymine (T10) is stabilized through direct hydrogen bonding with the triaminotriazine moiety of Z1.</p><!><p>The palindromic pentanucleotide 5'-GCTGC sequence in the crossover junction is held together by two stacked G:C pairs at the central core (GC-I, and GC-II) and two stacked G:C pairs on both sides of the intersection (GC-III and GC-IV). From the side view, two central stacked G:C pairs are rotated c.a. 40° with X-shape to form two non-planar minor groove aligned G·C·G·C tetrads with Watson-Crick-Franklin base pairing between the complementary residues and the N2 amino of guanines hydrogen bonded to the O2 of adjacent cytosines. Two stacked G:C pairs on the side rotate 50° with respect to the central stacked G:C pairs giving 2-fold symmetry and are interconnected by metal ion-mediated interactions (Figure S3). For the first time, four CoII ions were found to mediate the base pairing between N7 on guanine (G8) in chains A, B, C and D to N7 on guanine (G5) from the chains D, C, B and A, respectively, that further stabilize the crossover junction sites. The 2Fo-Fc electron density (contoured at 1.0 σ) clearly shows that each CoII ion has octahedral coordination between two guanosine nitrogen and four water molecules (Figure 3a). This type of four stacked G:C pair arrangement in the crossover junction core gives rise to a propeller-like structure. The current structure is mostly hydrated, and several water molecules mediate significant interactions to stabilize the residues in the central crossover junction (Figure 3b). Two water molecules W206 and W56 bridge the interactions between N3 on G5 and O4 on C6 in the central core (Figure 3c), whereas W18 and W19 mediate the hydrogen-bonding interaction between the side GC pairs with the central ones (Figure 3d and 3e).</p><!><p>In the current complex structure, four Z1 molecules were observed in an asymmetric unit intercalated through the minor groove side at the end of each pseudo-duplex arm. The close-up view of the Z1 binding site is shown in Figure 4a, which showed that the acridine moiety stacks with the triaminotriazine. The stacked-intercalator inserts into the 5'-CpT/TpG-3' step of the pseudo duplex where the acridine unit forms intramolecular stacking interactions with G11 base in the repeat sequence (Figure 4b). The intercalation of triaminotriazine causes the unpaired thymine (T4) to extrude out of the duplex and forms a complementary pairing to the other thymine (T10) through three hydrogen bond interactions. The flipping of thymine also causes the DNA backbone to bend c.a. 90° around the T4pG5 step that resulted in OP2 on guanine (G5) in such orientation that it forms a hydrogen-bonding interaction with one of the amino moieties of the triaminoazine ring (Figure 4a). In addition to these direct interactions between Z1 and DNA residues, three conserved water molecules were found to further stabilize the Z1-DNA complex (Figure 4c). LigPlot+ analysis19 showed that residue C3 and T4 of chain A, T10 and C11 of chain C, and G8 of chain D are in close van der Waals contact with the Z1 molecule in all four sites (Figure 4d). Altogether, these interactions contribute to the specific recognition of CTG repeats by Z1.</p><!><p>The stabilizing effects of Z1 binding to non-canonical duplex structures harboring one CTG repeat (AAT), two consecutive CTG repeats (ATT), two non-consecutive CTG repeats (TAT), and three consecutive CTG repeats (TTT) at a fixed Z1:DNA stoichiometry of 4:1 were tested using thermal duplex melting. We found that binding of Z1 leads to large stabilization effects on TTT (ΔTm = 16 °C) and TAT (ΔTm = 7.5 °C) oligonucleotides compared to AAT and ATT (Figure S4). Comparing the stabilization effects of Z1 on two CTG repeat DNAs, the ΔTm value of TAT increases significantly more than that of ATT (ΔTm = 4.7 °C) upon saturation with Z1 (10 μM), suggesting the binding of Z1 to neighboring CTG sites may cause steric hindrance and weaken the stabilizing effects of Z1 on DNA. These findings are consistent with the structural results showing that Z1 binds to TTT at both ends of DNA duplex and the central CTG is not involved in the interaction with Z1.</p><p>To understand whether the Z1 molecule binds at two CTG sites separated by at least one CTG site, the crystal structure of Z1 in complex with the palindromic DNA duplex harboring a central A:T base pair sequences, d(TTCTGCTGCTGAA/ TTCTGCAGCTGAA), denoted by Z1-TAT, at 1.65 Å resolution was solved for comparison (Figure S5). Each asymmetric unit of Z1-TAT complex contains two DNA duplexes bound by four Z1 molecules, in which one of the DNA duplex contains central A:T base pair and another possesses a central T:T base pair configuration (Figure S6). Upon superimposing the Z1-TTT and Z1-TAT complex structures shows an RMS deviation of 0.5 Å indicating both structures are identical globally with local differences (Figure S6a). Intriguingly, the Z1-TTT complex is more hydrated than that of Z1-TAT structure suggesting the importance of water in stabilizing the overall complexes (Figure S6b and S6c). Notable differences were found in the central T7-A7 base-pair geometries when comparing Z1-TTT to Z1-TAT. The T7:A7 base-pair shows less stacking interactions as the A7 was pushed outwards. The LigPlot+ analysis showed that the T7 of Z1-TTT complex has more van der Waals and water-mediated interactions with the flanking residues than A7 in the TAT complex (Figure S6d and S6e).</p><!><p>Because non-canonical structures of repetitive DNA sequences are hallmarks of many neurodegenerative diseases, small-molecule ligands targeting mismatch DNA can be a valuable tool in the diagnosis and treatment of neurological diseases.5, 20 Repetitive mismatches with polymer-like expansions and local instability can act as hotspots for small molecule recognition.21 Nakatani and coworkers showed that naphthyridine-containing compounds can recognize DNA mismatches in trinucleotide repeat DNA sequence with high affinity and specificity in vitro, and may induce repeat contractions in cells.22 Their findings led them to propose the use of naphthyridine analogs as a basis for the development of new types of therapeutic agents active against neurological disease.</p><p>Previous studies have shown that many DNA intercalators such as octahedral ruthenium complexes and actinomycin D can recognize DNA mismatches by extruding the mismatched base pairs and inducing a marked kink in the DNA structures.21, 23–24 Acridine-based compounds, including Z1, were rationally developed for targeting both d(CTG) and r(CUG) sites using structure-based design and for inhibiting both transcription of the expanded TNR of DM1 and inhibiting aberrant protein sequestration by the r(CUG) transcript.15, 25 The single stranded CTG repeat DNA can form stable, perpendicular, pseudo-duplexes when there are at least three repeating units. The present study demonstrates that the Z1 can tightly bind to T:T mismatches in CTG repeats and induce large scale structural deformation. The DNA backbone shows severe distortion in addition to thymine base flipping, resulting in a U-shaped structure (Figure 5a). Two U-shaped conformations form a marked crossover site of a -GCTGC- penta-sequence through the stacking, hydrogen bonding, and metal-mediated pairings to generate a four-way junction (4WJ).</p><p>The structures of other DNA four-way junctions have been previously observed to form complexes with small ligands such as psoralen and platinum-based compounds, however, the U-shaped structure described here is unique (Figure 5). Thus, Eichman et al. reported a hydroxymethyl-trimethylpsoralen (HMT) that covalently binds to DNA, inducing Holliday junction (HJ) formation by cross-linking the thymine bases across complementary strands in the X-stacked structure (Figure 5b).26 Recently, a platinum complex [Pt(H2bapbpy)]-(PF6)2 (where H2bapbpy is N-(6-(6-(pyridin-2-ylamino)-pyridin-2-yl)-pyridin-2-yl)pyridin-2-amine) has been shown to bind to the short oligonucleotides that form non-continuous pseudo duplexes (Figure 5c).27 The interface between the platinum complex and the terminals of inter-duplexes induces cytosine base flipping to generate a pseudo-4WJ like crossover through stacking and hydrogen bonding as observed in the crystal packing of the complex. On the other hand, the ligands recognizing DNA superstructures can be an attractive strategy in drug design rather than targeting standard duplex DNA. For example, Brogden et al. reported 6-carbon-linked bis(9-aminoacridine-4-carboxamide) ligand that can selectively recognize the antiparallel HJ DNA (Figure 5d).28 The ligand binds across the center of the junction to form a special topology where a flipped adenine base is replaced by acridine intercalators on either side of the crossover. The detailed structural analysis of high-resolution ligand-DNA complexes can be useful to the design of new compounds with selective binding to higher-order DNA that self-assemble into superstructures such as 4WJs or HJs.</p><p>The design of new chemical compounds with distinct physical and supramolecular properties that have the ability to induce formation of different types of DNA structures is becoming an attractive area of research.29–30 There is increasing evidence that suggests the non-classical DNA structures also possess important roles in biology.31–32 Although at this time it is unfeasible to ascertain whether the current structure observed in the solid state also form in vivo. The structures reported in this study expands the diversity of unusual DNA motifs induced by ligand and thereof offer a new tool for rational design of controlled DNA superstructures or sensors. Nonetheless, we have identified a G-CoII-G mediated pairings that stabilize the crossover junction sites. Such metal ion mediated base pairs composed of natural nucleotides have been studied extensively for applications in the development of novel therapeutics and various DNA nanoarchitecture designs. For instance, a new type of non-helical DNA structure driven by a heavy metal ion has been identified that has a potential application in designing nanostructures such as DNA 'tweezers', 'walkers' or 'gears'.33</p><p>Recently, Rousina-Webb et al. designed a metal ion selective DNA double crossover junction tile structure that has an application in designing robust, stable DNA-based material.34 Identifying the atomistic details of metal-mediated pairing will further the development of DNA nanotechnology. Inspired by the unexpected features of the current crystal structures, this work may also facilitate the development of more effective derivatives to target genomic mismatched bases. For example, modification of the triaminotriazine potion in the Z1 molecule may lead to derivatives with homo-purine mismatch specificities. In addition, the binding orientation of Z1 is such that the short linker is present in the minor groove, an appropriate position for specific interactions within the DNA duplex. The adjustment of the carbon chain length of the linker may affect the orientation of the acridine motif, whereas the modification of the acridine portion could affect the binding affinity to the DNA base pairs.</p><p>To summarize, recognition of T:T mismatches by Z1 results in the classic ligand binding-induced transformation, in this case triggering large-scale DNA deformations that ultimately lead to the formation of a non-canonical 4WJ. The ability to force DNA oligonucleotides to adopt a certain shape through sequence design and ligand binding to form supramolecular DNA may also have implications in the design of molecular devices such as ion sensors, nanowires and DNA magnets.34–35</p><!><p>The triaminotriazine-acridine based ligand, Z1, was synthesized as described previously.15 Single stranded synthetic DNA oligonucleotide sequences were commercially synthesized by MDBio Inc. with purification performed by polyacrylamide gel electrophoresis (PAGE). Absorbance measurements were carried out in a quartz cuvette using a JASCO V-630 UV/VIS spectrophotometer. The concentrations of oligonucleotides were determined by Beer's law (A = εbc, where A is the optical density at 260 nm, ε is the extinction coefficient, b is the cell path length (1 cm), and c is the DNA molar concentration). Oligomer extinction coefficients were estimated using tabulated values of monomer and dimer extinction coefficients with reasonable assumptions.36</p><!><p>The melting temperature (Tm) of the DNA oligonucleotides were analyzed using a JASCO UV/VIS spectrophotometer with 1 cm path length quartz cuvettes as previously described.17 Initially, the DNA oligomers (Table S3) were annealed by heating at 90 °C for 5 min to denature the strands and cooling on ice for 30 min. To ensure formation of the Z1-DNA complexes, 3 μM DNA oligomers were incubated with 12 μM Z1 in the presence of 12 μM CoII in MOPS buffer (20 mM MOPS, 300mM NaCl and 1 mM Na2EDTA, pH 6.5) at 4 °C overnight. The melting temperature curves were obtained by increasing the temperature from 4 °C to 95 °C at a rate of 1 °C/min and recorded every 0.5 min at 260 nm. The Tm values were determined from the observed curves using a polynomial curve fitting by Varian Cary Win UV Thermal application software (Ver. 3.0).</p><!><p>As the acridine moiety of Z1 possesses fluorescent properties, Job-type titration spectra were used to monitor the interactions between CTG DNA and Z1. The binding of Z1 to CTG DNA causes enhanced fluorescence emission exhibiting a maximum near 525 nm and a fluorescence intensity enhancement near 420 nm in the excitation spectrum with 525 nm emission monitoring. Samples of varying molar ratios of Z1:DNA were prepared with the total concentration held constant at 2 μM and incubated at 25 °C for 24 h. All spectra were recorded at 25 °C in 20 mM MOPS (pH 6.5), 300 mM NaCl and 1 mM EDTA. The spectra were measured at 25 °C with a JASCO model FP-4500 spectrofluorimeter.37 Experiments were performed using a 3 × 3 mm quartz cell cuvette with a 1 cm path length. Plots of fluorescence intensity versus molar ratio were obtained to determine binding stoichiometry.</p><!><p>To obtain the Z1-TTT crystals, 0.4 mM single-stranded d[BrUT(CTG)3AA] oligomer was pre-incubated with 0.7 mM ligand Z1 at 4 °C for 72 hours. Yellow-rhombic shaped crystals were grown after two weeks in a 3 μl drop containing 50 mM MES (pH = 6.5), 200 mM potassium chloride, 20 mM cobalt(II) chloride and 35% 2-methyl-2,4-pentanediol (MPD), equilibrated against 500 μl of 35% MPD using the sitting-drop vapor diffusion method. The Z1-TAT complex crystals were also obtained in similar conditions as described above where 0.2 mM d[TT(CTG)3AA] was mixed with an equimolar concentration of d(TTCTGCAGCTGAA) oligomer.</p><!><p>X-ray diffraction data of the Z1-TTT and Z1-TAT complexes were collected at beamlines TPS 05A and BL15A1 of the National Synchrotron Radiation Research Center (Hsinchu, Taiwan), respectively. The diffraction data for the Z1-TTT crystal in space group P21 with unit-cell parameters a = 57.2, b = 62.3, c = 29.7Å was collected at 100 K using the MX300HE detector at beamline 15A1. Single-wavelength anomalous diffraction (SAD) data was collected at a resolution of 2.1 Å from single peak wavelength using CoII as the anomalous scattering atom. The data with the anomalous signal was indexed, integrated, and scaled by HKL-2000, followed by CoII substructure localization using SHELX C/D/E.38 The resulting well-defined SAD electron density map was used to build initial models using the programs MIFit (Ver. 2010.10) and WinCoot (Ver. 0.8.4).39 The best built modeled structure of the Z1-TTT complex was used as a template to solve the phases of the Z1-TTT complex at a higher resolution of 1.55 Å. The structural refinements were performed using the PHENIX package (Ver. 1.14–3260).40 In addition, the diffraction data for the Z1-TAT crystal in space group P21 with unit-cell parameters a = 57.63, b = 62.09, c = 30.75 Å, was collected at 100 K on the MX300HS detector at beamline TPS05A. The phase for the Z1-TAT complex structure was determined by molecular replacement with Phaser MR in the PHENIX package suite (Ver. 1.14–3260) using the partial structure of the Z1-TTT complex as a template. The crystallographic and refinement statistics of these complexes are listed in (Table S4).</p>

PubMed Author Manuscript

Recombinant expression and phenotypic screening of a bioactive cyclotide against \xce\xb1-synuclein-induced cytotoxicity in baker\xe2\x80\x99s yeast

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown reduce the toxicity of human \xce\xb1-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1 to 5\xc3\x97104. This demonstrates the potential for using yeast to perform phenotypic screening of genetically-encoded cyclotide-based libraries in eukaryotic cells.

recombinant_expression_and_phenotypic_screening_of_a_bioactive_cyclotide_against_\xce\xb1-synuclein-

<p>Cyclotides are fascinating micro-proteins (≈30 residues long) present in plants from the Violaceae, Rubiaceae, Cucurbitaceae and more recently Fabaceae families.[1] They display various biological properties such as protease inhibitory, anti-microbial, insecticidal, cytotoxic, anti-HIV and hormone-like activities.[2] They share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology (Fig. 1A). This cyclic cystine knot (CCK) framework gives the cyclotides exceptional rigidity,[3] resistance to thermal and chemical denaturation, and enzymatic stability against degradation.[2] Interestingly, some cyclotides have been shown to be orally bioavailable,[4] and other cyclotides have been shown to cross the cell membrane through macropinocytosis.[5] Recent reports have also shown that engineered cyclotides can be efficiently used to target extracellular [6] and intracellular[7] protein-protein interactions. All of these features make cyclotides ideal tools for drug development to selectively target protein-protein interactions.[8]</p><p>Naturally occurring cyclotides are ribosomally produced in plants from precursor proteins[1b] and believed to be processed by specific proteases.[9] More than 200 different cyclotide sequences have been reported in the literature to date,[10] and it has been estimated by genomic analysis that ≈ 50,000 cyclotides may exist.[11] All naturally occurring cyclotides share the same CCK motif despite sequence diversity found in the loops decorating the cysteine-knot. Consequently, cyclotides can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.[12] Cyclotides can be chemically synthesized, thereby permitting the introduction of specific chemical modifications or biophysical probes.[13] More recently, cyclotides have also been biosynthesized in plant-derived cell cultures[14] and prokaryotic expression cells by making use of modified protein splicing units.[15] Cyclotides have been also shown to cross cellular membranes to target intracellular protein-protein interactions.[7] Altogether, these characteristics make cyclotides ideal substrates for in-cell molecular evolution strategies to enable generation and selection of compounds with optimal binding and inhibitory characteristics.</p><p>In-cell screening and selection methods of genetically-encoded cyclotide libraries provide several advantages over in vitro techniques: it ensures that hits are non-toxic, can bind the target in the appropriate cellular environment, are not rapidly degraded inside the cell, and possess high selectivity to work in living cells. In addition, this method also enables phenotypic screening for the rapid selection of novel bioactive compounds.</p><p>The use of an adequate microorganism that allows the production of large genetically-encoded libraries is key for the phenotypic screening of these type of libraries. The baker's yeast Saccharomyces cerevisiae has been used for decades as a versatile and robust model system for eukaryotic cellular biology.[16] For example, many proteins important in human biology, including cell cycle proteins, signaling proteins, and protein-processing enzymes, were first discovered by studying their homologs in yeast.[17] In addition, several human pathologies derived from protein misfolding have been successfully modeled in simple eukaryotic organisms such as yeast Saccharomyces cerevisiae.[18]</p><p>To test the feasibility of expressing folded cyclotides in S. cerevisiae, we used the cyclotide MCoTI-I (Fig. 1A). This cyclotide is a potent trypsin inhibitor (Ki ≈ 20 pM)[19] that is naturally found in the dormant seeds of Momordica cochinchinensis, a plant member of the Cucurbitaceae family.[20] Trypsin inhibitor cyclotides are interesting candidates for drug design because they show very low toxicities to mammalian cells and can be used as natural scaffolds to generate novel biological activities.[6–7, 13b, 21]</p><p>To express cyclotide MCoTI-I inside living yeast cells we made use of protein trans-splicing (PTS) to facilitate the intracellular backbone cyclization (Fig. 2). This process has been previously used to express small cyclic peptides[22] and more recently cyclotides[15a] in bacterial expression systems but never used before in a eukaryotic expression system to express large disulfide-containing cyclic proteins such as cyclotides. PTS-mediated backbone cyclization can be accomplished by rearranging the order of the intein fragments, i.e. by fusing the IN and IC fragments to the C- and N-termini of the linear polypeptide precursor to be cyclized (Fig. 1B). To boost the intracellular expression of folded cyclotide MCoTI-I in yeast we used the Nostoc puntiforme PCC73102 (Npu) DnaE split-intein. This DnaE intein has the highest reported rate of protein trans-splicing (τ1/2 ≈ 60 s) and has a high splicing yield.[23]</p><p>Accordingly, we designed the split-intein construct 1a (Fig. 1B), where the MCoTI-I linear precursor was fused directly to the N- and C-termini of the Npu DnaE IC and IN polypeptides, respectively. To facilitate backbone cyclization we used the native Cys residue located at the N-terminal position of loop 6 (Fig. 1). A His-tag was also added at the N-terminus of the construct to facilitate identification of the precursor and intein-containing byproducts of the cellular cyclization process.</p><p>Expression in yeast S. cerevisiae of cyclotide MCoTI-I using PTS was tested by employing two different high-copy 2µ episomal expression plasmids, pYES2/NT (under the control of a GAL1 inducible promoter) and p426GPD (under the control of a GPD constitutive promoter). Expression plasmids encoding the split-intein precursor 1a derived from plasmids pYC2/NT and p426GPD were transformed into S. cerevisiae strains INVSc1 and W303-1a, respectively, by electroporation. Expression of the MCoTI-precursor split-intein was accomplished for 48 h at 30° C in media containing either 2% galactose (inducible GAL1 promoter) or 2% glucose (constitutive GPD promoter). Under these conditions the precursor was expressed at relatively high levels in both cases, ≈ 10 mg/L (GAL1 promoter) and ≈ 7 mg/L (GPD promoter). In both cases the precursor was completely cleaved (Fig. 2A), indicating the intrinsic high reactivity of the split-intein construct to undergo protein trans-splicing. Next, we quantified the amount of natively folded MCoTI-I generated in-cell by LC-MS analysis using pure MCoTI-I as standard. Correctly folded MCoTI-cyclotides are able to bind trypsin with high affinity (Ki ≈ 20–30 pM). Therefore, this step can be used for affinity purification and to test the biological activity of the recombinant cyclotides. By either using the whole cell lysate or the fraction purified with trypsin-immobilized sepharose beads the LC-MS analysis revealed in both fractions the presence of a major peak that had the expected mass of the natively folded MCoTI-I cyclotide (Figs. 2B and S1). Quantification of the amount of cyclotide gave similar yields in both fractions ≈ 50 µg/L (GAL1 promoter) and ≈ 60 µg/L (GPD promoter), which correspond approximately to an intracellular concentration of approximately 450 nM and 660 nM, respectively. These results indicate that in cell produced cyclotide MCoTI-I is biologically active and therefore adopts a native cyclotide fold.</p><p>Encouraged by these results, we decided to express a bioactive cyclotide that could be used to perform phenotypic screening in yeast. S. cerevisiae has been shown to be a good model for many human diseases involving protein misfolding such as Parkinson's disease.[18, 24] The human protein α-synuclein (α-syn) is a small lipid-binding protein that is prone to misfolding and aggregation that has been liked to Parkinson's disease by genetic evidence and its abundance in the Parkinson's disease-associated intracellular aggregates known as Lewy bodies.[25] Overexpression of human α-syn in yeast S. cerevisiae leads to endoplasmatic reticulum stress, disruption of endoplasmic reticulum-Golgi vesicle trafficking, accumulation of lipid droplets, mitochondrial dysfunction, and ultimately cell death.[25–26] This cellular pathology mirrors many aspects of the dysfunction seen in neurons and glia cells of patients with Parkinson's disease.[27] In addition, genetic screening using a yeast synucleopathy model has produced inhibitors of α-syn cytotoxicy that resulted also effective in neuronal models.[24, 28]</p><p>To engineer the cyclotide MCoTI-I to inhibit α-syn-induced cytotoxicity in yeast S. cerevisiae, we used the sequence of cyclic peptide CP4 (cyclo-CLATWAVG), which was recently shown to reduce α-syn-induced cytotoxicity in a yeast synucleopathy model.[24] A CP4-derived linear peptide, in which the Cys residue was replaced by Ser, was grafted onto the cyclotide scaffold using loop 6 to provide cyclotide MCoCP4 (Fig.1). We have shown that loop 6 has the higher backbone mobility of the MCoTI-I cyclotide[3] and accordingly this loop has been shown to tolerate well the grafting of bioactive peptides.[6a, 7] Replacement of the Cys residue was done to facilitate the folding of MCoCP4. Cyclotide MCoCP4 was expressed using a high-copy 2µ plasmid under the control of a GPD constitutive promoter as described above for cyclotide MCoTI-I. Under these conditions, the MCoCP4 split-intein precursor 1b (Fig. 1B) was expressed with a yield similar to that of the MCoTI-I precursor (≈ 9 mg/L). As before, the precursor was completely processed in vivo (Fig. 3A). LC-MS analysis of the soluble cell lysate revealed the presence of a major peak with the expected mass for the natively folded MCoCP4 cyclotide (Figs. 3B and S3). Quantitative LC-MS analysis of the cell lysate provided an expression yield of 45 µg/L, which corresponds to an intracellular concentration of ≈ 390 nM. To confirm the identity of the cyclotide MCoCP4 expressed in yeast, this cyclotide was chemically produced using a GSH-induced cyclization-folding one-pot reaction as previously described.[6a, 7] The cyclization-folding reaction was very efficient and complete in less than 24 h (Fig. S6). Pure synthetic cyclotide MCoCP4 was shown to co-elute by HPLC-MS with yeast-produced MCoCP4 and adopt a native cyclotide fold by NMR (Table S2 and Figs. S7 and S9).</p><p>To test the cytoprotective activity of cyclotide MCoCP4 we used a yeast synucleopathy model previously described in the literature.[26, 29] This cellular model employs a GAL1 promoter to tightly control the expression of human α-syn encoded in the genomic DNA of the microorganism. In this way, α-syn can be expressed or totally repressed only when the yeast are grown on medium containing either galactose or glucose, respectively. Accordingly, we expressed the cyclotide MCoCP4 using a compatible µ episomal expression vector under the control of a GPD constitutive promoter using the yeast synucleopathy model described above. The inactive cyclotide MCoTI-I and cyclic peptide CP4 were also used as negative and positive controls, respectively. After transforming the cells with the plasmids encoding the different cyclic peptides, the yeast cells were grown first in medium containing 2% raffinose as carbon source and then serially spotted in media containing either 2% glucose (α-syn off) or 2% galactose (α-syn on) (Fig. 3C). Expression of the corresponding cyclotides under these conditions did not affect significantly their expression level. As expected, no cytotoxicity was observed when the cells were grown in the presence of 2% glucose (Fig. 3C), thereby indicating the total repression of α-syn expression as well as the lack of cytotoxicity of cyclotides MCoTI-I and MCoCP4. When cells were grown in the presence of galactose, which activates α-syn expression, cells coexpressing inactive cyclotide MCoTI-I were unable to grown at all dilutions tried in the assay (Figs. 3C and S8). As anticipated, coexpression of cyclic peptide CP4 was able to rescue the cytotoxic phenotype induced by α-syn therefore confirming the protective activity of this peptide.[24] Remarkably, cells expressing the engineered cyclotide MCoCP4 were also able to suppress the α-syn-induced cytotoxicity under the conditions used in this assay. The cytoprotective effect of MCoCP4 was similar to that of the peptide CP4. Preliminary results indicated that cyclic peptide CP4 can be cyclized by PTS using the Npu DnaE split-intein providing an intracellular concentration of ≈ 5 µM as quantified by LC-MS/MS (Fig. S2).</p><p>Finally, we decided to explore the feasibility of using the yeast synucleopathy model for the phenotypic screening of bioactive cyclotides against α-synuclein-induced cytotoxicity. For this purpose we generated a mixture of plasmids encoding MCoCP4 and MCoTI-I in a ratio of 1 to 5×104, respectively. This DNA mixture was transformed by electroporation to provide ≈ 107 transformants as determined by plating on medium containing 2% glucose. To perform the phenotypic screening the whole cell-based mixture was plated on media containing 2% galactose to activate the expression of α-syn and incubated for 3 days. Under these conditions ≈150 colonies were obtained (Fig. 3D). 30 different colonies were picked, the corresponding plasmids isolated and analyzed by DNA sequencing to confirm the identity of the encoded cyclotide. Almost all the colonies analyzed (27 of 30, 90%) provided DNA sequences encoding cyclotide MCoCP4 therefore demonstrating the feasibility of using yeast as eukaryotic platform to perform phenotypic screening of in-cell generated bioactive cyclotides. The appearance of a small amount of false positives probably stemmed from spontaneous genomic suppressor mutations, which are common in most yeast selection schemes.[24]</p><p>In summary we have shown that bioactive folded cyclotides can be produced in eukaryotic microorganisms such as yeast S. cerevisiae by PTS using highly efficient split inteins. This approach was successfully used for the production of a novel cyclotide (MCoCP4) that was able to inhibit α-syn-induced cytotoxicity in live yeast cells, therefore allowing us for the first time to perform a phenotypic screen to select cyclotide sequences with biological activity from inactive cyclotides in a yeast synucleopathy model. These exciting results open the possibility to perform in-cell phenotypic screens against other cellular pathologies using cyclotide-based libraries. Using eukaryotic microorganisms for screening purposes should provide a more biologically relevant cellular background for phenotypic screening but also facilitate the production of cyclotide-based libraries containing post-translational modifications such as phosphorylation and/or glycosylation, which are not available in bacterial expression systems. In addition, it should also allow screening cyclotides against proteins containing post-transtational modifications. Moreover, the use of the cyclotide molecular scaffold, which possesses unique pharmacological features such as extreme stability,[30] oral bioavailability[6b] and ability to cross cellular membranes,[5] should provide an extremely valuable screening platform for the selection of novel therapeutic leads.</p>

PubMed Author Manuscript

Parallel solid-phase synthesis of diaryltriazoles

A series of substituted diaryltriazoles was prepared by a solid-phase-synthesis protocol using a modified Wang resin. The copper(I)- or ruthenium(II)-catalyzed 1,3-cycloaddition on the polymer bead allowed a rapid synthesis of the target compounds in a parallel fashion with in many cases good to excellent yields. Substituted diaryltriazoles resemble a molecular structure similar to established terphenyl-alpha-helix peptide mimics and have therefore the potential to act as selective inhibitors for protein–protein interactions.

parallel_solid-phase_synthesis_of_diaryltriazoles

<!>Introduction<!><!>Synthesis of azide-functionalized Wang resins<!><!>Solid-phase synthesis of diaryltriazoles<!><!>Parallel synthesis of a compound library<!><!>Parallel synthesis of a compound library<!><!>Conclusion<!>General procedures<!>

<p>This article is part of the Thematic Series "Recent developments in chemical diversity".</p><!><p>The α-helix was the first-described secondary structure of peptides discovered by Linus Pauling in 1951 [1]. With about 30% of the amino acids in proteins being part of α-helices [2], it is the most common secondary structure found in proteins [3]. Protein–protein as well as protein–DNA and protein–RNA interactions often involve α-helices as recognition motifs on protein surfaces [4]. These helices are important targets for new drugs, but stabilization of the helix folding for small structures with less than 15 residues still remains a challenge [5–6]. Thus, new attempts have been made to design low-molecular-weight ligands that disrupt protein–protein interactions [7]. For example, fast proteolytic degradation observed with small peptide-based compounds [8], can be overcome by compounds stabilized by non-natural amino acids [9] or cross-linked between side chains and the backbone [10]. Replacement of the complete backbone by a nonpeptidic scaffold, which positions side chains in the typical i, i+3 and i+7 arrangement of an α-helix is another successful strategy [11]. Horwell pioneered this type of peptidomimetics and showed that 1,6-disubstituted indanes can imitate the helix residues i and i+1 [12–13]. Hamilton reported a 3,2′,2″-substituted terphenyl scaffold with a spatial orientation that mimics the i, i+3 and i+7 moieties on the surface of an α-helical peptide [14]. Inspired by the terphenyl-based α-helix mimetics 1, several related compounds containing three or more adjacent aryl rings (Scheme 1), such as 2, were reported [15]. However, the synthesis of substituted triaryl compounds can be tedious, and the predictability of their potency and selectivity as inhibitors is still limited. We have recently reported the synthesis of triazole-based α-helix mimetics 3 and 4 [16], which are efficiently available through azide–alkyne cycloadditions [17]. We now report the use of this chemistry to prepare libraries of potential inhibitors of protein–protein interactions.</p><!><p>Terphenyl scaffold 1 [13–14]; oxazole-pyridazine-piperazine 2 [14–15] and aryl-triazoles 3 and 4 [15–16] as α-helix mimetics.</p><!><p>Two azide-functionalized resins were prepared for the solid-phase synthesis of diaryl-triazoles. The commercially available 4-(bromomethyl)benzoic acid (5) was converted into azide 6 in anhydrous DMF with sodium azide under heating. Coupling to Wang resin in dichloromethane, by using DIC and DMAP as coupling reagents, gave resin 7 in quantitative yield (Scheme 2) [18]. Commercially available 4-azidobenzoic acid (8) gave resin 9 in an analogous esterification of a Wang resin.</p><!><p>Synthesis of azido-functionalized resins 7 and 9.</p><!><p>The conditions for the solid-phase synthesis of diaryltriazoles on functionalized Wang resin 7 were optimized by using five different alkynes 10a–e, containing acyclic or cyclic aliphatic moieties, simple arenes and 1-(but-3-yn-2-yl)-3-(4-chlorophenyl)-1-methylurea (10c) as an example of a more complex alkyne. The azide–alkyne [3 + 2] cycloaddition was catalyzed with copper(II) sulfate pentahydrate and L-ascorbic acid in DMF overnight at room temperature. A solution of EDTA was added to remove the remaining copper cations from the resin. Resin cleavage under acidic conditions with TFA in DCM gave compounds 11a–e in moderate to excellent overall yields of 57 to 90% (Table 1). Due to the solid phase synthesis protocol the crude material purity was typically high, ranging from 70 to 90%. Alkyne 10d and 10e bearing hydroxy groups were converted quantitatively, but elimination of water occurred in the presence of TFA. The dehydrated products were obtained in 57 and 71%. The remaining material was the corresponding hydroxylated product.</p><!><p>Copper-catalyzed [2 + 3] cycloadditions of resin-bound azide 7 with five terminal alkynes.</p><!><p>A larger compound library was prepared by using resins 7 and 9, 15 different terminal alkynes 10f–t and either copper or ruthenium-catalyzed [2 + 3] cycloadditions. The three reactions and the obtained products 11 (reaction 1), 12 (reaction 2) and 13 (reaction 3) are summarized in Table 2.</p><!><p>Copper-catalyzed [2 + 3] cycloadditions of resin bound azide 7 with five terminal alkynes. Compounds 13, with the exception of 13f, were only characterized during compound library synthesis, by HPLC–MS analysis.</p><!><p>Reaction 1 follows the established protocol and, gave after removal of the copper salts with a solution of EDTA and TFA cleavage, the corresponding products in good to quantitative yields (88–99%). Resin 9 was used in reaction 2 under otherwise identical reaction conditions. The use of an aromatic azide leads to more rigid products containing three adjacent aromatic rings: The central triazole and the phenyl ring of the benzoic acid as constant structural elements and the third ring consisting either of substituted benzenes, heteroarenes or a polycyclic aromatic compound. Lower product yields were obtained in this series of compounds, ranging from 21 to 63%. The lower reactivity of the aromatic azide and the increased steric demand may explain the decrease in yield in comparison to that of the former series of compounds. The formation of the compound 12o, bearing a particularly bulky substituent, was not observed. Replacing the copper(I) catalyst by a ruthenium(II) complex allows the preparation of regioisomers in reaction 3. Instead of the 1,4-disubstituted triazoles obtained from copper(I) catalysis, the complex pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride leads to 1,5-disubstituted triazole compounds [19]. 4-(Azidomethyl)benzoic acid functionalized Wang resin 7 and the alkynes 10f–t were reacted in DMF at 70 °C overnight, and the products 13b–o were obtained after TFA cleavage from the resin in moderate to good yields ranging from 43–96%. Only compound 13a could not be obtained. The proton NMR analysis allows us to clearly distinguish between 1,4- and 1,5-disubstituted triazoles due to a characteristic shift of the triazole proton resonance. The triazole proton of the 1,4-disubstituted ring in compound 11k shows a 1H NMR resonance at δ = 8.71 (400 MHz, DMSO-d6), while the resonance signal for the triazole proton of product 13f is observed at δ = 8.00 (400 MHz, DMSO-d6). Compounds 13, with the exception of compound 13f, were only characterized by mass spectrometry during the synthesis of the compound library.</p><p>The copper-mediated [2 + 3] cycloadditions are restricted to terminal alkynes. However, the ruthenium-catalysis allows the use of internal alkynes. In preliminary work, resin 7 was therefore reacted with internal alkynes 14a–c and pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride as catalyst in DMF at 70 °C overnight followed by TFA cleavage [20]. LC–MS analysis of the crude product revealed the formation of compounds 15a–c in high yields of 78–98% (Table 3).</p><!><p>Ruthenium-catalyzed [2 + 3] cycloadditions of resin-bound azide 7 with three disubstituted alkynes.</p><p>aYields based on LC–MS; compound 12b was used as standard.</p><!><p>Diaryltriazoles were obtained in an efficient three-step solid-phase procedure. Immobilization of aromatic azides on commercial Wang resin followed by copper(I)- or ruthenium(II)-catalyzed 1,3-cycloaddition and subsequent cleavage of the product from the resin gave the target structures in good to excellent yields with the possibility to introduce a wide variety of different substituents. The alternative use of copper or ruthenium catalysis for the on-bead cycloaddition gives regioisomeric products, which extends the diversity of the compound collection. The method may find application in the combinatorial search for selective protein–protein inhibitors. To that end, most of the compounds prepared herein were submitted to the Molecular Libraries Small Molecular Repository for ongoing inclusion in high-throughput screening activities.</p><!><p>GP 1 – Coupling of benzoic acid derivatives 6 and 8 on Wang resin: Wang resin (1 equiv) was preswollen in dichloromethane (0.8 mL/100 mg resin) for 2 h at room temperature. Subsequently, both coupling reagents N,N′-diisopropylcarbodiimide (3.5 equiv) and dimethylaminopyridine (0.5 equiv) were added. After the addition of the benzoic acid derivative 6 or 8 (2.5 equiv) the reaction mixture was stirred for 20 h at room temperature. The resin was first washed with dimethylformamide, methanol and dichloromethane (each solvent 3 × 0.8 mL/100 mg resin), and then dried in high vacuum for 3 h.</p><p>GP 2 – Huisgen 1,3-dipolar cycloaddition of solid-phase-immobilized azides with terminal alkynes by copper(I) catalysis: An azide-functionalized Wang resin 7 or 9 (1 equiv) was preswollen in dimethylformamide (1.5 mL/100 mg resin) for 2 h at room temperature. The copper(I) catalyst was prepared in situ by using L-ascorbic acid (0.5 equiv) as reducing agent and copper(II) sulfate pentahydrate (10 mol %). After the terminal alkyne (4 equiv) was added, the reaction mixture was stirred for 22 h at room temperature. The resin was washed with dimethylformamide, methanol and dichloromethane (each solvent 2 mL/100 mg resin). The remaining copper cations were complexed and removed by using a solution of ethylenediaminetetraacetic acid disodium salt. For this purpose, a 1:1 mixture of dimethylformamide and disodium EDTA (aq., sat.) was added to the resin and stirred for 10 min at room temperature. Again washing steps with water, dimethylformamide, methanol and dichloromethane (each solvent 3 × 2 mL/100 mg resin) were carried out.</p><p>GP 3 – Huisgen 1,3-dipolar cycloaddition of solid-phase-immobilized azides with terminal or internal alkynes by ruthenium(II) catalysis: The azide functionalized Wang resin (1 equiv) was preswollen in dimethylformamide (2 mL/100 mg resin) for 2 h at room temperature. Subsequently, the catalyst complex pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride, Cp·RuCl(PPh3)2, (5 mol %) and either a terminal or an internal alkyne (4 equiv) were added. After the reaction mixture was stirred for 22 h at 70 °C, the resin was washed with dimethylformamide, methanol and dichloromethane (each solvent 3 × 2 mL/100 mg resin).</p><p>GP 4 – Cleavage of solid-phase resin-bound molecules with TFA: The swollen resin was treated with a 1:4 mixture of trifluoroacetic acid and dichloromethane (1 mL/100 mg resin). After being stirred for 10 min at room temperature, the cleaved product was rinsed out of the resin using dichloromethane (1.5 mL/100 mg resin). The resin was treated once more with the 20% trifluoroacetic acid solution (1 mL/100 mg resin), stirred for 10 min at room temperature and washed with dichloromethane (3 × 1 mL/100 mg resin). The solvent was evaporated and the product was dried in high vacuum for 4 h.</p><p>4-(Azidomethyl)benzoic acid (6) [21]: The synthetic procedure leading to this literature-known compound was improved. 4-(Bromomethyl)benzoic acid (5, 1.2 g, 5.58 mmol, 1.0 equiv) and sodium azide (907 mg, 13.95 mmol, 2.5 equiv) were suspended in 25 mL of anhydrous dimethylformamide under a nitrogen atmosphere. After the reaction mixture was stirred for 15 h at 50 °C the solvent was evaporated. The colorless residue was dissolved in 90 mL of water and the solution was treated with 17 mL of hydrochloric acid (c 1 mol/L). The precipitate was separated with a Büchner funnel, dissolved in dichloromethane and dried over potassium sulfate. After filtration, and evaporation of the solvent, 4-(azidomethyl)benzoic acid (6, 880 mg, 4.97 mmol, 89%) was yielded as a colorless solid and dried in high vacuum overnight; mp 135.6–136.6 °C; 1H NMR (300 MHz, CDCl3) δ 4.46 (s, 2 H, H-6), 7.44 (d, 3JHH = 8.4 Hz, 2H, H-4), 8.14 (d, 3JHH = 8.3 Hz, 2H, H-3); 13C NMR (75 MHz, CDCl3) δ 54.3 (−, 1C, C-6), 128.0 (+, 2C, C-4), 129.1 (Cq, 1C, C-2), 130.8 (+, 2C, C-3), 141.5 (Cq, 1 C, C-5), 171.4 (Cq, 1C, C-1); IR (cm−1) : 2933 (w), 2880 (w), 2817 (w), 2656 (w), 2110 (m), 2086 (m), 1950 (w), 1682 (s), 1293 (s), 1239 (s), 707 (s), 545 (s); EIMS m/z: 177.0 (40) [M]+, 148.0 (80) [M − N2]+, 135.0 (100) [M − N3]+; Anal. calcd for C8H7N3O2: C, 54.24; H, 3.98; N, 23.72; found: C, 54.28; H, 4.25; N, 23.75.</p><!><p>Experimental details and spectra.</p>

PubMed Open Access

A comparison of 18F PET and 99mTc SPECT imaging in phantoms and in tumored mice

Our objective was to compare the performance of a microSPECT with that of a microPET in a Her2+ tumored mice using an anti-Her2 nanoparticle radiolabeled with 99mTc and 18F. Camera performance was first compared using phantoms; then a tumored mouse administered the 99mTc-nanoparticle was imaged on a Bioscan NanoSPECT/CT while another tumored mouse received the identical nanoparticle, labeled now with 18F, and was imaged on a Philips Mosaic HP PET camera. The nanoparticle was radiolabeled with 99mTc via MAG3 chelation and with 18F via SFB as intermediate. Phantom imaging showed that the resolution of the SPECT camera was clearly superior but even with 4 heads and multipinhole collimators, detection sensitivity was 15 fold lower. Radiolabeling of the nanoparticle by chelation with 99mTc was considerably easier and safer than manual covalent attachment of 18F. Both cameras provided accurate quantitation of radioactivity over a broad range. In conclusion, when deciding between 99mTc vs. 18F, an advantage rests with chelation of 99mTc over covalent attachment of 18F, achieved manually or otherwise, but with these small animal cameras, this choice also results in trading lower sensitivity for higher resolution.

a_comparison_of_18f_pet_and_99mtc_spect_imaging_in_phantoms_and_in_tumored_mice

INTRODUCTION<!>General experimental<!>Preparation of N-succinimidyl-4-[18F] fluorobenzoate<!>Preparation of the 18F labeled MORF nanoparticle<!>Preparation of the 99mTc labeled MORF nanoparticle<!>SPECT/CT and PET/CT phantom imaging<!>SPECT/CT and PET/CT animal imaging<!>Radiolabeling<!>SPECT and PET phantom Imaging<!>SPECT and PET animal Imaging<!>DISCUSSION<!>CONCLUSIONS<!>

<p>While the nuclear imaging modality of choice in the clinic is often PET rather than SPECT in part because of superior sensitivity, resolution and quantitative ability, it is unclear whether the same advantages apply to small animal imaging (1–6). This investigation was conducted primarily to compare the relative spatial resolution and detection sensitivity for 99mTc on a NanoSPECT/CT camera (Bioscan Inc., Washington D.C., USA) with 18F on a Mosaic HP PET (Philips Medical Systems, Inc., Cleveland, Ohio, USA). Several reports have appeared comparing radioactivity quantitation, imaging sensitivity and specificity of clinical SPECT and PET cameras in the diagnosis and monitoring of cancers and other disease states in patients (7–10). The number of comparison studies of SPECT and PET imaging of small animals using the same agent with different radiolabels is at present limited to 123I and 124I labeled metaiodobenzylguanidine(MIBG) for reporter gene imaging (11,12).</p><p>As a model radiopharmaceutical for this comparison, a delivery nanoparticle under development in this laboratory was used in which the biotinylated phosphodiamidate morpholino (MORF) oligomer (13), after radiolabeling with 99mTc or 18F, was linked to the biotinylated Trastuzumab (Herceptin™) antiHer2 antibody via streptavidin as shown in scheme 1. While the radiolabeling of the nanoparticle and its biodistribution was not an objective of this investigation and although the MORF nanoparticle has been repeatedly and successfully radiolabeled with 99mTc via MAG3 for antisense imaging (14), this report also describes the first labeling of an amine-derivitized MORF with 18F. Following construction of each Trastuzumab nanoparticle by addition of the biotinylated labeled MORF both 99mTc and 18F was administered to a mouse bearing Her2+ SUM190 xenografts and the animals imaged using the appropriate camera.</p><!><p>A phosphodiamidate morpholino (MORF) oligomer with the base sequence 5′-G CGTGCCTCCTCACTGGC and therefore antisense to the RIα mRNA was purchased with a biotin group on the 3′ equivalent end via a 6-aminohexanoic acid linker and a primary amine on the 5′ equivalent end (Gene Tools, Philomath, OR, USA). The S-acetyl NHS-MAG3 was synthesized in house and its structure confirmed by proton nuclear magnetic resonance and mass spectroscopy (15). The anti-Her2 Trastuzumab (Herceptin™ ) was obtained from Genentech Inc. (South San Francisco, CA, USA) as the clinical product. The Sulfo-NHS-LC-Biotin was purchased from Pierce (Rockford, IL, USA) and used to biotinylate the Trastuzumab antibody. Streptavidin was purchased from Sigma (St Louis, MO, USA). Standard chemicals were obtained from various suppliers and used without purification. The 99mTc pertechnetate was eluted from a 99Mo-99mTc radionuclide generator (Bristol-Myers Squibb Medical Imaging Inc., North Billerica, MA, USA). The 18F-fluoride was obtained on a QMA ion exchanging cartridge from PET-NET Solutions Inc. (Woburn, MA, USA). The human breast cancer cell line SUM 190 was purchased from Asterand Company (Detroit, MI, USA). Ethyl 4-(trimethylammonium)benzoate trifluoromethanesulfonate was synthesized in house following published procedures (16), and the structure confirmed by 1H NMR (300 MHz, D2O): d 1.18–1.23 (3H, t), 3.51 (9H, s), 4.20–4.27 (2H, q), 7.77–7.80 (2H, m), 8.04–8.07 (2H, m).</p><!><p>The amine-derivitized MORF was radiolabeled with 18F via N-succinimidyl-4-[18F] fluorobenzoate (18F-SFB) as intermediate, selected because of its high radiochemical yield and in vivo stability (17). The synthesis of 18F-SFB was achieved according to Cheng et al. (16).</p><p>The 18F-fluoride was eluted from the QMA ion exchanging cartridge using 1.5 mL of a mixed solution of Kryptofix 2.2.2 (1–2 mg) in 0.5 mL CH3CN and K2CO3 (10–12 mg) in 1 mL H2O. Of this, 0.5 mL (7.4×103 MBq) was injected into a 5 ml V-vial and the solution dried azeotropically by adding 0.5 mL of CH3CN and evaporating at 90o C under a stream of N2. Repeating the process two more times was necessary to obtain an anhydrous product. Ethyl 4-(trimethylammonium)benzoate trifluoromethane sulfonate, 10 mg in 200 μL anhydrous CH3CN was added to the vial containing the dried Kryptofix 2.2.2/K+ complex of [18F]F- and the solution heated to 90o C for 5 min. The ethyl ester was then hydrolyzed by adding 0.5 mL of 1 M NaOH and heating at 90o C for 5 min, before neutralizing with 0.8 mL of 1 M HCl. The neutral solution was diluted with 2 mL H2O and loaded onto an activated C18 Sep-Pak cartridge (Waters, Milford, MA, USA). The Sep-Pak cartridge was washed with 2 mL 0.1 M HCl to remove unlabeled 18F and the 4-[18F]fluorobenzoic acid (18F-FBA) was then eluted with 2 mL of CH3CN. The HPLC analysis, performed on a Vydac 218TP C18 5 μm column (Grace, Deerfield, IL, USA) using 1:1 0.08% TFA in CH3CN and 0.1% TFA in H2O as eluant at 1mL/min, showed the radiochemical purity of 18F-FBA to be over 98%.</p><p>To synthesize the active ester 18F-SFB, tetrapropylammonium hydroxide (15 μL, 1 M in H2O) and O-(N-succinimidyl) N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU, 10 mg) (Sigma-Aldrich) were added to the vial containing 18F-FBA in CH3CN and the vial sealed and heated at 90o C for 5 min. Acidification was instantaneous following addition of 3 mL of 5% HOAc followed by 6 mL of H2O. The mixture was loaded onto another activated C18 Sep-Pak cartridge. After washing with 10 mL of CH3CN/H2O (1:7, v/v) about 1.85×103 MBq of purified 18F-SFB was eluted with 2 mL CH2Cl2. The product was identified by comparison to a stable SFB sample by HPLC as above and was also shown to be over 99% radiochemically pure including radioactivity recovery.</p><!><p>The amine derivatived MORF oligomer (50 μg in 50 μl 0.3 M HEPES, pH 8.0) was added to the preparation vial containing 370 MBq 18F-SFB in 20 μL CH3CN and incubated at room temperature for 40 min with vigorously vortexing. The reaction solution containing the 18F-MORF was transferred to a 0.5 ml nonstick microfuge tube with 235 μg of streptavidin in 100 μl of normal saline slowly but with vigorous mixing. Because only about half the MORF obtained from the manufacturer was biotinylated, the MORF was added to the streptavidin at a 2 fold molar excess. The mixture was incubated at room temperature for 30 min. The Trastuzumab antibody was biotinylated as previously described and added at a 1:1 molar ratio to the MORF/streptavidin (13). After incubated at room temperature for an additional 30 min, the mixture was purified on an open G75 column with 0.1 M PBS as eluant. The fractions with peak radioactivity were combined.</p><!><p>The amine-derivitized MORF was conjugated with NHS-MAG3 as previously described (18). Because the preferred procedure for radiolabeling the MAG3-MORF with 99mTc involves heating at temperatures that could potentially denature streptavidin, the MORF was again radiolabeled before adding to streptavidin. The radiolabeling of the MAG3-MORF was achieved by adding about 407 MBq (120 μL) of 99mTc-pertechnetate generator eluant into a combined solution consisting of about 14 μg MORF in 26 μL of 0.25 M NH4OAc, 60 μL of 100 mg/mL Na2tartrate·2H2O in pH 9.2 labeling buffer (0.5 M NH4HCO3, 0.25M NH4OAc and 0.175 M NH3·H2O), and 15 μL of fresh 10 μg/μL SnCl2 dissolved in pH 8.7 labeling buffer. The final pH was about 8.5. After vortexing, the mixture was incubated at room temperature for 30 min followed by incubation at 95°C for 22 min. Purification was achieved by C18 Sep-Pak chromatography in which the first elution with 0.2 M NH4OAc removes radiolabeled pertechnetate and tartrate, the second elution with 40% CH3CN removes radiolabeled MORF while radiolabeled colloids are retained on the cartridge.</p><p>To prepare the nanoparticle, streptavidin (12.9 μL in PBS, 64.5 μg) was added to 50 μL of PBS in a 0.5 mL nonstick microfuge tube. After vortexing, the 99mTc labeled MORF solution was slowly added at a 2:1 molar ratio (again at a molar excess to compensate for approximately half the MORF that was received without a biotin group attached) with continuous stirring. After the addition, the solution was incubated at room temperature for 30 min followed by purification on an open G75 column. Fractions of 0.5 mL each were collected. To a combined fraction containing 4 μg streptavidin, 10 μg biotinylated Trastuzumab was added at a molar ratio of 1: 1, and, after incubated at room temperature for an additional 30 min, the product was drawn into a 1 mL U-100 Insulin Syringe for injection.</p><!><p>In any tomographic imaging approach, the distance of the object to be imaged from each pinhole, the diameter of the pinholes, the number of pinholes, the location of each pinhole and the number of projections all play important roles in determining detection sensitivity. In the case of the Mosaic HP PET animal camera, the detection sensitivity also depends upon the position of the object since sensitivity is maximum in the center of the FOV and decreases linearly down to zero at the edge of the FOV as more coincidences are lost when one or both of the annihilation photons escape.</p><p>Regarding resolution in pinhole SPECT imaging, the specifications of the multi-pinhole apertures, the intrinsic resolution of the crystal, the magnification employed in an imaging set up and the reconstruction settings combine to determine the reconstructed resolution. In the case of microPET imaging, in addition to the positron range that varies among PET imaging radionuclides, the reconstructed resolution depends on factors such as crystal pitch, non-collinearity of the annihilation photons caused by residual momentum of the positron, depth of interaction effects and the reconstruction methodology and settings (19).</p><p>Before proceeding to animals, resolution and detection sensitivities were compared using phantoms. To compare sensitivity, a 3 mL plastic syringe containing 18.5 MBq of 99mTc-pertechnetate in 2.5 mL H2O was inserted into a Lucite calibration phantom (Bioscan) with an inner and outer diameter of 1 and 2 cm respectively and the phantom was placed in the mouse bed of the NanoSPECT/CT camera. Images with a pixel size of 1 mm were recorded over 360o in 256x256 matrices with an acquisition time of 60 s per projection, resulting in a total of 24 projections per head over 30 min. The identical phantom was used to measure the PET sensitivity but with the syringe now containing 90 MBq of 18F in 2.5 mL H2O. The 18F phantom was put in the center of the Field of View (FOV) of the Mosaic camera and an emission-only acquisition was obtained at a 120 mm scan length. The total scanning time was 5 min. Images of coincidence counts with a pixel size of 1 mm were recorded in 128x128 matrices.</p><p>Because of the expected differences in resolution between the cameras, two phantoms (Data Spectrum Corp., Hillsborough, North Carolina, USA) of different sizes were required. The smaller phantom used with 99mTc was provided by Bioscan and was a similar but smaller version of the Jaszczak mini hot spot phantom, with hot rods of 1.2 to 1.7 mm. The larger phantom used with 18F was the Deluxe Mini Jaszczak phantom with hot rods of 1.2 to 4.8 mm. In each case the imaging protocols were similar to that described for the sensitivity measurements. The SPECT acquisition was obtained with 52 MBq of 99mTc in 24 min while the PET acquisition was obtained with 12 MBq of 18F in 5 min. Using a dose calibrator (Capintec Inc., NJ, USA) and a NaI(Tl) well counter, both calibrated for counts per minute per microcurie for both radionuclides as the standard of accuracy, radioactivity quantitation on both cameras was determined with five individual measurements for identical polyethylene tubes with a dimension of 12 mm and length of 75 mm containing from 0.74 to 9.25 MBq of 99mTc or 0.37 to 12.21 MBq of 18F.</p><!><p>All animal studies were performed with approval of the UMMS Institutional Animal Care and Use Committee. Two mice (30–40g, NIH Swiss, Taconic Farms, Germantown, NY, USA) bearing SUM190 (Her2+) tumors in one thigh were used when tumor size was just below 1 cm in any dimension. One animal was injected intravenously with 0.4 μg of 99mTc-MORF (13 MBq in 100 μl) as the MORF/Trastuzumab nanoparticle and tomographic imaging was performed at 3 and 9 h post administration on the NanoSPECT/CT camera. A CT acquisition was performed before each SPECT acquisition, at standard frame resolution, 45 kVp tube voltage and 500 ms of exposure time. About 4 min was required for each CT acquisition. The SPECT image parameters were again 1 mm/pixel, 256×256 frame size and 60 s per projection with 24 projections. Data were acquired in a step-and-shoot mode with the bed also stepping to include the whole body. Acquisition time was approximately 30 min. During imaging the animal was lightly anesthetized with 1.8% isoflurane in 1.5 L/min O2. The CT and SPECT reconstruction was performed using InVivoScope 1.37 software (Bioscan). The CT reconstruction was at standard resolution and SPECT acquisitions were reconstructed with a voxel size of 0.4 mm using the ordered subsets Expectation Maximization (OSEM) iterative reconstruction algorithm with 4 subsets and 6 iterations. During reconstruction high noise suppression with filtering was selected to achieve smooth and more artifact-free images. Finally, the SPECT/CT fusion image was obtained using the automatic fusion feature of the software. Region-of-Interest (ROI) analysis was also obtained using InVivoScope 1.37 software. The volume of interest (VOI) was obtained in the form of a cylinder, by first circling the ROI from the transverse profile, then selecting the length of the ROI from the maximum intensity projection.</p><p>Another tumored mouse was injected intravenously with 0.4 μg of 18F-MORF (0.22 MBq in 100 μl) as the identical MORF/Trastuzumab nanoparticle and PET imaging was performed at 2 and 6 h post administration on the Mosaic camera with 30 min acquisition time. The animal was lightly anesthetized during imaging as before. The PET mages were reconstructed without photon attenuation correction using the PETView program (Philips) with the fully 3D iterative reconstruction algorithm, giving a pixel size of 1 mm. Region-of-Interest (ROI) analysis was performed digitally using the Syntegra version 2.0j program (Philips). VOI was obtained from drawing the ROI in the slices of transverse profile, then interpolating the selected contours. After each PET acquisition, the mouse, immobilized on the Minerva bed (Bioscan) was transferred to NanoSPECT/CT camera for the CT acquisition and CT imaging and reconstruction was performed as before. The PET image DICOM files were transferred to the NanoSPECT/CT reconstruction workstation to provide the PET/CT fusion image.</p><!><p>The synthesis of 18F-MORF and the preparation of the MORF/Trastuzumab nanoparticle required about 4 h with a final yield of 0.55 MBq labeled to 1 μg of MORF (i.e. 0.55 MBq/μg) and in a nanoparticle with 9.5 μg streptavidin and 19 μg of Trastuzumab contained in 0.3 mL PBS.</p><p>The labeling of 99mTc-MORF and the preparation of the MORF/Trastuzumab nanoparticle was accomplished in 2 h with 13 MBq labeled to 0.4 μg of MORF (i.e. 33 MBq/μg) and in a nanoparticle with 4 μg streptavidin and 10 μg of Trastuzumab contained in 0.1 mL PBS. The radiochemical purity by size exclusion HPLC of both nanoparticles was over 99% as shown by a single peak and a radioactivity recovery of 100% in both cases.</p><!><p>The resolution obtainable by SPECT imaging of 99mTc on the NanoSPECT/CT camera and by PET imaging of 18F on the Mosaic HP PET camera are illustrated in Fig. 1. By recognizing the smallest visible size of hot rods in phantoms, the tomograms provide a value of 1.2 mm for the resolution of the SPECT image and a value of 2.4 mm for the resolution of the PET image.</p><p>While the spatial resolution of the NanoSPECT/CT camera was superior to that of the Mosaic HP PET camera, the opposite was true for sensitivity. Under conditions of this study, 2.47×108 counts were obtained in 5 min with 90 MBq of 18F to provide a sensitivity for the Mosaic HP PET camera of 9189 cps/MBq. By comparisons, 9.88×106 counts were obtained in 30 min with 18 MBq of 99mTc and therefore a sensitivity of 622 cps/MBq for the NanoSPECT/CT camera, a difference of about a factor of 15. However, in considering this comparison, it is important to appreciate that while the entire phantom was within the field of view at all times during acquisition on the Mosaic HP PET camera, this was not the case during acquisition on the NanoSPECT/CT camera. Because of the motion of both the gantry and the bed during acquisition, the SPECT sensitivity varies with the position of the phantom. This is illustrated in Fig. 2 in which the average counting rate from the four heads is presented individually for each of the 24 projections. From the 10th to 20th projection, the whole phantom was in the field of view, where the four detectors can see the total radioactivity of the phantom, resulting in maximum sensitivity for these projections.</p><p>Fig. 3 shows the agreement in radioactivity quantitation between well counting and PET (top panel) and SPECT (bottom panel) imaging. In both cases, the linear regression analysis generated a coefficient of determination (R2) of over 0.99.</p><!><p>The SPECT/CT fused images of the anterior (left panels) and left lateral (middle panels), are presented as maximum intensity projections in Fig. 4 from the 3 h (top row) and 9 h (bottom row) post injection acquisitions of one mouse. "Maximum Intensity Projections" (MIP) are defined by the manufacturer as that generated by all slices from all projections. The corresponding PET/CT fused images, also of the anterior (left panels) and left lateral (middle panels), are presented in Fig. 5 from the 2 h (top row) and 6 h (bottom row) post injection acquisitions of another mouse. All acquisitions required about 30 min.</p><p>Along with each image in both cases are presented a tomographic slice through the tumored thigh at the same level in both animals (right panels). Tumor accumulation in the left thigh is prominent in all images. Comparison of the early and late SPECT images provide evidence of rapid blood clearance, especially from the heart and carotid arteries, and slower clearance from the liver, kidneys and especially tumor. The pharmacokinetic behavior of the MORF/Trastuzumab two component nanoparticle is consistent with that previously reported for this 99mTc labeled nanoparticle (14). Comparison of the early and late PET images provides evidence of rapid blood clearance, especially from the heart and carotid arteries, and possibly slower clearance from the liver and especially tumor, although animal-to-animal differences cannot be excluded.</p><p>The percent of injected dosage in the whole body, kidneys, liver and in tumor, were estimated by quantitating the SPECT and PET acquisitions at both time points for each animal. Other organs were not included in this quantitation measurements either because of low accumulations or because of interference.</p><p>The results are presented in the histograms of Fig. 6 (SPECT) and Fig. 7 (PET). A comparison of the quantitation results show that the decay corrected whole body 99mTc radioactivity decreased from 8.66 MBq (66 % ID) to 7.28 MBq (56% ID) from 3 to 9 h while the 18F radioactivity decreased from 0.16 MBq (71% ID) to 0.15 MBq (66% ID) from 2 to 6 h. The results also show similar radioactivity accumulations at both time points in tumor, however, heart and liver shows much higher radioactivity accumulations of 18F. Surprisingly, the kidney accumulations are obviously much lower in the mouse receiving the 18F agent.</p><!><p>The superior sensitivity and excellent resolution of clinical PET compared to clinical SPECT cameras has advanced the field of nuclear medicine and the increasing availability of commercial PET clinical cameras provides opportunities to exploit new radiopharmaceuticals labeled with PET radionuclides for oncologic, cardiac, neurologic, etc. imaging (3). However, the large difference in subject size between human patients and small animals requires a different physics for the optimization of cameras performance. We therefore asked whether the advantages of PET over SPECT tomographic imaging in the clinic extend to the imaging of small animals such as tumored mice. Although not the object of this investigation, because of differences in camera performance, we also asked whether labeling our delivery nanoparticle with 18F would offer advantages over labeling with 99mTc. Because of the large size of the nanoparticle, the pharmacokinetics was expected to be largely radiolabel independent.</p><p>Apart from camera performance, there clearly are advantages to radiolabeling with 99mTc, usually the preferred SPECT radionuclide, compared to radiolabeling with 18F, often the preferred PET radionuclide (20–22). For those radiopharmaceuticals not commercially available and therefore requiring in-house preparation, radiolabeling by chelation with 99mTc will in all cases require less time and effort and less personnel exposure than radiolabeling with 18F. Use of a remote 18F automated synthesis apparatus would significantly increase the efficiency of the synthesis and decrease personal exposure but the effort would still not approach the simplicity of chelation labeling as shown in this research in the labeling of the MORF oligomer with 99mTc and 18F. To our knowledge, this investigation is the first to compare the Bioscan NanoSPECT/CT and Philips Mosaic HP PET cameras by imaging mice administered the same agent but with different radiolabels.</p><p>We recognize that arriving at a meaningful comparison between PET and SPECT imaging is complicated by the distinctly different detection physics involved. Regarding camera performance, whereas clinical PET cameras are reported to provide superior spatial resolution compared to SPECT cameras at least in the case of PET radionuclides such as 18F with relatively low maximum beta energies, the opposite was found to be the case for the small animal cameras of this investigation. A comparison of the PET and SPECT phantom images in Fig. 1 clearly shows the latter to provide superior spatial resolution. The NanoSPECT is a conventional gamma camera configured as a small animal imaging system using, in this case, four multi-pinhole aperture plates each with 9 pinholes of 1.4 mm rather than conventional parallel hole or fan-beam collimators. The superior spatial resolution is also evident in a comparison of the whole body images shown in Figs. 4 and 5. However, in agreement with the experiences with clinical cameras, the higher detection sensitivity of PET cameras extended to small animal cameras as well. Under the conditions of this study, two sources positioned in identical geometry in both cameras provided approximately 15 fold higher counts in the Mosaic HP PET camera compared to the NanoSPECT/CT camera. Accordingly, the images of Figs. 4 and 5 were obtained in animals injected with 13 MBq of 99mTc and only 0.22 MBq of 18F requiring 30 min for both SPECT and PET acquisition.</p><p>Since this investigation was more concerned with comparing camera performance than with comparing the pharmacokinetics of the two radiolabeled nanoparticles, only two tumored mice were imaged, relying upon the results of the phantoms studies to compare resolution, sensitivity and accuracy. Accordingly, the animal results are subject to uncertainties related to animal-to-animal variations. This may explain the small but important differences observed in the pharmacokinetics of the two nanoparticles. While tumor accumulations were identically high due to the targeting of the Her2+ SUM190 xenografts by the antiHer2 antibody within each nanoparticle, accumulations of 18F were higher in blood (heart) and liver at both time points. These differences were unexpected since the large size of the nanoparticle was expected to mask small differences in chemical properties related to the different labeling methods.</p><!><p>Radiolabeling of the MORF oligomer within the nanoparticle by chelation with 99mTc was considerably more efficient than radiolabeling with 18F via the manual synthesis with 18F-SFB. Thus when deciding between 99mTc vs. 18F as the radiolabel for the nanoparticle (and other similar biomolecules) for imaging on the NanoSPECT/CT and Mosaic HP PET small animal imaging cameras, while an advantage rests with chelation of 99mTc over covalent attachment of 18F, the choice of 99mTc trades lower sensitivity for higher resolution.</p><!><p>A tomographic image of a phantom with hot rods of 1.2 mm to 1.7 mm obtained on the NanoSPECT/CT small animal imaging camera (left panel) and a tomographic image of another phantom with hot rods of 1.2 mm to 4.8 mm obtained on the Mosaic HP PET (right panel) small animal imaging camera.</p><p>Detection sensitivity in cps/MBq vs. projection number showing variation due to the motion of both the gantry and the animal bed during imaging on the NanoSPECT/CT camera.</p><p>Radioactivity quantitation comparison between well counting and PET (top panel) and SPECT (bottom panel) imaging. Solid line connecting data points drawn by a linear regression analysis.</p><p>SPECT and fused CT projections obtained by imaging one mouse bearing a SUM190 tumor in the left thigh at 3 h (top row) and again at 9 h (bottom row) post injection of 100 μL (13 MBq) of 99mTc labeled nanoparticle with each acquisition requiring 30 min. Each row presents an anterior (left panel) and left lateral projection (middle panel), both Maximum Intensity Projections (MIP) and a transverse slice (right panel) of the acquisition centered on the tumor.</p><p>PET and fused CT projections obtained by imaging one mouse bearing a SUM190 tumor in the left thigh 2 h (top row) and again at 6 h (bottom row) post injection of 100 μL (0.22 MBq) of 18F labeled nanoparticle with each acquisition requiring 30 min. Each presents an anterior (left panel) and left lateral projection (middle panel), both Maximum Intensity Projections (MIP) and a transverse slice (right panel) of the acquisition centered on the tumor.</p><p>The percent injected dose (%ID) in the whole body, tumor and three normal organs at 3 and 9 h post administration of the 99mTc-MORF/Trastuzumab nanoparticle to a tumored mouse. Radioactivity quantitation was accomplished by using InVivoScope software.</p><p>The percent injected dose (%ID) in the whole body, tumor and three normal organs at 2 and 6 h post administration of the 18F-MORF/Trastuzumab nanoparticle to a tumored mouse. Radioactivity quantitation was accomplished by using Syntegra version 2.0j program.</p><p>Showing the preparation scheme for 99mTc and 18F labeling streptavidin nanoparticles.</p>

PubMed Author Manuscript

Optimization of phenolics and flavonoids extraction conditions of Curcuma Zedoaria leaves using response surface methodology

This study focused on maximizing the extraction yield of total phenolics and flavonoids from Curcuma Zedoaria leaves as a function of time (80–120 min), temperature (60–80 °C) and ethanol concentration (70–90 v/v%). The data were subjected to response surface methodology (RSM) and the results showed that the polynomial equations for all models were significant, did not show lack of fit, and presented adjusted determination coefficients (R2) above 99%, proving their suitability for prediction purposes. Using desirability function, the optimum operating conditions to attain a higher extraction of phenolics and flavonoids was found to be 75 °C, 92 min of extraction time and 90:10 of ethanol concentration ratios. Under these optimal conditions, the experimental values for total phenolics and flavonoids of Curcuma zedoaria leaves were 125.75 ± 0.17 mg of gallic acid equivalents and 6.12 ± 0.23 mg quercetin/g of extract, which closely agreed with the predicted values. Besides, in this study, the leaves from Curcuma zedoaria could be considered to have the strong antioxidative ability and can be used in various cosmeceuticals or medicinal applications.

optimization_of_phenolics_and_flavonoids_extraction_conditions_of_curcuma_zedoaria_leaves_using_resp

Background<!><!>Fitting the response surface models<!><!>Fitting the response surface models<!>Response surface analysis<!><!>Effects of process variables on the total phenolics content (TP)<!><!>Effects of process variables on the total flavonoids content (TF)<!><!>Conclusions<!>Raw materials<!>Plant extraction<!>Experimental design<!><!>Determination of total phenolic content<!>Determination total flavonoids content<!>Statistical analysis and optimization<!>Verification of models

<p>Plants are a substantial source of natural antioxidants. Active compounds present in natural antioxidants such as phenolic, carotenoids, flavonoids, folic acid, benzoic acid, and tocopherol are secondary metabolites of the plants which can provide various potential treatment and prevention of cancer, cardiovascular diseases, neurodegenerative diseases and etc. [1, 2].</p><p>Phenolics or polyphenols, including flavonoids, have received greater attention because they are often identified as biological response modifiers and have various functions such as metal chelators and free radical terminators [3, 4]. The bioactive compounds present in these compounds provide a variety of physiological functions, for instance, antimicrobial, antiallergenic, anti-inflammatory, and antimutagenic effects [5]. Moreover, it has been reported that the active compounds found in phenolic acids (caffeic, chlorogenic acid, benzoic acid) and flavonoids (catechin, quercetin, rutin) are potent antioxidants because they have all the right structural features for free radical scavenging activity [6, 7].</p><p>Curcuma zedoaria (Christm.) Roscoe. from Zingiberaceae family is popularly known as white turmeric, zedoaria or gajutsu [8]. This medicinal herb is largely found in East-Asian countries including Malaysia, Indonesia, China, India, Japan, Vietnam and Bangladesh [9]. Traditionally, zedoaria is hugely consumed as a spice, a flavoring agent, a tonic, a treatment for menstrual disorders, vomiting, toothache and it is also made into perfume [10, 11]. A study done by Angel et al. [12] reveals that zedoaria plants have a certain camphoraceous aroma and enormous functional active compounds such as essential oils, phenolics, and flavonoids which are strong components of anti-oxidant agent [12]. Meanwhile, Srivastava et al. [13] reported that Curcuma zedoaria is closely related to Curcuma longa. Therefore, the correlative isolated active compounds found in zedoaria such as curcumin, demethoxycurcumin and bisdemethoxycurcumin could be effectively used as antioxidant and anti-inflammatory, similar to Curcuma longa which is popularly used as antioxidant, antiulcer, anti-inflammatory, etc. Moreover, in vivo studies reported that the rhizomes of the plant possess potent antioxidant activity which exhibited higher radical scavenging activity [14].</p><p>The extraction of antioxidant compounds is a crucial process to determine the quantity and type of bioactive compounds, each with different therapeutic properties that will be extracted out. According to Aybastier et al. [15] many factors contribute to the efficiency of extractions such as the type of solvent, the concentration of solvent, temperature, time, pH and solid–liquid ratios. Response surface methodology (RSM) is a powerful mathematical technique being widely used in many industries for technological operations to optimize the experimental conditions. RSM is also useful to maximize or minimize various independent variables as it evaluates the effects of multiple factors and their respective interactions on one or more response variables simultaneously. Besides, RSM not only serves as a visual aid to have a clearer picture about the effects of various factors on extraction but also helps to locate the region where the extraction is optimized.</p><p>Therefore, the optimum extraction conditions (time, temperature and solvent ratio) to obtain the highest amount of phenolic and flavonoid compounds from Curcuma zedoaria leaves was identified using RSM technique. Despite numerous studies on rhizomes of zedoary which investigated its antioxidant activity, the leaves of the plant literally have not gained enough recognition and study to the best of our knowledge. In addition, Chanda and Nagani [16] reported that leaves, in general, are selected for the evaluation of total antioxidants activity due to high content of bioactive compounds.</p><!><p>The experimental data obtained for the three responses based on the CCD matrix</p><!><p>The final empirical regression model of their relationship between responses and the three tested variables for phenolic and flavonoid contents could be expressed by the following quadratic polynomial equation [Eqs. (1–2)]:1\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$egin{aligned} Phenolic \, content &= 122.36 + 5.74X_{1} + 2.03X_{2} + 4.10X_{3} \ &\quad - 4.11X_{1} X_{2} - 1.62X_{1} X_{3} -2.77X_{2} X_{3} \& \quad + 1.34X_{1}^{2} - 1.19X_{2}^{2} - 3.55X_{3}^{2} \end{aligned}$$\end{document}Phenoliccontent=122.36+5.74X1+2.03X2+4.10X3-4.11X1X2-1.62X1X3-2.77X2X3+1.34X12-1.19X22-3.55X32 2\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$egin{aligned} Flavonoid \, content &= 6.23 - 0.013X_{1} - 0.016X_{2} + 0.091X_{3} \ & \quad - 0.08X_{1} X_{2} - 0.064X_{1} X_{3} + 0.039X_{2} X_{3}\ &\quad imes 0.05X_{1}^{2} + 0.021X_{2}^{2} - 0.070X_{3}^{2} \end{aligned}$$\end{document}Flavonoidcontent=6.23-0.013X1-0.016X2+0.091X3-0.08X1X2-0.064X1X3+0.039X2X3×0.05X12+0.021X22-0.070X32where X 1 is the temperature, X 2 is the time and X 3 is the ethanol concentration ratio. A negative sign in each equation represents an antagonistic effect of the variables and a positive sign represents a synergistic effect of the variables.</p><!><p>Analysis of variance (ANOVA) for the model</p><!><p>The performance of the models was also checked by calculating the determination coefficients R 2, adjusted R 2, regression (p value), regression (F-value), lack of fit (p-value), coefficient variation (C.V%) and probability values related to the effect of the three independent variables. Based on the result, the coefficient of determination R 2 is defined as the ratio of the explained variation to the total variation in total phenolic and total flavonoid contents were R 2 = 0.9993 and R 2 = 0.9952 respectively showing a good fit model. The closer R 2 value to unity, the better and significant empirical model fits the actual data. Furthermore, the calculated adjusted R 2 values for studied responses variables were higher than 0.80, hence there is a close agreement between the experimental results and the theoretical values predicted by the proposed models. The coefficients of variations (C.V) for total phenolic and flavonoid contents were 0.24 and 0.17 respectively, which indicates that a relatively lower value of CV showed a better reliability of the response model. It was observed that the lack of fit gave no indication of significance (p < 0.05) for all the models tested, thus proving that the satisfactory fitness of the response surface model was within the chosen range and significant (p < 0.05) to the factors effect.</p><p>Based on analysis of ANOVA, any terms from quadratic polynomial coefficients model, large F-values and a small p-values indicated a more significant effect on the respective response variables. The 3-D surface plots of the fitted polynomial regression equations were generated by the software to better visualize the interaction effect of independent variables on responses.</p><!><p>Temperature, time and ethanol concentration are the main factors that affect the extraction condition of the maximum total phenolics and flavonoids content. This section discusses how these conditions work on natural antioxidants extraction. Three-dimensional model graphs were plotted as shown in the respective figures. The response surface plots of the model were done by varying two variables, within experimental range under investigation and holding the other variables at its central level (0 levels).</p><!><p>Response surface plots for the effects of temperature, time and ethanol concentration on total phenolic contents of Curcuma zedoaria leaves extracts. a Temperature versus time. b Ethanol concentration versus temperature. c Time versus ethanol concentration</p><!><p>The surface plot in Fig. 1a demonstrates the function of temperature (X 1) versus time (X 2) effect on total phenolic contents at fixed ethanol concentration (80:20). It was observed that increasing the extraction temperature and time resulted in higher phenolic content in Curcuma zedoaria leaves. The maximum amount of phenolics can be achieved at the highest temperature of 75–80 °C at the shortest extraction time of 80–100 min. Nevertheless, when the temperature was kept at the highest level of 80 °C with longer extraction time at 120 min, they did not show any significant improvement in TP extraction as the value continuously dropped. This agreed with the working high temperature employed in this study which required short periods of time to avoid the degradation of the phenolic compounds. At short periods of time, the temperature enhanced the extraction process but for relatively long periods, the effect is inverted and the phenolic compounds are threatened by oxidation or degradation [17]. Moreover, according to Vajić et al. [18] prolonged time of extraction enhances phenolic solubility due to Fick's second law of diffusion which predicts that equilibrium of extraction will be achieved after a certain time. These results are similar to a study reported by of Rajha et al. [19] which showed the total phenolics from grape byproducts increased with the increment of temperature and reduction of time.</p><p>Figure 1b depicts the effects of temperature (X 1) versus ethanol concentration (X 3) at constant extraction time 100 min. The surface plot reveals that the maximum phenolic content can be achieved at highest ethanol concentrations (90:10) as compared with low ethanol concentrations (70:30) at fixed extraction temperature. The higher phenolic content could be explained by the natural polarity of the solvents used [20]. Ethanol and water were used in this study because they are safer to handle as compared to other organic solvents and more importantly, they are acceptable for human consumption. Samuagam et al. [21] stated that a suitable solvent ratio is able to improve the efficiency of extraction. The surface plots also reveal that by increasing the extraction temperature to higher levels, the amounts of phenolic gradually dropped and this might be explained by the fact that the final equilibrium between the solvent concentrations in the plant matrix and the temperature will be achieved after a certain concentration level [22]. This phenomenon is similar to a phenolic study from lettuce by-products which can be explained by the use of higher temperature and adequate solvent concentrations which may cause softening of plant tissue, resulting in enhanced diffusion rate and increase in the production of phenolic compounds. However, after a certain level, it will subsequently decline and remain constant as the extraction has completed and they have achieved their equilibrium state [23]. Therefore, the maximum total phenolic content in Curcuma zedoaria leaves can be obtained with optimum ethanol concentration and an extraction temperature of approximately 80–85 v/v% and 75–80 °C respectively.</p><p>The response surface plot as a function of time (X 2) versus ethanol concentration (X 3) at constant temperature 70 °C is presented in Fig. 1c. The surface plots revealed that the higher TP contents can be obtained when conducted at increasing ethanol concentration at fixed extraction time. Based on the result at constant extraction time of 120 min, 90% of ethanol concentrations yielded the most TP as compared with 70% ethanol concentrations. However, longer extraction time degrade the phenolic activity in Curcuma zedoaria leaves. Therefore the optimum extraction of phenolic can be obtained when conducted at a range of 80–90 v/v% and 100 min of ethanol concentrations and extraction time respectively. Beyond this optimal, the TP content declined. These overall results of phenolic content indicate a similar trend as observed in the phenolic content of tea (camellia sinensis L.) fruit peel by Xu et al. [24] where the TP contents increased with increasing the independent variables ethanol concentration and processing time until a maximum amount of phenolic was reached, thereafter, the amount subsequently declined rapidly as reaction has completed.</p><!><p>Response surface plots for the effects of temperature, time and ethanol concentration on total flavonoid content of Curcuma zedoaria leaves extracts. a Temperature versus time. b Ethanol concentration versus temperature. c Time versus ethanol concentration</p><!><p>The 3D shows the response surface plot as a function of temperature (X 1) versus time (X 2) at fixed extraction ethanol concentration (80:20) as shown in Fig. 2a. Response surface plot showed that extraction temperature exhibited a weaker effect whereas extraction time represented a relatively significant effect on the flavonoids yield. An increase in the yield of flavonoid could be significantly achieved with the increase of extraction time, at any level of extraction temperature. Therefore, the optimum amount of flavonoid was achieved in this study at 65–70 °C and 90–100 min of extraction time. However, the results of the present research for time and temperature were different compared with other studies [4, 19]. This difference could be the due to differences in the type of material, considering some plants may synthesize and accumulate the different amount of secondary metabolites (flavonoids) and also the optimization extractions range used in the study.</p><p>The 3D surface plots in Fig. 2b shows the interaction between extraction temperature (X 1) and ethanol concentration (X 3) at the fixed 100 min. Statistical analysis reveals that the most significant with p < 0.0001 in TF was ethanol concentration. According to Bazykina et al. [25] flavonoids and their glycosides are thought to be efficiently extracted from plant materials by ethanol solvent. It was observed that the value of TF increased when ethanol concentration was increased from 70 to 90 v/v% at fixed 60 °C extraction temperature. In contrast, increasing the extraction temperature at highest ethanol concentrations resulted to decreased, TF values. This phenomenon can be explained by the higher movement of the particles which causes plant tissue to rupture and hence allowing higher solubility of solvent until it starts to degrade to a lower value as it had achieved the stable state [26]. The results obtained for flavonoids are in agreement with the previous report from Cryptotaenia japonica hassk by Lu et al. [27] where the flavonoid content increased when the temperature of extraction increased to below 70 °C and exhibited a decreasing trend above the optimum level of temperature. Thus, as mentioned earlier the optimum extraction temperature for maximum flavonoid content was at 65–70 °C with 85–90 v/v% ethanol concentrations.</p><p>Figure 2c illustrates the response surface plot between the extraction time (X 2) and ethanol concentration (X 3) at constant extraction temperature (70 °C). The response surface plots demonstrated that the value of TF obtained in Curcuma zedoaria leaves mainly depended upon ethanol concentrations. An increase in ethanol concentration promoted the breakdown of the cell membrane that enhanced the permeability of the solvent into a solid matrix. In this study, highest flavonoids content can be achieved when conducted at highest ethanol to water ratio (90:10) as compared with (70:30) with increasing extraction time. A great increase in the yield also resulted when extraction time was increased in the range of 80–120 min. However, the time curve started to level off at 100 min, which indicated that 100 min were required to achieve maximum flavonoids activity.</p><!><p>Comparison between the predicted and experimental values for antioxidants from extracts of Curcuma zedoaria leaves</p><!><p>Response surface methodology (RSM) and a design called central composite design (CCD) were successfully developed to determine the optimum process parameters and the second order polynomial models for predicting responses were obtained. The best combination of extraction temperature, time and ethanol concentrations were found to be 75 °C with 92 min and 90:10 ethanol to water ratio which rendered a mean phenolic content of 125.75 ± 0.17 mg/g GAE and 6.12 ± 0.23 mg/g QE of total flavonoid content from experimental run and thus indicated good antioxidant activities from the leaves of Curcuma zedoaria.</p><!><p>Curcuma zedoaria leaves were collected from a local farmer in Kedah, Malaysia. The chemicals, sodium carbonate, aluminium chloride, ethanol was purchased from J. Kollin Chemicals, Germany. Folin-Ciocalteu's phenol reagent, gallic acid and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemical reagents used in this study were of analytical grade class.</p><!><p>The air-dried leaves of Curcuma zedoaria plant were cut into pieces and ground into powder form using a mechanical blender. About 0.5 g of powdered leaves were exactly weighed into a 150 mL round bottomed flask and mixed with ethanol. The extraction process was performed using a reflux systems equipped with a temperature controller and digital timer. The extract was then filtered through normal filtration using Whatman filter paper and vacuum-dried in a rotary evaporator, at 40 °C until the excess solvent was completely removed.</p><!><p>The optimization of the extraction conditions from the Curcuma zedoaria leaves was established by using response surface methodology (RSM). This powerful mathematical and statistical technique is useful for modeling and analysis of problems in which a response is influenced by several independent variables and the objective is to find the relationship between the factor and the response to optimize the conditions. A design expert software Version 7.0.0, (Stat ease Inc., Minneapolis, USA) was used in this study. The experimental plan was carried out based on three factor/five level design referred to as rotatable central composite design (CCD). The selection of CCD as the experimental design is because it is more precise for estimating factor effects [28]. Hence, the interaction effect between factors can be evaluated and optimized in the full factor space.</p><!><p>Independent test variables and their coded and uncoded value used for CCD matrix</p><!><p>The total phenolic compounds in Curcuma zedoaria leaves was developed using the method of Singleton and Rossi [29] with minor modifications. For each sample, 100 μL (1 mg/mL) of the sample extract was mixed with 50 μL Folin-Ciocalteu's reagent (2 N) previously diluted with 7.9 mL distilled water. After 4 min, 1.5 mL of 7.5 w/v% sodium carbonate solution was added to the mixture and incubated in the dark room at room temperature for 2 h. The absorbance values of the sample were measured at 765 nm using a UV–VIS microplate reader. Standard of gallic acid with different concentrations (25–1000 μg/L) was prepared in this study to generate a standard calibration curve. The samples were calculated based on the standard calibration curve and were expressed as mg gallic acid equivalent (mg/g GAE).</p><!><p>The content of flavonoid in the studied leaves extract was determined using spectrophotometric method [30]. From each sample, 100 μL (1 mg/mL) were mixed with 2% AlCl3 and incubated for 15 min at room temperature. The absorbance was measured at λ = 406 nm. The same procedure was repeated for the standard solution of quercetin at different concentrations (25–250 μg/mL) and the calibration line was obtained. Based on the measured absorbance, the concentration of flavonoids was calculate (mg/mL) on the calibration line and the content of flavonoids in extracts was expressed in terms of quercetin equivalent, QE (mg of quercetin/g of extract).</p><!><p>Best fitted model of response can be achieved by highlighting these statistical parameters including the adjusted multiple correlation coefficients (adjusted R 2), multiple correlation coefficients (R 2), coefficient variation (C.V%), lack of fit, regression F-value and regression p-value by using analysis of variance (ANOVA). This statistical approach was used to summarize the results obtained under all experimental conditions with a confidence interval of 95% set to test the significant effect of the factors and their interaction. The optimal extraction conditions were selected based on the condition of achieving the highest total phenolics and flavonoids content in Curcuma zedoaria leaves by using the desirability function approach in design expert software. The fitted polynomial equation was expressed in the form of three-dimensional surface plots in order to illustrate the relationship between responses and the experimental variables used.</p><!><p>The optimal conditions for the extraction of the total phenolic and flavonoid content from Curcuma zedoaria leaves, in terms of extraction temperature, time and ethanol concentrations, were determined by comparing the actual experimental values with predicted value from the final response regression equations. Besides, a few random extraction conditions were prepared in order to validate the models. This action is of utmost importance to confirm the adequacy of the final reduced models.</p>

PubMed Open Access

Atomistic simulation of lipid and DiI dynamics in membrane bilayers under tension

Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from \xe2\x88\x922.6 mN m\xe2\x88\x921 to 15.9 mN m\xe2\x88\x921. Simulations showed that the bilayer thickness decreased linearly with tension consistent with volume-incompressibility, and this thinning was facilitated by a significant increase in acyl chain interdigitation at the bilayer midplane and spreading of the acyl chains. Tension caused a significant drop in the bilayer\'s peak electrostatic potential, which correlated with the strong reordering of water and lipid dipoles. For the low tension regime, the DPPC lateral diffusion coefficient increased with increasing tension in accordance with free-area theory. For larger tensions, free area theory broke down due to tension-induced changes in molecular shape and friction. Simulated DiI rotational and lateral diffusion coefficients were lower than those of DPPC but increased with tension in a manner similar to DPPC. Direct correlation of membrane order and viscosity near the DiI chromophore, which was just under the DPPC headgroup, indicated that measured DiI fluorescence lifetime, which is reported to decrease with decreasing lipid order, is likely to be a good reporter of tension-induced decreases in lipid headgroup viscosity. Together, these results offer new molecular-level insights into membrane tension-related mechanotransduction and into the utility of DiI in characterizing tension-induced changes in lipid packing.

atomistic_simulation_of_lipid_and_dii_dynamics_in_membrane_bilayers_under_tension

Introduction<!>Simulation methodology<!>Results and discussion<!>Tension induces bilayer thinning and interleaflet interdigitation<!>Tension reduces lipid acyl chain packing and order<!>Tension reduces the electrostatic potential barrier through lipid/water dipole reordering<!>Moderate tension increases lipid lateral diffusion by increasing free-area, but free-area theory does not hold for large tensions<!>Changes in lipid packing are reflected in changes in DiI diffusion and rotation<!>DiI sensitivity to membrane tension may be revealed in fluorescence lifetime measurements<!>Conclusions<!>

<p>Mechanical forces modulate cell growth, differentiation, signal transduction, transport, and migration, through biochemical signaling pathways1 which may be related to membrane molecular organization and dynamics.2,3 For example, lateral membrane tension causes conformational changes in integral membrane proteins,3 and affects membrane permeability,4,5 lipid lateral diffusion,2,6 and organization of lipid rafts.7,8 These effects are believed to be mediated by bilayer thickness changes that result in lipid phase separation or hydrophobic mismatch between the lipid acyl chains and transmembrane region of proteins, leading to distortion of the lipid bilayer and concomitant protein conformational changes.9–11</p><p>Despite the importance of lipid dynamics in cell signaling, to date the only experimental studies quantifying the relationship between lipid dynamics and force have been conducted in sheared endothelial cells2,6,12 and in hair cells.13,14 In these studies, a lipoid dye, such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 9-(dicyanovinyl)-julolidine (DCVJ), or di-8-ANEPPS, was used to infer lipid dynamics from fluorescence intensity or fluorescence recovery after photobleaching (FRAP). Because these studies probed lipid dynamics indirectly and because the precise membrane tensions, at the molecular level, were unknown, there is a need to quantify directly the relationship between membrane tension and lipid dynamics.</p><p>The most prominent methods to assess lipid dynamics, including FRAP, fluorescence correlation spectroscopy (FCS), fluorescence anisotropy, and fluorescence lifetime imaging15–17 probe membrane lipid dynamics by analyzing the dynamics of lipophilic fluorescent dyes (e.g. DiI, 1,6-diphenyl-1,3,5-hexatriene (DPH), and Laurdan). In particular, DiI is popular because of its structural similarity to phospholipids and its ability to selectively partition into different lipid phases (gel or fluid) depending on the matching between the length of its alkyl chains and the lipid acyl chain length.18 Spectroscopic investigations employing DiI have been used to study membrane organization and dynamics.19,20 The fluorescence lifetime of DiI depends on the accessibility to water21 and on the viscosity of the local microenvironment,19 offering a useful tool to detect lipid rafts in cells and phase separation in model membranes. However, proper interpretation of these fluorescence measurements requires precise knowledge of location, orientation, and interactions of dye with lipids and water, which are difficult to obtain experimentally.22,23 Examples of the utility of using molecular dynamics (MD) simulation as a tool to answer these questions include predictions of the location of drug-like small molecules in lipid bilayers along with validation by small-angle neutron scattering experiments.24,25</p><p>The aim of this computational modeling study was to determine the effects of membrane tension on mechanotransduction-related structural and dynamical properties of the bilayer. In addition, we wished to understand the fidelity with which DiI, a popular membrane probe, reflects lipid dynamics, so that DiI photophysics could be used as a readout for tension effects on stressed membranes. To accomplish this goal, we performed a series of atomistic molecular dynamics (MD) simulations of fluid-phase dipalmitoylphosphatidylcholine (DPPC)/DiI bilayers under various physiological tensions. The main readouts from this study are as follows. First, we characterized the effects of tension on bilayer thickness, acyl chain packing, interdigitation, and electrostatic potential. Second, we determined the relationship between area-per-lipid and lipid lateral diffusion, and compared these results to predictions from free-area theory. Third, we compared the DiI probe dynamics to the dynamics of the native lipids, leading to an analysis of the relationship between lipid packing and fluorescence lifetime of DiI in terms of hydration and local viscosity.</p><!><p>Force field parameters for DPPC and DiI-C18 were identical to Berger et al.26 Bond lengths and bond angles of the DiI's headgroup were obtained from X-ray crystallography data of a structurally similar carbocyanine dye.27 Simple point charge (SPC) model was used for water.28 The partial charges for the DPPC molecules were identical to those described by Chiu et al.,29 whereas, the partial charges on the DiI molecule were obtained by performing ab initio quantum mechanical calculations using Gaussian 03 software package with the Hartree–Fock method and the 6–31G basis set using the charge partitioning scheme of Merz–Kollman.22,29</p><p>A simulation box of well-equilibrated pure DPPC bilayer consisting of 128 DPPC molecules and 3655 water molecules was obtained from Tieleman and Berendsen.28 The DiI-C18 dye was incorporated in the DPPC bilayer by replacing two DPPC molecules with a single DiI-C18, in each leaflet. To ensure electrical neutrality, two chloride ions were added to the system. To make sure that the equilibrium position of the dye was independent of its initial position, the dye headgroups were placed at random z-locations (above and below the lipid–water interface) and the system was equilibrated. Initial configurations of the simulation box with varied area-per-lipid (α) were constructed by scaling the original system, while maintaining constant volume, and the systems were equilibrated as described below. Membranes were simulated at seven different area-per-lipid values ranging from 0.635 to 0.750 nm2, corresponding to tensions ranging from −2.6 to 15.9 mN m−1. Final production runs were performed on NPzAT ensemble. This ensemble was chosen in order to conveniently apply tension by prescribing area per lipid. Other ensembles such as NPT or NPzgT ensembles could have been chosen and these would give equivalent structural and dynamical properties.28,30</p><p>Molecular dynamics simulations were carried out using the GROMACS software package (version 3.3.2).31,32 Pre-equilibration of energy minimized structures was performed under NVT conditions, at 323 K, for a simulation time of 1 ns, allowing for removal of any overlaps or defects caused by DiI placement and the application of tension. Subsequent equilibration of the structures was performed under NPzAT conditions, at 323 K and 1 bar normal pressure, for a total simulation time of 100 ns. Final production runs were carried out under NPzAT conditions for an additional 100 ns for each system configuration. Periodic boundary conditions were applied in all three coordinate dimensions. The temperature of the production runs was 323 K, which is above the gelto-liquid phase transition temperature, Tm, of DPPC (~315 K). Temperature and pressure were controlled using Berendsen's weak coupling method with the time constants set to 0.1 ps and 1.0 ps respectively.33 Semi-anisotropic scaling was used for pressure coupling with zero compressibility in xy-plane to maintain the area constant. The LINCS algorithm was used to constrain the bond lengths, allowing for larger time steps than if the bonds were unconstrained.31,32 The Particle-Mesh Ewald (PME) method was used for electrostatic interactions, with a direct-space cutoff of 1 nm,34,35 and cubic interpolation (PME order = 4) for the calculation of long-range interactions in reciprocal space, with a Fourier transform grid of 0.12 nm maximum. Despite its computational cost, PME was chosen because it allows for proper electrostatics in systems with charged molecules and ions.36,37 The Lennard-Jones interactions were cutoff (shifted and truncated) at 1.0 nm. A time-step of 2 fs was used with a leap-frog integration algorithm for the equations of motion, accommodating bond constraints and weak coupling to constant T and P baths.31,32</p><!><p>In this study, we addressed how membrane tension or, equivalently, how lipid packing affects structural and dynamical properties of a fluid-phase DPPC bilayer by performing a series of united-atom molecular dynamics (MD) simulations. Various physicochemical properties of the bilayer were analyzed as a function of area-per-lipid (α). We included DiI-C18 in our simulations to facilitate the interpretation of lipid packing and dynamics from single-molecule fluorescence measurements of lateral and rotational diffusion, and fluorescence lifetime of DiI, a popular membrane probe. Perturbative effects of DiI on the lipid bilayer were studied previously by Gullapalli et al.22 Results are divided into five subsections: structural changes of lipid bilayer under tension, tension-induced changes in lipid order, electrostatic potential and lipid/water dipole ordering, lipid lateral diffusion and free-area theory, and sensitivity of DiI dynamics to membrane tension.</p><!><p>Surface tension was estimated from the pressure tensor, as described in ref. 38. As expected, the surface tension increased linearly with an increase in area, from −2.6 mN m−1 at α = 0.635 nm2 to 15.9 mN m−1 at α = 0.750 nm2, above which rupturing of the bilayer was observed (rupture data not shown). While this rupture tension is in good agreement with values from micropipette aspiration of lipid vesicles (ranging from 10 to 20 mN m−1)5 MD simulated rupture and experimental rupture tensions often differ because rupture/pore tension depends strongly on the loading rate, which is inherently larger in MD simulations.39 Thus within the range of tensions simulated in this study, pore formation or rupture cannot be observed in the size/time scales studied here.40 Zero surface tension corresponded to α = 0.646 nm2, close to the experimental value of 0.64 nm2 for DPPC.41 In addition, the area compressibility modulus calculated from the tension–area plot38 was 105 mN m−1, in good agreement with the previous simulation value of 107 mN m−1 for DPPC bilayer at 50 °C.38 Experimentally, a compressibility modulus of 234 mN m−1 was reported for DMPC lipid at room temperature.42 Using an identical force field to the current simulations, Lindahl and Edholm43 reported a simulated value of 250–300 mN m−1 for a larger membrane patch (1024 lipids), suggesting that the lower value in the current study is likely due to the finite-size effect.5 Considering the empirical nature of the force field parameters, these results indicate that the simulation methodology is sufficiently accurate in determining the microscopic and macroscopic properties of the lipid bilayer over an extended range of simulated tensions.</p><p>Bilayer thickness, defined as the distance between water and lipid density crossover points on either side of the bilayer, was directly computed from the mass density profiles (Fig. 1).23 The bilayer thickness decreased linearly with increases in area-per-lipid, consistent with volume-incompressibility (Table 1). The density profile of the bilayer is highly reminiscent of a confined film36,37—rather than a constant density bulk fluid—and thus changes in bilayer thickness are expected to result in structural reorientations within the bilayer.44 In support of this interpretation, it was observed that increasing the surface area resulted in a decrease of the lipid density at the headgroup region and a concurrent increase in the local density at the mid-plane of the bilayer (Fig. 1). This indicates increased interdigitation of the acyl chains of the opposing leaflets due to extension of the chains beyond the bilayer mid-plane. Increased interdigitation has physiological implications; for example, acyl interdigitation has been proposed to result in the formation of membrane micro-domains.45 Also, interdigitation of the acyl chains can alter the hydrophobic interactions and lateral pressure profile of the bilayer, which in turn can alter protein conformation.46,47 Spreading of the acyl chains also takes place upon decrease in bilayer thickness (see below).</p><!><p>The effect of membrane tension on lipid chain order was determined by computing the order parameter of sn-1 and sn-2 chains separately, as a function of the chain carbon atom number, in accordance with our previous publication.22 The absolute value of the order parameter, |SCD|, can vary from 0.5 (high ordering) to 0 (low ordering). Order parameter profiles for the sn-1 and sn-2 chain as a function of area-per-lipid are shown in Fig. 2A and 2B. For α = 0.635 nm2, SCD versus carbon chain number exhibited the typical trends with a plateau region near the headgroup (with an average order of 0.2 ± 0.02) which gradually dropped to near zero at the terminal methyl groups of the tails. These values are in good agreement with the values reported previously from experiments48 and simulations.26,35 Increasing the surface area caused a significant decrease in the order parameter values of both sn-1 and sn-2 chains throughout the length of the carbon chain. Changes in the order parameter with tension were smallest (~30%) near the headgroup region and largest (~50%) at the terminal tail region.</p><p>Changes in the SCD order parameter resulted from the combined effect of tension on the acyl chain dihedrals and on the chain tilt angle.49 To determine the relative magnitude of tension-induced changes in these parameters, we calculated the average trans fraction of the chain dihedrals, and the average angle between the end-to-end acyl chain vectors. In general, the average trans dihedral fraction was largest near the headgroup region and decreased towards the terminal tail region (a strong drop was observed in the last dihedral of both the acyl chains). Although measurable decreases in the average trans fraction of the chain dihedrals were observed with increased area-per-lipid, these changes were small (<3%, shown in Fig. 2C) and there was no change in the qualitative trend. This result strongly suggests that the acyl chain configuration is not markedly affected by the application of tension. On the other hand, when we analyzed the distribution of angles between the end-to-end sn-1 and sn-2 chain vectors, shown in Fig. 2D, the peak angle gradually increased from 36 degrees (for α = 0.635 nm2) to 57 degrees (for α = 0.750 nm2), with applied tension. This result clearly denotes that a significant "spreading" of the chains takes place with increases in the area-per-lipid. We can conclude that the tension-induced decrease in the SCD order parameter is primarily due to an increase in the spreading of the acyl chains, i.e., changes in the acyl chain orientation within the bilayer, rather than due to any changes in the acyl chain conformations (i.e. trans and gauche fractions). Similar molecular shape changes of lipid were simulated previously in the outer leaflet of high curvature liposomes.50</p><!><p>The electrostatic potential at the lipid–water interface results from the orientation of the water and lipid dipoles. To determine this electrostatic potential, the ensemble-averaged charge density along the z-axis was computed. Fig. 3A depicts the individual contributions of lipids and water to the total charge density. Due to symmetry, only half of the simulated membrane is shown in the figure. Compensation of the dipole potential of the lipid molecules by water resulted in a negative potential in the bulk water with respect to the bilayer interior. The corresponding electrostatic potential with respect to the center of bilayer was computed by double integration of the time-averaged charge density, ρ(z), using Poisson's equation: (1)V(z)=φ(z)−φ(0)=−1ε0∫0zdz′∫0z′ρ(z″)dz″ where ϕ(z) and ρ(z) are the time-averaged dipole potential and the charge density, respectively, as a function of the distance normal to the bilayer, and ε0 is the permittivity of vacuum. ϕ(0) is the dipole potential at the bilayer center that acts as a reference point. The resulting potential profiles for different values of the area-per-lipid, are shown in Fig. 3A. In the case of α = 0.635 nm2, the potential difference between the center of the bilayer and water was −615 mV. This value is consistent with previous simulations of DPPC bilayers.22 Experimentally, values for the potential difference vary from −200 to −575 mV for various phosphocholine–water interfaces.35 With increasing lateral tension, the following effects in the electrostatic potential profiles were observed (illustrated in Fig. 3A): First, the potential difference between the bilayer interior and bulk water decreased from 615 mV (for α = 0.635 nm2) to 588 mV (for α = 0.750 nm2). Similar results have been reported by Skibinsky et al., where lowering of surface tension by addition of trehalose induces an increase in electrostatic potential.51 Second, the positive potential barrier at the lipid–water interface, which originates from the strong ordering of water molecules around the phosphoryl groups of the lipids, was reduced in magnitude and shifted in position away from the bilayer center. The height of the potential barrier with respect to bulk water is shown in Table 1. The potential barrier reduced by ~80 mV at the highest tension simulated. These tension-induced changes in electrostatic potential at the lipid–water interface may have physiological significance, as a decrease in the electrostatic potential of ~30 mV can cause significant increase in ion transport across the lipid bilayer.52,53</p><p>The ordering of the water dipole was quantified by calculating the time-averaged projection of the water dipole unit vector onto the interfacial normal (z-axis, Fig. 3B). A value of 〈 cos θ〉 = −0.5 corresponds to perfect ordering of the water dipoles parallel to the membrane normal, whereas a value of 〈 cos θ〉 = 0 corresponds to random water dipole orientation (disordered state). In general, high ordering of the water dipoles along the membrane normal is observed near the phosphorous atoms of the lipids.35 Away from the interface, the ordering persists in the z-axis until the point where the lipid density becomes zero. Increasing the membrane tension resulted in decreased water dipole ordering around the lipids, as seen by an increase in the 〈cos θ〉 minimum. This decreased water dipole ordering correlatesi well with the reduced potential barrier at the lipid–water interface and explains the previously observed changes in the electrostatic potential profile at the lipid–water interface. Also, a slight shift was observed in the location of the water dipole where ordering towards the membrane surface was highest (Fig. 3B). This shift correlates well with the spatial shift observed in the electrostatic potential profile.</p><p>The ordering of the lipid dipoles at the lipid–water interface was quantified by calculating the angular distribution of the P–N vector with respect to the bilayer normal (Fig. 3C). In general, the peak of the P–N vector angle was around 90°, indicating that the P–N vector was aligned parallel to the lipid–water interface. Increasing the area-per-lipid resulted in a slight decrease of the peak angle value, as evidenced by the peak shifts of the P–N vector from 90° (for α= 0.635 nm2) to 82° for the largest tension simulated (for α = 0.750 nm2) (Fig. 3C). That is, increases in tension resulted in a tilt in the P–N vector towards the bilayer normal. Considering both the water and lipid dipole orientations, we can conclude that both water and lipid dipole reordering contribute to the observed changes in electrostatic potential profiles with tension. Based on these results we predict that mechanical stretching of the lipid bilayer can result in significant changes in small-molecule diffusion through the bilayer, due to reduced resistance to diffusion accompanying reductions in electrostatic potential. This novel mechanism of mechanosensitivity of cell membranes is worthy of further simulation and experimental testing.</p><!><p>Lateral diffusion coefficients (D) were computed from the mean-squared displacement (MSD) of the center-of-mass (COM) motion of the molecules. The MSD was ensemble averaged and calculated for multiple time-origins, and D was quantified through Einstein's equation: (2)D=limt→∞12dt〈[r→1(t+t′)−r→1(t′)]2〉 where, ri are the x,y positions of the center of mass of a lipid i at a given time t′ and after a time interval t (i.e., at time t + t′); d is the dimensionality of the motion considered (here d = 2 for the in-plane lateral diffusion); the brackets denote ensemble average (over molecules and time) and also over multiple time origins t′. The MSDs were corrected for the COM motion of the membrane (i.e. removing any net leaflet translation). MSDs of DPPC at different area-per-lipid values are shown in Fig. 4A.</p><p>Lipids exhibit two different types of in-plane motion, a "rattling-in-cage" motion at short time-scales (<1 ns), and translation via "hopping" diffusion at longer time scales (>10 ns), as shown in the inset of Fig. 4A.54 Long-time diffusion coefficients were quantified by fitting the MSD curves to a linear function at long times (10 to 70 ns). Experimental values of the lateral diffusion coefficient of lipids in fluid-phase membrane bilayers range from 1.5 × 10−12 m2 s−1 to 6 × 10−12 m2 s−1 depending on the method.22 The simulation-measured diffusion coefficient of DPPC at α = 0.635 nm2 was 8.1 × 10−12 m2 s−1, which is close to the values obtained using fluorescence correlation spectroscopy.20 According to the free-area theory,55 lipid hopping from one cage to another depends on the availability of a void space (larger than a critical size) next to the molecule. Opening up of a void space occurs occasionally due to random density fluctuations. Free-area theory of lipid lateral diffusion has been shown to fit well with experimental diffusion data obtained as a function of temperature and cholesterol concentration in lipid bilayers.56,57 However, the predictive ability of the model has been challenged because of the large number of fitting parameters required.58 Lipid lateral diffusion coefficient (D) according to free-area theory is given by the equation, (3)ln(D)=ln(gdu)−γacaf where, g is a geometric factor (~1/4), d is diameter of the cage, u is the gas kinetic constant, γ is the free area overlap factor (0.5 to 1), ac is the critical area required for lipid diffusion, and af is the free area defined as the difference between average molecular area (α) and the van der Waals area of the lipid (a0). Note that the critical area is not the same as the van der Waals area. Fitting parameters include maximum diffusion coefficient (i.e.Dmax = gdu, from eqn (3)), critical area of the lipid, and free-area overlap factor (γ). Typical values of a0 and ac for phosphocholines are 0.42 nm2 and 0.48 nm2 respectively.59 The Dmax can be approximated by KBT/f, where f is the friction coefficient given by 4πηR for a spherical particle of radius R in a medium of viscosity η.</p><p>According to eqn (3), free-area theory predicts a linear relationship between ln(D) and 1/af with a constant slope for lipid monolayers in the area-per-lipid range of 0.50 nm2 to 0.90 nm2.59 From our simulations, however, the plot of ln(D) vs. 1/af (Fig. 4B) over the range of area-per-lipid tested, resulted in two distinct linear regimes. The slope of the curve, β = −γac, measured at smaller area-per-lipid (0.635 to 0.700 nm2) was −0.34, which is comparable to that measured in various fluid-phase lipid monolayers (summarized in Table 1 of ref. 59). However, the slope of the curve at larger area-per-lipid (0.700 to 0.750 nm2) was −1.2. Since there is no evidence of phase transition, explanation of a nonconstant slope must be due to changes in interleaflet friction and/or molecular shape. Indeed, experimental studies showing that lipid diffusion is substantially higher in monolayers (D ≈ 20 × 10−12 m2 s−1) compared to bilayers (D ≈ 6 × 10−12 m2 s−1), as measured using FCS20,59 suggest that changes in interleaflet interactions explain discrepancies between low and high tension slopes.</p><p>We now explore the idea of non-constant friction and molecular shape further. First, fitting of diffusion data to free-area theory is typically done by assuming that the Dmax is constant for a given lipid, i.e. the friction coefficient is assumed constant through the entire range of area-per-lipid values. However, Dmax obtained from extrapolating the linear fits to 1/af = 0 in Fig. 4C shows that Dmax is not constant and increases at high tensions. This suggests that the friction on the lipid molecules is decreased significantly at high tensions. This is supported by other observations in this study, namely, increase in disorder of the water molecules at the lipid headgroups (Fig. 3C) and increased disorder of terminal tail regions of lipid acyl chains (Fig. 2A and 2B). The decrease in headgroup and inter-leaflet friction might account for the dramatic increase in diffusion coefficient at higher tensions compared to the values predicted by free-area theory. Second, a significant increase in the slope of ln(D) vs. 1/af at high tensions also indicates an increase in critical area of the molecule and/or the free-area overlap factor. However, changes in these parameters are not sufficient to explain changes in diffusion. Specifically, although free-area theory can be used to fit the diffusion data at large tensions, the fits result in a β of −1.2. To obtain this slope either the overlap factor (γ) or the critical area (ac) must be significantly greater than 1. But the overlap factor, by definition, lies between 0 (no overlap of free area) and 1 (complete overlap). Similarly, the critical area, the minimum area needed for diffusion, is normally held constant (at 0.48 nm2) but could increase if the molecular shape changes. Increase in critical area of the molecule at high tensions is evident from the observation that significant spreading of the acyl chains occurs at high tensions (Fig. 2D) but it will never increase sufficiently to yield a β of −1.2. Other local-density/free-area theoretical approaches, which have been developed for polymer and oligomer systems, can yield quantitative agreement with the D values obtained from the simulations,60,61 however these models introduce additional adjustable parameters, which may be difficult to define properly for the lipid bilayer system. We conclude that a new theory for lipid diffusion is needed that takes into account changing friction and molecular shape with tension.</p><!><p>Experimentally, membrane dynamics are often assessed using measurements of dynamics of fluorescent probe molecules.15,17,19,20 Such spectroscopic measurements assume that the probe molecules faithfully reflect lipid dynamics. Interpretation of the obtained data necessitates knowledge of the microenvironment factors such as hydration and viscosity, which are dictated by the location and orientation of the chromophore. We discuss the sensitivity of fluorescence dynamics of DiI to lipid packing and compare DiI dynamics to the native lipid dynamics. Key properties pertaining to DiI are summarized in Table 2.</p><p>The lateral diffusion coefficient of DiI has been shown to be in the same range but slightly lower than that of DPPC.22 In this study, we could not test the sensitivity of long-time lateral diffusion coefficient of DiI to lipid packing due to lack of sufficient statistics; there exist only two DiI molecules in the simulation box compared to 124 DPPC molecules. Alternatively, we computed the short-time diffusion (cage diffusion) of DiI and DPPC as a function of tension, and compared their MSDs at time t = 200 ps (Tables 1 and 2, respectively). The MSD of DiI was lower than that of DPPC at all tensions. The free area of the DPPC bilayer is smaller near the lipid headgroup region and increases considerably in the hydrophobic region of the bilayer.62 Despite the fact that DiI was located near the lipid acyl chain region (Table 2),22 DiI exhibited slower diffusion than DPPC. This is most likely due to the rigid and bulky structure of the DiI headgroup. Nevertheless, MSD of both DPPC and DiI scaled linearly with increases in tension and exhibited equal sensitivity. Considering that DiI exhibits hopping translation similar to DPPC at long time scales (inset of Fig. 4A), diffusion of DiI at longer times will likely also scale similarly to DPPC. Direct evidence for this would require simulating substantially larger systems, which is beyond the capabilities of our computational facilities. Nevertheless, based on the above observations, we conclude that the lateral diffusion mechanism of DiI is similar to that of the native lipid and that tension induces increases in DiI diffusion that are quantitatively similar to lipid diffusion.</p><p>We further assessed the rotational dynamics of DiI and DPPC by computing the rotational autocorrelation function of DiI's orientation vector (vector joining the two indole rings) and of the P–N vector of DPPC, respectively (Fig. 5). On average, the orientation vector of DiI was parallel to the lipid–water interface to within a few degrees, and was independent of tension-induced changes in lipid packing (Table 2). The rotational correlation function, C(t), is defined as C(τ) = 〈P2(cos(θ(τ)))〉, where θ(τ) is the angle between the orientation vectors separated by a time interval `τ', P2 is the second Legendre polynomial, and 〈 〉 represents the ensemble average. Rotational autocorrelation curves of DPPC and DiI for different values of tension are shown in Fig. 5A. In most cases, the rotational correlation of DiI did not decay completely to zero suggesting that either DiI does not undergo isotropic rotation or, more probably, our simulation time was not sufficient to reach the asymptotic limit. A double-exponential function of the form F(ft)=F0+∑i=12e−tτ, commonly used to fit the experimental rotation data, was used here to determine the rotational relaxation times63 (Table 2). The fast and slow relaxation times represent the wobbling motion and overall rotation, which were in the range of 0.8 to 1.1 ns and 6.1 to 9.7 ns, respectively, for DiI. These results are in good agreement with experimentally-determined rotational correlation times of DiI–C12 (shorter chain length than DiI–C18) in fluid-phase DOPC bilayer.64 In comparison, the fast and slow relaxation times of DPPC were on the order of 0.1 to 0.3 ns and 1.7 to 3.8 ns, respectively (Table 1), and were smaller compared to DiI by a factor of 2 to 3. Both slow and fast relaxation times of DPPC and DiI decreased linearly with increased area-per-lipid with equal sensitivity, as inferred from the linear fits shown in Fig. 5B. These observations show that even though the rotational relaxation times of DiI do not match those of DPPC, their trend with membrane tension suggests they are a sensitive indicator of lipid packing.</p><!><p>Although the present classical molecular dynamics simulations cannot simulate fluorescence, which is a quantum mechanical process, they do enable one to assess the local physical factors that govern fluorescence. In general, fluorescence lifetime of carbocyanine chromophores is sensitive to water accessibility and to the local microviscosity. Cyanine dyes exhibit weak fluorescence in water and a dramatic increase in quantum yield upon incorporation into lipid membranes.21 Viscosity-dependent fluorescence lifetime of cyanine dyes has been shown to be related to changes in the trans–cis photoisomerization dynamics of the central methine bridge.65,66 Moreover, Packard and Wolf have shown that fluorescence lifetime of DiI increases with an increase in order of the lipid acyl chains.19</p><p>We assessed chromophore hydration by counting the average number of water molecules in the first shell of DiI's head-group. This hydration number can be accurately computed from the radial pair-distribution function of DiI-nitrogens and water-oxygens. Hydration of the chromophore under different membrane surface areas varied between 4.7 and 5.6 water molecules with no particular trend with tension (Table 2). This observation is in concert with the other trends observed here; namely, since the location of the dye with respect to lipid–water interface does not change with increased area-per-lipid (Table 2) and water does not penetrate appreciably beyond lipid headgroups (Fig. 1), no marked change in the DiI hydration is expected. On the other hand, measurable decreases in the rotational relaxation times of DiI were observed with increased tension, which is indicative of decreased viscosity near the DiI's headgroup region. These results indicate that changes in fluorescence lifetime of DiI due to membrane order19 are most likely due to changes in the viscosity near the headgroup, rather than due to changes in hydration. In summary, these results suggest that fluorescence lifetime of DiI may be a sensitive indicator of tension-induced decreases in lipid packing in membranes.</p><!><p>Lateral tension-induced changes in membrane organization and dynamics play an important role in transforming mechanical signals into biochemical signals at the cell surface. Despite the known significance of membrane tension, very little is known at a molecular level about the effects of tension on membrane organization. The goal of this study was to provide insights into the effects of membrane lateral tension on lipid structure and dynamics through atomistic molecular dynamics simulations of a fluid-phase lipid bilayer under a broad range of tensions (from zero to values just below rupture tension). Quantitative agreement of the simulation findings with available experimental values indicates that the simulation methodology used was robust and accurate in determining equilibrium properties of the lipid bilayer.</p><p>Key findings from the simulations are as follows. First, physiologically relevant tensions in the range of 0–15 mN m−1 caused decreases in bilayer thickness in a linear fashion consistent with volume-incompressibility. Second, tension induced a significant increase in acyl chain interdigitation and a decrease in lipid order. Third, tension induces a significant decrease in electrostatic potential barrier (up to 80 mV at the highest tension), due to decreased ordering of both water and lipid dipoles. Fourth, the observed lateral diffusion coefficient of DPPC cannot be described satisfactorily using the free-area theory, across all tensions applied, due to a significant change in molecular shape and friction at high tensions. Finally, DiI has systematically lower lateral and rotational diffusion coefficients compared to DPPC, but the increase in each with tension is quantitatively similar for DiI and DPPC. Similarly, fluorescence lifetime of DiI, which depends on lipid order near the headgroups, appears to be a good indicator of tension in membranes.</p><p>These results have potential physiological implications. For instance, hydrophobic mismatch between lipids and proteins causes opening and closing of transmembrane stretch-activated ion channels.67 Alternatively, forces may cause changes in the electrostatic potential of the bilayer, which in turn affects membrane channel conductance, ion and water transport through the lipid bilayer, protein conformation, and kinetics of membrane-bound enzymes.68 For example, decrease in dipole potential leads to a decrease in dissociation of gramicidin channel dimers leading to increased sodium ion permeability.53,69 Altered lipid mobility, due to force-induced changes in lipid packing, can also lead to changes in protein molecular mobility and change the kinetics of enzymatic reactions that require protein complex formation (e.g. dimerization).70,71 Force-induced changes in lipid mobility are also associated with regulation of mitogen activated protein (MAP) kinase activity.2,6 To explain the relationship between lipid mobility and membrane protein-mediated signaling, Nicolau et al.72 proposed that a local decrease in lipid viscosity, reflected in lipid mobility, temporarily corrals membrane proteins and increases their residence time and interaction kinetics leading to initiation of MAPK signaling pathways once a threshold residence time is reached.73 Studies on model membranes have demonstrated that membrane tension promotes formation of large domains from micro-domains in order to minimize line tension developed at microdomain boundaries,7,8 and there exists a critical pressure at which lipid phase separation into liquid-ordered and liquid-disordered domains is observed.74 Taken together, these studies point to changes in bilayer structure and dynamics as a mechanism of force-induced biochemical signaling.</p><p>Future research will be needed to develop a new theory for tension-diffusion relationship that takes into account frictional and molecular shape changes. The current simulations not only provide additional quantitative insights into some of the well-studied bilayer properties (e.g. bilayer thickness, diffusion coefficient), but also lead to novel hypotheses related to membrane-mediated mechanotransduction in cells (e.g. interdigitation and electrostatic potential) that can be tested experimentally.</p><p>In addition, we tested which DiI fluorescence spectroscopic properties have potential as reporters of membrane tension effects on lipids. We observed that although DiI exhibited slower lateral and rotational diffusion compared to DPPC, its lateral and rotational diffusion increased with tension in a manner quantitatively similar to DPPC. This suggests that changes in DiI dynamics are good indicators of membrane tension. We also showed that hydration of the dye does not vary with packing, whereas the local viscosity experienced by the dye changes significantly. These results support the utility of DiI as a reporter of lipid packing and validate the use of DiI to label membrane cellular microdomains based on underlying heterogeneity in lipid order. Thus these findings offer new insights into the interpretation of fluorescence dynamics of DiI and lipids in lipid bilayer systems.</p><!><p>Mass density profiles of lipid (solid), water (dashed), and DiI-C18 (dotted) across the lipid bilayer at selected values of area-per-lipid (the center of the bilayer was set at z = 0, DiI density is at 20× for clarity). A snapshot of the simulation box is also shown (DPPC—grey, DiI—red, water—purple, and DPPC phosphorous atoms are shown in green).</p><p>(A and B) Deuterium order parameter (SCD) versus chain position for sn-1 and sn-2 acyl chains of the DPPC molecules for different values of area-per-lipid. (C) Dihedral trans fraction of the lipid acyl chain dihedrals, averaged over sn-1 and sn-2 chains. (D) Distribution of the end-to-end acyl–acyl angle of the DPPC lipids.</p><p>(A) Charge density profile of the lipid bilayer is shown on the left, including the contributions from lipid and water. On the right are the respective electrostatic potential profiles as a function of area-per-lipid. (B) Ordering of water dipoles with respect to bilayer normal at different area-per-lipid. (C) Angular distribution of DPPC P–N vector with respect to the normal of the bilayer. In (A) and (B) the abscissa is normalized by the size of the simulation box for each α, and only half of the simulation box is shown, due to symmetry (z = 0 denotes the center of the bilayer).</p><p>(A) Mean square displacements (MSD) of lipid molecules under different tensions. Representative xy-trajectories of DPPC and DiI molecules are shown in the inset (α = 0.635 nm2). (B) The plot of ln(D) vs. 1/af, where two different linear regimes were identified, represented by solid lines, with slopes β. Error bars represent standard errors, n = 124.</p><p>(A) Rotational autocorrelation curves of DiI (dotted) and DPPC (solid) under different tensions. (B) The corresponding rotational relaxation times of DiI and DPPC, τ1 (fast) and τ2 (slow), obtained from fit with bi-exponential decay functions. Solid lines are linear fits of the data. 95% confidence interval of τ1 and τ2 of DiI are ±0.03 and ±0.25 ns respectively. 95% Confidence interval of τ1 and τ2 of DPPC are ∓0.01 and ±0.25 ns respectively.</p><p>Summary of structural and dynamical properties of the lipid bilayer as a function of area-per-lipid</p><p>Reported mean standard errors (S.E.) are upper limits of all the simulations.</p><p>95% confidence intervals obtained from curve-fitting.</p><p>Location, orientation, hydration, and dynamics of DiI</p><p>Positive values indicate DiI location below the lipid–water interface.</p><p>A bimodal distribution of dye orientation was observed, with one dye molecule temporarily trapped in a metastable configuration with an average orientation of around 60 degrees. For this reason, the lateral and rotational dynamics are not reported.</p><p>The values in parenthesis indicate full width half maximum (FWHM) of the distribution.</p><p>95% confidence intervals obtained from curve-fitting.</p>

PubMed Author Manuscript

Development of a new biocathode for a single enzyme biofuel cell fuelled by glucose

In this study, we reported the development of Prussian blue (PB), poly(pyrrole-2-carboxylic acid) (PPCA), and glucose oxidase (GOx) biocomposite modified graphite rod (GR) electrode as a potential biocathode for single enzyme biofuel cell fuelled by glucose. In order to design the biocathode, the GR electrode was coated with a composite of PB particles embedded in the PPCA shell and an additional layer of PPCA by cyclic voltammetry. Meanwhile, GOx molecules were covalently attached to the carboxyl groups of PPCA by an amide bond. The optimal conditions for the biocathode preparation were elaborated experimentally. After optimization, the developed biocathode showed excellent electrocatalytic activity toward the reduction of H 2 O 2 formed during GOx catalyzed glucose oxidation at a low potential of 0.1 V vs Ag/AgCl, as well as good electrochemical performance. An electrocatalytic current density of 31.68 ± 2.70 μA/cm 2 and open-circuit potential (OCP) of 293.34 ± 15.70 mV in O 2 -saturated 10 mM glucose solution at pH 6.0 were recorded. A maximal OCP of 430.15 ± 15.10 mV was recorded at 98.86 mM of glucose. In addition, the biocathode showed good operational stability, maintaining 95.53 ± 0.15% of the initial response after 14 days. These results suggest that this simply designed biocathode can be applied to the construction of a glucose-powered single enzyme biofuel cell.

development_of_a_new_biocathode_for_a_single_enzyme_biofuel_cell_fuelled_by_glucose

<!>Materials and methods<!>Instrumentation.<!>Electrochemical measurements.<!>Results and discussion<!>Morphological study.<!>Stability study of the biocathode.<!>Conclusion

<p>With the growing demand for green electrical energy generation technologies, scientists are making great efforts to develop fuel cells (FCs), which are considered as one of the most promising alternative sustainable energy sources due to their renewable and environmental protection characteristics 1 . Unlike conventional FCs, which utilize the oxidation of fuels (H 2 , ethanol, or methanol) on an anode and reduction of an oxidant on a cathode employing a noble metal catalyst 2 , biological fuel cells (biofuel cells, BFCs) convert chemical energy into electrical energy by using organic fuels (sugars, alcohols, organic acids) produced during metabolic processes and a biological catalyst, which is usually either a microorganism or an enzyme. An enzymatic biofuel cells (EBFCs) are type of BFCs that use purified redox enzymes immobilized on an anode and/or on a cathode to achieve electrocatalytic reactions 3 . Enzymes are highly specific to their respective substrate and typically operates in mild conditions. Therefore, BFCs are an attractive alternative when it is not possible to use high temperatures or where harsh reaction conditions are undesirable. Moreover, enzymes immobilized on the electrode surface allow membrane-less configuration of EBFCs, opening up opportunities for the development of miniaturized systems for powering electronic devices 4 and self-powered electrochemical biosensors, the main advantage of which is a simplified two-electrode system without an external power supply 5 . In addition to lactate 6 , cholesterol 7 , ethanol 8 and other EBFCs, special interest in recent years has been focused on the development of membrane-less EBFCs that can deliver electrical energy using oxidation of glucose at an anode and O 2 9,10 or H 2 O 2 11,12 reduction at a cathode. Glucose and O 2 are an ideal source of fuel and oxidizer because they are found in all organic tissues and can be constantly replenished during metabolic processes 13 . EBFCs that use enzymatic reactions on both electrodes have also been researched and published 14,15 . Researchers expect that in the future, miniature membrane-less EBFCs will supply energy to implantable medical devices, like insulin pumps, hearing devices, bone stimulators or pacemakers, and will also be used as self-powered biosensors, which, using an analyte as a fuel, will be able to supply themselves with energy, and at the same time to determine the amount of an analyte 16 . Implanted self-powered biosensors could be used to measure various substances that cause heart disease or cancer, as well as blood glucose 17,18 . To use EBFCs for these purposes, they should be small and light, operate at Over the last decade, the performance of EBFCs has greatly improved by using various nanomaterials, such as carbon nanotubes (CNTs), graphene oxide (GO), noble metal nanoparticles or conjugated polymers (CPs). These materials often have good biocompatibility, also electrical conductivity and large surface area. Their use allows to improve the efficiency of electron transfer and a magnitude of the generated electrical signal and often provides a stable matrix for enzyme immobilization. Due to the large surface area, nanomaterials can increase enzyme loading, furthermore, to improve the activity and stability of immobilized enzymes, thus improving the performance of EBFCs. Among the nanomaterials mentioned, CPs and CPs-based nanocomposites have gained the considerable attention of many scientists. For example, Haque and co-workers 19 reported a glassy carbon electrode modified with a conducting composite consisting of chitosan, reduced GO, polyaniline (PANI), ferritin and glucose oxidase (GOx) as a potential bioanode for glucose EBFC. The bioanode was capable to generate a current of 3.5 mA/cm 2 at 20 mM of glucose. The performance was improved due to the porosity and large surface area of the composite material, which allows the immobilization of a larger amount of enzyme and facilitates the diffusion of glucose. Although the bioanode generated a lower current signal, it nevertheless had a high operational stability and maintained 95% of the initial response after one week. Kang and co-workers 20 proposed a glucose/O 2 EBFC based on glassy carbon electrodes modified with a novel three-dimensional PANI and CNTs composite with rhizobium-like structure. The composite was prepared by in-situ polymerization of aniline monomers around and along the functionalized CNTs and then carbonized at a high temperature was used as a substrate for immobilization of GOx (anode) and laccase (Lac) (cathode). The EBFC was performed with a maximum power density of 1.12 mW/cm 2 at 0.45 V. Moreover, three fabricated EBFCs connected in series were able to light up a yellow light-emitting diode (LED) whose turn-on voltage was about at 1.8 V. Later Kang and co-workers 21 reported glucose/O 2 EBFC based on GOx and Lac immobilized on carbonized rectangular polypyrrole tubes. A nickel foam was utilized as the substrate electrode. The open-circuit voltage The opencircuit of the designed EBFC reached 1.16 V and the maximum power density was measured to 0.350 mW/cm 2 at 0.85 V. Three of the fabricated EBFCs connected in series were able to light up a white LED whose turn-on voltage was about at 2.4 V for more than 48 h.</p><p>Most of the glucose EBFCs utilize glucose-oxidizing enzyme (GOx or glucose dehydrogenase) on a bioanode combined with oxygen reducing enzymes (commonly bilirubin oxidase or Lac) on a biocathode. Biocathodes based on immobilized peroxidase (PO) 14,22 and biocathodes in which GOx is combined with PO that catalyses the reduction of H 2 O 2 , produced during glucose oxidation on GOx modified electrodes 23 , have also been published. Such systems have a drawback: the use of two enzymes, which makes the system more complex and increases the cost. In addition, enzymes used may have different optimal operating conditions. These drawbacks can be avoided by designing a so-called single enzyme EBFC, in which the same enzyme is used for the anodic and cathodic reactions. The present paper describes the fabrication and investigation of a novel biocathode in the construction of which an "artificial PO" Prussian blue (PB) was used instead of PO. According to the mechanism of H 2 O 2 reduction on PB modified electrodes, PB is electrochemically reduced to form Prussian white (PW), which catalyses the reduction of H 2 O 2 at low potential 24 and PW is oxidized to PB again. Due to the reversible electrochemical redox ability of PB, it acts as a renewable catalyst throughout the electrochemical process 25 . Although PO-like property of PB has been studied for the design of biosensors 5,26 and FCs 27,28 , it has not yet been used in the construction of a biocathode for a single enzyme EBFC, whose anodic and cathodic reactions would be both based on the processes biocatalysed by GOx. This enzyme immobilized on the bioanode and biocathode would catalyze the oxidation of glucose to H 2 O 2 , which would be reduced on the surface of the biocathode. To design such biocathode, graphite rod (GR) electrode was coated with a composite of PB particles embedded in the PPCA shell (GR/PB-PPCA) and an additional layer of PPCA (GR/PB-PPCA/PPCA) by cyclic voltammetry (CV). Finally, GOx was covalently linked to the carboxyl groups of the PPCA (GR/PB-PPCA/PPCA-GOx). Immobilized GOx acted as a catalyst that oxidizes glucose by molecular O 2 , PB, meanwhile, acted as an electrocatalyst for the reduction of H 2 O 2 formed during the enzymatic reaction. To achieve the best performance of the biocathode, preparation conditions were optimized by evaluating the generated signal to glucose. After optimization, the performance of the biocathode was investigated.</p><!><p>Chemicals. GOx from Aspergillus niger (freeze-dried powder 360 U/mg protein), iron (III) chloride (FeCl 3 ) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Carl Roth GmbH. Potassium hexacyanoferrate (III) (K 3 [Fe(CN) 6 ]) were from Sigma-Aldrich. Pyrrole-2-carboxylic acid (PCA) and D-(+)-glucose monohydrate (C 6 H 12 O 6 × H 2 O) were obtained from Alfa Aesar GmbH. Hydrogen peroxide (H 2 O 2 ) was obtained from Chempur and N-hydroxysuccinimide (NHS) from Merck. All chemicals were of analytical grade. All aqueous solutions were prepared in ultra-high quality (UHQ) water, which was obtained using the DEMIWA rosa 5 water purification system (WATEK, Czech Republic). In addition, glucose solution was prepared at least 24 h before use to allow glucose to mutarotate and to reach equilibrium between α-and β-forms. 40.0 mg/mL solution of GOx was freshly prepared in sodium acetate-phosphate buffer solution composed of 0.05 mM CH 3 COONa, 0.05 mM Na 2 HPO 4 and 0.05 mM NaH 2 PO 4 (A-PBS) and rapidly used. 0.5 M solution of PCA was prepared in ethanol absolute.</p><!><p>All electrochemical experiments as well as electrochemical synthesis of the PB-PPCA/ PPCA composite were performed using a computer-controlled potentiostat/galvanostat Autolab PGSTAT30 (Eco Chemie, Netherlands) driven by NOVA1.9 software. The voltammetric and amperometric cell was com-GR electrode pre-treatment and preparation of the biochatode. GR electrodes were prepared by breaking a graphite rod (15.0 cm in length, 3.0 mm in diameter and 99.999% purity, Sigma-Aldrich) into smaller rods of the required length. The broken rods were mechanically polished using very fine (P320) and finally ultrafine grit (P2000) sandpaper until the working surface of the electrode is completely smooth, and then were polished using a sheet of paper, washed with UHQ water, then ethanol and dried at room temperature. Finally, the side surface of the rod was isolated with a silicone tube so that only the working surface of the GR electrode was in contact with the solution in the electrochemical cell. The working surface area of GR electrodes thus prepared was 0.0707 cm 2 .</p><p>Pre-treated GR electrodes were used to perform electrochemical synthesis of the PB-PPCA/PPCA composite using CV. Under the optimized modification conditions, the pre-treated GR electrode, together with the reference and auxiliary electrodes, was immersed in an electrochemical cell filled with 5 mL of a solution consisting of 100.0 mM HCl, 100.0 mM KCl, 1.0 mM FeCl 3 , 1.0 mM K 3 [Fe(CN) 6 ] and 35.0 mM PCA. The potential was then scanned for 50 consecutive cycles in the range of potentials from − 0.4 to + 1.0 V at a scan rate of 0.1 V/s. During this process, a composite of PB particles embedded in the PPCA shell (PB-PPCA) was formed on the GR electrode surface (GR/PB-PPCA). After synthesis of the PB-PPCA composite, the GR/PB-PPCA electrode was washed well with UHQ water and immersed in an electrochemical cell filled with 5 mL of A-PBS buffer solution with 0.1 M KCl additive (A-PBS-KCl), pH 6.0, and containing 200.0 mM PCA. The potential was then scanned for 5 consecutive cycles in the range of potentials from − 0.4 to + 1.0 V at a scan rate of 0.1 V/s. During this process, an additional layer of PPCA was formed on the GR/PB-PPCA electrode (GR/PB-PPCA/PPCA).</p><p>To modify the GR/PB-PPCA/PPCA electrode with GOx, the electrode was immersed in a tube filled with a mixture of 0.4 M EDC and 0.1 M NHS solutions in a ratio of 1:1 and left for 30 min at ambient temperature. The electrode was then removed from the mixture, washed with UHQ water, and immersed immediately in a 40 mg/mL GOx solution in A-PBS, pH 4.0, stirring the solution gently from time to time. The GOx was attached covalently by an amide bond to the electrode surface (GR/PB-PPCA/PPCA-GOx). Finally, the GR/PB-PPCA/ PPCA-GOx electrode was washed with UHQ water and to remove non-covalently bound enzyme was conditioned in A-PBS-KCl, pH 6.0, for 15 min, stirring the solution gently from time to time. The prepared GR/ PB-PPCA/PPCA-GOx electrodes were stored in closed test tubes above a drop of A-PBS-KCl, pH 6.0, at + 4 °C temperature until used in the experiments.</p><!><p>Electrochemical characterization of bare GR and modified GR electrodes was performed by CV, amperometric and potentiometric techniques. For half-cell measurements, threeelectrode cell was used for CV and amperometric measurements, while two-electrode cell was used for potentiometric measurements at open circuit or at an external load of 476 kΩ. A-PBS-KCl buffer solution, pH 6.0, was used as the electrolyte solution. The solution in the cell was continuously stirred with a magnetic stirrer at 450 rpm during amperometric and potentiometric measurements. Meanwhile, CV measurements were performed without stirring; stirring was turned on only after the addition of glucose to mix the solution. Amperometric current dependence of the biocathode on glucose concentration was studied at + 100 mV vs reference electrode. After stabilization of the background current or potential (base line), in the amperometric and potentiometric measurements respectively, a solution of glucose was injected in the electrochemical cell. The biocathode-generated signal was expressed as the change in cathodic current (ΔI) or the change in potential (ΔE) calculated from the signal recorded by the addition of glucose minus the baseline signal. During the CV measurements, the potential was scanned in the range of potentials from − 0.2 to + 0.5 V at a scan rate of 0.1 V/s or other as required, and the peak current was monitored. The results of all experiments were represented as a mean value of three independent measurements.</p><!><p>In this research, a novel GR/PB-PPCA/PPCA-GOx biocomposite based biocathode was developed. CV was used for the electrochemical synthesis of the composite consisting of PB particles embedded in a PPCA shell (PB-PPCA) and for the formation of an additional PPCA layer over PB-PPCA (PB-PPCA/PPCA). To synthesize PB-PPCA on top of a GR electrode (GR/PB-PPCA), the electrode was immersed in an electrochemical cell filled with a solution consisting of HCl, KCl, FeCl 3 , K 3 [Fe(CN) 6 ] and PCA, and polymerization was performed. The GR/PB-PPCA electrode was then immersed in an electrochemical cell filled with A-PBS-KCl buffer solution containing PCA and an additional PPCA layer (GR/PB-PPCA/PPCA) was synthesized. Finally, using activation of carboxyl groups of PPCA by a mixture of EDC and NHS, GOx molecules were linked to the PPCA via amide bonds (GR/PB-PPCA/PPCA-GOx). The design concept and operation of the biocathode are shown in Fig. 1. The operation of the biocathode can be explained by the electrocatalytic activity of PB towards to the reduction of H 2 O 2 formed during GOx catalyzed oxidation of glucose. Fe(III) of PB after receiving the electron is electrochemically reduced to form PW, which has a high reduction activity 25 . The H 2 O 2 formed during enzymatic reaction is reduced by PW, and the PW is reoxidized to PB. Due to the reversible redox activity of PB, it acts as a renewable catalyst throughout the bioelectrochemical process. The amount of H 2 O 2 formed during GOxcatalyzed reaction depends on the glucose concentration, thus the current or potential signal generated by the biocathode due to H 2 O 2 reduction dependent on the glucose concentration. biocathode, its preparation conditions were optimized by estimating the magnitude of the generated current signal to glucose. Since the GR electrode was coated layer by layer with PB-PPCA/PPCA composite, the electrochemical synthesis conditions of PB-PPCA were first optimized. During optimization, PB-PPCA electrodes prepared under different conditions were modified with an additional layer of PPCA and immobilized GOx under constant conditions. The amperometric response of the GR/PB-PPCA/PPCA-GOx biocathodes to glucose in A-PBS-KCl buffer solution, expressed as cathodic current change (ΔI), was then investigated. Figure 2A shows the experimental results obtained during the optimization of FeCl 3 and K 3 [Fe(CN) 6 ] concentration. As can be seen, the current signal increased with increasing equimolar concentrations of FeCl 3 and K 3 [Fe(CN) 6 ] up to an optimal concentration of 1.0 mM. Meanwhile, the optimal PCA concentration was found to be 35.0 mM (Fig. 2B). It is likely that when the concentrations of FeCl 3 and K 3 [Fe(CN) 6 ] are too high and the PCA is too low, PB particles from the resulting PB-PPCA composite can diffuse into the solution, thus reducing the current generated by the biocathode. On the other hand, at too high concentration of PCA, a thick layer of PPCA is formed. Due to the low conductivity of this layer, the current generated is also reduced.</p><p>The current response to glucose was also depended on the potential scan rate (Fig. 2C) and the number of potential scans (Fig. 2D). The highest current response of the biocathode after addition of glucose was registered when a potential scan rate of 0.1 V/s and 50 potential scans were used. During polymerization, a thick polymer shell is formed by applying more potential scans and higher scan rate. Meanwhile, with less potential scans and lower scan rate, the electrode surface may be inefficiently coated by PB-PPCA composite. This, as can be seen from the results, also affects the current generated by the biocathode.</p><p>During the optimization of the deposition of the additional PPCA layer, the dependence of the current signal on PCA concentration and potential scan rate was also observed. The highest current signal was registered when 200 mM of PCA (Fig. 3A) and 5 potential scans (Fig. 3B) were used. Such results are related to the thickness of the resulting additional PPCA layer. The higher the PCA concentration and the number of potential scans, the thicker the additional PPCA layer. The thicker layer causes a decrease in the current generated by the biocathode. On the other hand, with a fuller and more uniform coating, more GOx molecules can be attached to the electrode surface. Based on the results of this study, 200 mM PCA and 5 potential scans were selected as optimal conditions for the electrochemical polymerization of the additional PPCA layer on the GR/PB-PPCA electrode.</p><p>The concentration and pH of the GOx solution used for biocathode preparation were also optimized. In this work, a covalent amide coupling technique using a mixture of EDC and NHS was used for GOx immobilization. To find the most suitable pH, after activation of the PPCA carboxyl groups, the GR/PB-PPCA/PPCA electrodes were immersed in GOx solution in A-PBS buffer with a certain pH from 4.0 to 7.0. The current response of the prepared biocathodes to glucose was then investigated. As can be seen from the results presented in Fig. 3C, the magnitude of the current signal was dependent on the pH, and the highest current signal was registered at pH 4.0. These results were consistent with those obtained for GR electrodes modified with a nanobiocomposite composed of poly(1,10-phenanthroline-5,6-dione), PPCA, gold nanoparticles and GOx 29 , and can be explained by the pre-concentration of the enzyme near the electrode surface at this pH, resulting in a higher amount of immobilized GOx. Very similar results were obtained when the influence of the concentration of GOx solution used during immobilization on the magnitude of the current signal generated by the biocathode was investigated. The increase in the enzyme concentration resulted in an increase in the current response of the biocathode to glucose and the highest response was recorded then the highest GOx concentration of 40 mg/mL was used (Fig. 3D). Because 40 mg/mL is a sufficiently high concentration, the effect of higher concentrations was not studied and this GOx concentration was chosen for use in biocathode preparation.</p><p>Electrochemical behaviour of the biocathode. Electrochemical behaviour was studied by recording cyclic voltammograms after an appropriate step of the biocathode preparation process. Figure 4A shows the corresponding cyclic voltammograms registered for GR, GR/PB, GR/PB-PPCA, GR/PB-PPCA/PPCA, and GR/ www.nature.com/scientificreports/ PB-PPCA/PPCA-GOx electrodes. As can be seen, in the voltammogram recorded for the GR electrode, there are no oxidation and reduction current peaks (redox peaks) in a potential range between − 0.2 and + 0.5 V vs Ag/ AgCl. Meanwhile, for the other electrodes studied, a pair of well-defined redox peaks due to the electrochemical reaction of high-spin ferric ions in PB (Fe 2+ /Fe 3+ transition) 24 were recorded in this potential range. The positions of the characteristic redox peaks and the distances between them are given in Table 1.</p><p>The occurrence of characteristic redox peaks and the increase in peak current compared to the GR electrode indicate successive deposition of PB on all modified electrodes. The observed redox peaks potential values were similar to those described in the literature for PB modified electrodes [30][31][32] . In addition, as can be seen from Fig. 4A, the cyclic voltammograms of the GR/PB-PPCA, GR/PB-PPCA/PPCA and GR/PB-PPCA/PPCA-GOx electrodes showed a significant increase in redox peaks intensity compared to the GR/PB electrode. These results demonstrated that the presence of PCA in the electropolymerization solution increases the amount of PB on the electrode surface due to its distribution inside the polymer matrix. Meanwhile, the decrease in the intensity of the GR/PB-PPCA/PPCA redox peaks compared to GR/PB-PPCA could be explained by the formation of an additional PPCA layer. Because electropolymerization was carried out in an aqueous medium without removal of oxygen, the PPCA layer formed is low conductive or even non-conductive 29 . Therefore, the formation of an additional layer of PPCA causes a decrease in the intensity of the redox peaks. For the GR/PB-PPCA/PPCA-GOx, an even greater decrease in redox peaks intensity was observed due to nonconducting nature of enzyme 33,34 immobilized on the surface. In addition, as shown in Fig. 4A, the presence of 10.0 mM glucose in A-PBS-KCl buffer solution caused an increase in GR/PB-PPCA/PPCA-GOx reduction peak current by approximately 29 μA (black line). This indicates that the H 2 O 2 formed during the enzymatic glucose oxidation reaction was electrochemically reduced on the surface of the biocathode.</p><p>The cyclic voltammograms of the GR/PB-PPCA/PPCA-GOx biocathode in A-PBS-KCl, pH 6.0, at different potential scan rates are shown in Fig. 4B. As can be seen, the intensity of the redox peaks varied with the potential scan rate and was directly proportional to the scan rate (Fig. 4C). The linear relationship between oxidation (I ox ) www.nature.com/scientificreports/ and reduction (I red ) current peaks and potential scan rate and the ratio of I ox /I red almost equal to unity, revealed the quasi-reversible and surface-confined 32 electrochemical behaviour of the PB in PB-PPCA/PPCA-GOx biocomposite, in which PB is reduced to PW and is re-oxidized to PB.</p><!><p>The surface morphology of bare GR and GR at different stages of the modifying process (GR/PB, GR/PB-PPCA, GR/PB-PPCA/PPCA, and GR/PB-PPCA/PPCA-GOx) was studied using SEM at 3 kV accelerating voltage, 50,000 magnification and 0.8 nA current. The SEM images in Fig. 5 clearly demonstrate changes in surface morphology during GR modification.</p><p>As can be seen, the GR surface is quite smooth with minor defects occurring during surface polishing. (Fig. 5A). Meanwhile, a completely different surface morphology was observed for the other electrodes. Cubic PB structures of approximately 100 nm in size are observed on the surface of GR/PB (Fig. 5B), similar to those published by other authors 32,35 . However, as can be seen, the surface coating is very uneven with large, uncoated GE areas. Not uniform coating may be related to the removal of PB particles from the surface during synthesis and electrode washing after synthesis. Meanwhile, the presence of PPCA in the synthesis solution resulted in a much better and more uniform coating with a higher amount of PB on the electrode surface (Fig. 5C). However, the characteristic cubic structure of PB was no longer visible. This change in morphology may be related to the disruption of the growth of PB particles into the cubic framework due to the spatial limitations resulting from the embedment of PB particles into the growing PPCA shell 36 . PB-PPCA coating showed an irregular globular morphology, which was in agreement with other reports for PB and polymer composites 36,37 . A rougher coating with larger structures compared to GR/PB-PPCA as well as globular surface morphology was observed for GR/ PB-PPCA/PPCA (Fig. 5D). This indicates that PB-PPCA was coated with an additional layer of PPCA. Similar surface morphology, with slightly larger structures was observed for GR/PB-PPCA/PPCA-GOx (Fig. 5E). The results of surface morphology studies confirmed the assumption that PB was incorporated into PPCA during the formation of the PB-PPCA layer, and an additional PPCA layer was formed on the surface of the PB-PPCA. As can be seen, a linear increase in biocathode response was observed with increasing glucose concentration up to about 10.00 mM. With further increase in glucose concentration, a non-linear increase in the biocathode response was observed up to 98.86 mM, and then the recorded signal became saturated because the catalytic reaction was inhibited at higher glucose concentrations. As a result, the amount of H 2 O 2 formed during the enzymatic reaction became constant. The maximal current density of 85.86 ± 6.30 μA/cm 2 , the potential of 221.03 ± 13.90 mV, and the OCP of 430.15 ± 15.10 mV at 98.86 mM glucose were recorded. Meanwhile, at 10.00 mM glucose, the recorded current density was 31.68 ± 2.70 μA/cm 2 , the potential was 150.73 ± 6.70 mV and the OCP was 293.34 ± 15.70 mV. The performance of the developed biocathode was comparable to the previously reported biocathodes. Some of them are listed in Table 2..</p><!><p>To investigate the stability of the developed biocathode, the current signal generated by it was monitored over a period of 14 days. The measurements were carried out in A-PBS-KCl buffer solution, pH 6.0, containing 10.0 mM of glucose. Between measurements, the biocathode was stored at + 4 °C in a closed vessel above a drop of A-PBS-KCl buffer solution, pH 6.0. As can be seen from the experimental data presented in Fig. 7, current generated by the GR/PB-PPCA/PPCA-GOx biocathode after addition</p><!><p>In this study, a graphite rod electrode modified with a biocomposite composed of Prussian blue, poly(pyrrole-2-carboxylic acid) and glucose oxidase (GOx) was proposed as a potential biocathode for glucose-powered single enzyme biofuel cell. PPCA allowed covalent immobilization of GOx, PB, meanwhile, exhibited electrocatalytic activity to the reduction of H</p>

Scientific Reports - Nature

Effects of Modulating M3 Muscarinic Receptor Activity on Azoxymethane-Induced Liver Injury in Mice

Previously, we reported that azoxymethane (AOM)-induced liver injury is robustly exacerbated in M3 muscarinic receptor (M3R)-deficient mice. We used the same mouse model to test the hypothesis that selective pharmacological modulation of M3R activity regulates the liver injury response. Initial experiments confirmed that giving a selective M3R antagonist, darifenacin, to AOM-treated mice mimicked M3R gene ablation. Compared to vehicle controls, mice treated with the M3R antagonist had reduced survival and increased liver nodularity and fibrosis. We next assessed AOM-induced liver injury in mice treated with a selective M3R agonist, pilocarpine. After pilocarpine treatment, stimulation of post-M3R signaling in the liver was evidenced by ERK and AKT activation. In contrast to the damaging effects of the M3R antagonist, administering pilocarpine to AOM-treated mice significantly attenuated hepatic stellate cell activation, collagen deposition, bile ductule proliferation, and liver fibrosis and nodularity. As anticipated from these findings, livers from pilocarpine-treated mice exhibited reduced expression of key players in fibrosis (\xce\xb11 collagen, \xce\xb1-smooth muscle actin, TGF-\xce\xb21, PGDF, TGF-\xce\xb21R, PGDFR) and decreased mRNA levels for molecules that regulate extracellular matrix formation (TIMP-1, TIMP-2, MMP-2, MMP-13). Cleaved caspase-3, nitrotyrosine and BrdU immunostaining provided evidence that pilocarpine treatment reduced hepatocyte apoptosis and oxidative stress, while increasing hepatocyte proliferation. Collectively, these findings identify several downstream mechanisms whereby M3R activation ameliorates toxic liver injury. These novel observations provide a proof-of-principle that selectively stimulating M3R activation to prevent or diminish liver injury is a therapeutic strategy worthy of further investigation.

effects_of_modulating_m3_muscarinic_receptor_activity_on_azoxymethane-induced_liver_injury_in_mice

1. Introduction<!>2.1. Animals<!>2.2. Experimental Designs<!>2.3. Liver Histology and Morphometry<!>2.4. Immunohistochemistry (IHC)<!>2.5. Quantitative RT-PCR (qPCR)<!>2.6. Statistical Analysis<!>3.1. Treatment with darifenacin worsens the severity of AOM-induced liver injury<!>3.2. Treatment with pilocarpine stimulates post-M3R signaling<!>3.3. Treatment with pilocarpine attenuates gross liver injury and the likelihood of developing ascites<!>3.4. Treatment with pilocarpine reduces liver fibrosis<!>3.5. Treatment with pilocarpine reduces ductular proliferation<!>3.6. Treatment with pilocarpine alters the expression of tissue inhibitors of metalloproteinasese (TIMP) and matrix metalloproteinases (MMP)<!>3.7. Treatment with pilocarpine attenuates hepatocyte apoptosis, and TNF-\xce\xb1 and FasL expression<!>3.8. Treatment with pilocarpine promotes hepatocyte restoration<!>3.9. Treatment with pilocarpine reduces hepatic nitrosative stress<!>4. Discussion

<p>In both humans and animal models, cholinergic input, primarily from the vagus nerve, modulates liver injury responses. In rodents, for example, vagus nerve transection reduces oval cell reaction after galactosamine-induced liver injury, and attenuates hepatocyte proliferation and liver regeneration following partial hepatic resection [1-3]. In contrast, vagus nerve stimulation in rats attenuates hepatic ischemia-reperfusion injury [4]. In humans, denervated liver grafts used for transplantation exhibit reduced ductular reaction compared to innervated native livers [3]. Although acetylcholine-induced activation of hepatic muscarinic and nicotinic receptors has been implicated, the mechanisms underlying cholinergic regulation of liver injury are uncertain [3, 5]. Muscarinic cholinergic G protein-coupled receptors are expressed widely and modulate a variety of biological functions [6, 7]. Of five muscarinic receptors designated M1R-M5R, M3R is the subtype primarily expressed in human and rodent liver [3, 8]. Although M3R expression and activation was shown to promote cell survival in various organ systems, its role in regulating liver injury is incompletely understood.</p><p>Administration to rodents of high-dose azoxymethane (AOM), an ingredient of cycad palms and a by-product of oxidation of the industrial compound methylamine, induces acute hepatic failure, whereas repetitive low doses induce chronic liver injury that mimics human disease [9-15]. AOM-induced changes in stellate cell activation, collagen deposition, and expression of cytokines and regulators of the extracellular matrix are similar to those observed in other chemically-induced chronic liver injury models [16]. Using mice with genetic ablation of Chrm3, the gene encoding M3R, we showed that M3R-deficiency augments AOM-induced liver nodularity, fibrosis, bile ductular proliferation, oval cell expansion and hepatocyte apoptosis [17]. Compared to wild-type mice, treating M3R-deficient mice with AOM markedly increased liver injury and reduced animal survival [17]. Treatment with scopolamine butylbromide, a non-selective muscarinic receptor antagonist, also worsened AOM-induced liver injury but the effects were much less severe than those seen with Chrm3 gene ablation [17], perhaps because of confounding effects of this agent on other muscarinic receptor subtypes.</p><p>Based on these observations we hypothesized that selective modulation of M3R activity alters the liver injury response, thereby identifying M3R as a novel therapeutic target. The goal of the present study was to test this hypothesis using the AOM-induced liver injury model. First, we examined the actions of an M3R-selective antagonist, darifenacin, anticipating that, like Chrm3 gene ablation, this agent would greatly increase the severity of AOM-induced liver injury. Then, we examined the effects of a selective M3R agonist, pilocarpine, on AOM-induced liver injury in mice, anticipating that this would have the opposite action and ameliorate AOM-induced liver injury. In our previous studies inhibition of M3R activation preceded (M3R-deficient mice) or coincided (scopolamine butylbromide treatment) with induction of liver injury (AOM administration). To more closely mimic the clinical setting in which treatment is instituted only after liver injury is recognized, in the present study we investigated the effects of modulating M3R activity after AOM treatment was completed. To gain insight into the molecular mechanisms underlying the effects of M3R activation in the liver, we examined stellate cell activation, collagen deposition, bile ductule and hepatocyte proliferation, hepatocyte apoptosis and the expression of key molecules regulating liver fibrosis and extracellular matrix formation. Our results show that treatment with pilocarpine attenuates AOM-induced liver injury, thereby providing proof-of-principle for the therapeutic potential of selective activation of M3R in chronic liver injury.</p><!><p>Animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the U.S. National Academy of Sciences (National Institutes of Health publication 86-23, revised 1985) and were approved by both the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine and the Research and Development Committee at the VA Maryland Health Care System. All mice [genetic background: 129S6/SvEv X CF1 (50%:50%), Taconic Labs, NY] were housed under identical conditions in a pathogen-free environment with a 12:12-hour light/dark cycle and free access to standard mouse chow and water. Mice were acclimatized for two weeks prior to any treatment.</p><!><p>The actions of darifenacin, an M3R-selective antagonist, and pilocarpine, an M3R-selective agonist, were studied in the AOM model of liver injury; 6-week-old male mice treated with intraperitoneal AOM (10 mg/kg once each week for 6 weeks; Midwest Research Institute, Kansas City, MO). After a one-week interval, to deliver test agents, mice were implanted subcutaneously with osmotic mini-pumps (Alzet model 2006, DURECT Corporation, Cupertino, CA). In the first treatment group mice were treated with darifenacin (3 mg/kg/day; US Biological, Swampscott, MA; n = 19 mice) or vehicle (50% DMSO, n = 20 mice). In the second treatment group mice were treated with pilocarpine (2.5 mg/kg/day; Sigma-Aldrich; n = 14 mice) or vehicle (PBS; n = 14 mice). To achieve 14 weeks of treatment, after the first 7 weeks pumps were removed and replaced with new pumps freshly-loaded with test agents.</p><p>Mouse weight and mortality were recorded weekly. Twenty-one weeks after the first AOM dose surviving mice were euthanized. Additional control mice that did not receive AOM were euthanized at the same time. To assess hepatocyte proliferation, two hours before euthanasia, mice were injected with 50 mg/kg 5-bromo 2'-deoxyuridine (BrdU; Sigma, St. Louis). At euthanasia, two investigators masked to study group graded gross liver appearance (0, normal; 1, mild liver surface nodularity; 2, intermediate liver surface nodularity; 3, marked nodularity and/or ascites) [17]. Livers were removed, weighed and fixed in 4% para-formaldehyde. For later measurement of mRNA levels, a portion of liver was stored in RNAlater.</p><!><p>Paraffin-embedded liver sections were stained with hematoxylin and eosin (H&E) and assessed for fibrosis using the Ishak fibrosis scale by a pathologist (WT) masked to study group [18]. Hepatic collagen was quantified using morphometric analysis of Sirius red-stained sections. Briefly, after dewaxing and hydration, liver sections were stained with picroSirius red solution for 1 h followed by washing in acidified water. Resulting sections were dehydrated, mounted and examined using a Nikon i80 photo-microscope (100 × total magnification). Ten different areas were examined from each section. Since the degree of Sirius red staining measured by the saturation of the red channel correlates well with chemically-determined collagen content and morphometrically-determined fibrosis, fibrosis was assessed as the proportion of summed pixels per unit area of liver section (in arbitrary units) determined using Image Pro-plus software (version 5.0; Media Cybernetics, Silver Spring, MD).</p><!><p>IHC was performed utilizing the avidin-biotin reaction with vectastatin elite ABC kit (Vector Labs, Burlingame, CA) per manufacturer's recommendations. After deparafinization, hydration and endogenous peroxidase blockade (10% H2O2), heat-induced antigen retrieval was performed using citrate buffer. Sections were incubated at room temperature with 5% normal goat serum (20 min), avidin and biotin blocking reagent (15 min each), and washed 3 times with 0.1% Tween-20 in PBS between each step. After overnight treatment with primary antibody at 4°C, sections were incubated with biotinylated secondary antibody (30 min) followed by incubation with streptavidin-HRP (30 min). Further, sections were stained and then counterstained with DAB (2 min) and hematoxylin (4 min), respectively. Hepatocyte proliferation and apoptosis were assessed using primary antibodies against BrdU (BD Bioscience, San Jose, California; dilution 1:100) and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA; dilution 1:100), respectively. Ductular proliferation was assessed using anti-EpCAM antibodies (Abcam, Cambridge, MA; dilution 1:100). To assay BrdU-stained nuclei, at least 1000 hepatocyte nuclei were counted at 200X magnification. Cleaved caspase-3 staining was assessed using Image Pro-plus software. Bile ductule cells were defined as EpCAM-stained cuboidal cells forming ductular structure with lumens. At least 5 fields were examined at 200X magnification and results expressed as the number of bile ducts/high-power field (HPF). To determine the impact of treatments on oxidative stress and activation of post-M3R signaling pathways, livers sections were stained for 3-nitrotyrosine (3-NT, 1:200) (EMD Millipore), phosphorylated extracellular signal-regulated kinases (p-ERK, 1:100) (Cell Signaling Technology, Beverly, MA) and p-AKT (1:50, Cell Signaling Technology, Beverly, MA). Liver sections stained for 3-NT and p-ERK were graded semi-quantitatively by masked investigators for: 0-occasional, 1-minimal, 2-moderate and 3-extensive staining. p-AKT-stained hepatocytes were counted at 200X magnification by examining at least 5 fields per section.</p><!><p>Liver samples were homogenized using a Polytron PT2100 homogenizer (Kinematica, Switzerland) and RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) per manufacturer's instructions. cDNA was synthesized using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Assays were performed in 96-well plates using Sybr green master mix (12.5 μl, Qiagen, USA), template (1 μl), each primer (1 μL, 10 pmol/μl) and nuclease-free water (9.5 μl). Primer sequences are listed in Table 1. The following two-step thermal cycling profile was used for qPCR analysis (StepOnePlus™ Real-Time PCR, Applied Biosystems, USA): Step I (cycling): 95°C for 5 min, 95°C for 10 s and 60°C for 30 s for 40 cycles. Step II (melting curve): 60°C for 15 s, 60°C 1 min and 95°C for 30 s. To determine fold changes in mRNA expression, the ΔΔCt method was used.</p><!><p>Data are expressed as mean ± the standard error (SE). To determine statistical significance, analysis was performed using Student's unpaired t-test (normally distributed data) or the Mann-Whitney U test (nonparametric data). Nominal data were analyzed using a χ2 test with Fisher's test. Correlations were determined using Spearman's test. Survival was analyzed using the Kaplan-Meier method. Significance was defined as P < 0.05.</p><!><p>Initially, to improve our understanding of the specific role of M3R activation, we determined whether a selective M3R antagonist, darifenacin, mimicked the actions of Chrm3 gene ablation on AOM-induced liver injury [17]. Figure 1A illustrates our study design (details in Methods). As shown in Fig. 1B-D, compared to vehicle controls (50% DMSO), darifenacin-treated mice gained less weight, were more likely to die before attaining the study end-point and had reduced liver-weight-to-body-weight ratios. Additionally, darifenacin-treated mice had increased liver nodularity (Fig. 1E), a nearly four-fold increase in fibrosis measured using Sirius red staining (Fig. 1F) and increased Ishak fibrosis scores (not shown). Our previous study indicated that inhibiting M3R activation promotes AOM-induced ductular proliferation [17]. Thus it was also not surprising that we observed a greater than two-fold increase in ductular proliferation in darifenacin-treated compared to vehicle-treated mice (Fig. 1F). Collectively, these findings suggest that darifenacin treatment closely mimics the pattern of AOM-induced liver injury seen with Chrm3 gene ablation than treatment with the non-selective muscarinic receptor antagonist, scopolamine butylbromide [17].</p><!><p>Next, we determined the effects of a selective M3R agonist, pilocarpine, on AOM-induced liver injury in mice. Based on our results with darifenacin, we anticipated that M3R activation would have the contrary effect and ameliorate AOM-induced liver injury. Figure 2A illustrates our study design (details in Methods). First, to confirm that treatment with pilocarpine stimulated M3R activation, we examined the phosphorylation of two key signaling molecules downstream of the receptor, ERK and AKT [19, 20]. As shown in Fig. 2 B and C, at the end-point of the study, immunohistochemical analysis revealed significantly greater ERK and AKT activation in liver sections from pilocarpine-treated compared to control mice, thus providing evidence that pilocarpine activated post-M3R signaling.</p><!><p>During the course of the study, there were no significant differences in the weights of PBS- and pilocarpine-treated mice (Fig. 3A) or in their liver-weight-to-body-weight ratios (Fig. 3C). Compared to control mice, pilocarpine-treatment was associated with a trend towards improved survival (Fig 3B; P = 0.2) and reduced risk of developing ascites (Fig. 3D; P = 0.08). However, liver nodularity was significantly reduced in pilocarpine-treated compared to PBS-treated mice (Fig. 3E).</p><!><p>To determine the effect of pilocarpine treatment on AOM-induced liver fibrosis, we analyzed Sirius red- and H&E-stained liver sections. Representative photographs of Sirius red-stained liver sections are shown in Figure 4A. Pilocarpine-treated mouse livers had significantly reduced collagen deposition (morphometric analysis of Sirius red-stained sections) and Ishak fibrosis scores (H&E-stained sections) compared to those from PBS-treated mice (Fig. 4A and B). Strong correlation was observed between liver nodularity and Sirius red-stained area (r=0.56, P <0.01). In concert with these changes, livers from pilocarpine-treated mice had significantly reduced expression of mRNA for α1-collagen and α-smooth muscle actin (α-SMA) (Fig. 4C and D). These findings validated our assessment of liver injury by gross examination. qPCR analysis of mRNA isolated from mouse livers revealed that compared to those from PBS-treated mice, liver sections from pilocarpine-treated mice had modest, but significant, reductions in the expression of TGF-β1, TGF-β1R, PDGF and PDGFR (Fig. 4E), key mediators of hepatic stellate cell activation [21]; there was no effect on TGF-β2 expression (not shown).</p><!><p>To determine the effect of pilocarpine on ductular proliferation, we examined EpCAM-stained liver sections and counted bile ducts in five or more randomly-selected high-power fields. The numbers of ductules in livers from pilocarpine-treated mice were significantly reduced compared to those from PBS-treated mice (Fig. 4F). These findings indicate that stimulating M3R activation after AOM induction of liver injury attenuates ductular proliferation. There was strong correlation between ductular proliferation and liver fibrosis; bile ductule number in EpCAM-stained liver sections correlated with Sirius red staining (r=0.52, P <0.01). Thus, data from our previous [17] and current studies indicate that the state of M3R activation modulates AOM-induced ductular proliferation.</p><!><p>Liver fibrosis results from an imbalance between matrix production and degradation. TIMPs and MMPs, which derive primarily from activated stellate cells, are major regulators of extracellular matrix deposition and reorganization. Therefore, we determined the effect of pilocarpine treatment on expression of mRNA for TIMPs and MMPs that are recognized as playing major roles in matrix reorganization in cirrhosis. As shown in Fig. 5A, pilocarpine treatment markedly reduced expression of TIMP-1 and TIMP-2, and MMP-2 and MMP-13; mRNA expression of each of these genes was reduced by at least 50%.</p><!><p>Hepatocyte apoptosis is the principal initiator of liver fibrosis. In liver injury, TNF-α induces hepatocyte apoptosis and primes quiescent hepatocytes for replication [16, 22, 23]. Hence, we assessed the effect of pilocarpine treatment on hepatocyte apoptosis, using immunohistochemical staining for cleaved caspase-3 as a marker, and also measured levels of TNF-α and FasL mRNA. As shown in Fig. 5B, cleaved caspase-3 staining was significantly reduced in pilocarpine-treated compared to PBS-treated mice. In addition, pilocarpine treatment significantly reduced hepatic mRNA levels of both TNF-α and FasL (Fig. 5C).</p><!><p>Hepatocyte proliferation is an important restorative response in liver injury. In the absence of injury, hepatocytes remain quiescent. Previously, we showed that immunochemical staining of liver sections from untreated mice revealed rare BrdU-stained nuclei, whereas sections from AOM-treated mice had evidence of increased hepatocyte proliferation [17]. To characterize further the effects of pilocarpine on hepatocyte restoration following injury, we examined BrdU-stained liver sections. Compared to those from PBS-treated mice, livers from pilocarpine-treated mice had a modest, but statistically significant, increase in the numbers of BrdU-stained hepatocytes (Fig. 6A). These findings suggest that pilocarpine-induced M3R activation strengthens the restorative response to liver injury by stimulating hepatocyte proliferation.</p><!><p>Finally, to gain additional mechanistic insight into the therapeutic potential of activating M3R to modulate toxic liver injury, we evaluated the effects of pilocarpine treatment on peroxynitrite generation, an indicator of nitrosative stress that plays a role in hepatocyte apoptosis and fibrogenesis [24-27]. As shown in Fig. 6B, we found that treatment with pilocarpine reduced 3-nitrotyrosine staining in AOM-treated mouse livers (~50% reduction in 3-nitrotyrosine staining). This finding indicates that pilocarpine treatment reduces AOM-induced nitrosative stress in mouse liver.</p><!><p>In the present study, by showing that darifenacin, the most M3R-selective antagonists available [28], augmented AOM-induced liver injury, we validated our previous findings with M3R-deficient mice and mice treated a broad-spectrum muscarinic receptor antagonist scopolamine butylbromide [17]. More importantly, we demonstrate the therapeutic potential of stimulating M3R activation to mollify liver injury. Specifically, we found that treating mice with pilocarpine, an agonist with the greatest affinity for M3R among the five muscarinic receptor subtypes [29, 30], robustly attenuated AOM-induced liver injury as evidenced by: 1) reduced liver nodularity, fibrosis and ascites; 2) reduced hepatocyte apoptosis and increased hepatocyte proliferation; 3) reduced ductular proliferation; 4) down-regulated expression of α1-collagen, α-SMA and other initiators and perpetuators of hepatic fibrosis; 5) reduced expression of liver injury cytokines e.g. TNF-α; 6) reduced oxidative stress and 7) increased activation of hepatocyte survival signaling, e.g. increased activation of ERK and AKT. Our findings with pilocarpine support further investigation of the therapeutic potential of M3R activation to prevent or treat reduce chronic liver injury.</p><p>The role of cholinergic input in regulating liver injury was previously evaluated by other investigators using vagus nerve transection or stimulation, experimental approaches with important disadvantages. Acetylcholine, the major neurotransmitter released by the vagus, activates both muscarinic and nicotinic receptors; both receptor classes are expressed in the liver [5, 8]. Also, in addition to acetylcholine, the vagus releases other neurotransmitters that may modulate injury responses (e.g. vasoactive intestinal peptide) [31]. Hence, approaches utilizing selective M3R ablation and inhibition [17], or selective M3R activation as shown herein, provide a clearer representation of the role that muscarinic receptors play in regulating liver injury. Additionally, our experimental strategy suggests that pharmacological modulation of M3R activation after liver injury (that is, after completion of AOM treatment) alters the liver injury response, again speaking to its therapeutic potential.</p><p>DMSO, the vehicle for darifenacin, is reported to attenuate concanavalin-, thioacetamide- and acetaminophen-induced acute liver injury, and thioacetamide-induced liver fibrosis [32-35]. By inhibiting NF-kB activation, DMSO, a highly effective free-radical scavenger, reduces the transcription of inflammatory cytokines, particularly TNF-α, thereby modulating immune cell function [35-38]. Despite these potentially protective effects of DMSO, darifenacin strongly augmented AOM-induced liver injury; a finding that reveals the major regulatory role of M3R in liver injury. To avoid masking therapeutic benefit with pilocarpine, we used a different vehicle, phosphate-buffered saline, for experiments with the M3R agonist.</p><p>Pilocarpine treatment reduced fibrosis in conjunction with markedly attenuating expression of markers of stellate cell activation, i.e. α1-collagen and α-SMA. Pilocarpine-treated mice had reduced hepatic expression of ligands and receptors that promote stellate cell activation and proliferation, i.e. TGF-β1, PDGF and their receptors. In AOM-treated mice, increased hepatic expression of extracellular matrix regulators such as TIMP-1 and TIMP-2, and MMP-2 and MMP-13, consistent with other models of chronic liver injury, was markedly blunted by pilocarpine treatment [39]. These data indicate that one mechanism whereby treatment with pilocarpine attenuates chronic liver injury is by reducing fibrogenesis.</p><p>Activation of M3R was shown to promote survival and reduce apoptosis in other cell types [6]. Since ongoing hepatocyte apoptosis is a strong contributor to hepatic fibrosis and cirrhosis [40], and rodent hepatocytes express M3R, we assessed hepatocyte survival in our treatment groups. AOM-treated mice that received pilocarpine demonstrated reduced hepatocyte apoptosis and reduced expression of liver injury cytokines, TNF-α and FasL. Moreover, livers from pilocarpine-treated mice showed increased hepatocyte proliferation and activation of ERK and AKT, key pro-survival ligands downstream of M3R activation.</p><p>The data shown here provide insight into the mechanisms whereby M3R activation reduced chronic liver injury. However, our study does have limitations. Pilocarpine crosses the blood-brain-barrier [41] and central nervous system (CNS) effects modulating the liver injury response cannot be excluded. In this context, it is reassuring that AOM-treated mice that received darifenacin had more severe AOM-induced liver injury, similar to that observed previously with M3R ablation [17]; darifenacin has minimal brain penetration. This makes it unlikely that M3R expressed in the CNS plays an important role in the liver injury response. Collectively, our findings suggest that M3R activation within the liver itself is most important for regulating injury.</p><p>In chronic liver diseases, ductular reaction correlates with fibrosis [42, 43]. Recent studies suggested that ductular reaction can also drive fibrosis [44, 45]. Chobert et al. showed that CK-19-positive cells express TGF-β1, which may promote stellate cell activation and hepatic fibrosis [45]. Previously, we reported strong correlation between CK-19 and Sirius red staining [17]. In the present study we observed strong correlation between Sirius red and EpCAM staining. Taken together, our previous and current findings are consistent with the notion that ductular proliferation plays an important role in fibrogenesis [45]. Since bile ductular cells express M3R, we speculate that the modest reduction in ductular proliferation seen in pilocarpine-treated mice results from a combination of pilocarpine-induced M3R-mediated proliferation and reduced compensatory reaction due to augmented hepatocyte proliferation and hepatic parenchyma restoration.</p><p>In conclusion, the key observation in our study is that pharmacological activation of M3R attenuates chronic liver injury. This effect is associated with reduced hepatocyte loss and fibrogenesis, and increased hepatocyte survival. While our study provides mechanistic insights into M3R-mediated regulation of liver injury, additional studies are required to gain a better understanding of M3R-mediated regulation of inflammatory cytokines. Our findings should encourage the development and evaluation of M3R-based therapeutic approaches to prevent liver injury and promote repair.</p>

PubMed Author Manuscript

Metal\xe2\x80\x93organic frameworks constructed from monomeric, dimeric and trimeric phenanthroline citrate zinc building units

Adduct of mononuclear and dinuclear citrate zinc complex [Zn(Hcit)(phen)(H2O)][Zn2(Hcit)(phen)2(H2O)3]\xc2\xb713.5H2O (1) and its aggregate [Zn3(Hcit)2(phen)4]n\xc2\xb714nH2O (2) (H4cit = citric acid, phen = 1,10-phenanthroline) were synthesized in weak acidic solutions. The former was obtained from the reaction of zinc nitrate, citric acid and phenanthroline in a molar ratio of 3 : 2 : 3, while a slightly excess of phenanthroline results in the formation of the polymeric product 2 in a molar ratio of 3 : 2 : 4. Transformation of 1 to 2 was finished by the reaction of 1 with an equimolar of phenanthroline in 72% yield. Reverse conversion of 2 to 1 is obtained in 77% yield, showing an equilibrium between 1 and 2. Neutral compound 1 consists of one monomeric anionic unit [Zn(Hcit)(phen)(H2O)]\xe2\x88\x92 and one dimeric cationic unit [Zn2(Hcit)(phen)2(H2O)3]+ that connect each other by strong hydrogen bonds [O6\xe2\x8b\xafO4w 2.636(2); O7\xe2\x8b\xafO3w 2.630(3) \xc3\x85]. In 2, the citrate ligand links each trinuclear unit [Zn3(Hcit)2(phen)4] to generate an infinite 1D chain that extents into a 3D supramolecular structure by intra- and inter-molecular hydrogen bonds. Moreover, 1 and 2 exhibit strong fluorescence at room temperature.

metal\xe2\x80\x93organic_frameworks_constructed_from_monomeric,_dimeric_and_trimeric_phenanthroline_

<p>Carboxylate–metal complexes have been investigated over the last few years due to their interesting coordination chemistry, unusual structural features, remarkable physical and chemical properties, and potential applications as dyes, extractants, drugs, pesticides, catalysts, [1–4]. The rational design and synthesis of novel metal carboxylates with structural diversity are still of great challenge. Several factors such as the coordination nature of metal ions [5], counteranions [6], solvents [7], metal-to-ligand ratio [8], pH value [9], and reaction temperature [10] have been found to influence the structures of metal-organic coordination compounds. Hydroxypolycarboxylic acids can act not only as hydrogen-bond acceptors, but also as hydrogen-bond donors to form extended structures by means of additional hydrogen-bond interactions. Citric acid, an α-hydroxytricarboxylic acid, has been widely known for its abundance in physiological fluids and its chemical versatility toward transition metal ions. The diverse coordination modes of citrate contribute largely to the formation of various structural types for transition metal citrates, such as mononuclear, dinuclear and polynuclear structural units [11–15]. 1,10-Phenanthroline is an example of N-donor chelating bidentate ligand. It can efficiently provide π-π stacking interactions in either intra- or intermolecular mode. This feature contributes the stability of structures as well as the formation of extended supramolecular structures [16]. These two ligands can simultaneously coordinate with metal ions and provide further intermolecular interactions that allow the formation of supramolecular arrays. In addition, d10 metal (such as ZnII and CdII) complexes exhibit not only appealing structures but also photoluminescent properties [17,18]. In this paper, we reported the syntheses, spectroscopic properties, crystal structures and interconversion of two citrate phenanthroline mixed-ligand complexes [Zn(Hcit)(phen)(H2O)][Zn2(Hcit)(phen)2(H2O)3]·13.5H2O (1), [Zn3(Hcit)2(phen)4]n·14nH2O (2) with different metal-to-ligand molar ratio.</p><p>The syntheses of 1 and 2 were carried out in warm aqueous solutions [19]. The synthetic conditions for 1 and 2 are very similar except for the different metal-to-ligand molar ratio. The reaction with Zn(NO3)2/H4cit/phen molar ratio of 3 : 2 : 3 results in the formation of 1, but the reaction with ratio of 3 : 2 : 4 forms a new coordination polymer 2 with 1D chain. Moreover, polymer 2 could be obtained in 1 : 1 : 1 ratio. Further standing of the reaction mixture will lead to crystallization of 1 on the surface of 2. Compound 1 also crystallized out from the filtrate after 2 was removed, indicating an equilibrium between the two complexes. Transformation of 1 to 2 could be finished by the reaction of 1 with an equimolar of phenanthroline in 72% yield at pH 5.1. Meanwhile, 2 could be transformed to 1 with the addition of zinc nitrate and citric acid at pH 5.3, as shown in Scheme 1. The interconversion between 1 and 2 showed that the metal-to-ligand molar ratio plays a key role in the formations of citrate phenanthroline zinc complexes [19].</p><p>The crystal structure of 1[20] consists of a monomeric anionic unit [Zn(Hcit)(phen)(H2O)]−(1a), a dimeric cationic unit [Zn2(Hcit)(phen)2(H2O)3]+(1b) and crystallized water molecules, as shown in Figures 1a and 1b. Zinc ions are six-coordinated and show similar distorted octahedral coordination geometries [ZnO4N2]. Zn1 and Zn2 are surrounded by three oxygen atoms from α-hydroxy, α-carboxy and β-carboxy groups of citrate, two nitrogen atoms from phenanthroline and one water molecule. Zn3 ion is surrounded by two nitrogen atoms from phenanthroline and two water molecules, and the two oxygen atoms from one β-carboxy group of citrate, containing a weak coordinated Zn3–O14 bond [2.747(2) Å]. The other Zn–O distances range from 2.006(2) to 2.263(2) Å, and the Zn–N distances vary from 2.112(2) to 2.158(2) Å (Table S1). These bond distances around Zn ions are comparable to those previously reported for zinc(II) complexes of α-hydroxycarboxylate and N-donor ligands (Table S3) [13,21–25]. Careful comparisons show that zinc complex [Zn2(Hmal)(phen)Cl]n (H3mal = malic acid) reported recently contains an unusual coordination of malate with α-alkoxy, α-carboxy and β-carboxy groups, in which the α-hydroxy group is deprotonated [13].</p><p>The Zn2 and Zn3 cations in 1 are connected by citrate ligand to form a dimeric unit [Zn2(Hcit)(phen)2(H2O)3]+(1b). The discrete monomeric unit [Zn(Hcit)(phen)(H2O)]−(1a) and dimeric unit [Zn2(Hcit)(phen)2(H2O)3]+(1b) are linked together by strong double hydrogen bonds between the uncoordinated β-carboxy group in 1a and the coordinated water molecules O3w and O4w in 1b [O6⋯O4w 2.636(2), O7⋯O3w 2.630(3) Å]. The monomeric unit 1a is connected each other by strong interlaced hydrogen bonds between the uncoordinated water molecule O1w and the oxygen atom O2 of coordinated α-carboxy group [O1w⋯02a 2.678(2) Å, a -x, 1 – y, 1 – z], which forms a hexameric zinc unit. The oxygen atom O5 of β-carboxy group in 1a further forms strong hydrogen bond with the uncoordinated water molecule O2w in 1b [O5⋯O2wb 2.727(3) Å, b 1 + x, y, z]. This results in the formation of an infinite chain along b-axis (Fig. S1).</p><p>Structural analysis [20] reveals that 2 is a one-dimensional coordination polymer with trinuclear subunits. In the asymmetric unit of 2, there are three zinc atoms, two citrate ligands, and four phenanthroline ligands (Fig. 2a). The Zn1 atom is six-coordinated by four oxygen atoms and two nitrogen atoms in a distorted octahedral coordination geometry. Among the above-mentioned oxygen atoms, three are from α-hydroxy, α-carboxy and β-carboxy groups of one citrate anion, and one is from β-carboxy groups of the other citrate ligand, with Zn–O distances ranging from 2.009(2) to 2.230(2) Å. The two nitrogen atoms are from one phenanthroline ligand with Zn–N distances ranging from 2.126(2) to 2.170(2) Å. The Zn2 center is five-coordinated by two nitrogen atoms from one phenanthroline ligand [Zn–N 2.081(2)–2.126(2) Å], two oxygen atoms from hydroxy and α-carboxy groups of one citrate ligand [Zn–O 2.001(2)–2.143(2) Å], and one oxygen atom from the birdging β-carboxy group of the other citrate ligand [Zn2–O5 = 1.970(2) Å] in a distorted trigonal bipyramidal geometry. Interestingly, there exists a strong intramolecular hydrogen bond between the coordinated hydroxy oxygen atom O8 and the oxygen atom O13 of uncoordinated β-carboxy group in the citrate ligand [O8⋯O13 2.533(3) Å]. The Zn3 center is six-coordinated by four nitrogen atoms from two chelate phenanthroline [Zn–N, 2.120(2) to 2.170(2) Å] and two oxygen atoms from the β-carboxy group of one citrate ligand [Zn–O, 2.155(2) to 2.240(2) Å] in a distorted octahedron. Especially, two oxygen atoms of one β-carboxy group coordinate to Zn(II) ion forming a four-member ring with a O11–Zn3–O12 angle of 59.50(7)°. The trinuclear units are futher expanded to form a 1D chain by the linkage of monodentate β-carboxy groups of the citrate (Fig. 2b). The bond distances around all zinc atoms are comparable to those of zinc(II) complexes with α-hydroxycarboxylate and N-donor ligands previously reported (Table S3) [22,24,26,27]. The citrate ligand exhibits an interesting connection mode with zinc ions. One citrate group acts as a tridentate-monodentate ligand bridging three Zn ions, while the other acts as a bidentate-bidentate ligand bridging to the two Zn ions. In addition, the 1D chains are connected by intermolecular hydrogen bonds to generate a 3D supramolecular structure (Fig. S2).</p><p>The IR spectra of 1 and 2 (Fig. S3) show characteristic bands of carboxylate groups. The peaks at 1624, 1604, 1585 and 1428, 1412, 1386 cm−1 for 1, 1624, 1603, 1587 and 1428, 1394, 1385, 1369 cm−1 for 2 correspond to νasym and νsym of the coordinated carboxy groups, respectively. The absence of IR absorption bands around 1700 cm−1, attributed to protonated carboxylic acid group, indicates the full deprotonation of citric acid in 1 and 2, as revealed by the structural analysis. The IR spectra of 1 and 2 are similar, however, there exist considerable differences in several regions, as shown in Fig. S4. Based on these differences, the reversible transformation between 1 and 2 are traced by IR spectra respectively (Fig. S5 and S6). The results show that the conversion from 1 to 2 processes much quickly than that from 2 to 1. The adduct of monomer and dimer 1 can be transformed into coordination polymer 2 in 5 hrs at 60 °C, while the reverse conversion from 2 to 1 is finished in more than two days at 60 °C.</p><p>Thermogravimetric analysis (TGA) was performed to gauge the thermal stability of the complexes. The experiments were performed on the crystal samples of 1 and 2 under nitrogen atmosphere with a heating rate of 10 °C/min−1 (Fig. S7 and S8). The weight losses of 1 corresponding to the release of the lattice and coordinated water molecules are observed from 35 and 155 °C. The anhydrous composition begins to decompose at 200 °C. The TGA curve of 2 shows it undergoes dehydration between 33 and 135 °C. The decomposition of the anhydrous composition occurs at 225 °C.</p><p>The solid-state fluorescent properties of 1 and 2 were investigated at room temperature. The emission spectra are given in Fig. 3. Upon excitation at 335 nm, 1 exhibits strong fluorescent emission bands at 372 and 385 nm. Upon excitation at 338 nm, 2 exhibits strong fluorescent emission bands at 378 and 397 nm. Compared to the free phen ligand (362 and 380 nm, λex = 340 nm) (Fig. S9), the emission bands of 1 and 2 are red shifted (10 and 5 nm for 1, 16 and 17 nm for 2). As reported in the literatures [28,29], these emission bands of 1 and 2 are probably assigned to intraligand fluorescent emission. In addition, the red-shifts of the emissions may be attributed to effect of metal coordination [30].</p><p>In summary, two zinc(II) compounds have been synthesized from the reactions of zinc nitrate with citrate and phenanthroline in aqueous solution. The structural analysis displays that sligtly changing the metal-to-ligand molar ratio leads to the different structures. 1 is an adduct with a monomeric anionic unit [Zn(Hcit)(phen)(H2O)]− and a dimeric cationic unit [Zn2(Hcit)(phen)2(H2O)3]+, which are linked by strong hydrogen bonds. 2 exhibits a one-dimensional coordination polymer chain structure with trinuclear subunits, linked by the monodentate β-carboxy groups of citrate ligands. In addition, 1 and 2 can be converted reversably and exhibit strong fluorescence at room temperature in the solid state.</p>

PubMed Author Manuscript

Thermodynamic insights into the entropically driven self-assembly of amphiphilic dyes in water

Self-assembly of amphiphilic dyes and p-systems are more difficult to understand and to control in water compared to organic solvents due to the hydrophobic effect. Herein, we elucidate in detail the self-assembly of a series of archetype bolaamphiphiles bearing a naphthalene bisimide (NBI) p-core with appended oligoethylene glycol (OEG) dendrons of different size. By utilizing temperaturedependent UV-vis spectroscopy and isothermal titration calorimetry (ITC), we have dissected the enthalpic and entropic parameters pertaining to the molecules' self-assembly. All investigated compounds show an enthalpically disfavored aggregation process leading to aggregate growth and eventually precipitation at elevated temperature, which is attributed to the dehydration of oligoethylene glycol units and their concomitant conformational changes. Back-folded conformation of the side chains plays a major role, as revealed by molecular dynamics (MD) and two dimensional NMR (2D NMR) studies, in directing the association. The sterical effect imparted by the jacketing of monomers and dimers also changes the aggregation mechanism from isodesmic to weakly anticooperative.

thermodynamic_insights_into_the_entropically_driven_self-assembly_of_amphiphilic_dyes_in_water

Introduction<!>Molecular design and synthesis<!>Temperature-dependent self-assembly and morphology of NBI aggregates<!>Thermodynamic proling of NBI self-assembly<!>Structural characterization via molecular dynamics (MD) and 2D NMR studies<!>Conclusions<!>Conflicts of interest

<p>Self-assembled nano-and mesoscale structures play a major role in nature and particularly in living organisms. [1][2][3][4] The sophisticated functionalities of these structures is imputable to the interplay of hydrogen bonds (H-bonds) 5 as well as solvophobic effects, 6 derived to the unique role of water as a solvent. 4,7 The desire to mimic and understand such naturally occurring self-assembled systems has prompted the investigation of various amphiphilic/bolaamphiphilic molecules consisting of non-polar hydrophobic cores attached with water solubilizing side chains. 8 Through these investigations, a wide variety of nanometric supramolecular aggregates of different morphologies (tubular, brillar, micellar, vesicular) has been prepared via exploring solvophobic effects, [9][10][11] H-bonding, [12][13][14][15] electrostatic screening, 16,17 metal-ion coordination, [18][19][20] variation of hydrophilic/hydrophobic balance, 21,22 and co-solvent modulation. [23][24][25] However, different from the very intensively conducted studies on the enthalpic and entropic contributions that govern supramolecular host-guest complex formation in water, [26][27][28] studies devoted to an in-depth understanding of the thermodynamic prole of self-assembly processes of p-amphiphiles in water remain scarce. 29 Nevertheless, such an understanding is warranted not only from a supramolecular design perspective, but also in therapeutic, and materials sciences.</p><p>In this direction, we have identied p-conjugated cores of perylene bisimide dyes appended with six oligoethylene glycol (OEG) chains as very useful amphiphilic molecules, whose selfassembly processes can be easily followed by various spectroscopic and microscopic techniques. [30][31][32][33] But only very recently we unveiled our serendipitous discovery that the self-assembly of OEG substituted perylene bisimide (PBI) derivatives in water is not driven by enthalpic dispersion and electrostatic forces as in organic solvents, 31 but by entropic factors, albeit the process can be shied to an enthalpic route by the addition of only small amounts of an organic co-solvent. 34 We attributed this intriguing behaviour to the exclusion of water molecules from the OEG side chains which leads to a dominant entropic contribution to the self-assembly in pure aqueous environment, which was also later demonstrated for other dye assemblies by Ghosh et al. 35 Whilst these interesting results warrant further studies, our PBI systems aggregated too strongly in water, evading an in-depth thermodynamic characterization including isothermal titration calorimetry (ITC). Due to the smaller pcore, naphthalene bisimides (NBIs) appeared to be more promising because their self-assembly requires higher concentrations which is benecial for methods like NMR and ITC.</p><p>Herein, we report our detailed studies on the self-assembly of a series of naphthalene bisimides functionalized with OEG chains of different glycol units (NBI 1: n ¼ 2, NBI 2: n ¼ 3, and NBI 3: n ¼ 4) in water (Fig. 1a). By means of UV-vis spectroscopy and ITC studies, we have achieved the dissection of entropic and enthalpic contributions to their self-assembly. Remarkably, we found that enthalpy, entropy and free energy changes of NBIs 1-3 aggregation in water strongly depend on the interaction of water molecules with the ether oxygens and accordingly on the OEG chain length (Fig. 1b). Additional structural investigations by molecular dynamics (MD) and 2D-NMR techniques revealed back-folding of glycol chains with sequestration of the NBI p-cores from water, to be of importance as well.</p><!><p>In our previous work, the larger p-core of PBI prevented the quantication of aggregation parameters in pure water due to its pronounced association tendency. 34 Hence, for the current study, we have employed the smaller naphthalene homologue in lieu of perylene to assert moderate aggregation constants in water. Following the same design principles, bolaamphiphilic derivatives NBI 1-3 were prepared with OEG-substituted brushes at both ends of the p-core (Fig. 1a). The number of glycol units per chain was systematically increased from three to four and ve for NBI 1, NBI 2 and NBI 3, respectively.</p><p>The synthesis of amphiphilic brush substituents was carried out by a two-fold, one-pot Sonogashira reaction via coupling of 2-bromo-1,3-diiodo-5-nitrobenzene with respective glycol chain substituted with a propargyl unit. 34 Subsequent reduction of the triple bonds and the nitro group in H 2 atmosphere at high pressure in the presence of 10% Pd on carbon yielded the corresponding amino derivatives. Finally, these compounds were treated with naphthalene-1,4,5,8-tetracarboxylic dianhydride in acetic acid to obtain the bolaamphiphilic NBI derivatives. The synthetic details and characterization data for all new compounds are reported in the ESI. †</p><!><p>To investigate the aqueous self-assembly behaviour of NBIs 1-3, initially we performed temperature-dependent UV-vis experiments below the cloud point (vide infra). For comparison, rst we measured the absorption spectra of the NBIs in CHCl 3 (Fig. S1 †). In an organic solvent of intermediate polarity like CHCl 3 , NBI 1 exists in monomeric form and shows an absorption maximum at 381 nm. In water, even at a dilute concentration of 9.7 Â 10 À5 M, this absorption maximum is shied to 364 nm, suggesting an H-type aggregated state (Fig. 2a). While monitoring the temperature-dependent UV-vis spectra of NBI 1 in a range of 10 C to 50 C in water, we observed a decrease in the ratio of these two vibronic bands, with a concomitant hypochromic shi. This clearly indicates an increase in degree of aggregation with increasing temperature. 13 Similarly, both NBI 2 and NBI 3 exhibit an inverse temperature response where the aggregation is favoured at elevated temperatures (Fig. S2 †). Unlike the majority of supramolecular systems which disassemble upon heating, we have previously observed that bolaamphiphilic PBIs attached with OEG brushes show an inverse temperature response, where aggregation is favoured at higher temperatures. 34 This unique thermodynamic signature is now also manifested in the current NBI series as corroborated by temperature-dependent UV-vis experiments. It is noteworthy that the spectral changes upon aggregation are by far less pronounced for NBIs compared to PBIs due to the much smaller transition dipole moment of their S 0 / S 1 transition.</p><p>At higher temperatures, we observed the phase separation of the NBIs from the aqueous phase. This is attributed to the lower critical solution temperature (LCST) phenomenon which is typical for OEG appended systems. 36 The specic temperature denoting the onset of this precipitation, called cloud point (CP), can be determined by monitoring the transmittance at a wavelength where the molecule does not absorb (here 800 nm). The phase separation from the binary solution is characterized by an abrupt drop in transmittance. The knowledge of CP is quintessential for our self-assembly studies since it sets the upper limit for the temperature window where aggregation can be monitored. Furthermore, it gives clue towards the amount of water molecules forming H-bonds to OEG chains, as the CP increases with extent of hydration. NBI 1, containing the shortest glycol chain, shows a CP of 59 C at a concentration of 1 Â 10 À3 M in water, while NBI 2 and NBI 3 show phase separation at 78 and 88 C, respectively, at the same concentration (Fig. 2b). Since the clouding is mainly associated with the dehydration of glycol units, an increase in the CPs suggests an increase in the extent of hydration with elongation of glycol chains.</p><p>In order to characterize the morphology of the aggregates formed below CP, stock solutions of NBIs in water at 22 C were spin coated onto silicon wafer treated with argon plasma and visualized using atomic force microscopy (AFM). The microscopy images for NBI 1 obtained by tapping mode reveal short nanorods with a diameter of $2 nm and an average size distribution of 20-45 nm, suggesting a one dimensional (1D) self-assembly (Fig. 2c). The presence of anisotropic aggregates was further conrmed via DLS measurements which showed size dependence upon variation of the scattering angle (Fig. S3a †). 37 Similarly, morphological investigations performed on NBI 2 as well as NBI 3 suggested nano-rod like selfassembled species with a diameter of $2 nm (Fig. S4 †).</p><!><p>In an attempt to obtain a comprehensive thermodynamic prole for the self-assembly of NBIs 1-3 in water, we explored concentration-dependent UV-vis studies below CP to monitor their transformation from monomers to 1Daggregates. Fig. 3a displays the spectral changes observed in our concentrationdependent experiment performed on NBI 1 at 25 C.</p><p>It was observed that with an increase in concentration, the absorption maximum shis to 364 nm compared to the monomeric absorption maximum (381 nm), correlating to the spectral changes observed in temperature-dependent measurements. This suggests the formation of an H-type excitonically coupled stack. 38 Moreover, the transition from the monomeric to aggregated state is characterized by the presence of two isosbestic points (324 nm and 394 nm), implying an equilibrium between monomeric and aggregated species.</p><p>Fig. 3a inset shows the corresponding plot of the degree of association (a agg ) versus the logarithm of concentration. It was observed that the best t for the data points was obtained with an isodesmic model, i.e. an equal association constant for each monomer addition. 39 From this, the logarithm of the association constant, log K ass ¼ 3.8, and the standard Gibbs free energy of association ðDG ass Þ of À21.9 kJ mol À1 was estimated for NBI 1 (at 25 C). From the concentration-dependent UV-vis studies, the critical aggregation concentration (CAC) of 0.33 mM was also determined for NBI 1 at 25 C (Fig. S9a †). 14 To delve deeper into the understanding of thermodynamic parameters associated with the self-assembly, we performed the same experiment at different temperatures, from 10 to 50 C (Fig. S5 †).</p><p>Previously, the van't Hoff equation has been successfully utilized to derive standard enthalpy ðDH ass Þ and standard entropy ðDS ass Þ changes of self-assembly by assuming a linear relationship of the natural logarithm of aggregation constants with respect to temperature. 40,41 However, this method is only valid when the enthalpy and entropy changes remain constant with changes in temperature. 42 Processes in water, however, are usually associated with wide uctuations in these parameters, thus impeding an accurate description of the self-assembly process. 29,43,44 This limitation can be surpassed by taking the heat capacity changes into account. One such modication is Clarke-Glew method, where the isobaric temperature dependence of rate constants is described around a reference temperature, q. 45,46 This approach allows the calculation of the change in heat capacity at constant pressure, DC p , which is inaccessible by the van't Hoff equation due to its inherent assumptions. According to the simplied form of Clarke-Glew method (also referred as extended/integrated van't Hoff equation), the change in association constant with respect to temperature can be expressed by eqn (1). ln ½KðTÞ ¼ ln ½KðqÞ þ DHðqÞ R</p><p>where ln [K(T)] is the natural logarithm of the equilibrium constant at temperature T, ln [K(q)] is the natural logarithm of the equilibrium constant at the reference temperature q, DH(q) is the enthalpy change at the reference temperature, and DC p is the change in heat capacity at constant pressure.</p><p>While plotting the natural logarithm of the association constant versus the inverse of temperature, indeed a much better t is obtained with the non-linear Clarke-Glew equation as compared to the van't Hoff equation (Fig. 3b). Accordingly, a standard enthalpy of 11.2 kJ mol À1 and a heat capacity change of À289 J mol À1 K À1 can be calculated for the self-assembly of NBI 1. With an elevation in temperature, an increase in aggregation strength is observed, quantitatively supporting our temperature-dependent UV-vis measurements. Furthermore, the negative slope of the curve suggests the endothermic nature of self-association over a broad temperature range, which is hence enthalpically disfavoured.</p><p>Similarly, concentration-dependent UV-vis experiments were conducted for NBI 2 and NBI 3 at different temperatures (Fig. S6 and S7 †). In both cases, we observed that the mechanism of selfassembly differs from the isodesmic model and is better described by a weak anti-cooperative process with a cooperativity factor of s ¼ 2 and s ¼ 3 for NBI 2 and NBI 3, respectively. By tting the data according to the Goldstein-Stryer model 47 utilized for (anti)cooperative aggregation processes, a logarithm of the association constant, log K ass ¼ 3.3, and a standard Gibbs free energy, DG ass ¼ À18:8 kJ mol À1 was determined for NBI 2 at 25 C, suggesting a weaker aggregation tendency as compared to NBI 1. Using the Clarke-Glew plot, a standard enthalpy change of 18.1 kJ mol À1 is calculated, which shows that the selfassembly of NBI 2 is enthalpically more disfavoured than NBI 1 (Fig. S8a †). NBI 3 exhibited the weakest aggregation tendency of all three derivatives, with log K ass ¼ 2.8 and a standard Gibbs free energy, DG ass ¼ À16:4 kJ mol À1 at 25 C. The selfassembly, in this case, is disfavoured by a standard enthalpy ðDH ass Þ of 23.2 kJ mol À1 (Fig. S8b †). Furthermore, the CAC estimated for NBI 2 (1.6 mM) and NBI 3 (3.5 mM) at 25 C conrms the decreasing tendency of aggregation while increasing the glycol chain length from NBI 1 to NBI 3 (Fig. S9b and c †).</p><p>The thermodynamic signature at 25 C obtained for the three derivatives is represented in Fig. 4, which depicts that the selfassembly for all the NBI derivatives in water is enthalpically disfavoured and entropically driven. Furthermore, this penalty in the standard enthalpy of association ðDH ass Þ and the gain in standard entropy of association ðDS ass Þ is augmented as the OEG chain length is increased from NBI 1 to NBI 3. Since our CP measurements suggest an increase in hydration with chain elongation, this trend can be attributed to the increased number of water molecules that are removed for well-hydrated monomer units upon aggregation. On the other hand, the aggregation tendency decreases with an increase of glycol units as reected by increased DG ass values. In order to validate the thermodynamic parameters obtained by our UV-vis experiments, we resorted to an independent technique to derive the enthalpy, entropy and free energy of association. This technique is given with an ITC dilution experiment that allows direct determination of enthalpy and gathers insight into its temperature dependency, which is inaccessible via other methods. Even though ITC is well established for natural 48,49 and synthetic host-guest interactions, 28,[50][51][52] the advent of this technique to probe self-assembly is quite recent. 34,[53][54][55] In a typical ITC dilution experiment, aliquots of a concentrated solution of the aggregated species is titrated into the pure solvent taken in the cell. The dissociation of the aggregate is then accompanied by non-constant heat signals along with constant heat of dilution. 53 From this, enthalpy and other thermodynamic parameters can be determined. Fig. 5a shows the evolution of heat per injection of a concentrated aqueous NBI 1 solution (c ¼ 5.2 Â 10 À3 M) into pure water at 25 C leading to its disassembly, which depicts an exothermic heat ow, i.e., the dis-assembly process is enthalpically favoured.</p><p>The corresponding enthalpogram could be well tted to an isodesmic model (Fig. 5b). 56,57 A standard enthalpy change of À13.8 kJ mol À1 for dis-assembly (or +13.8 kJ mol À1 for the corresponding self-assembly) and logarithm of the association constant, log K ass ¼ 3.8 at 25 C was determined for NBI 1, which is indeed in good concordance with the previously obtained values from UV-vis experiments (vide supra). Also, a CAC value of 0.21 mM was deduced for NBI 1 from the aforementioned ITC dilution experiment (Fig. S10 †). 35 The accompanying heat of dilution estimated from the overall heat evolved during injection of NBI 1 is provided in Table S1. † Accordingly, different from our previous study of a strongly aggregating PBI, 34 here we could for the rst time quantify the entropically driven self-assembly thermodynamics in water and derive values for DH ass and K ass of high accuracy. The thermodynamic parameters obtained by both these methods are tabulated in Table 1.</p><p>Successively, to understand the inuence of temperature on the enthalpy of self-assembly, we repeated the ITC dilution experiment at different temperatures, from 10 to 50 C (Fig. S11 †). It was observed that with increasing temperature, the enthalpy of association for NBI 1 is concomitantly decreased (Fig. 5c). It is expected that an elevation in temperature decreases the H-bond strength between the water molecules and OEG chains, 58 thus reducing the enthalpic penalty associated with the dehydration of water molecules during selfassembly. The increased aggregation tendency of these systems at higher temperatures could be traced to this easiness in the release of H-bonded water molecules. The resulting heat capacity change for NBI 1 aggregation was quantied as À280 J mol À1 K À1 using eqn (2),</p><p>Similar dilution experiments in pure water were also performed for NBI 2 and NBI 3 at 25 C (Fig. S12 and S13 †). Here also the dilution experiments revealed exothermic signals for disassembly, accordingly the self-assembly is endothermic. Intriguingly, in both cases, we observed heat signals associated with two distinct processes (Fig. S12b and S13b †). Such two-step processes with similar heat signature have been previously reported for host-guest studies of ions with macrocycles which follow negative cooperative mechanism. 59 We assume that since the aforementioned derivatives aggregate via a weak anticooperative mechanism, the rst injections might represent the dissociation of fully aggregated aliquots into monomers whereas the latter injections show the dissociation into the dimeric species. Unfortunately, the currently available model was not able to describe these processes and hence hampers the accurate determination of aggregation parameters for NBI 2 and NBI 3. Furthermore, the lack of saturation at the end-point of dilution experiment due to lower aggregation tendency impedes the estimation of CAC for NBI 2 and NBI 3 via ITC.</p><p>Thus, we could independently conrm by both UV-vis studies and ITC dilution experiments that the self-assembly of NBIs 1-3 in water is entropically driven and primarily attributable to the release of water molecules from the glycol units.</p><p>Here the length of the OEG side chains plays a prominent role for both the enthalpic and entropic contributions to the aqueous self-assembly of our NBI series. In order to obtain deeper insights into the role of molecular structure in orchestrating this specic aggregation trend in water, structural attributes, especially the conformational nature of glycol units have to be investigated in detail.</p><!><p>Aer procuring quantitative information about the thermodynamic signature associated with the self-assembly, we pondered upon the role of molecular structure in directing the association process. For this, we employed all atoms molecular dynamics (MD) simulations in pure water on NBI 1 and NBI 3 which have the shortest and the longest OEG chains, respectively, in the series. Interestingly, both NBIs show a back-folded conformation of glycol chains around the naphthalene core in the monomeric form (Fig. 6a and d). A similar observation was previously discussed by Meijer and Pavan et al. and has been attributed to the shielding of the hydrophobic surface from the surrounding bulk water. 12,60 Fig. 6b and e show the density proles of C-atoms of OEG chain over the aromatic cores for NBI 1 and NBI 3. As seen clearly, the preferred orientation of side chains during the MD regime resides over the core instead of extending into the bulk water.</p><p>Next, two such pre-equilibrated monomers were immersed into a periodic simulation box lled with explicit water molecules and allowed to equilibrate over MD regime. The distance between the two monomers (0.4 nm) suggests an explicit p-p stacking, with a rotational offset of 10 (Fig. S15a and S16a †). A snapshot from the trajectory of NBI 1 stacking depicts that the glycol chains still prefer a back-folded orientation in the aggregated state (Fig. S14a †). However, the tail density is now more distributed around the p-core, suggesting that some of the back-folding was replaced in order to accommodate the incoming monomer (Fig. 6c). This release of ordered chains might contribute to the conformational entropy of side chains, aiding overall entropy of the association, along with the removal of hydrated water molecules.</p><p>Similarly, for NBI 3, stacking interactions were studied via MD simulations (Fig. S14b †). Here we see again that the glycol chains are folded over the naphthalene core, in both monomeric and dimeric form. However, due to the increased length of OEG units, the density of back-folded conformation is concomitantly higher as compared to NBI 1 (Fig. 6f). The rotational offset for the NBI 3 stack ($60 ) is signicantly larger compared to NBI 1, which could be rationalized by the steric hindrance of back-folded glycol chains (Fig. S16b †).</p><p>To experimentally verify the presence of back-folding as suggested by MD simulations and to unravel the aggregate structure, we conducted detailed one-dimensional (1D) and two dimensional (2D) NMR studies. The 1 H NMR spectrum of NBI 1 in CDCl 3 shows well resolved sharp signals corresponding to the monomeric state (Fig. 7a). In contrast, the naphthalene core protons are signicantly broadened as well as up-eld shied in D 2 O, indicating an aggregated state aided by p-p stacking. Insights into the aggregate structure were probed subsequently via 1 H-1 H Rotating Frame Overhauser Effect Spectroscopy (ROESY). Fig. 7c and d show selected regions of superimposed ROESY and COSY spectra of NBI 1 in D 2 O. Nuclear Overhauser Effect (NOE) correlations could be observed between the naphthalene core protons (H a ) and the glycol protons (H e /H e 0 ) which is in compliance with the back-folded conformation of side chains. The coupling between the naphthalene core protons and phenyl protons suggests a slightly rotated offset between NBI 1 monomers in the stacked conformation as predicted by MD simulations. A tentative assignment of NOE correlations with a snapshot from MD regime of NBI 1 is given in Fig. 7e. Similarly, for both NBI 2 and NBI 3, through-space interactions could be traced between glycol chain protons (H e / H e 0</p><p>) and core protons (H a ), thus corroborating the presence of back-folded conformations in these systems (Fig. S17 and S18 †) and validating the structural predictions from MD simulations.</p><p>In the thermodynamic analysis of the current system, we have observed that the elongation of glycol units from NBI 1 to NBI 3 is associated with a nearly ten-fold decrease in association constant and a concomitant drop in the magnitude of free energy. Combined results from MD simulations and NMR studies suggest that the back-folding of glycol chains is orchestrating this effect. Furthermore, the change of aggregation mechanism from isodesmic to weak anti-cooperative can also be attributed to the more pronounced jacketing of monomer and dimer species by the longer OEG chains.</p><p>In addition, our results also relate to the studies on biomolecules. Here, previously it was observed that the substitution of proteins with polyethylene glycol (PEG) results in a decrease in binding affinity due to the interactions between PEG chains and the active site of the protein. 61,62 Meijer et al. predicted that this could be ascribed to the back-folding of the glycol chains operative in water. 63 Our current studies prove that the back-folding indeed interferes with the association process by shielding the hydrophobic surface from the surrounding bulk water. Accordingly, we can conclude that OEG and PEG chains play a pivotal role in directing the thermodynamics of aggregation in water.</p><!><p>In this contribution, three archetype bolaamphiphilic naphthalene bisimides were studied to derive an understanding of the different factors that contribute to the entropically driven self-assembly of bolaamphiphilic moieties substituted with OEG units in water. By utilizing UV-vis and ITC dilution experiments, we have successfully dissected the thermodynamic parameters of the aggregation process. The entropically favoured nature of the self-assembly is attributed to the release of water molecules from the glycol units which is enthalpically penalized. Further, we were able to show that a thermodynamic tuning of p-core aggregation in water can be achieved by modulating the length of solubilizing OEG chains. With elongation of the side chains, the enthalpic as well as entropic parameters also increase, attributed to an increment in dehydrated water molecules upon aggregation. However, this augmentation in their length hinders the self-assembly via a back-folding process as revealed by MD simulations and 2D-NMR studies, resulting in a decrease of the magnitude of Gibbs free energy and deviation from the isodesmic mechanism. Our current study sheds light into the fundamental aspects of bolaamphiphilic aggregation in water and opens up a strategy for more predictable aqueous self-assembly processes of oligo-and polyethylene glycol functionalized amphiphilic molecules.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Hydrofluoromethylation of alkenes with fluoroiodomethane and beyond

A process for the direct hydrofluoromethylation of alkenes is reported for the first time. This straighforward silyl radical-mediated reaction utilises CH 2 FI as a non-ozone depleting reagent, traditionally used in electrophilic, nucleophilic and carbene-type chemistry, but not as a CH 2 F radical source. By circumventing the challenges associated with the high reduction potential of CH 2 FI being closer to CH 3 I than CF 3 I, and harnessing instead the favourable bond dissociation energy of the C-I bond, we demonstrate that feedstock electron-deficient alkenes are converted into products resulting from net hydrofluoromethylation with the intervention of (Me 3 Si) 3 SiH under blue LED activation. This deceptively simple yet powerful methodology was extended to a range of (halo)methyl radical precursors including ICH 2 I, ICH 2 Br, ICH 2 Cl, and CHBr 2 F, as well as CH 3 I itself; this latter reagent therefore enables direct hydromethylation. This versatile chemistry was applied to 18 F-, 13 C-, and D-labelled reagents as well as complex biologically relevant alkenes, providing facile access to more than fifty products for applications in medicinal chemistry and positron emission tomography.

hydrofluoromethylation_of_alkenes_with_fluoroiodomethane_and_beyond

Introduction<!>Results and discussion<!>Conclusions

<p>The introduction of uoroalkyl groups has garnered signicant interest in medicinal chemistry, enabling the modulation of biological and physicochemical properties of lead candidates for drug discovery. [1][2][3] Whilst the elds of radical tri-uoromethylation and diuoromethylation have been extensively explored, [4][5][6][7][8][9][10] the uoromethyl radical has received far less attention. [11][12][13] This is unexpected as the uoromethyl group features frequently in pharmaceutical drugs, more oen to improve metabolic stability by serving as a bioisosteric replacement of functional groups responsible for poor performance. 14,15 In recent years, several reagents for the generation of the CH 2 F radical have been developed. [16][17][18][19][20] Oen, efficient activation of these reagents requires harsh reaction conditions, such as elevated temperatures, strong oxidants, or strong reductants. Furthermore, many of these reagents are either expensive, highly toxic or non-commercial, requiring multistep syntheses for their preparation. As part of our growing interest in developing "minimalistic" procedures for the late-stage hydrouoroalkylation of alkene-containing biologically active molecules, [21][22][23] we sought to develop an operationally simple method for the direct hydrouoromethylation of alkenes, as an attractive strategy for the introduction of this motif to C(sp 3 )enriched backbones (Scheme 1).</p><p>In 2020, an indirect method for the hydrouoromethylation of alkenes was developed by Aggarwal and co-workers; 13 this elegant multi-step procedure starts with the conversion of alkenes into boronic esters, subsequent treatment at low temperature (À78 C) with in situ formed uoroiodomethyl lithium to generate uoroboronic esters, and a nal protodeboronation. Our aim was to develop a one-step method that avoids operational complexity and over-engineering, ideally using uoroiodomethane which is a non-ozone depleting, easy to handle and inexpensive commercial CH 2 F radical precursor. We noted that uoroiodomethane has found applications as an electrophilic or nucleophilic uoromethylation reagent as well as in cross-coupling reactions, [24][25][26][27] but has not been explored in the context of radical chemistry.</p><p>The high reduction potential of CH 2 FI (E red ¼ À2.19 V vs. saturated calomel electrode (SCE) in MeCN), 28 much closer to MeI (E red ¼ À2.39 V vs. SCE in MeCN) 28 than CF 3 I (E red ¼ À1.22 V vs. SCE in MeCN), 29 encouraged the implementation of an activation pathway exploiting instead the favourable bond dissociation energy (BDE) of C-I (BDE (FH 2 C-I) ¼ 233 kJ mol À1 ) versus C-F (BDE (IH 2 C-F) ¼ 460 kJ mol À1 ). 30 Since the pioneering work of Chatgilialoglu, 31 tris(trimethylsilyl)silane (TTMSS) has found ample applications as a powerful tool for mild radical generation via the activation of alkyl halides. [32][33][34][35][36] In addition, TTMSS has valuably complemented Giese-type reactions, a commonly exploited platform for late-stage functionalisation, by providing a suitable alternative to traditional toxic tin-based reagents. 37 Consequently, we envisioned that the supersilyl radical (TMS) 3 Sic would be well suited to release cCH 2 F from CH 2 FI. Subsequent Giese-type addition of cCH 2 F to the electron-decient alkene would generate a carbon-centered radical intermediate. Hydrogen-atom transfer (HAT) between this electrophilic species and hydridic (TMS) 3 SiH would afford the desired hydrouoromethylated product, and (TMS) 3 Sic entering chain propagation. Initiation for this process would be triggered by photolytic C-I cleavage of FH 2 C-I. 38 This method offers the prospect of being applicable to a range of other halocontaining alkyl radicals, provided that competitive hydrogen atom abstraction with (TMS) 3 SiH does not occur prior to Giese addition. Herein, we report the realisation of this strategy with a wide range of haloiodomethanes for the direct hydrohalomethylation of electron-decient alkenes including biologically relevant molecules. The method was extended to 18 F-hydrouoromethylation and hydromethylation with iodomethane along with ve of its D and 13 C isotopomers.</p><!><p>Preliminary experiments were conducted with N-phenyl acrylamide (1a) (Table 1). 39 Various combinations of silanes and solvents revealed that the desired hydrouoromethylated product (2a) was obtained in 71% with (TMS) 3 SiH in MeCN at room temperature under blue light irradiation for 16 h (entry 1). 40 The addition of fac-Ir(ppy) 3 (0.5 mol%) did not lead to signicant improvement (entry 2). The simpler protocol was therefore retained for further investigations. Control experiments indicate that the reaction was not effective in absence of light (entry 3), and unsuccessful in absence of silane or in presence of the radical scavenger TEMPO (entries 4 and 5). No deuterium incorporation was observed in the product when the reaction was performed in CD 3 CN. 40 These data corroborate our proposed radical chain propagation mechanism, initiated by blue-light homolysis of the CH 2 F-I bond. 38 Giese addition of the uoromethyl radical to an electron-decient alkene furnishes an electrophilic carbon-centered radical intermediate, capable of undergoing HAT with (TMS) 3 SiH. The resulting silyl radical enables chain propagation by abstracting iodine from CH 2 FI to afford (TMS) 3 SiI along with cCH 2 F. 40 With the optimised reaction conditions in hand, we sought to explore the scope of this hydrouoromethylation protocol (Scheme 2A). Various functional groups, such as methoxy, nitrile, halide, ketone, ether, amide, ester, aniline, and sulfone were tolerated. The addition of fac-Ir(ppy) 3 (0.5 mol%) led to higher yields for selected substrates. 40 N-Aryl acrylamides bearing electron-withdrawing and electron-donating groups afforded the desired products in moderate to excellent yields (2a-d). The hydrouoromethylation of N-heteroaryl acrylamides, such as pyridyl and benzothiazyl was also successful (2e, 2f). Alkenes substituted with sulfones and esters were competent substrates generating 2g and 2h in moderate yield. As deuteration can improve metabolic stability, 41 we investigated the hydrouoromethylation of a deuterated alkene (1i) that was successfully converted into [D 3 ]2i. The gem-disubstituted alkene 1j provided 2j in 64% yield. Pleasingly, the internal alkene 1k was reactive under our reaction conditions and afforded uoromethylcyclobutane 2k in moderate yield. This result is signicant as 1,2-disubstituted uoroalkyl cyclobutanes currently require multiple steps for their preparation. 42 A non-Scheme 1 Hydro(per)fluoromethylation of alkenes. This work: direct silyl radical-mediated hydrofluoromethylation of electron-deficient alkenes and extension to numerous hydro(halo)methylation reactions. cyclic trisubstituted alkene afforded the product in 57% yield (2l). Styrene derivatives such as 1m and 1n afforded the desired products in synthetically useful yields (2m, 2n). Our protocol is amenable to scale-up as demonstrated by the 10 mmol scale hydrouoromethylation of N-benzylmaleimide affording 2o in 88% yield. The synthesis of uorinated pyrrolidine 2p, amine 2q, alcohol 2r and carboxylic acid 2s was performed in two steps, offering a pathway to diversify the range of products within reach from CH 2 FI. The late-stage hydro-uoromethylation of complex biologically active molecules was considered next. The anti-cancer drug ibrutinib as well as estrone, tyrosine and ethacrynic acid derivatives afforded the desired hydrouoromethylated products in good yields (2t-w).</p><p>The tolerance of functional groups was investigated with a robustness screening. 40 These experimental data provide an overview of the many heteroarenes (e.g. pyridazine, 1,3,5triazine, indole, benzothiazole or oxazole) that are tolerated under the optimised reaction conditions. Whilst additives containing nucleophilic functional groups such as alcohols and anilines were tolerated, side reactivity arising from nucleophilic substitution was observed. 40 Competitive alkylation was suppressed when using 1.0 equivalent of CH 2 FI, albeit at the expense of reduced yield for the hydrouoromethylated product. Aliphatic amines were tolerated but yields did not exceed 30%. 40 The hydro-uoromethylation of alkenes not bearing electron-withdrawing groups was possible albeit signicantly less efficient. 40 With a protocol relying on the favourable C-I bond dissociation energy and considering the importance of homologation in medicinal chemistry, 43 we considered the generation of products from a series of homologated uoroiodoalkanes (Scheme 2B). 44,45 Hydrouoroalkylation of alkenes 1g, 1j and 1l provided effortlessly the homologous series of products 3d-i. Specically, the uoroethyl radical was efficiently generated applying similar silyl radical activation, and 3a was isolated in good yield. The introduction of the uoroethyl radical was successfully performed on linear terminal, gem-disubstituted, and trisubstituted alkenes (3d, 3f, 3h). The method was further extended to uoroiodopropane as shown with the synthesis of 3e, 3g, and 3i. Precursors featuring additional uorine atoms were less suitable with the diuoroethylated product 3b isolated in 30%, and no product observed when attempting to prepare the hydrotriuoroethylated product 3c. Increased uorine content enhances radical electrophilicity, thereby encouraging undesired H-atom abstraction from (TMS) 3 SiH. 40 Given the success of our protocol, we further investigated the applicability of our method for the generation of [ 18 F]CH 2 F radical from [ 18 F]CH 2 FI (Scheme 2C). [46][47][48] Compounds labelled with the radioisotope F-18 are important for applications in Positron Emission Tomography (PET). [49][50][51][52][53] The synthesis of [ 18 F] CH 2 FI in high molar activity (A m ) is well-established and has been automated. [54][55][56][57] To date, this labelled reagent is mainly employed for the electrophilic 18 F-uoromethylation of phenols. 58,59 We now demonstrate that [ 18 F]CH 2 FI is well suited for [ 18 F]CH 2 F radical chemistry. Specically, Ibrutinib, an estrone, a tyrosine, and an ethacrynic acid derivative underwent 18 F-hydrouoromethylation in radiochemical yields up to 81% ([ 18 F]2t-w). This reaction was best performed for 20 minutes at ambient temperature in the presence of fac-Ir(ppy) 3 under bluelight irradiation. This method offers an alternative to nucleophilic 18 F-uorination with [ 18 F]uoride for precursors that are either unstable, require complex multiple steps synthesis, or lead predominantly to elimination products. Haloiodomethanes other than uoroiodomethane were also considered as they would allow for the one-step introduction of reactive halomethyl groups to alkenes (Scheme 2D). Controlled activation of reagents such as ICH 2 X (X ¼ Cl, Br, I) would enable their use for example as cCH 2 + synthon. To date, only few examples for the generation and use of halomethyl radicals have been reported. [60][61][62][63][64] When diiodomethane was employed under the standard reaction conditions, N-benzylmaleimide underwent hydroiodomethylation in 62% yield (4a). Similarly, hydrobromomethylation (from dibromomethane or bromoiodomethane), hydrochloromethylation (from chloroiodomethane), and hydrobromouoromethylation (from dibromouoromethane) provided the corresponding halomethyl alkanes in moderate yields (4b-4d). 23,65 Other alkenes afforded the hydrochloromethylated products in moderate yields (4e-4g).</p><p>Although full conversion of starting material was observed for these reactions, purication via silica gel chromatography led to elimination, which is reected in the lower yield for these compounds upon isolation.</p><p>Competition experiments were performed to calibrate the reactivity of uoroiodomethane versus other alkyl iodides (Scheme 3). When equimolar amounts of iodomethane and uoroiodomethane were subjected to the standard reaction conditions, product resulting from uoromethyl radical addition was obtained in 74% yield (2n), along with 25% of the hydromethylated product 5a. When the reaction was carried out with equimolar amounts of iodoethane, products 2n and 6 were formed in close to 1 : 1 ratio. Additional competition experiments showed that the iso-propyl and tert-butyl adducts (7, 8) were formed preferentially over the hydrouoromethylated product. The reactivity of these alkyl iodides therefore decreases in the following order: tBuI > iPrI > CH 2 FI $ EtI > MeI.</p><p>A notable outcome of this study was the observation that net methane addition across the double bond took place with iodomethane. Currently, protocols for the generation of the methyl radical from iodomethane (BDE CH3-I ¼ 239 kJ mol À1 , E red ¼ À2.39 V vs. SCE in MeCN) remain underdeveloped. 34,66 In recent years, the methyl radical has been generated from numerous precursors. [67][68][69][70][71] The formation of the methyl radical oen requires harsh reaction conditions, limiting the applicability of these protocols. Furthermore, the use of the methyl radical towards application to isotopic labelling is far from trivial. Iodomethane, on the other hand, can provide effortless access to a variety of useful isotopologues that would otherwise be beyond reach. The straightforwardness of our protocol prompted us to optimise the hydromethylation of alkenes using iodomethane as methyl radical precursor (Scheme 4). We noted signicant gas release when applying our reaction conditions, attributed to methane resulting from competitive HAT between the methyl radical and MeCN (BDE NCCH2-H ¼ 389 kJ mol; BDE CH3-H ¼ 439 kJ mol À1 ). 69 A screen of solvents, reactants stoichiometry and photocatalysts allowed for hydromethylation to occur in up to 93% yield (5a). 40 Under the optimised reaction conditions consisting of 4.0 equivalents of MeI, 3.0 equivalents of (TMS) 3 SiH and 1,2-diuorobenzene as solvent, in combination with photocatalyst MesAcrBF 4 (0.5 mol%), the hydromethylation of various alkenes took place in good to excellent yield (5b-f). Considering that bioactive compounds containing stable heavy isotopes are useful for example as mass spectroscopy standards, 41,72 the hydromethylation of an ethacrynic acid derivative was performed with CH 3 I, CH 2 DI, CHD 2 I, CD 3 I, 13 CH 3 I, and 13 CD 3 I. All six isotopologues (5h-5m) were obtained in moderate yield. [73][74][75]</p><!><p>In conclusion, the rst direct hydrouoromethylation of a broad range of electron-decient alkenes has been developed using uoroiodomethane. Mechanistically, the process harnesses known principles; so its core value is rooted in its immediate synthetic power. With the current global necessity "to do more with less", this minimalistic and mild chemical method stands out as it is operationally simple with the supersilyl radical precursor (TMS) 3 SiH being the only chemical required in addition to the reaction partners. The mild reaction conditions are compatible with complex biologically active molecules such as Ibrutinib. The methodology was successfully adapted for the 18 F-labelling of complex alkenes, and offers a new C-CH 2 18 F disconnection strategy for radiotracer development. The method was extended to additional uoroiodoalkanes enabling facile product homologation, as well as multiple (halo)methyl radicals including the methyl radical itself and ve of its D and 13 C isotopomers.</p>

Royal Society of Chemistry (RSC)

A photoacoustic-fluorescent imaging probe for proteolytic gingipains expressed by Porphyromonas gingivalis

Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. This probe harnesses the intramolecular dimerization of peptide-linked cyanine dyes to induce fluorescence and photoacoustic off-states. Upon proteolytic cleavage by Argspecific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU/mL bacteria in vitro. RgpB activity was imaged in the subgingival pocket of porcine jaws with 25 nM sensitivity. The diagnostic efficacy of the probe was evaluated in gingival crevicular fluid (GCF) samples from subjects with (n = 14) and without (n = 6) periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson's r = 0.71). The highest activity was seen in subjects with the most severe disease. Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, demonstrating relevance and potential utility for animal models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer's disease.

a_photoacoustic-fluorescent_imaging_probe_for_proteolytic_gingipains_expressed_by_porphyromonas_ging

Introduction<!>3E-F, Fig. S9).

<p>Periodontitis is a chronic inflammatory disease that affects 46% of adults in the United States and generates billions of dollars per year in direct costs [1] . The pathogenesis of the disease remains an active research topic; however, it is principally associated with a dysbiotic oral microbiome and the accompanying immune response [2] . Periodontitis-associated bacteria reside in the subgingival crevice, and their presence in biofilms and gingival crevicular fluid contribute to degradation of host tissue and deepening of the periodontal pocket [3] . When untreated, periodontitis causes oral pain, tooth loosening, and tooth loss. Furthermore, the long-term loading of the immune system has been linked to increased risks for cardiovascular disease [4] , pre-term birth [5] , cancer [6] , and even dementia [7] .</p><p>Periodontal health is measured via periodontal probing and clinical examination with metrics that include the pocket depth, clinical attachment level, bleeding on probing, tooth mobility, and inflammation. Together, these metrics are used to form a diagnosis.</p><p>In general, this established practice is functional and affordable, but pocket depth and clinical attachment level measurements suffer from relatively high inter-examiner error due to differences in probing force/angulation while also causing patient discomfort. Moreover, these techniques largely assess the effects of disease rather than using molecular diagnostics for precision health. Therefore, new techniques to detect disease at the point-of-care-particularly with utility for imaging and identification of disease at the molecular levelremain an unmet need in the field of oral health.</p><p>Many of the periodontal pathogens that have been linked to disease are anaerobic, such as Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis [8] . Among this "red complex", P. gingivalis is the most well-characterized: Its presence in subgingival plaque has been correlated with disease progression in longitudinal human studies [9] . As a function of their anaerobic metabolism, these pathogens secrete protease virulence factors that degrade extracellular proteins and modulate the host immune response [10] . P. gingivalis, in particular, is known to secrete proteases called gingipains that exhibit trypsin-like activity [11] . Indeed, P. gingivalis and gingipain proteases have attracted attention both as diagnostic and therapeutic targets. A variety of naturally derived and synthetic gingipain inhibitors have been reported in the literature while demonstrating evidence for potential treatment of periodontal disease though clinical trials have been relatively rare [12] . Intriguingly, evidence of gingipains has been identified in the post-mortem brains of patients with Alzheimer's disease (AD) and are the target of an ongoing AD clinical trial for a small molecule gingipain inhibitor [13] . A parallel research effort is targeting P. gingivalis directly with an antibody therapy [14] .</p><p>From a diagnostic perspective, advances in gingipain detection have included the development of substrates and paper-based assays for in situ analysis [15] , a plasmonic nanosensor [16] , and a gingipain-responsive/drug-loaded hydrogel [17] . The goal of this study was to develop an activatable probe for gingipains with utility for in vivo imaging-such work was motivated by its potential as a clinical tool for periodontal diagnosis and as a research tool for investigation of the role of gingipains in periodontitis and other diseases.</p><p>Photoacoustic imaging is particularly attractive because it augments the existing strengths of ultrasound-good tissue penetration, low cost, and real-time monitoring. It can use both exogenous and endogenous contrast based on optical absorption. Many small molecule and nanoparticle contrast agents have been engineered for photoacoustic imaging and activatable probes for molecular imaging are particularly desirable [18] . Further, the applications of acoustic imaging and nanoscale materials have been expanding [19] but they have not yet been combined for oral imaging. In previous work, we introduced a dye-peptide scaffold that exploits the intramolecular coupling of cyanine dyes to generate photoacoustic and fluorescent signal upon proteolysis by trypsin. Here, we leveraged this approach to create an activatable photoacoustic and fluorescent molecular imaging agent for gingipain proteases.</p><p>To select a gingipain-cleavable peptide substrate, we first applied a structural model of peptide-protein affinity to screen a series of pentapeptides for their affinity to the Arg-specific cysteine protease gingipain R (RgpB, PDB: 1CVR) [20] . The RgpB protease is composed of a 435-residue, single-chain polypeptide that forms a catalytic domain and an immunoglobulin-like domain [21] .</p><p>The peptide candidates were generated with three constraints: a five-residue length, an arginine at the third residue (P1), and a lysine at the fifth residue (C-terminus, P2'). The peptide length was restricted to facilitate intramolecular interaction between N and C terminal dyes while reducing the likelihood of cleavage by off-target proteases. The central arginine was necessary for cleavage by RgpB, and the Cterminal lysine was chosen for its reactive free amine. These conditions allowed us to generate 8,000 possible sequences that were screened for affinity to RgpB using an open-source structural model (PepSite 2.0) based upon a library of known peptide-protein complexes [20b] . The results were plotted as the inverse p-value to signify relative affinity (Fig. 1A) where the p-value represents the statistical significance for the overall score of a given binding site defined by Petsalaki et al. [22] . Of these peptides, the top result that did not contain a cysteine (excluded to reduce effects from dithiol coupling) was APRIK (p-value 0.0266) and was selected for probe synthesis. Additionally, the median result (TTRIK (p-value: 0.1866)) and last result (EEREK (p-value: 0.6872)) were synthesized and 3 conjugated with dyes to serve as experimental controls for the model predictions (Fig. 1B). Visualization of the APRIK-RgpB interaction demonstrated that the peptide was predicted to bind the catalytic domain of RgpB (Fig. 1C). The three candidate peptides were used to synthesize homodimer probes [Cy5.5]2[APRIK], [Cy5.5]2[TTRIK], and [Cy5.5]2[EEREK], referred throughout as C2A, C2T, and C2E, respectively (Fig. S1). RP-HPLC retention times for the conjugates decreased slightly from C2A (11.8 min) to C2T (11.7 min) to C2E (10.9 min), corresponding to the increasing hydrophilicity of the residues in each peptide (Fig. S2A-C); the structures of the probes were confirmed with ESI-MS (Fig. S2D-F).</p><p>The absorbance maxima of the conjugates in water were blueshifted relative to their spectra in DMSO (Fig. 1E)-a solvatochromic effect indicative of aromatic dye stacking [23] . This blue shift confirmed intramolecular dye coupling, i.e., DMSO promotes intramolecular separation of the dyes by neutralizing their attractive π-π interactions, thus mimicking the effect of proteolytic cleavage of the peptide linker. Indeed, the fluorescence of the intact conjugates was also selfquenched but was activated upon incubation with RgpB: We measured the fluorescence from each conjugate at a range of concentrations with constant RgpB and observed stronger foldenhancement for C2A/C2T than C2E (Fig. 1F). While C2A and C2T performed similarly in this comparison, we selected C2A for further development due to its higher predicted affinity for RgpB and higher signal to background ratio at concentrations > 6 μM. The decreased activation at higher concentrations was caused by increased selfquenching of the probes, though this is dependent upon the amount of DMSO in the mixtures. Upon incubation of C2A with RgpB, the absorbance maxima of the dyes at 680 nm were recovered with increasing concentrations of protease (Fig. 2A). The fluorescence emission at 700 nm was also proportionally enhanced (Fig. 2B). The fluorescence limit of detection was 1.1 nM (linearity 0 -5 nM) (Fig. 2C). Additionally, the photoacoustic intensity of the samples excited at 680 nm was proportional to their absorption (Fig. 2A, D), and the photoacoustic limit of detection was 10 nM RgpB.</p><p>To further verify the probe's sensitivity and selectivity for gingipains associated with P. gingivalis, we grew and isolated bacterial supernatants from both P. gingivalis and another oral anaerobe, F. nucleatum (Fig. 3A). F. nucleatum is a good negative control because it is also commonly identified in the gingival sulcus but is a saccharolytic and commensal bacterium known to not secrete gingipains [24] . These anaerobes were first grown on blood agar and enumerated from liquid suspensions via optical density after development of standard curves (Fig. S3, Fig. S4). The presence of Arg-specific gingipain in the P. gingivalis cultures was confirmed with a commercially available enzyme-linked immunoassay (ELISA) kit (Fig. S5); in addition, activity was measured by incubation with a commercially available fluorescent substrate, Boc-Phe-Ser-Arg-AMC, as previously described [25] (Fig. S6). Then, upon incubation of the C2A probe with P. gingivalis supernatant, we directly observed cleavage of intact C2A (TR = 21.2 min) into Cy5.5-APR (TR = 17.2 min, [M+2H] 2+ = 412.91 m/z) and IK-Cy5.5 (TR = 18.2 min, [M+2H] 2+ = 454.93 m/z) fragments with HPLC and ESI-MS (Fig. S7), thus demonstrating the expected activity of Arg-gingipain in the bacterial supernatant and intended cleavage of C2A. Indeed, the probe activated fluorescence 135-fold over the course of 2 hours, corresponding to enhanced emission at 700 nm and absorbance at 680 nm (Fig. 3B, Fig. S8); this activation was reduced by 97% upon coincubation with leupeptin-a known gingipain inhibitor [26] (Fig. 3B). The fluorescence was not activated by F. nucleatum. As with fluorescence, we observed an increasing trend for the photoacoustic intensities of the samples excited at 680 nm, thus demonstrating selective photoacoustic imaging of gingipains from P. gingivalis (Fig. 3C-D). The limits of detection for the bacteria were tested via serial dilution of the supernatants in broth and determined to be 4.4E4 CFU/mL via fluorescence and 4.1E5 CFU/mL via photoacoustics (Fig.</p><!><p>To date, reported strategies for measurement of gingipain activity have used in vitro detection methods, including a nanobody immunoassay [15a] , an electrochemical biosensor [27] , fluorogenic dipeptides [15b] , peptide-functionalized magnetic nanobeads [28] , and refractometry of protein-functionalized gold nanoparticles [16] . These have reported detection limits of 7.81E6 CFU/mL bacteria, 5E5 CFU/mL bacteria, 1E5 CFU/mL bacteria, 49 CFU/mL bacteria, and 4.3 nM Kgp (CFU/mL not reported), respectively. While the C2A probe has comparable sensitivity to these in vitro sensors (fluorescence LOD: 4.4E4 CFU/mL and 1.1 nM RgpB, photoacoustic LOD: 4.1E5 CFU/mL and 15 nM RgpB), it is the first reported gingipain probe suitable for photoacoustic imaging while also achieving a dual-modal fluorescence readout, with applicability for in vivo oral photoacoustic imaging, a technique that is gaining preclinical traction [29] . The added value of imaging is the monitoring of disease progression or response to therapy with the spatial integration of anatomic markers of disease. Indeed, to characterize the imaging performance of the C2A probe in relevant oral anatomy, it was used to resolve the periodontal pocket/gingival sulcus of intact porcine jaws with photoacousticultrasound imaging (Fig. 4). Here, buffer, C2A, and C2A + RgpB (25 and 50 nM), were irrigated sequentially into the gingival sulcus of the second molar of a porcine mandible (n = 3). 3D photoacousticultrasonographs of the tooth/gingiva were generated (Fig. 4A, left) and anatomical markers were readily resolved in the midsagittal cross sections (Fig. 4A, right), including the gingival margin (GM, pink) and alveolar bone crest (ABC, teal). The uncleaved C2A probe did not possess significantly more photoacoustic signal (red) than buffer alone (Fig. 4B). However, C2A activated with 25-50 nM RgpB generated clear and increasing subgingival photoacoustic signal (Fig. 4C-E, yellow boxes), representing the subgingival distribution of RgpB-cleaved probe. In addition, spectral imaging could distinguish the imaging signal from cleaved C2A (< 750 nm) from the relatively flat spectra from supragingival signal caused by tooth staining (Fig. 4F-G). Overall, this experiment demonstrates the ability to image the spatial distribution of subgingival gingipain activity in relation to key landmarks of oral anatomy while achieving low nanomolar sensitivity.</p><p>In a study by Guentsch et al., ELISA was used to identify micromolar concentrations of gingipain in gingival crevicular fluid (GCF) collected with paper point sampling from patients with periodontal disease [30] . This is well above the low nanomolar detection limits of C2A for RgpB: Therefore, to evaluate the diagnostic efficacy of the C2A probe in clinically relevant samples, we collected GCF from 40 tooth sites in a set of 20 subjects, comprising both healthy patients and individuals with symptoms of periodontal disease sampled at a dental clinic. The GCF samples were assayed with both qPCR and C2A via fluorescence to measure the number of P. gingivalis cells and proteolytic gingipain activity, respectively. Of these, 25% (10/40) contained P. gingivalis via qPCR and these were considered positives (Fig. 5A). Gingipain activity via C2A fluorescence was correlated with the PCR results (Pearson's r = 0.71, Fig. 5B), albeit with lower sensitivity: Fluorescence activation was observed in 5/10 of the positives and 2/30 of the negatives, corresponding to a detection rate of 50% and a false positive rate of 6.67% (Fig. 5A). However, the higher sensitivity of qPCR was expected given its inherent signal amplification mechanism. Another difference is that while qPCR may reflect the amount of live and dead cells, it is not a measurement of the active gingipain activity that is evidenced to play a direct role in the pathogenic process of periodontal disease [31] . The activity data was also analyzed with respect to disease severity for each tooth site (Fig. 5C). Interestingly, gingipain activity was primarily observed in the GCF from Class III sites (with the greatest total facial CAL). Though half of these sites did not exhibit gingipain activity, these results support the hypothesis that local gingipain activity may contribute to more severe periodontal damage.</p><p>Lastly, the potential role of P. gingivalis and gingipains in neurological pathologies, especially Alzheimer's disease, is of mounting research interest [13,32] . Photoacoustic imaging is well-suited for real-time imaging and monitoring of murine disease models, and thus we performed proof of concept imaging of cleaved and uncleaved probe in extracted murine brains (fixed in 1% agar). The C2A probe was first incubated with RgpB at increasing probe concentrations to confirm cleavage at sufficient concentrations for imaging in animal tissue (Fig. S10A-B), and the highest tested concentration (30 μM) was chosen for injection (Fig. S10C). Subsequently, aliquots of buffer, C2A, and C2A + RgpB (pre-incubated and monitored for 2 h) were injected into the lambda points of respective brains (Fig. S10D)these were then imaged in 3D with a photoacoustic-ultrasound scanner at 680 nm using sonography gel for acoustic coupling. Negligible photoacoustic signal was detected in the buffer-injected brain (Fig. 6A), while minor background was observed for the uncleaved probe (Fig. 6B). The strongest signal was detected from the brain injected with C2A + RgpB, visible in axial, coronal, and sagittal cross-sections of the tissue (Fig. 6C). Further, spectral photoacoustic imaging of the injected brains allowed signal from C2A to be distinguished from background by its characteristic absorption/photoacoustic peak in the near infrared (Fig. S11). These experiments demonstrate that the C2A probe could have value as a research tool for gingipain imaging in more complex models of infection for Alzheimer's disease pathogenesis. Future studies may integrate the probe with in vivo models of P. gingivalis infection, though potential limitations include issues that affect many smallmolecule photoacoustic probes, including low signal to background ratio in blood at low concentrations and photoinstability associated with the dissociation of conjugated π electrons following absorption [23,33] . Nevertheless, proof-of-concept imaging utility was demonstrated in the oral cavity and brain parenchyma using resected porcine jaws and murine brains, respectively. Lastly, in future efforts to improve sensitivity to P. gingivalis, a lysine residue could be included in the peptide linker for cleavage by Lys-gingipain (Kgp), in addition to D-amino acids for increased bacterial specificity [34] .</p><p>In summary, a molecular imaging probe, C2A, was designed and synthesized to harness the intramolecular dimerization of peptide-linked cyanine dyes to induce fluorescence and photoacoustic off-states. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), 5-fold photoacoustic enhancement and >100-fold fluorescence enhancement was achieved with detection limits of 1.1 nM RgpB and 4.4E4 CFU/mL bacteria. RgpB activity was imaged in the subgingival pocket of porcine mandibles with 25 nM sensitivity. The diagnostic efficacy of the probe was evaluated in gingival crevicular fluid (GCF) samples from subjects with (n = 14) and without (n = 6) periodontal disease; activation correlated to qPCR-based detection of P. gingivalis (Pearson's r = 0.71), and activity was highest in subjects with the most severe disease progression. Lastly, photoacoustic imaging of RgpB-cleaved probe was demonstrated in murine brains ex vivo, thus demonstrating future utility for imaging studies of general infection by P. gingivalis.</p>

ChemRxiv

The Effects of Tamoxifen on Immunity

Tamoxifen is a widely known anti-estrogen which has been employed in adjuvant treatment of early-stage, estrogen-sensitive breast cancer for over 20 years. Less well known are the effects of tamoxifen on immune function, which we discuss here. We review the growing body of evidence which demonstrates immunomodulatory effects of tamoxifen, including in vitro and in vivo studies as well as observations made in breast cancer patients treated with tamoxifen. Taken together these studies suggest that tamoxifen is capable of inducing a shift from cellular (T-helper 1) to humoral (T-helper 2) immunity. Interestingly, the immunomodulatory effects of tamoxifen appear to be independent of the estrogen-receptor and may be mediated through the multi-drug resistance gene product, Permeability-glycoprotein, for which a role in immunity has recently emerged. We furthermore discuss the clinical implications of the immunomodulatory effects of tamoxifen which are twofold. First, tamoxifen may be utilized in the treatment of immune-mediated disorders, particularly of those arising from aberrant T-helper 1 cell activity, including allograft rejection, Crohn\xe2\x80\x99s disease, and Th1-mediated autoimmune conditions such as diabetes mellitus, scleroderma, and multiple sclerosis. Second, given that cellular T-helper 1 immunity is targeted against cancer cells, the tamoxifen-induced shift away from cellular immunity represents a significant step in fostering a cancerogenic environment. This may limit the anti-cancer effects of tamoxifen and thus explain why tamoxifen is inferior compared to other anti-estrogens in preventing disease recurrence in early-stage breast tumors.

the_effects_of_tamoxifen_on_immunity

INTRODUCTION<!>PHARMACOLOGY OF TAMOXIFEN<!>TAMOXIFEN AS AN IMMUNE MODULATOR<!>CLINICAL IMPLICATIONS<!>CONCLUSION<!>

<p>A fundamental milestone in the treatment of breast cancer was the discovery that adjuvant anti-estrogen therapy increases survival in women with estrogen-sensitive tumors. For over two decades tamoxifen had been the first-line anti-estrogen for adjuvant hormonal treatment of breast cancer in post-menopausal women and was recently replaced by a different class of anti-estrogens, the aromatase inhibitors [1–3]. Whilst its role in breast cancer treatment may be diminishing, a growing body of evidence has defined a novel role for tamoxifen as an immune modulator. Here, we review the effects of tamoxifen on immunity, discuss underlying pharmacological mechanisms and explore potential clinical applications.</p><!><p>Tamoxifen, first described 1967 as a potential contraceptive agent [4], belongs to a class of molecules called triphenylethylenes derived from the estrogen agonist diethylstilbestrol which was first synthesized in 1938 [5]. The structure of tamoxifen, which is closely related to that of clomiphene, is depicted in Fig. (1). The stilboestrol-like backbone with a basic side chain is thought to mediate the anti-estrogenic effects [6]. The pharmacological targets of tamoxifen include the estrogen receptor (ER), the multi-drug resistance gene product, Permeability-glycoprotein (P-glycoprotein) [7], and the recently discovered 7-transmembrane G protein-coupled receptor 30 [8].</p><p>Tamoxifen is metabolized in the liver into a variety of metabolites which are mainly estrogenic [9]. Interestingly, one of its major metabolites, 4-hydroxytamoxifen, is thought to be mutagenic by forming DNA adducts which may mediate the carcinogenic effects of tamoxifen [5].</p><p>The anti-estrogen effects of tamoxifen are mediated through the ER, which is present in the cytosol but also within cell membranes [6]. Whilst in breast tissue the action of tamoxifen on the ER is predominantly inhibitory, tamoxifen stimulates the ER in other tissues. Estrogen agonism and antagonism give rise to a variety of beneficial and adverse effects including: protection of osteoporotic bone, postmenopausal symptoms, an increased risk of endometrial cancer, thromboembolism, and strokes [5,6]. In addition to its action on the ER, tamoxifen is an inhibitor of P-glycoprotein, a 170-kDa protein located both within the cell membrane and in the cytosol [10]. P-glycoprotein is a member of the ATP-binding cassette superfamily of active transporters [11], and it has been proposed that tamoxifen inhibits P-glycoprotein through interference with its ATPase activity [12]. Traditionally associated with multi-drug resistance of certain mammalian solid tumors and hematological malignancies, a role for P-glycoprotein in immunity has recently emerged [13]. Its function in various immune cells has been demonstrated, including lymphocytes and dendritic cells [14–17]. The role of P-glycoprotein in immunity is particularly interesting for the present discussion interesting for the present discussion because P-glycoprotein inhibition may account for some of the effects of tamoxifen on immunity.</p><!><p>Yearlong clinical experience with tamoxifen suggesting it has no apparent effects on immunity is backed by studies into the effects of tamoxifen on immunity in breast cancer patients. A number of such studies from the 1980s and early 1990s failed to reveal any influence of the drug on immune function [18]. However, more recent evidence from investigations in humans, in animal models as well as observations from in vitro studies indicate the contrary (Table 1).</p><p>Rotstein et al. and subsequently Robinson et al. reexamined the effects of tamoxifen on immunity in breast cancer patients and demonstrated modulatory effects [19,20]. Rotstein et al. studied the function of peripheral lymphocytes derived from breast cancer patients (n=23) treated with tamoxifen for 1.5 to 2 years [19]. They observed that lymphocytes from tamoxifen-treated women, compared to those from healthy controls, showed significantly reduced Natural Killer activity against a human leukemia cancer cell line (K562) whilst exhibiting a higher proliferation response in the presence of a mitogen (concanavalin A). Robinson et al. studied the effects of tamoxifen on immunity in patients with bilateral breast cancer (n=21) who were in remission and had completed radiotherapy and chemotherapy at least one year prior to the study. They observed that the relative proportion and absolute number of CD4 lymphocytes was reduced in tamoxifen treated patients, compared to untreated breast cancer patients and to healthy controls [20]. Moreover, in vitro proliferation of lymphocytes derived from tamoxifen treated patients was decreased. Finally, in vitro Natural Killer cell activity, which is increased in untreated patients, returned to levels of healthy controls in patients treated with tamoxifen. In addition to these two studies in breast cancer patients, two case reports point towards tamoxifen-mediated modulation of immune function in humans [21,22]. De et al. have presented a patient suffering from corticosteroid-resistant Riedel's disease, a rare chronic inflammatory disease of the thyroid gland, in whom remission was induced and maintained by tamoxifen [21]. Similarly, Sereda and Werth reported successful treatment with tamoxifen of a dermatomyositis rash which was difficult to control with conventional systemic immunosuppressants [22].</p><p>Further evidence demonstrating tamoxifen-mediated immunomodulation stems from a variety of in vivo models including: mice developing autoimmune diseases akin to systemic lupus eryhthematosus [23–26]; murine experimental autoimmune encephalomyelitis [27]; and experimental autoimmune uveitis in rats [28].</p><p>In experimental systemic lupus erythematosus mice (NZBxNZW F1 mice) tamoxifen was shown to increase survival; at six months of age all mice treated with tamoxifen were alive whereas 40% of untreated mice had died [23]. Other beneficial effects of tamoxifen treatment in these mice were reduced thrombocytopenia and proteinuria, less advanced renal disease (on histopathological examination) and diminished production of IgG3 auto-antibodies (against nuclear extracts and DNA) [23]. In another mouse model of experimental systemic lupus eryhthematosus (16/6 idiotype-induced disease in BALB/c female mice) tamoxifen has been shown to delay the onset disease [24]. Furthermore, tamoxifen-treated mice developed a milder disease phenotype with normal leukocyte and platelet numbers and no renal immune complex deposition. Similarly, in NZB/W F1 mice, which also develop a lupus-like disease, tamoxifen increased survival, reduced proteinuria and lessened histopathological renal disease [25]. Moreover, tamoxifen decreased numbers of splenic B-cells and serum levels of tumor necrosis factor receptors in these mice. In another mouse model of lupus, the MRL-1pr/1pr mice, tamoxifen again increased survival rates and disease severity as evidenced by diminished proteinuria, auto-antibodies (anti-double stranded DNA) and lymphadenopathy [26].</p><p>An intriguing in vivo observation was made by Bebo et al. who studied the effects of tamoxifen in experimental autoimmune encephalomyelitis of mice [27]. They found that tamoxifen induced a T-helper cell 2 (Th2) bias with increased Th2 transcription factors in cultures of myelin-specific lymphocytes, which suggests that tamoxifen may have differential effects on cellular (T-helper 1, Th1) and humoral (Th2) immunity. Other effects of tamoxifen treatment in autoimmune encephalomyelitis mice that Bebo et al. observed include: reduction of symptoms and the degree of demyelination; suppression of T-cell production stimulated by myelin; impairment of the ability of dendritic cells to stimulate myelin-specific T-cells. Differential effects of tamoxifen on Th1 and Th2 immunity has also been reported by de Kozak et al. who studied the effects of tamoxifen in experimental autoimmune uveoretinitis of rats [28]. The investigators found that intraocular injection of tamoxifen-loaded nanoparticles significantly inhibited onset of disease and induced a shift from a Th1- to a Th2-mediated immune response. Tamoxifen decreased gamma interferon (a Th1 cytokine) production by inguinal lymph node cells, reduced their Th1-mediated delayed hypersensitivity response and induced an antibody class switch indicative of a Th2 response. Interestingly, the immunomodulation by tamoxifen was not fully reversed by concomitant intraocular injection of 17beta-estradiol, suggesting that tamoxifen modulates immune function in part through an estrogen-independent mechanism.</p><p>That tamoxifen modulates immunity through an estrogen-independent mechanism has also been demonstrated by Komi et al. who studied the effects of tamoxifen on function of human monocyte-derived dendritic cells, key regulator cells of the immune system, in vitro [29]. Compared to control cells, dendritic cells cultured in the presence of tamoxifen developed a distinct phenotype (CD14−, CD1a−, CD80−, CD86+). Furthermore, tamoxifen-treated dendritic cells were inferior in inducing proliferation amongst allogeneic T-cells and in producing interleukin-12 upon stimulation. Interestingly, blockade of the estrogen receptor did not reverse or affect the action of tamoxifen on dendritic cells suggesting that tamoxifen exerts its effects on dendritic cells through an estrogen-independent mechanism. In a different series of experiments [30], Komi et al. demonstrated that tamoxifen also modulated the differentiation into dendritic cells of synovial fluid macrophages, obtained from the synovial fluid of patients suffering from rheumatoid arthritis. Macrophages treated in vitro with tamoxifen developed a phenotype (CD14−, CD1a−, CD80−, CD86+) similar to that of tamoxifen-treated dendritic cells [29], and the ability of tamoxifen-treated macrophages to induce proliferation of allogeneic T-cells was reduced.</p><p>Taken together, the evidence from human, in vivo and in vitro studies presented here suggests that tamoxifen modulates immune function with differential effects on Th1 and Th2 immunity, which may be mediated through an estrogen-independent mechanism. As discussed above, tamoxifen is an established inhibitor of P-glycoprotein which has recently been shown to regulate immunity [14–17]. Thus, it is conceivable that the immunomodulatory effects of tamoxifen are at least in part mediated through P-glycoprotein, which is supported by work from our laboratory [15,17].</p><p>Studying the in vitro effects on human lymphocytes of tamoxifen and the anti-P-glycoprotein monoclonal antibody (Hyb-241), we demonstrated that both tamoxifen and Hyb-241 blocked P-glycoprotein, as assessed by P-glycoprotein mediated efflux of calcein-AM dye [15]. We then showed that both tamoxifen and Hyb-241 inhibited alloantigen-induced T-cell proliferation in a dose-dependent manner and reduced cytokine production measured in supernatants of the lymphocyte cultures (interleukin-2, tumor necrosis factor-alpha, gamma-interferon). In a different study, we repeated the aforementioned experiments of Komi et al. and substituted tamoxifen with the pharmacological P-glycoprotein inhibitor PSC833 [17]. We were able to reproduce the findings of Komi et al. and showed that P-glycoprotein blockade has the same effects on dendritic cell phenotype and function as tamoxifen did in the study of Komi et al. [29]. Interestingly, we also observed differential effects of P-glycoprotein inhibition of Th1 and Th2 immunity. Dendritic cells treated with the P-glycoprotein inhibitor PSC833 lost the ability to induce Th1 responses whilst retaining the ability to stimulate Th2 cells. For instance, when mixed with lymphocytes in vitro, control dendritic cells predominantly activated gamma-interferon secreting Th1 lymphocytes. In contrast, dendritic cells treated with the P-glycoprotein inhibitor mainly stimulated interleukin-5-secreting Th2 lymphocytes. Thus, similar to the effects of tamoxifen in murine experimental autoimmune encephalomyelitis and in experimental autoimmune uveoretinitis of rats, P-glycoprotein inhibition induced a shift from Th1 to Th2 activity in vitro.</p><p>Despite this intriguing in vitro evidence, the effectiveness of P-glycoprotein as an immune modulator in the clinic remains to be demonstrated. Tamoxifen may be an attractive P-glycoprotein inhibitor for clinical use given its favorable safety profile compared to most existing pharmacological P-glycoprotein inhibitors which have exhibited unacceptable toxicity in clinical trials [28]. Tamoxifen may also be superior to specific monocloncal antibodies in targeting P-glycoprotein dependent-immune mechanisms because as a lipophilic substance it would reach the intracellular compartment where P-glycoprotein is located within dendritic cells [10], which are key target cells of immune therapy.</p><p>Whilst we propose that tamoxifen exerts its immunomodulatory effects through P-glycoprotein, it is possible that these effects are mediated through other pathways. For example, it has been proposed that tamoxifen may modulate nuclear factor kappa beta, a key regulator of immune function, which is structurally and functionally closely related to the estrogen receptor [5]. However, this hypothesis remains to be explored experimentally. Furthermore, a novel 7-transmembrane G protein-coupled receptor 30 that binds both estrogen and tamoxifen has recently been discovered [8]. Its function is currently being defined, and it may be worth investigating whether it is linked into immune regulatory networks.</p><!><p>The clinical implications of the effects of tamoxifen on immunity are twofold. First, tamoxifen may be utilized in the treatment of immune-mediated disorders. We speculate that particularly Th1-mediated disorders would be amenable to treatment with tamoxifen, including allograft rejection, Crohn's disease, and Th1-mediated autoimmune conditions such as diabetes mellitus, scleroderma, and multiple sclerosis. Major clinical benefits would be gained if these immune disorders, which are traditionally treated with systemic immunosuppressants, responded to tamoxifen which has a comparably favorable safety and adverse effect profile. However, the long-term use of tamoxifen would be limited by its adverse effects, especially the aforementioned increased risk of developing endometrial cancer. It would therefore be important to develop tamoxifen analogues without substantial adverse effects for the treatment of chronic immunological diseases.</p><p>Second, the effects of tamoxifen on immunity may explain why compared to aromatase inhibitors tamoxifen is inferior in preventing disease recurrence in breast tumors [1–3]. The inferiority of tamoxifen remains elusive, particularly in the treatment of breast cancer requiring chemotherapy, since as a P-glycoprotein inhibitor tamoxifen would be expected to enhance the effectiveness of chemotherapy. Based on our review one may speculate that the inferiority of tamoxifen is due to a shift away from anti-cancer cellular immunity, which represents a significant step in fostering a cancerogenic environment.</p><!><p>Here we have discussed the growing body of evidence demonstrating a role for tamoxifen as an immune modulator. Whilst we propose that the effects of tamoxifen on immunity are mediated at least in part through P-glycoprotein, more research is required to fully understand the mechanisms underpinning the immunomodulatory effects of tamoxifen. Furthermore, because the clinical studies of immunity in breast cancer patients receiving tamoxifen were conducted in the 1980s and early 1990s it would be important to reinvestigate immunity in these patients with the whole range of modern immunological research methods. Finally, given the beneficial safety profile of tamoxifen, particularly when used short-term, it would seem appropriate to take findings from the laboratory to the bedside and to test tamoxifen in a variety of immune-mediated conditions. Tamoxifen, which has once failed as a contraceptive and then revolutionized the treatment of breast cancer, may re-emerge as a novel modulator of the immune system.</p><!><p>Chemical structure of tamoxifen.</p><p>The Effects of Tamoxifen on Immunity</p><p>Symbols ↑ = increase; ↓ = decrease.</p>

PubMed Author Manuscript

Multi-Target Dopamine D3 Receptor Modulators: Actionable Knowledge for Drug Design from Molecular Dynamics and Machine Learning

Local changes in the structure of G-protein coupled receptors (GPCR) binders largely affect their pharmacological profile. While the sought efficacy can be empirically obtained by introducing local modifications, the underlining structural explanation can remain elusive. Here, molecular dynamics (MD) simulations of the eticlopride-bound inactive state of the Dopamine D3 Receptor (D3DR) have been clustered using a machine learning-based approach in the attempt to rationalize the efficacy change in four congeneric modulators. Accumulating extended MD trajectories of receptor-ligand complexes, we observed how the increase in ligand flexibility progressively destabilized the crystal structure of the inactivated receptor. To prospectively validate this model, a partial agonist was rationally designed based on structural insights and computational modeling, and eventually synthesized and tested. Results turned out to be in line with the predictions. This case study suggests that the investigation of ligand flexibility in the framework of extended MD simulations can assist and inform drug design strategies, highlighting its potential role as a powerful in silico counterpart to functional assays.

multi-target_dopamine_d3_receptor_modulators:_actionable_knowledge_for_drug_design_from_molecular_dy

Introduction<!>Ligand Docking and MD simulations<!>Cluster analysis<!>Principal Component Analysis (PCA) in the space of dihedral angles<!>Synthesis and Pharmacological evaluation of Compound 5<!>Analysis of the clustering graph in the training set (Compounds 1-4)<!>Antagonist-induced conformations<!>Partial agonist-induced conformations<!>Agonist-induced conformations<!>Design, MD analysis and biological evaluation of the predicted compound 5<!>Dihedral PCA<!>Conclusions<!>Molecular Dynamics Setup<!>Cluster Analysis<!>General methods and abbreviations

<p>The pharmacological properties of a drug are commonly considered to be continuous functions in chemical space: small changes in chemical structure lead to small differences in a compound's pharmacological profile. However, recent evidence has highlighted the existence of discontinuities: sometimes, small structural changes lead to large differences in one or more features. Activity cliffs are the best characterized form of discontinuity, [1] but this concept can be extended to the study of other relevant properties. GPCRs have emerged as a target class whose modulators explore rugged chemical landscapes. Small variations in GPCR binders can lead to significant changes in the efficacy, with or without affecting binding affinity. [2] This is particularly important in drug discovery because agonists, antagonists, or inverse agonists can all be therapeutically relevant, depending on the receptor and pathological framework. In some cases, the efficacy can be tuned by adopting empirical synthetic strategies. However, the underlying structural mechanisms have often remained elusive. GPCRs exist in an equilibrium ensemble of metastable conformations whose stabilization, following ligand binding, is crucial to eliciting a particular response. [3][4][5] The energy difference among states is often minimal, which likely explains how small structural variations in a ligand could affect the receptor's conformational ensemble. Here, we tested the possibility of using MD simulations and cluster analysis in a comparative fashion, to rationalize and predict how local structural variations affect the efficacy of four modulators which we had previously reported as multi-target binders of Fatty Acid Amide Hydrolase (FAAH), an enzyme involved in the endocannabinoid signalling pathway, and dopamine D3 receptor (D3DR). [6] Both proteins have been independently studied for nicotine addiction. Despite these targets being biochemically and structurally unrelated, we were able to conceive molecules with high affinity for both of them. Yet, accurately predicting efficacy at D3DR remained problematic. In this context, computational methods improving the design of multi-target directed ligands (MTDLs) holds great potential toward the development of efficient drugs against tobacco dependence.</p><p>Here, we selected a training set of four compounds that, despite their high structural similarity, show increasing levels of efficacy at D3DR without any relevant change in affinity. Compound 1 (Figure 1) is a neutral antagonist that bears: i) a 2,3-dichloro substitution on the pendant aromatic ring of the piperazine; ii) an unsaturation in the butyl/(E)-but-2ene linker, and iii) a carboxamide substituent in the distal ring of the biphenyl group. In the presence of a saturated linker, 2 behaves like a partial agonist, eliciting only 56% of the inhibitory response. The removal of the carboxamide in 3 does not alter the efficacy (65%) profile with respect to 2 (58%), despite a moderate decrease in activity. Compound 4 differs in features i) and ii), and is therefore an almost full agonist with 88% efficacy compared to the effects of 300 nM of dopamine on cAMP inhibition [6][7][8]. To derive our model and to understand which structural features affect efficacy, we attempted to interpret how the conformational behavior of each ligand within the binding site induces or stabilizes different interactions in residues H349 6.55 , Y365 7.35 and S193 5.43 , at the same time destabilizing the inactive crystal structure of D3DR (the superscript number indicating conserved positions according to Ballesteros and Weinstein numbering) [9].</p><p>H-bond networks involving conserved pairs of amino acids in positions 6.55, 7.35 and 5.43 have been subject of several studies on the D2-like dopamine receptors sub-family (D2, D3 and D4). In the homologous (78% sequence homology) dopamine D2 receptor (D2DR) in complex with agonists and partial agonists, [10][11][12][13] these interaction networks have been associated with low-energy patterns and functional bias. Three conserved functional serine residues on TM5, i.e., S192 5.42 , S193, 5.43 and S196 5.46 are crucial in the GPCR activation pathway that involves the formation of H-bonds between ligand, water, and receptor. [10,14] The inward movement and anticlockwise rotation (from the extra cellular point of view) of TM5 is required to orient the serines toward the binding site.</p><p>However, the serine in position 5.43 is only secondarily involved in catechol binding and has been found to establish favorable agonist-induced H-bonds with H393 6.55 in D2DR. [10,[12][13] In H393 6.55 A and H393 6.55 F mutants, a 28-fold drop in dopamine binding affinity has been observed, which correlated with reduced efficacy and confirmed the role of an aromatic residue with H-bonding capabilities in that position. [11] In the eticlopride-bound D3DR crystal structure, Y365 7.35 is fixed in a stable interaction network involving H349 6.55 and I183 on the extracellular loop 2 (EL2). [15] This interaction is absent in the D4 Dopamine Receptor (D4DR) structure complexed with nemonapride, where a valine substitutes the tyrosine in position 7.35.[16] Y365 7.35 V D3DR mutants show decreased constitutive activity of the receptor, while V430 7.35 Y cross-mutation causes the opposite effect in D4DR, highlighting that a H-bond network involving the two residues plays a functional role in regulating specific response at D3 receptor subtype. Furthermore, in microsecond-long MD simulations on an active D2DR homology model in complex with dopamine, the χ1 torsion (C-CA-CB-CC) of H349 6.55 mainly adopted three specific dihedral angle values, namely -60°, 60°, and 140°. [12] While these angles could all be induced in the presence of the natural substrate, MD simulations with known partial agonists revealed that, in that case, the χ1 dihedral angle of H349 6.55 was mainly stabilized at 60° or 140°. [13] These different orientations influence the interactions and the dynamics of the D2DR-G protein interface.</p><p>Therefore, the H-bond patterns established by H349 6.55 with Y365 7.35 and S193 5.43 , hereafter referred to as interaction pattern 1 and interaction pattern 2, respectively, have been proposed to play a crucial role in modulating the response of D2-like sub-family of dopamine receptors in presence of molecules with different pharmacological profiles.</p><p>Based on the insights gained from our studies, we designed, synthesized and tested a novel D3DR partial agonist (compound 5 in Figure 1) with dual FAAH/D3DR affinity and the sought efficacy profile.</p><!><p>Receptor coordinates of the human D3DR crystal structure in complex with eticlopride (PDB ID: 3PBL) [15] were retrieved from the PDB and used for docking and refinement procedures implemented in the ICM software suite. [17] Hydrogen atoms were added.</p><p>Polar hydrogen atoms and the positions of asparagine and glutamine side chain amidic groups were optimized and assigned the lowest energy conformation. After optimization, histidines were automatically assigned the tautomerization state that improved the hydrogen bonding pattern. Finally, the original ligand was deleted from the holo structure.</p><p>Binding sites were identified with the IcmPocketFinder tool as implemented in ICM3.7.3.</p><p>[18] The tolerance value was set equal to 4.6. The macro provides a mesh associated with every detected pocket. The graphical object closest to the co-crystallized ligand position was selected. All the residues with at least one side chain non-hydrogen atom within 3.5 Å of the selected mesh were considered part of the pocket. Ligands were assigned the right bond orders, stereochemistry, hydrogen atoms, and the most abundant protonation states predicted at pH 7.4. Each ligand was assigned the MMFF force field atom types and charges. [19] The docking engine used was the Biased Probability Monte Carlo (BPMC) stochastic optimizer, as implemented in ICM3.7 (Molsoft LLC, San Diego, CA -USA). [18] The ligand binding site at the receptor was represented by precalculated 0.5 Å spacing potential grid maps, representing van der Waals potentials for hydrogen and heavy atom probes, electrostatics, hydrophobicity, and hydrogen bonding. The van der Waals interactions were described with a smoother form of the 6-12 Lennard-Jones potential with the repulsive contribution capped at 4.0 kcal/mol. The electrostatic contribution was buffered, artificially increasing the distance between oppositely charged atoms to avoid their collapse when the electrostatic attractive energy prevailed on the softened van der Waals repulsion. The molecular conformation was described with internal coordinate variables. The adopted force field was a modified version of the ECEPP/3 force field with a distance-dependent dielectric constant. [20] Given the number of rotatable bonds in the ligand, an adaptive algorithm (thoroughness 3.0) calculated the basic number of BPMC steps to be carried out. The binding energy was assessed with the standard ICM empirical scoring function. [21] For each ligand, the best scoring pose was selected as representative of the ligand-bound conformation.</p><p>The D3DR in the inactive crystallized structure was properly refined and minimized after ligand placement, and chosen as a starting structure for MD simulations. Here, we focused on the ability of our compounds to destabilize the crystallized inactive state of the D3DR, triggering the onset of early local events connected to full structural transitions. In fact, large scale conformational changes remain out of reach for an antagonist-bound initial state, even with microseconds of MD trajectories. By comparing different ligands bound to the same inactive structure, we did not have to model an active state by homology. Furthermore, small differences (5-10 folds) in energy have been found between D3DR conformations with high and low affinity for agonists. [22] In the crystal structure, the long intracellular loop 3 (IL3) (R222-R318) involved in G-protein binding is not solved but substituted by T4-lysozime for stability reasons. [15] As in previously reported long MD simulations of β2-adrenergic GPCR, [23] we did not attempt to model IL3, since IL3 does not affect ligand binding. [24][25] In this study, we adopted the same strategy for four reasons: i) ligand binding to D3DR is not significantly modified by the presence of guanyl nucleotides (G-shift), meaning that G-protein binding and activation has only a weak influence on the orthosteric binding pocket; [22] ii) D3DR expressed in E. Coli has shown similar ligand binding capabilities in the presence and absence of Gi/o; [25], and iii) modeling extended protein loops does not ensure the reproduction of salient features of these domains. [26] Using the membrane embedding procedure implemented in BiKi LifeSciences (http://www.bikitech.com/), [27] we enclosed each complex in a simulation box of 8x8x10 nm containing 182 molecules of palmitoyl oleoyl phosphatidylcholine (POPC) lipids. Systems were prepared as described in the Supplementary Material. Rather than simulating multiple replicates, we preferred to carry out single MD simulations accumulating 3.05 µs on each system. This choice allowed us to increase the probability to observe rare events in the inactive structures of the receptor, enabling systematical comparisons of ligand-induced states on longer time-scales. To avoid any memory of the initial structure, we discarded the first 50 ns from each production run; the analyses were performed on 30000 snapshots extracted from each trajectory.</p><!><p>Conformers were pooled together based on their shared scaffold (Figure S1) and clustered by k-medoids algorithm (see description of the algorithm in the Supplementary Material). [28] To simplify the analysis and get more interpretable and robust results, three clustering rounds were performed, merging compounds 1, 2, 4 in the first run, and 1, 3, 4 in the second run. Similarly, compounds 1, 5, 4 were merged in a meta-trajectory to study ligands partitioning in presence of the predicted ligand 5.</p><!><p>To get a per ligand retrospective characterization of the space sampled by compounds 1-5, we built a dataset such that each line represented a ligand, while the columns (the features) were obtained by a processing of dihedral angles time data (30000 values x ligand). In detail, we first computed the sinuses and cosines of each angle for each ligand to correctly take into account the periodicity of the variables. Next, for each dihedral angle time series (either the sin/cos values) we computed a histogram with nb bins. This binning allowed us to get a discrete version of the distribution function that, at equilibrium, must be stationary. Hence, each ligand was represented by a column of ni entries where ni = 3*nb*2. The multiplier by three derives from the number of angles analyzed, while the multiplier by two derived from the sinus and cosine representations.</p><p>On this matrix we performed PCA analysis.</p><!><p>Description of synthesis and structural characterization of compound 5 are reported in the dedicated section of the Supplementary Material. The pharmacological profile of the molecule was assessed based on the ligands' inhibiting effects on cAMP accumulation via activation of Gi protein, as described in details in ref. [6][7][8].</p><!><p>Compounds 1-4 were docked into the binding site of D3DR inactive structure (PDB ID: 3PBL) (see Material and Methods). [15] Similarly to the eticlopride-bound structure, all the best-scoring ligand poses reproduced the driving interaction between the basic nitrogen of the piperazine ring and the side chain of D110 3.32 . As expected, given the similarity, all molecules docked consistently and in qualitative agreement with the binding mode proposed by Chien and colleagues for D3-selective derivatives. [15] In Figure 2, the clustering results on the 1, 2, 4 set are reported as a graph. Bent and extended conformations of the ligands were isolated from each trajectory but with varying frequencies. In the graph, clusters display a selective enrichment in one or more ligands (Table S1) with different efficacy profiles and are connected through a heterogeneously populated hub node mainly characterized by bent poses (cluster 0, Figure 2c). The insets 2a, 2b, 2d-2f in Figure 2 highlight global differences in ligand scaffold orientations in the binding site (cyan licorice) relatively to the representative conformation of the hub node (gold licorice). Cluster 1 and cluster 3 were almost exclusively populated by compound 1, with 10145 and 7624 members (Figure 2a-b). The substituted 2,3dichlorophenyl ring and the trans double bond in the linker led to extended rearrangements that could be found in these clusters and that were stable in time. For example, the conformation observed in cluster 1 only appeared after 1.85 µs but was thereafter stably preserved until the end of the simulation. Lacking both the unsaturation in the linker and substituents in the pendant aromatic ring, 4 sampled the broader portion of the conformational space, showing substantial enrichment in node 7 (2005 members, bent conformation, Figure 2f) and almost exclusively populating cluster 5 (Figure 2d) and cluster 8. In the trajectory, the bent conformation associated with cluster 7 appeared after 100 ns and lasted for an additional 400 ns. Agonist-specific conformations associated with cluster 5 (9672 members) and cluster 8 (6778 members) appeared late in the simulation (around 2 µs) and were stably preserved. Compound 2 mostly interconverted between bent conformations of cluster 4 (10305 members) and cluster 6 (11773 members) (Figure 2e). Through MD simulations and cluster analysis carried out on the second dataset (compounds 1, 3, 4) we: i) assessed the robustness of the algorithm in reproducing agonist-selective and antagonist-selective clusters obtained in the first group; and ii) investigated the influence of the carboxamide substitution at the biphenyl group on the dynamics of 2 and 3. The topology of the graph obtained for the set formed by 1, 3, and 4 (Figure S2) is consistent with the one reported in Figure 2, robustly returning a similar partition (Table S2), in line with the overlapping efficacies of 2 and 3 (see discussion in the Supplementary Material).</p><p>Taken together, our results suggest that different efficacy profiles could be linked to preferential stabilization of ligand-specific bent and extended conformations. Whereas compound 1 stabilized extended rearrangements, the two partial agonists 2 and 3 preferentially bound the receptor with exclusive bent conformations (clusters 4 and 6) without never transitioning into agonist-/antagonist-specific nodes. Similarly, the full agonist 4 was able to enrich exclusive clusters, but it preserved the unique feature of switching between selective bent (cluster 7) and extended conformations (clusters 5 and 8). At the receptor level, ligand-induced changes involved functional residues as H349 6.55 , Y365 7.35 , S193 5.43 and extracellular loops 2 and 3 (EL2, EL3) conformations. To compare our results with relevant findings on D2-like sub-family of DRs, [10][11][12][13][15][16] we monitored the status of ligand-induced networks and local interaction patterns (1 and 2) involving the phenyl ring of the ligands, H349 6.55 , Y365 7.35 , and S193 5.43 , in each system (Figure 3). Also, we collected the values of χ1 dihedral angles in H349 6.55 and S193 5.43 , which recent studies have linked to the earliest stages of the activation process. The fluctuations of these internal variables are summarized in Figure 3 and they will be separately discussed and compared in the light of the experimental efficacies of the individual compounds. Cluster graph of the conformations explored by 1 (magenta), 2 (green), and 4 (blue). Each node represents a conformation. The size of each node is proportional to the cluster population. Each node is colored according to the relative cluster enrichment. Edges define transitions between clusters. In the insets (a-f), representative medoids (cyan) from each cluster are shown in complex with their corresponding D3DR conformation, and compared to "hub" medoid pose (gold) in cluster 0. Red circles on TM3, TM5, TM6, and TM7 indicate diagnostic residues D110 3.32 , S193 5.43 , H349 6.55 , and Y365 7.35 . EL2 connects TM4-TM5; EL3 connects TM6-TM7. (a, f) H-bond distance H349 6.55 (N)-Y365 7.35 (H); H-bond distances indicating the interaction pattern 1 are calculated between the hydrogen on the epsilon nitrogen atom of H349 6.55 and the phenolic hydrogen atom of Y365 7.35 ; (b, g) aromatic interaction distance H349 6.55 -phenyl ring (ligands 1-5) calculated between rings' centers of mass. (c, h) H-bond distance H349 6.55 (N)-S193 5.43 (H); H-bond distances connoting the interaction pattern 2 are calculated between the epsilon-bound hydrogen atom of H349 6.55 and the oxygen atom of S193 5.43 side chain; (d, i) χ1 dihedral angle of S193 5.43 . (e, l) χ1 dihedral angle of H349 6.55 . Color codes for ligands 1-5 are consistent with Figures 2 and S2.</p><!><p>Antagonist-specific clusters 1 and 3 were both characterized by an extended conformation of the common core (Figure 4) and stabilized D3DR in a closed state due to concerted motions of TM6-TM7 toward TM1-TM2 (compare TM7 in Figures 2a-c).</p><p>This shift caused EL2 to come in close contact with EL3 (Figure 2a). H349 6.55 and Y365 7.35 remained around 6 Å apart. In line with recent work on antagonist-bound D3DR structures, H349 6.55 and S193 5.43 were found at around 8 Å apart, indicating that the antagonist stabilized longer TM5-TM6 interface distances. [29] Therefore, neither interaction pattern 1 (Figure 3a, magenta line) nor interaction pattern 2 (Figure 3c, magenta line) were ever observed. Instead, they were replaced by stable H-bond gating bridges established by H349 6.55 and Y365 7.35 with EL2 residues, as I183 and S182 (Figure 4 and Figure 5a, c). In cluster 3, the antagonist further stabilized Y365 7.35 orientation via T-shape p-p interactions with the biphenyl moiety (Figure 4b). The antagonist limited the fluctuations of EL2, stabilizing the loop in a conformation that sealed the binding site from above (Figure 2a), as also observed from the lowest average number of waters surrounding the ligand along the trajectory (Table S3 and Figure S3). This shielding process was enhanced by the van der Waals interactions established by I183 side chain in the phenylpiperazine binding site. Our findings are in agreement with recently reported MD simulations and mutagenesis studies, which recognize EL2 as a crucial element in GPCR activation, [30][31] and I183 as an important residue for antagonist binding. [32] The key difference between the two antagonist clusters was in the orientation of the 2,3-dichlorophenyl ring relative to membrane plane. While cluster 3 identified a perpendicular orientation (Figure 4a), cluster 1 displayed a parallel one (Figure 4b). The 2,3-dichlorophenyl group established very weak aromatic and hydrophobic interactions with H349 6.55 (distance ≈ 6.5 Å, magenta line in Figure 3b). After 2 µs, the transition from cluster 3 to cluster 1 occurred and the distance between the imidazole ring in H349 6.55 and the ligand further increased, reaching an average value of 7.5 Å. This change was induced by the rotation of the C(Cl)CNC dihedral angle (see Figure S1 and Figures 2a, 4b) observed in cluster 1, where the unfavorable interaction with H349 6.55 was replaced by a T-shape aromatic stacking with the side chain of F188 5.38 . This phenylalanine made an inward rotation into the binding site, which was followed by the formation of p-p interactions with 1. Contacts established with position 5.38 have been associated with β-arrestin activation in 5-HT2B and D2DR. [33][34] When the antagonist stabilized in cluster 1, rotation of the phenylpiperazine core also induced F346 6.52 side chain to shift outward at the interface of TM5-6, where optimized parallel stacking between the ring of 1 and the side chains of F197 5.47 , F338 6.44 , and F346 6.52 was observed.</p><p>In agreement with our observations, such outward rotation opens a "cryptic pocket" which was found to be crucial in explaining the efficacy of a D3DR antagonist. [32] In contrast, Michino and coworkers have recently observed that F346 6.52 rotation toward TM5-TM6 interface facilitates the inward motion of TM6 and can be considered a signature of partial agonists-driven destabilization of the inactive state of D3DR. The authors observed how the aromatic residue pointed toward the receptor core in presence of an antagonist, hindering TM6 movement. [29] Our results showed that F346 6.52 sampled both orientations, pointing toward receptor core when 1 was in cluster 3 and toward the TM5-TM6 interface when the antagonist populated cluster 1. On the same line, along the trajectory, the χ1 dihedral angle of H349 6.55 showed small fluctuations around 60° (Figure 3e) while S193 5.43 mainly oriented outward (-50°) (Figure 3d). In recent MD studies on dopamine-bound active model of D2DR, H349 6.55 χ1 value could indeed be sampled, but with much lower frequency with respect to values conducive to a fully substrate-activated state of the receptor (-60°), whereas S193 5.43 χ1 value stabilized around 160°. [12]</p><!><p>In 2 and 3, a partial agonist efficacy profile was associated with a different behavior, as a consequence of the increased ligand flexibility. Namely, bent conformations of 2 observed in nodes 0, 4, 6, and 7 were stabilized by T-shape stacking between the ligand 2,3-dichlorophenyl ring and F346 6.52 . This residue pointed toward the receptor core, as in the receptor crystallographic structure, without rotating outward. This is particularly relevant in light of the results recently reported by Ferruz and coworkers, [32] who have shown how inward/outward conformations of F346 6.52 could have a crucial role in D3DR response to ligands of varying efficacies (see the Supplementary Material for detailed discussion of partial agonist 3). In our simulations, partial agonist binding dynamics did not lead to antagonist-induced H-bond bridges (Figure 5). Conversely, we could observe EL2 displacement (Figure 2e and Figure S2a), enhancing water influx into the orthosteric binding pocket relatively to 1 (Figure S3 and Table S3). Compounds 2 and 3 facilitated this process preserving a dynamic coupling of TM6-TM7 interface. Indeed, distances between interacting atoms in H349 6.55 and Y365 7.35 were substantially shorter in these trajectories than in the antagonist-bound receptor, dropping on average by 2 to 4 Å. The 2,3-dichlorophenyl ring of 2, as also seen for 1, made only weak T-shape p-p interactions with H349 6.55 (Figure 3b). Furthermore, in contrast to the antagonist, bent conformations of compounds 2 and 3 formed a parallel stacking interaction with the side chain of Y365 7.35 , promoting the breaking of the H349 6.55 -I183 and Y365 7.35 -S182 Hbonds and the formation of the interaction pattern 1 (Figure 6 and Figure S4). For compound 2, the break occurred more frequently in the pair Y365 7.35 -S182 (Figure 5c). Indeed, in cluster 4, this ligand stabilized also a partially open state of the gate, retaining H349 6.55 in proximity of both Y365 7.35 and I183 (Figure 6a). Accordingly, the H349 6.55 -S193 5.43 distance was on average longer for partial agonists-bound receptor, with values fluctuating in the range of 4-10 Å (Figure 3c).</p><p>Partial agonists could block progression toward a fully active state by preventing stable TM5-TM6 interface contacts. [13] Thus, none of the simulated partial agonists was able to establish significant H-bond interactions between residues involved in interaction pattern 2. Our results agree with recent MD simulations of D3DR-ligand complexes, where bent poses have been associated with partial agonism. [29] In line with these findings, in selective clusters 4 and 6 (Figure 2 and Figure S2) the distance between TM5 and TM6 increased, while tightening interactions at TM6-TM7 interface could be observed. The formation of the interaction pattern 1 in partial agonists simulations stabilized this inter-helical rearrangement. Inward rotation of the χ1 angle of the H349 6.55 in cluster 6 did not break pattern 1 (Figure 3a, e, 6b) and induced substantial side-bending of TM6. The analyzed binding modes were in very good agreement with the effect induced by the partial agonist FAUC350 on the same dihedral angle of H349 6.55 in the active ternary model of D2DR, where the ligand promoted coupling of TM6-7 and formation of the H349 6.55 -Y365 7.53 H-bond. Interestingly, this study reports that differential stabilization of inter-helical interaction patterns in the G-protein-bound model of D2DR is responsible for loosening intracellular coupling between the G-protein and D2DR, likely identifying structural patterns at the basis of partial agonism at the highly homologous D2DR. [13]</p><!><p>In analogy with partial agonists, the almost full agonist 4 (hereafter simply referred to as agonist) initially induced interaction pattern 1 in the representative bent conformation of cluster 7 (Figure 3a and Figure 7a), while also inducing TM6-TM7 coupling. The gating H-bond interaction between Y365 7.35 and S182 was not formed when 4 populated this cluster, again indicating that TM6-TM7 coupling promoted receptor opening (Figure 5c). Consistently, EL2 displacement was observed and this was, in turn, conducive to a pronounced increase in solvation (Figure S3 and Table S3). These results are in line with NMR studies on rhodopsin in which H-bond network reorganization between EL2 and TM4-6 has been coupled to EL2 displacement from the binding site during substrate-induced activation. [30][31] In cluster 7, the pendant phenyl ring in the arylpiperazine moiety of 4 and the side chain of H349 6.55 formed an aromatic T-shape p-p interaction which was uniquely preserved by the agonist along the whole trajectory. Indeed, the distance between the two rings was stably preserved at approximately 5 Å (Figure 3b). The dihedral angle of S193 5.43 frequently rotated inward, around 160° (Figure 3d), i.e. the value observed for dopamine in the active model of D2DR. [12][13] Our simulations revealed that interaction pattern 1 destabilized after 500 ns (Figure 3a) and the trajectory evolved toward the conformations that populate cluster 5 and cluster 8.</p><p>These agonist-selective extended conformations caused the largest increase in the H349 6.55 -I183 distance (Figure 5a), which further allowed waters to reach the pocket (Table S3 and Figure S3). Besides the aromatic interactions with H349 6.55 , the phenyl ring of 4 established tight parallel stacking with F346 6.52 . While H349 6.55 side chain maintained its initial orientation (χ1 ≈ 60°, Figure 3e), F346 6.52 side chain underwent an exclusive rotation into the binding site in the opposite direction with respect to what was observed for 1, and pointed toward the intracellular region of the receptor. The ability to maintain stable interactions with H349 6.55 and F346 6.52 was a unique feature of 4. The ligand-induced rotameric state of F346 6.52 was observed only in response to the shift of the phenyl ring of the agonist at the TM5-TM6 interface, where it preserved an orientation perpendicular to membrane plane (Figure 7b). Inward rotation of F346 6.52 observed in our simulations minimized the steric hindrance that hampers TM6 inward motion, which is a crucial event in the destabilization of the receptor inactive state. The biphenyl ring interacted with Y365 7.35 pushing TM7 toward TM5-6. Furthermore, stable interactions of the phenylpiperazine ring of 4 with H349 6.55 at TM5-TM6 interface brought the histidine to point toward TM5 with higher frequency. In this conformation, H349 6.55 and S193 5.43 were only 2 to 4 Å apart, a rearrangement never observed in ligands with lower efficacy (Figure 3c and Figure 7b). The interaction pattern 2 first appeared in the trajectory around 1 µs, remained stable for roughly 100 ns, and was later transiently re-visited, as observed in Figure 3c (spikes in the blue line). The agonist, probably due to the persistent memory of the initial inactive state of D3DR, was not able to induce a complete rotation of H349 6.55 and S193 5.43 χ1 torsions toward -60° and 160°, respectively, which are the values that fully optimize the H349 6.55 -S193 5.43 interaction in dopamine-bound D2DR. [12] However, even without stably preserving interaction pattern 2, in our simulations of D3DR in complex with 4, a series of agonist-specific changes in the interaction networks, which did not occur in antagonist-and partial agonist-bound complexes, could be observed. In particular, compound 4 selectively enriched bent and extended clusters, in which it was the only ligand able to preserve tight interaction with the H349 6.55 . In doing so, the agonist stabilized the interaction pattern 1 for 500 ns and also induced transient formation of the interaction pattern 2 for a maximum time of 100 ns, complementing the shift of H349 6.55 at TM5-TM6 interface. Such changes promoted abrupt opening of the extracellular portion of receptor and temporarily increased the contacts between TM5 and TM6. Maintaining aromatic interactions with F346 6.52 in a uniquely inward-rotated state contributed to this scenario, reducing the hindrance at the interface.</p><!><p>Taken together, our results provided the structural basis for understanding the varying efficacy of the 1-4 series. The 2,3-dichloro substitution and the butyl/(E)-but-2-ene linker were both needed to obtain a full antagonist, i.e. a molecule that was unable to promote any of the agonist-stabilized interaction patterns and that preserved D3DR in a closed configuration. Conversely, the removal of chloride atoms and the introduction of a flexible butyl linker led to an almost full agonist activity for 4. Compound 4 was able to uniquely establish long-lasting contacts with functional histidine in position 6.55, orienting this residue to establish both of the investigated interaction patterns, albeit to different extents. Interestingly, the partial agonist properties of semi-flexible 2 and 3 (bearing the 2,3-dichloro substitutions, but a saturated butyl linker) were explained according to their ability to stably induce only one of the intermediate interaction schemes, thus hampering but not completely blocking the cascade of events that concurred in the perturbation of the inactive receptor state. These results are in good agreement with the structure-efficacy relationship previously reported for other series of compounds. [13] To gain confidence on this model, we designed a new compound introducing on the shared scaffold local modifications that, based on our understanding of the structureefficacy relationship, were likely conducive to a partial agonist profile. Two main aspects were considered in the design of the new ligand. First, our MD studies tried to provide a rationale for previous studies on a series of structurally related phenylpiperazine derivatives, which showed that the simple replacement of a butyl linker with a butyl/(E)but-2-ene in two identical ligands could transform an agonist into a partial agonist, and a partial agonist into a full antagonist. [35] We reasoned that the introduction of a butyl/(E)but-2-ene linker in 4 could reduce ligand flexibility, and in doing so, it could prevent the stabilization of both patterns. Second, we reduced the molecular weight of the new compound by removing the carboxamide substituent in the meta position of the biphenyl group. Our SAR studies on O-aryl carbamate derivatives revealed that this substituent influences the affinity for the receptor but not the efficacy of the ligand. [6,8] Insights gained from our simulations revealed that removing this moiety allowed 3 to sample a wider set of conformations, which, however, resulted in the induction of the interaction pattern 1, that is, the hallmark of partial agonists' profiles (see the Supplementary Material and Figure S2). The designed compound 5 is shown in Figure 1.</p><p>The conformations obtained from 3.05 µs of MD simulations were then subjected to the previous analysis, merging the trajectories of compounds 1, 5, and 4 to assess how the new ligand conformations partitioned in the presence of our reference agonist and antagonist. The obtained clusters are reported in Figure 8. As in previous cases, the graph highlighted a hub cluster, cluster 0, which was almost equally populated by the three ligands (Table S4). In analogy with 3, thanks to the increased flexibility in the analyzed dataset (1, 5, 4), the hub node was characterized by extended and not bent conformations (Figure 8a). The featuring binding modes of 5 grouped in cluster 4 and cluster 6 (Figure 8b-c), with 9688 and 8429 conformations, respectively. An interesting difference between the partitions was the relative abundance of 5 in the agonist-selective cluster 7 (Figure 8). This was three times greater than 4 (6463 vs. 2173 members) (Table S4). In the previous analysis, this cluster contained only 569 conformations of 3 and 126 of 2.</p><p>Our model identified this medoid as a crucial intermediate for establishing interaction pattern 1 in the agonist simulation. Interaction scheme 1 was never formed in cluster 4 (Figure 9a). It only appeared after 1.2 µs when 5 transitioned first to cluster 7 (refer to medoid in Figure 7a) and then to cluster 6 (Figure 9b). Indeed, this pattern was stably preserved until the end of the simulation (Figure 3f), with H349 6.55 -Y365 7.35 distance fluctuating in the range 2-4 Å. While the histidine kept a stable χ1 angle of 60° (Figure 3l), following the formation of the interaction pattern 1 and population of the agonist-like cluster 7, we observed inward rotation of S193 5.43 in TM5 (from 60° to 180°) (Figure 3i).</p><p>In line with results on 2 and 3, the interaction pattern 2 was never formed in presence of 5 (Figure 3h), as the distance between H349 6.55 and S193 5.43 was stably over 4 Å for the whole trajectory. In clusters 4, 6 and 7, the receptor was found in an open state with TM6-7 being dynamically coupled through stabilization of the interaction pattern 1 (see also TM6-TM7 in Figure 8b-c). The average number of solvent molecules around the ligand was comparable to 2-3 and in between the values calculated for 1 and 4 (Table S3 and See Figure 2 for graph description. In the insets (a-c), representative medoids (cyan) of each cluster are shown in complex with their corresponding D3DR conformation and compared to the most populated "hub" medoid pose (gold) in cluster 0. Indeed, bending of compound 5 in cluster 4 caused the breaking of the H-bond between Y365 7.35 and S182. Later in the trajectory, the ligand stabilized in the bent orientation of cluster 6, where both gating interactions were broken (Figure 5b, d) and interaction pattern 1 was stably induced (Figure 3f and Figure 9) In analogy with 3, in cluster 6, the lack of the carboxamide substituent in the distal phenyl ring of the biphenyl group induced the ligand to drift deeper into the pocket (Figure 8c and Figure 9b), compromising the aromatic interaction between the ligand phenyl ring and H349 6.55 (Figure 3g). Ligand 5 interacted with F338 6.44 , F197 5.47 , and F346 6.52 at the interface of TM5-TM6, inducing outward rotation of F346 6.52 side chain and causing an increase in distance between these two helixes. To release the steric clash between F346 5.47 and F338 6.44 , TM5 slightly rotated clockwise, inducing EL2 to partially extend over the binding site (see relative orientations of TM5 and EL2 in cluster 4 to 6 in Figure 8b, c). These results are in agreement with the recently proposed mechanism of D3DR activation by a phenylpiperazine series of partial agonists. [29] In contrast, recent MD studies on D3DR antagonists from Ferruz and colleagues have associated the outward rotation of F346 6.52 side chain to the formation of the cryptic pocket responsible for antagonist-like responses. [31] Our observations suggest that the interaction of partial agonists, like 3 and 5 with this cryptic site could actually be responsible for antagonistlike properties, resulting in hampered activation of D3DR and partial agonism profiles.</p><p>However, MD simulations of partial agonist 2 showed that this compound stabilized the rotameric state observed for F346 6.52 in the eticlopride-bound D3DR, [15] suggesting that both conformations are likely to reduce the efficacy depending on the preferred bent and extended binding mode. In other words, we propose that an antagonist would likely elicit no response if an outward rotation of the F346 6.52 occurs in D3DR. When a rigid ligand binds the receptor in an extended mode, it blocks solvent access, preventing the formation of any interaction pattern. In contrast, in presence of semi-flexible partial agonists, which stabilize a more open conformation of the binding site, an outward rotation of the active site phenylalanine could still concur to destabilization of the inactive state. Interestingly, when our flexible agonist increased receptor solvation, assuming bent and extended conformations, F346 6.52 rotated in an unexpected direction, orienting its side chain toward the center of the helical bundle. Tight binding of H349 6.55 was also found to be uniquely preserved in the full agonist simulation, where both interaction patterns were visited. In contrast, this residue seems not crucial for antagonism. In partial agonists simulations, weak interactions with this residue were responsible for the induction of just one of the functional interaction schemes involved in D3DR activation.</p><p>Importantly, the partition for compounds 1 and 4 was very robust. This is because, despite changing the initial set of conformations, medoid positions along the trajectory did not change. Indeed, we found that clusters 1, 3, 5, 7, and 8 were consistent with those already identified in the two previous sets of compounds. According to our model, compound 5 behaved as a partial agonist, destabilizing the inactive state, promoting receptor opening via TM6-TM7 coupling and binding site solvation. In line with other partial agonists, it induced only one of the two interaction patterns associated with agonist-like properties.</p><p>To validate our hypothesis, 5 was synthesized and its experimental efficacy was tested in vitro for its ability to inhibit accumulation of cAMP. [6][7][8] Compound 5 revealed partial agonist properties when compared to the effects elicited by 300 nM of dopamine (D3DR efficacy: 60%). Moreover, dropping the terminal carboxamide led to a D3DR 23 nM EC50. As expected, this is weaker than that of 2 and 3. However, it is a promising value in light of the reduced molecular weight.</p><!><p>To further characterize our results, we also performed a retrospective compoundbased PCA analysis of torsional angles (Figure 10). Namely, we attempted to identify key torsional angles in the analyzed series of compounds. The dataset was obtained by extracting the values of three dihedral angles from 1-4 (Figure S1) over the entire 12 µs (120000 data points) of aggregate production runs. Next, data were binned in a reduced number of representative histograms. We performed a dimensionality reduction through dihedral principal component analysis (PCA) on this space and projected on the three main components to help visualize our ligands. Compound 5 was then embedded in the space identified by the four known compounds. The two distances obtained by averaging those from 1 and 4 were 1.43*10 4 and 1.46*10 4 for compound 2 and compound 5, respectively, making them equidistant from the two reference points. These results were robust to changes in the initial number of bins. Compound 5 actually stabilized in a specific orientation, which, as for 2 and 3, was somewhere in between the agonist and antagonist ones. This suggests that 5 could possess the same functionality of 2 and 3 at D3DR. Overall, although limited to a dataset of only four compounds, we found this (retrospectively applied) analysis useful in obtaining a quick and concise understanding of the simulations, recapitulating insights obtained with more complex analytical frameworks. The highly symmetric behavior of 2,3 and 5 when compared to 1 and 4 suggests that this could be an effective vector space where the efficacy profile of new compounds belonging to this series could be prospectively characterized. This also make us optimistic that a similar framework could be duplicated for other series and, possibly, other targets.</p><!><p>In summary, we used MD simulations to rationalize the experimentally observed efficacies of O-aryl carbamate derivatives. Our comparative analyses on the destabilizing effect of our ligands on the D3DR inactivated structure, we got evidence that structurally similar molecules can engage in subtly different interaction patterns and that these are, in turn, conducive to different efficacy profiles. The conformational changes reported from our simulations could be only related to the destabilization of the D3DR reference state, albeit known to be connected to initial steps in the concerted process of receptor activation. We found that the extent of these conformational changes was helpful in discriminating between ligand efficacies, and could therefore be of great help in designing a new ligand with a tailored pharmacological profile. Compound 5 was designed based on SER data to further test the consistency of the simulative outcome and was eventually synthesized and tested. As expected, this ligand behaved as a partial agonist. In due time, and in light of the ever-increasing computational power available to the scientific community, this work could pave the way to a more systematic application of MD as the in silico counterpart of functional assays, much as docking and free-energy methods can be regarded as the in silico counterpart of binding assays.</p><!><p>The membrane protein complex was then solvated with an average of 12300 water molecules (TIP3P model). Force fields available in AMBER 14 were used to parameterize protein, [1] lipids, and ligands, corresponding to ff14SB, [2] lipid14, [3] and GAFF, [4] respectively. Point charges for ligands</p><p>were derived from the electrostatic potential calculated after geometry optimization at the Hartree-Fock level of theory with a 6-31G* basis set, following the RESP procedure as implemented in Antechamber. Simulations were performed on GPU-equipped workstations with Gromacs 4.6.7 MD engine. [5] In detail, the MD protocol encompassed three steps: minimization, equilibration, and production. Each system was minimized for 5000 steps and then thermalized to 300 K in different phases. Temperature was raised to 300 K in 300 ps within the NVT ensemble, in three consecutive increments of 100 K lasting 100 ps each. Then, volume and density were allowed to equilibrate in NPT ensemble at 300 K and target pressure of 1 bar for 200 ps. Lipids, ligands, and water molecules were equilibrated first, applying position constraints only to protein backbone (1000 kJmol -1 nm -2 ) in NVT steps. During the NPT equilibration, protein structure constraints were removed to allow relaxation at 300 K. Production runs were performed in NPT conditions with semi-isotropic pressure control, using Parrinello-Rahman barostat; temperature was kept at 300 K using v-rescale thermostat.</p><p>A cut-off of 11 Å was used to switch off van der Waals interactions, while Particle Mesh Ewald was used to calculate electrostatics of the system, with a spacing of 1.6 Å. Finally, a 2 fs time-step was used to accumulate 3.05 µs of simulated time for each of the five systems, for a total of 15.25 µs.</p><p>Dumping time was set equal to 100 ps. Our analysis covered the last 3 µs of collected statistics for each of the investigated complexes.</p><!><p>We used a variant of k-means algorithm, namely k-medoids, [6] as implemented in the BiKi LifeSciences suite. [7] Generally, k-means generates an artificial mean structure, identified by coordinates that have minimal sum of squared deviations from a cluster center. The algorithm minimizes a distance-based cost function which is the sum of squared errors (SSE) as defined in Eq.</p><p>(1):</p><p>In Eq. ( 1), k is the number of clusters, n is the total number of conformations, x is the ith element of the cth cluster and m is the mean of the cth cluster. In each step, centroids are randomly chosen, closest objects are grouped around them, and SSE is calculated. Then, new arbitrary k medoids are chosen, clustering is performed again, and the new SSE is compared with the previous one in an iterative fashion until the difference between the previous and the present cycle cannot be further reduced, and medoid positions do not change anymore. While efficient in terms of computational time, k-means clustering is sensitive to outliers and to the initialization procedure for the random search of medoids. K-medoids is similar to k-means, as it is a medoid-centered algorithm, but instead of taking means as the centroid of the cluster, k-medoids assigns to centroids a physical meaning, identifying them as real objects in the data set. The new medoids are the most centrally located objects of each cluster. This modification introduces the possibility of returning to minimize the real sum of dissimilarities (distances) between the objects x in a cluster and their medoid m, which is a real representative conformation of the cluster. In other words, in Eq. ( 1), the difference is an absolute distance from a reference point and not a distance from the mean. Moreover, it overcomes some limitations of the classical k-medoids algorithm, which, as k-means-like algorithms, randomly select the initial medoids. This procedure affects computational efficiency and makes the results dependent on the choice of k. This k-medoids version provides a method to select the initial k medoids. The distances dij are first calculated between every pair of i and j objects, and a distance matrix is created once. Then, a variable for each j object, vj, is calculated as in Eq. ( 2):</p><p>The values obtained for each j object are sorted in ascending order and the first k medoids with minimal values of v are considered as initial cluster centers. This makes the algorithm deterministic because the initial k medoids are always those that minimize the total distance to all other objects i.e.</p><p>the most central ones. Also, medoids are updated, finding a new one for each cluster that minimizes the total distance to any other objects in the cluster. The main advantages of this procedure are the ability to work with medoids that can be associated with sampled conformations, and to use an objective function based on absolute distances to refine the quality of the clustering. Moreover, the algorithm is robust to outliers because the most centered conformations are selected as the initial 5 medoids. In our systems, trajectories were concatenated based only on the common scaffold shared by the five ligands (Figure S1). The choice of the representative medoids was performed based on RMSD-based threshold between medoids in a given partition. The number of clusters was considered meaningful of sampling diversity if the difference between the medoids was more than 3Å. Automated column chromatography purifications were performed using a Teledyne ISCO apparatus (CombiFlashTM Rf) with pre-packed silica gel columns of different sizes (from 4 g to 120 g).</p><!><p>Mixtures of increasing polarity of cyclohexane and ethyl acetate or dichloromethane and methanol were used as eluents. Preparative TLCs were performed using Macherey-Nagel pre-coated 0.05 mm TLC plates (SIL G-50 UV254). Hydrogenation reactions were performed using H-CubeTM continuous hydrogenation equipment (SS-reaction line version), with disposable catalyst cartridges (CatCartTM) preloaded with the required heterogeneous catalyst. Microwave heating was performed using ExplorerTM-48 positions instrument (CEM). NMR experiments were run on a Bruker Avance III 400 system (400.13 MHz for 1H, and 100.62 MHz for 13C), equipped with a BBI probe and Zgradients. Spectra were acquired at 300 K, using deuterated dimethylsulfoxyde (DMSO-d6) or deuterated chloroform (CDCl3) as solvents. Chemical shifts for 1H and 13C spectra were recorded</p>

ChemRxiv

The Effects of Secondary Oxides on Copper‐Based Catalysts for Green Methanol Synthesis

AbstractCatalysts for methanol synthesis from CO2 and H2 have been produced by two main methods: co‐precipitation and supercritical anti‐solvent (SAS) precipitation. These two methods are compared, along with the behaviour of copper supported on Zn, Mg, Mn, and Ce oxides. Although the SAS method produces initially active material with high Cu specific surface area, they appear to be unstable during reaction losing significant amounts of surface area and hence activity. The CuZn catalysts prepared by co‐precipitation, however, showed much greater thermal and reactive stability than the other materials. There appeared to be the usual near‐linear dependence of activity upon Cu specific area, though the initial performance relationship was different from that post‐reaction, after some loss of surface area. The formation of the malachite precursor, as reported before, is important for good activity and stability, whereas if copper oxides are formed during the synthesis and ageing process, then a detrimental effect on these properties is seen.

the_effects_of_secondary_oxides_on_copper‐based_catalysts_for_green_methanol_synthesis

<!>Introduction<!>Results and Discussion<!>Co‐precipitated catalysts<!><!>Co‐precipitated catalysts<!><!>Co‐precipitated catalysts<!><!>Co‐precipitated catalysts<!><!>Co‐precipitated catalysts<!><!>Co‐precipitated catalysts<!>Supercritical anti‐solvent (SAS) precipitation catalysts<!><!>Supercritical anti‐solvent (SAS) precipitation catalysts<!><!>Supercritical anti‐solvent (SAS) precipitation catalysts<!><!>Supercritical anti‐solvent (SAS) precipitation catalysts<!><!>Supercritical anti‐solvent (SAS) precipitation catalysts<!>Conclusions<!>Materials<!>Co‐precipitated catalysts<!>Supercritical anti‐solvent precipitation catalysts<!>XRD<!>Surface area measurements<!>Catalytic methanol synthesis<!>Conflict of interest

<p>J. S. Hayward, P. J. Smith, S. A. Kondrat, M. Bowker, G. J. Hutchings, ChemCatChem 2017, 9, 1655.</p><!><p>In recent years the continuing rise in atmospheric anthropogenic carbon and the ensuing effects that this engenders on the climate have led to increasing efforts towards capture, sequestration, and utilisation of carbon dioxide. Given a general societal trend towards greener energy and fuel sources, the utilisation of CO2 as an abundant carbon source has become more and more relevant. The hydrogenation of CO2 to produce methanol for use as both a fuel and a chemical precursor is one possible route to this goal. The concept of a cycle using CO2 and methanol in such a manner is generally attributed to Olah et al.,1 and is referred to as the anthropogenic carbon cycle. One of the attractive facets of this method is the possibility of a truly green fuel; CO2 can be captured from sources such as power stations and combined with hydrogen generated from a renewable source, such as electrolysis of water where the electricity is supplied from solar power. The synthesis of green methanol in this way can be regarded as a way of storing H2, effectively for storing renewable energy chemically, in addition to the production of a fuel.</p><p>Global methanol production is in the region of 80 Mt per annum.2 Industrially, methanol is produced from a mixture of carbon monoxide, carbon dioxide, and hydrogen (syngas) at elevated pressures and moderate temperatures. It has been shown that carbon dioxide is the carbon source at the molecular scale, producing methanol and water. Carbon monoxide is present to convert the water produced into CO2 and H2 via the water‐gas shift reaction. These reactions are represented by Equations (1), (2):(1)CO2+3H2→CH3OH+H2O(methanolsynthesis) (2)CO2+H2→H2O+CO(reversewater-gasshift)</p><p>The current industrial synthesis is from a syngas, deriving from fossil fuels, which has a mix of CO and CO2, but the main synthesis route is as in Equation (3), with little water production:(3)CO+2H2→CH3OH</p><p>However, the reaction studied herein differs from this system in that there is no CO present, since we are testing the possibility of using recaptured CO2 and renewable hydrogen, as opposed to fossil fuel generated syngas. This means that without CO being present to enable overall water‐gas shift, our system will contain a significantly higher proportion of water vapour than a system running syngas.</p><p>The catalysts used for this reaction are composed of copper, zinc, and alumina, and are based on the catalysts originally designed by ICI during the 1960s.3, 4 The optimisation of these catalysts was performed long before modern techniques made it possible to understand the fundamentals of the active sites and reaction mechanism. In recent years there have been increasing numbers of studies into these fundamental aspects of this catalysis,5, 6 with the focus generally tending towards the simpler binary system of Cu/ZnO. From this has risen the consensus that the methanol productivity is strongly correlated with the specific copper surface area of a catalyst,7, 8 and that other factors such as the oxidation state of the copper,9 and the copper–zinc interaction,10, 11 also have an effect.</p><p>The optimised industrial catalyst is granted excellent copper surface area by way of the structure of the precursor material from which it is derived. Such catalysts are synthesised by co‐precipitation of metal nitrate salts with sodium carbonate to produce a hydroxycarbonate precursor phase, which is then calcined to form CuO and ZnO. It is known that the most active catalysts are formed from a precursor consisting predominantly of zinc‐substituted malachite phases. When properly prepared, the specific structure of the final catalyst is defined by this precursor phase, leading to a material with the desired high copper surface area and good copper–zinc interaction.12</p><p>Whilst copper comprises the active metal in the catalysts, the role of the zinc is less clear,13 and as such there have been many investigations into copper‐based catalysts with alternative secondary metal oxides such as magnesia,14 ceria,15, 16 and zirconia.17, 18 Of particular note are studies that show Cu/MgO catalysts as having higher copper surface areas, and yet having lower methanol activity,14 which runs counter to the accepted stance of copper surface area being directly linked to such activity. The issue seems to stem from the nature of the standard catalyst, the synthesis of which has been highly optimised in terms of pH, temperature and ageing times. Such catalysts are generally precipitated in the range of pH 6–7, and have been shown to lose activity if precipitated at higher pH ranges.12 However, the precipitation of magnesium nitrate requires a pH in the region of 9. Thus it is difficult to deconvolute whether the negative effect on activity is an artefact of the substituted oxide or the pH of synthesis.</p><p>Supercritical anti‐solvent (SAS) precipitation presents an interesting way to approach this problem, as the procedure rapidly precipitates material without the need for a base. It has been shown that this method can produce copper–zinc catalysts with high copper surface area, and that these catalysts are active for methanol synthesis and water‐gas shift reactions.19 A wide range of materials can be precipitated in this manner, in all cases without the requirement of a specific precipitating agent. This allows us to sidestep the need for specific pH ranges found in co‐precipitation, allowing us to remove it as a factor.</p><p>In this study we report the changes in methanol synthesis activity for copper catalysts synthesised with various secondary oxides using both co‐precipitation and SAS techniques. The changes were monitored through reactivity measurements, and through assessment of the copper surface area and particle size both before and after exposure to reaction conditions. Through this we hope to discover what factors result in activity loss in the co‐precipitated catalysts, and to investigate which metal oxides are capable of producing active, stable catalysts when the negative effects of high‐pH co‐precipitation are removed.</p><!><p>This study was conducted using Cu/MxOy catalysts, where M=Mg, Zn, Mn, or Ce. Based on the results of previous studies a molar ratio of 70:30 between copper and the secondary oxide was chosen. This amount has been shown to be close to the limit of incorporation of zinc into a malachite structure,20 and as such was used as a standard to which the other catalysts were held.</p><!><p>The catalysts were prepared as described in the Experimental Section. The surface areas of the materials produced are shown in Table 1.</p><!><p>Co‐precipitated catalyst details.</p><!><p>X‐ray diffraction (XRD) of the precursor phases revealed patterns consistent with those of malachite21 in the cases of all CuZn‐CP catalysts, as shown in Figure 1. The other precursors showed broader diffraction peaks. CuMg‐CP and CuCe‐CP show two major diffraction peaks consistent with CuO, with CuMg‐CP having low, broad peaks consistent with malachite. The small peak at 33° in CuCe‐CP may be residual malachite, and this material also shows broad peaks at angles consistent with CeO2. Based on these observations it would seem that the hydroxycarbonate phase is formed initially in all cases, as evidenced by the blue coloured material often reported in such cases. The colour change towards green in the CuZn and CuMn catalysts can be attributed to the formation of the malachite phase, whereas the darkening in colour of the CuMg and CuCe catalysts can be attributed to the formation of copper oxide phases. This cannot solely be attributed to the effects of the pH, as the CuZn catalysts prepared at higher pH do not show these phases. Therefore, this would seem to be an effect of the oxide, although possibly this is in combination with the elevated pH.</p><!><p>XRD patterns of co‐precipitated catalyst precursors.</p><!><p>The work of Fujita et al.22, 23 showed that a calcination temperature of 330 to 350 °C is sufficient to form the final catalysts. Based on this, all catalysts were calcined at 330 °C for 3 h in flowing air, with a thermal ramp rate of 5 °C min−1. The catalysts were uniformly brown after this calcination step, with the exception of CuCe‐CP, which presented a slightly grey hue.</p><p>XRD of the calcined catalysts gave similar patterns for all the materials except for CuCe‐CP (Figure 2). All had peaks consistent with copper oxide, but with the CuCe‐CP material having additional peaks consistent with CeO2. With the exception of the appearance of a small, sharp peak at 36°, the precursor and calcined versions of CuCe‐CP are very similar. The precursor and calcined versions of CuMg‐CP are also highly similar, with the calcined version losing the small, broad peaks associated with malachite. This is consistent with copper oxide already being formed during drying in these materials. The surface areas of the materials after calcination decreased by less than 10 % from the values found in the precursors.</p><!><p>XRD patterns of co‐precipitated catalysts after calcination.</p><!><p>The catalyst samples were tested for methanol synthesis as described in the Experimental Section, and the only significant products seen were CO and methanol. The CuZn catalysts generally appeared to undergo a strong initial deactivation, but were stable after 3 h. The other secondary oxides displayed varying behaviour, which will be discussed below. Of the co‐precipitated catalysts, copper–zinc showed the highest activity towards methanol production, and also showed the lowest amount of deactivation (Table 2). A trend amongst the copper–zinc catalyst was also evident; increasing pH lowered CO2 conversion and methanol selectivity, while those at lower pH preparation tended to show a lower degree of deactivation. CuZn 6.5‐CP lost only 5 % CO2 conversion from 1–8 h, compared with 7 % for CuZn 9‐CP and 10 % for CuZn 10‐CP. CuMn‐CP and CuMg‐CP had similar, if not higher, copper surface areas than the CuZn‐CP catalysts before reaction, but were not as active. CuMg‐CP gave good CO2 conversion and excellent methanol selectivity, but continued to deactivate after 3 h, stabilising after 6 h. CuMn‐CP appears to have gained activity over time, but a full time on‐line reaction study showed a slightly more complicated effect. CuMn‐CP started with the low activity shown above, and appeared to immediately start deactivating. However, after about 2 h, it began to show a marked increase in activity over the next hour, with both CO2 conversion and methanol selectivity rising rapidly to approximately 6.3 % conversion and 63 % selectivity. It maintained this activity for about 2 h before undergoing a rapid deactivation. Of particular interest is that the overall CO production rate changed only slightly during this time, implying that the increased CO2 conversion was primarily driven by a large increase in methanol selectivity, and that the deactivation occurred in the reverse manner. This would seem to indicate that species or active sites are briefly formed on CuMn catalysts that are highly active, but highly unstable. A repeat of this test over a longer time period (16 h) showed that the deactivation continued beyond 8 h, with the material having apparently stabilised after about 11 h, at which time it displayed CO2 conversion of <1 %. CuCe‐CP deactivated steadily, stabilising only in the final hour of testing. Whilst it was the only catalyst to increase methanol selectivity steadily, it does not seem to be a viable catalyst due to high deactivation and low activity.</p><!><p>Catalytic activity and copper surface areas of co‐precipitated catalysts.</p><!><p>The relationship of the copper surface areas to activity is interesting (Figure 3 and Table 2). Initial copper surface areas appear to match trends in the activity quite well, with the notable exception of CuMg‐CP. This catalyst possesses higher copper surface area than any of the others, and yet has lower activity. However, if one considers the post‐reaction copper surface areas, there is a more evident trend. Here, the higher surface areas correspond to higher activities with the exception of the CuMn‐CP sample. However, the CuMn‐CP catalyst was observed to be deactivating rapidly at the termination of the reaction, and the depressurising and cooling steps before recovery of the catalyst take approximately an hour. It may be that the CuMn‐CP catalyst continued to deactivate through this time. It appears that, although conversion has generally diminished, the intrinsic per site activity has increased after 8 h running, evident in the data of Figure 3. It is likely that this is due to a morphology change of the Cu particles, perhaps such that the Cu−ZnO interaction is not lost as much as the Cu surface area.</p><!><p>The dependence of catalyst activity on the Cu metal surface area for co‐precipitated catalysts. The lines are a guide for the eye. The red data points are for initial conversion and pre‐reaction metal area, whereas the blue data points relate the final conversion and metal surface area measured post‐reaction.</p><!><p>Thus, the value of the copper–zinc catalysts would appear to be a combination of high initial copper surface area and their ability to better retain this surface area during reaction conditions.</p><!><p>Supercritical anti‐solvent precipitations were carried out as described below and produced very fine powder, which was either blue or green depending on the secondary oxide (Table 3).</p><!><p>Supercritical anti‐solvent precipitation catalyst details.</p><!><p>XRD analysis on the precursor phases showed them to be highly amorphous/nanoparticulate (Figure 4). It is difficult to thus draw any conclusions about the materials formed, but these observations are in line with those reported for the synthesis of supercritically prepared georgeite19, 24 It is possible that the other oxides form similar amorphous materials as an effect of the extremely rapid precipitation step found in SAS precipitations.</p><!><p>XRD patterns for SAS catalyst precursors.</p><!><p>XRD analysis on the calcined SAS materials (Figure 5) showed a number of similarities to the CP materials. CuCe shows a small diffraction peak in the region of copper(II) oxide, and shows broad reflections consistent with the presence of CeO2. The other materials all appear to be copper oxide, as was observed in the CP materials. However, whereas the CP materials all displayed a distinctive double peak, the CuZn‐ and CuMg‐SAS show a single, broader reflection. This is indicative of smaller crystal domains in the material, which is likely to be an effect of the highly amorphous precursor being unable to generate long‐range order upon calcination. Unlike the co‐precipitated catalysts, the SAS catalysts displayed a far more significant loss of surface area upon calcination, with all losses being in the region of 30 %. A notable exception to this is CuMg‐SAS, which lost less than 5 %.</p><!><p>XRD patterns of SAS catalysts after calcination.</p><!><p>Catalytic testing and copper surface area measurements were carried out in an identical manner to those described for the co‐precipitated materials.</p><p>The reactivity behaviour of the SAS catalysts can be seen to be significantly different from that of the co‐precipitated materials, with the possible exception of the CuCe‐SAS material, which in both cases shows decreasing activity but increasing methanol selectivity (Table 4). These catalysts all displayed stronger initial deactivation than their co‐precipitated counterparts, but were all stable after 5 h. The CuZn‐SAS material is of particular interest. In keeping with evidence that increased copper surface area is directly linked to increased CO2 conversion (Figure 6), it is the most active catalyst when based on the results taken after 1 h. It remains active for approximately 4 h, but loses methanol selectivity as it does so. After this point, it begins to rapidly lose activity and continues to lose selectivity, stabilising after 5 h. Cu SSA measurements show a significant drop as a result of the reaction.</p><!><p>Catalytic activity and copper surface areas of SAS catalysts.</p><p>The dependence of catalyst activity on the Cu metal surface area. The lines are a guide for the eye, but indicate linear behaviour, though here the number of data points is limited. The red data points are for initial conversion and pre‐reaction metal area, whereas the blue data points relate the final conversion and metal surface area measured post‐reaction.</p><!><p>The CuMn‐SAS sample once again displayed a more complex behaviour than is suggested. Initial results shown here are at t=1 h, but data recorded before this point show CuMn‐SAS to be highly active, more so than CuZn‐SAS. However, it immediately deactivates, losing over 60 % of its activity in the initial hour. After 4 h it has almost completely deactivated and is predominantly selective towards the production of CO. The copper surface area measurements show a significant loss of surface area throughout the reaction, which is likely to be the cause of this deactivation. It is of note, though, that the initial activity is significantly higher than the pre‐reaction Cu SSA of the CuMn‐SAS sample would suggest. It is possible that this is a similar effect to that which was seen for CuMn‐CP, but without the induction period. The highly mixed and amorphous structure of the CuMn‐SAS could be very active initially, but then undergoes severe deactivation for the same reasons as before. This would seem to be borne out by the dramatic drop in copper surface area.</p><p>CuMg‐SAS displayed behaviour entirely contrary to the CP equivalent, proving to be the most stable of the SAS catalysts in terms of both CO2 conversion and selectivity. It does not appear to display such rapid deactivation, nor the switching to CO production of its CP counterpart, and after 8 h is comparable in activity to CuZn 6.5‐CP, which is due to its higher selectivity. Whilst it is shown to lose a significant amount of copper surface area, the loss is nowhere near as severe as in the cases of the other SAS materials.</p><!><p>A number of conclusions can be drawn from the results herein. One of the initial questions was to what extent, in co‐precipitated catalysts, is he activity of copper‐based catalysts determined by the pH of precipitation and to what extent is it affected by the secondary oxide. Based on the results shown, we can say that both play a role in the activity of the catalyst. CuZn‐CP catalysts were more active than all of the other secondary metals at the equivalent pH. From the results of the CuZn materials we see that increased pH leads to decreased activity. A difference in precipitation behaviour was seen as well; whereas the CuZn‐CP catalysts form zincian malachite at all three pH values, when the zinc is replaced with magnesium or ceria at elevated pH it leads to the direct formation of copper oxides during the ageing step of the synthesis, as confirmed by XRD. The better performance of the Zn materials is probably due to the formation of this phase.</p><p>CuMn‐CP showed interesting behaviour, in that there appeared to be an induction period where the activity increased, reaching a plateau for a time before rapidly deactivating. In many ways this mirrors the findings of Helveg et al.,25 who showed that copper–zinc catalysts will display similar tendencies depending on the oxidising or reducing nature of the atmosphere. Although the gas mixture is highly reducing due to 60 % H2, the oxidising nature of the atmosphere increases with increasing steam content.26 As H2O is a by‐product of both the methanol and reverse water‐gas shift reactions which occur, it would seem that the catalyst generates an active phase which is then adversely affected by the increased water content that this improved activity engenders. This then leads to the severe deactivation seen.</p><p>Once the results of the SAS catalysts are factored in, more conclusions can be drawn. Whilst for the CP materials increased pH leads to lower activity, the SAS results show that this is not the sole determinant. The activities still do not correlate exactly with the Cu SSA, as the CuMg has a higher area than CuZn. This shows that there is indeed an additional effect from the secondary oxide beyond the simple improvement of the active metal surface area, and that the relatively lower activity of the CuMg catalysts is not only a result of the higher precipitation pH required in CP.</p><p>Further investigation of these effects are required to ascertain which properties of the secondary oxides are affecting the Cu. H2‐TPR could be useful to investigate the reducibility of the catalysts, and CO2‐TPD can be used to assess changes in the basicity of the catalysts.</p><p>The CuZn‐SAS catalyst shows a similar deactivation to that reported before, although it does not show the initial induction period. Interestingly, the CuMn catalyst appears to have very similar behaviour, although it deactivates even more swiftly. Both CuZn‐SAS and CuMn‐SAS suffer a particularly pronounced loss of Cu surface area during the reaction, with CuMn‐SAS falling to the lowest value of any tested catalyst. Based on these overall results, it would seem that Mn and Zn behave in a broadly similar manner when paired with Cu, but that Zn is the better choice due to increased stability of the supported Cu metal.</p><p>CuMg catalysts proved interesting, as they were the only instance in which the SAS material was more stable than the CP material. This is seen in both the activity data and the copper surface area data, and could be down to a number of factors. The CuMg‐CP material showed evidence of CuO formation during the initial precipitation, and whilst this material had a high copper surface area it swiftly deactivated under reaction conditions. This behaviour was not observed in the SAS material, implying that the formation of the CuO phase was not conducive to retention of the high copper surface area even though it generated a high initial value. The amorphous SAS precursor, however, led to a material more stable than its CP counterpart or any other SAS prepared material. This may be due to the properties of MgO itself, which is not reported to form strong interactions with Cu (unlike zinc) and does not have a variety of possible oxidation states (unlike manganese and ceria).</p><p>When taken as a whole, the results strongly imply that whilst the initial copper surface area is important, the ability to retain this surface area whilst under reaction conditions would appear to be key. Further, the idea that copper surface area is directly correlated to methanol activity may not be easily applicable to materials using different secondary oxides. An excellent example of this lies in the CuMg catalysts. CuMg‐CP has a higher initial copper surface area than its CuZn‐CP equivalent, but its rapid deactivation means that the post‐reaction area value shows a truer measure of its activity. The same is true of CuMg‐SAS and CuZn‐SAS. In this instance the CuMg‐SAS has the lower initial surface area, but proves to be the more active catalyst in the long run due to its stability. This focus on stability appears to be a strength of the co‐precipitated CuZn materials, which displayed the lowest amount of deactivation.</p><p>Thus, the stability of the materials, and their effectiveness as catalysts, can be attributed to a number of factors beyond initial copper surface area. The formation of the malachite phase seems to be especially important in coprecipitation; CuZn and CuMn‐CP catalysts form this phase, and were significantly more active than their amorphous SAS counterparts. This phase appears to grant a greater degree of stability to the resulting catalysts. Where materials did not form this phase, they were all found to be less stable. This effect cannot be attributed to the presence of zinc as the secondary oxide, as the CuZn‐SAS catalyst was highly unstable. By contrast, the formation of the CuO phase during precipitation was indicative of a poor catalyst.</p><p>The results obtained using the SAS‐prepared catalysts help to back up the benefits of the malachite phase, but also show that for some materials the pH is a significant factor. CuMg is a good example of this; neither the CP nor the SAS catalyst form the malachite phase, but the elevated pH led to the formation of the undesirable CuO phase during co‐precipitation. Where this phase was not observed, in the SAS material, the catalyst was far more effective. This was not the case for the CuCe materials, which were less effective regardless of preparation method. This indicates that the choice of oxide is highly relevant.</p><p>Overall, the results seem to show that when considering co‐precipitation, CuZn catalysts appear to be significantly better due to a number of benefits granted by the precursor phase. CuMn catalysts behave in a similar manner, but deactivate more rapidly. When the materials were prepared by a method which leads to a highly amorphous precursor, other oxides become viable. CuMg seems in particular to be hampered by the high pH needed for precipitation. Once this limitation was removed, it proved to be an effective catalyst. This could potentially be of use as other precipitation methods are investigated.</p><p>Another important conclusion is the apparent confirmation of the work of Hadden et al.,27 who suggested that the correlation between copper surface area and activity was only valid between families of catalysts prepared with similar method. This is borne out in our results, as the higher surface area materials do not always prove to be the most active, and nor is the copper surface area across the range of oxides always directly proportional to the activity. We can extend these conclusions to account for the post‐reaction surface area losses. It seems that different preparation conditions, methods, and secondary oxides strongly influence the rate of initial deactivation of the catalysts, which is a key factor in their activity after stabilisation.</p><!><p>Copper(II) acetate monohydrate (puriss. p. a., ≥99.0 %), zinc(II) acetate dihydrate (puriss. p. a., ≥99.0 %), manganese(II) acetate tetrahydrate (99+%) copper(II) nitrate hemipentahydrate, zinc(II) nitrate hexahydrate, manganese(II) nitrate tetrahydrate, cerium nitrate, magnesium(II) nitrate hexahydrate, sodium carbonate, and cerium acetylacetonate hydrate were all purchased from Sigma–Aldrich. Magnesium(II) acetate tetrahydrate (analytical) was obtained from Amresco. Ethanol (absolute 99.8 %, Certified AR) was purchased from Fischer Scientific and CO2 (CP grade) was provided by BOC. All purchased materials were used as received. Deionised water was provided in‐house.</p><!><p>The co‐precipitated catalyst precursors were synthesised by co‐precipitation of metal salts using a Toledo Metrohm autotitrator.</p><p>Copper nitrate hemipentahydrate (Cu(NO3)2⋅2.5 H2O), zinc nitrate hexahydrate (Zn(NO3)2⋅6 H2O), and aluminium nitrate nonahydrate (Al(NO3)2⋅9 H2O) were dissolved in deionised water to create a mixed‐metal solution with a total molar concentration of 0.25 M. Additionally, a base solution was created by dissolving sodium carbonate (Na2CO3) in deionised water to give a concentration of 1.5 m Na2CO3.</p><p>A small aliquot (20 cm3) of the mixed metal solution was added to the reaction vessel, which was stirred continuously. The amount of liquid was chosen such that it was sufficient to cover the pH probe. This initial aliquot was brought to pH 6.5 by the addition of the base solution until the target pH was reached.</p><p>Subsequently, the mixed metal solution was added to the vessel at a rate of 5 cm3 min−1 with continuous stirring. Concurrently, base solution was added at a sufficient rate to ensure that the reaction mixture maintained a constant pH of 8. Once all of the mixed metal solution was added, the pH was monitored and controlled for a further 10 min to ensure complete precipitation of the material. Thereafter, the precipitate was allowed to age in solution at 65 °C for 3 h.</p><p>This precipitate was filtered under suction and washed with water to remove excess sodium salts. This material was then dried at 110 °C for 16 h before being calcined at 325 °C (thermal ramp rate 5 °C min−1) in static air for 3 h to produce 2.5–3 g of the catalyst.</p><!><p>A mixed solution of Cu(OAc)2⋅H2O (4.1561 mg mL−1) with either Zn(OAc)2⋅2 H2O (1.9584 mg mL−1), Mg(OAc)2⋅4 H2O (1.9132 mg mL−1), Mn(OAc)2⋅4 H2O (2.1866 mg mL−1), or Ce(acac)3⋅x H2O (3.9026 mg mL−1) was prepared in a 5 vol % H2O/ethanol mixture (1000 mL) to give a nominal Cu:X molar ratio of 70:30. SAS preparation was performed using apparatus manufactured by Separex. Liquefied CO2 was pumped to give a flow rate of 6.5 kg h−1 and the whole system was pressurised to 110 bar and held at 40 °C. Initially, pure solvent (5 vol % H2O/ethanol) was pumped through the fine capillary into the precipitation vessel, with a flow rate of 6.5 mL min−1 for 15 min, in co‐current mode with scCO2 in order to obtain steady state conditions inside the vessel. After this initial period, the flow of liquid solvent was stopped and the mixed metal solution was delivered at a flow rate of 6.5 mL min−1. This gave a scCO2/mixed metal solution molar ratio of 22:1. The system pressure and temperature were maintained and the preparation conditions were carefully controlled. Leak checks were also periodically carried out throughout the procedure using snoop solution. When all the mixed‐metal solution had been processed, a drying step was carried out. This was achieved by pumping pure ethanol at 6.5 mL min−1 co‐currently with scCO2 for 30 min, before leaving with just scCO2 to pump for a further 60 min. This was to wash the vessel in case residual solvent condensed during depressurisation and partly solubilised the prepared materials. When the drying step was complete the scCO2 flow rate was stopped, the vessel was depressurised to atmospheric pressure and the precipitate was collected. Experiments were conducted for approximately 5 h which resulted in the synthesis of ca. 2.5–3 g of solid.</p><!><p>Powder X‐ray diffraction measurements were performed using a PANalytical X′pert Pro diffractometer with Ni filtered CuKα radiation source operating at 40 kV and 40 mA. Patterns were recorded over the range of 10–80° 2θ using a step size of 0.016°. All patterns were matched using the ICDD database.</p><!><p>Cu surface area analysis was carried out on a Quantachrome ChemBET chemisorption analyser equipped with a thermal conductivity detector (TCD). Calcined samples (50 mg) were reduced to catalysts using 10 % H2/Ar (30 mL min−1) with heating to 140 °C at 10 °C min−1, and then to 225 °C at 1 °C min−1. For Cu surface area analysis, catalysts were cooled to 65 °C under He for N2O pulsing. 12 N2O pulses (113 μl each) were followed with 3 N2 pulses for calibration. The amount of N2 emitted was assumed to amount to half a monolayer coverage of oxygen and that the surface density of Cu is 1.47×1019 atoms m−2.</p><!><p>The catalytic performance of the catalysts for CO2 hydrogenation was determined in a fixed‐bed continuous‐flow reactor. The catalyst (0.2 g, 425–600 μm) was placed in a stainless steel tube reactor with an internal diameter of 4.57 mm. Prior to the reaction, the catalysts were prereduced in a flow of 5 % H2/He (30 mL min−1) for 1 h at 225 °C under atmospheric pressure. The reactor was then allowed to cool to room temperature before gas flow was switched to the reactant mixture (CO2:H2:N2 20:60:20 molar %). The pressure was increased to 20 bar using a backpressure regulator before the flow was set to 12.5 mL min−1 to give a GHSV of 1000 h−1. The reactions were conducted at 225 °C. All post‐reactor lines and valves were heated at 110 °C to avoid product condensation. The gas products were analysed via online gas chromatography using an Agilent 7890 system with a flame ionisation detector (FID) and TCD. Nitrogen was used as an internal standard. Samples were taken every 15 min over the course of 8 h. CO2 conversion was calculated by the change in moles of CO2 compared to calibration runs. The selectivities of methanol and CO represent the respective carbon molar % of the products. In all cases, methanol and CO were the only products observed.</p><p>After the reaction, the reactor was depressurised and left under flowing helium (10 mL min−1) until cool. The catalyst was then recovered from the tubes and subjected to another set of copper surface area measurements as above. The reduction step was performed again to minimise the effects of passivation caused by contact with air during transit.</p><!><p>The authors declare no conflict of interest.</p>

PubMed Open Access

Critical assessment of staining properties of a new visualization technology: a novel, rapid and powerful immunohistochemical detection approach

Immunohistochemical staining of tissue sections is a vital technique in pathological diagnostics and theranostics. Several kinds of detection systems are available—each of them with their advantages and disadvantages. Here we present the results of a study assessing a prototype immunohistochemical detection technology (PIDT) for visualization of antigens in tissue sections. Different tumor tissues (n = 11) were stained with selected antibodies (n = 30) and a subset of these under different fixation conditions. The staining properties were assessed according to six staining quality parameters (signal distribution, intensity, tissue and slide background, acutance, clarity of details, and subcellular morphological details), and the results were compared with those of a well-established detection system (EnVision FLEX). Overall, both detection methods revealed good to optimal results regarding the evaluated parameters even under unfavorable fixation conditions. However, with the prototype detection technology a quicker turnaround time was reached primarily due to shorter primary antibody incubation times. Moreover, PIDT-stained tissues showed higher signal intensity and a uniform signal distribution over the tissue slide, still, with well-preserved tissue morphology and without impairing the gradation of staining intensity of different cell types. In particular, the prototype detection technology performed better in poorly or delayed fixed tissue. In situations where rapid and profound results are in demand, and particularly in the context of a small laboratory setting, this prototype detection technology could be a useful addition to the established detection systems.Electronic supplementary materialThe online version of this article (10.1007/s00418-020-01906-5) contains supplementary material, which is available to authorized users.

critical_assessment_of_staining_properties_of_a_new_visualization_technology:_a_novel,_rapid_and_pow

<!>Introduction<!>Study design<!>Pre-analytic treatment<!>Staining procedures<!>Staining evaluation<!>Run time calculation<!>Statistical analyses<!>Results and discussion<!>Short primary antibody incubation times lead to significantly reduced assay run time especially with low numbers of slides<!><!>PIDT displays superior signal homogeneity and comparable signal intensity to EnVision FLEX<!>PIDT and EnVision FLEX show comparable very low background, EnVision FLEX with higher scores for acutance and morphological details<!><!>PIDT and EnVision FLEX are comparably robust to fixation differences<!><!>PIDT and EnVision FLEX are comparably robust to fixation differences<!>

<p>Any listing of the virtues of immunohistochemistry would be incomplete if it did not include the visual pleasure derived from examination of this material (…). There is undoubtedly an aesthetic component to the practice of histology (Juan Rosai, in (Dabbs 2013))</p><!><p>Immunohistochemistry (IHC) is a method to localize specific antigens in tissues with the purpose to characterize distinct cell types, mainly for diagnostic and research purposes. Additionally, the field of theranostics—which is composed of the terms therapy and diagnostics and implicates that pathologists serve oncologists and their patients with accurate tissue diagnostics that drive therapies (Dabbs 2013)—would be inconceivable without reliable and rapid immunohistochemical techniques.</p><p>Since Coons et al. described the principles of IHC (Coons et al. 1942), the technology has undergone a range of modifications, including enzymatic labeling (Avrameas 1972) before the first application in routinely processed formalin-fixed, paraffin-embedded (FFPE) tissues (Taylor and Burns 1974). Subsequently, modifications have continued to improve IHC, including the development of different amplification methods such as tyramide (Toda et al. 1999) and polymer-based labeling systems (Bisgaard et al. 1993; Heras et al. 1995; Bisgaard and Pluzek 1996; Sabattini et al. 1998). The availability of a wide range of visualization alternatives with different properties is exactly why the selection of an appropriate visualization system is a key element in obtaining optimal staining performance for the given purpose (Giorno 1984; Cordell et al. 1984; Skaland et al. 2010).</p><p>The aim of this study was to critically assess a not yet commercially available prototype immunohistochemical detection technology (PIDT) on routinely processed tissue samples. The PIDT builds on an improved tyramide system (Lohse et al. 2014). By use of DAB as a crosslinker in a first deposition step, a very weak primary DAB stain is formed that anchors multiple accessible FITC reporters for a second DAB stain with anti-FITC/HRP. This assures that the stains remain crisp and localized, while still allowing strong amplification with short incubation times. Although there are more steps in the staining procedure, each step, including primary antibody, can be kept at 1–8 min, allowing faster staining on Autostainer Link 48 up to 12–18 slides after which the single robot dispenser becomes the time-limiting factor.</p><p>For this purpose, immunohistochemical stainings were performed on tumor and control tissue sections with the novel technology using the well-established EnVision FLEX visualization system (Sabattini et al. 1998) as a reference system. The staining results were evaluated according to a defined scoring system and data were compared statistically.</p><!><p>Buffered 4% formalin-fixed and paraffin-embedded tissues were used unless otherwise noted. All stains were performed in a routine laboratory setting. All antibodies were ready-to-use and provided by Agilent Technologies. The reactivity of each antibody was tested with appropriate control tissues (e.g. tonsil, appendix, bone marrow, tumors) as used routinely by the Laboratory of Immunohistochemistry of the Department for Pathology, University Medical Center Freiburg, and recommended by Dako's "Atlas of Stains" (Dako 2012). For all comparisons, the PIDT stains were compared to EnVision FLEX stains, provided by Agilent technologies.</p><p>Initially, a phase 1 study was designed to evaluate workflow aspects (incubation times and run-time calculations) and to get a first impression of the staining performance of PIDT using a panel of 30 different primary antibodies reacting with nuclear, cytoplasmic, and membranous antigens. Antibodies used for subtyping epithelial, mesenchymal, and hematological tissues were chosen to cover all major purposes of routine applications. Sections from formalin-fixed, paraffin-embedded tissues of five different tumors were stained with each antibody and both visualization systems (see Suppl. Table 1), totaling 300 sections. After optimization of the protocols using PIDT, a subset of 11 antibodies were selected for phase 2 to further evaluate the general staining characteristics. The staining results of each slide were evaluated and scored as described below.</p><p>To test the robustness of PIDT towards different fixation conditions, four samples of fresh tonsil tissue were separately fixed in 4% formalin under defined fixation conditions: under-fixation (4–6 h), standard-fixation (24–48 h), weekend-fixation (48–78 h), over-fixation (7 days), fixation under frozen section conditions (tissue was frozen, thawed, and fixed for 16–18 h), and delayed-fixation (16–18 h fixation after tissue dried out for 2 h). After paraffin embedding, a tissue microarray (TMA) was created containing all four cases with all six different fixation conditions. Each tissue core was 2 mm in diameter. TMA sections were stained using nine different antibodies (BCL6, CD2, CD10, CD20, CD23, CD68, CK-Pan, Ki-67, and p63).</p><p>To evaluate the signal quality in poorly fixed tissues, samples from larger resection specimens of mastectomies and pneumectomies with estrogen receptor- or TTF-1-positive breast and lung adenocarcinomas, respectively, were used. These sections were all stained with Ki-67, estrogen receptor, or TTF-1, respectively.</p><p>To test if PIDT is able to display distinct differences in signal intensity among different structures within one tissue, the following stainings were performed: Pan-cytokeratin on small-cell lung cancer and CD23 on chronic lymphatic leukemia tissue sections. The stains were evaluated regarding the typically dot-like and—compared to non-neoplastic epithelium—attenuated pan-cytokeratin signal pattern of the tumor cells as well as the attenuated CD23 signal intensity of chronic lymphatic leukemia cells compared to non-neoplastic follicular dendritic cells.</p><p>Finally, evaluation of the staining performance in bone marrow biopsies was included in the study as this tissue type represents a special challenge for immunohistochemical staining. Follicular lymphoma-infiltrated bone marrow biopsies were fixed in 4% buffered formalin, subjected to decalcification in a mixture of 10% ethylenediamine tetraacetic acid disodium salt (Serva) and 3.3% tris-(hydroxymethyl) aminomethane (AppliChem) in dd H2O at pH of 7.0–7.2 overnight at room temperature and subsequently embedded in paraffin. The biopsies were stained with BCL6, CD10, and CD79a antibodies.</p><!><p>Target retrieval of tissue sections for PIDT staining was done using a single target retrieval solution (TRS), while pretreatment for Envision FLEX staining was performed with either high or low pH TRS according to validated protocols used by the Department for Pathology, University Medical Center Freiburg. De-paraffinization, rehydration, and epitope retrieval were performed with the PT Link, Pre-Treatment Module for Tissue Specimens (Agilent Technologies).</p><!><p>All stainings were performed using the Autostainer Link 48 (Agilent Technologies) automated staining platform. For the PIDT, a staining protocol was established (see suppl. Table 2). The appropriate incubation time was optimized for each primary antibody. Validated protocols from the Department for Pathology were used for EnVision FLEX stainings. Appropriate control tissues were included in every run. The stainings with PIDT and EnVision FLEX were performed separately in different runs to be able to assess workflow aspects. Additionally, for each staining run with the PIDT, one slide of consecutively cut sections of tonsil tissue stained with Ki-67 was included for assessment of staining reproducibility.</p><!><p>The specific staining pattern of each antibody used as the basis for the evaluation was defined according to David Dabbs' "Diagnostic Immunohistochemistry"(Dabbs 2013) and the criteria for optimal staining of the NordiQC (https://www.nordiqc.org). A total of six staining quality parameters to be assessed separately were defined: homogeneity of the specific staining across the tissue, the intensity of the specific signal, tissue background, acutance of the specific signal, clarity of cellular or subcellular morphological details, and slide background. Each parameter was assessed semi-quantitatively using a scale of 3 for optimal, 2 for good, 1 for borderline, and 0 for poor results.</p><p>The evaluation was performed by a pathologist who was blinded with respect to which visualization system (PIDT vs EnVision FLEX) was used. A mean value based on the individual scores from the five different tissue sections stained was calculated for each antibody and visualization system. The mean values were used to compare the two visualization systems.</p><p>For the reproducibility testing using Ki-67-stained slides, the six parameters were evaluated, and the percentage of nuclear positive cells out of 300 cells was calculated. The mean values of the parameters and the percentage of nuclear positive cells were compared among the staining runs.</p><!><p>The duration of each run of the staining was measured from start of barcode reading to completion of final hematoxylin wash, and the number of loaded slides was annotated.</p><!><p>For statistical analyses of data, GraphPad Prism 6 Software (GraphPad Inc.) was used. The scores of the two visualization systems were compared and the statistical significance of differences between the two systems was calculated by use of a paired two-tailed t test. When more than two groups were compared a one-way ANOVA test was used. A p value of < 0.05 was considered to be statistically significant.</p><!><p>In this study, we assessed the properties of a prototype immunohistochemical detection technology (PIDT) by Agilent Technologies in a routine setting. We compared the staining quality and their laboratory workflow impact using different tumor tissues and selected antibodies to compare immunohistochemical staining between the established EnVision FLEX system and PIDT. The staining properties were scored for defined parameters and the results were compared between the two detection systems.</p><p>The results show that both techniques provided a high staining quality with only marginal differences in specific staining details. PIDT was found to provide a more homogeneous distribution of staining intensity across the tissue and, in bone marrow biopsies, a signal enhancement, which led to an increase in staining quality.</p><!><p>Optimization of the protocol for PIDT demonstrated that very short incubation times for the primary antibody were required: only 1 min incubation time for 12 of the primary antibodies and 2 min for nine of the antibodies. The longest incubation time was 8 min among the 30 antibodies. A considerable advantage of PIDT was the short incubation time of the primary antibody of 1–2 min for most antibodies resulting in a slide number-dependent run time decrease of 2.75× (up to five slides) and 1.8× (up to ten slides). A total of 76 staining runs were conducted, 53 runs with PIDT and 23 runs with EnVision FLEX. The average total staining run time on Autostainer Link 48 was 58 min (range 30–109 min) for the PIDT and 111 min (range 89–149 min) for EnVision FLEX with statistically significant difference (p < 0.0001). The difference in total staining run time depended on the number of slides included in the respective staining runs where the assay run time is shown for the following groups of slides: low volume (1–6 slides), medium volume (7–12 slides), and high volume (13–19 slides). With an increasing number of slides, the mean staining time per run increased significantly with PIDT (low: 40 min, medium: 57 min, and high: 77 min; p < 0.0001), whereas the staining run time with EnVision FLEX changed only to a minor extent (low: 102 min, medium: 109 min, and high: 120 min; p = 0.0518). Comparison of slide run time within each group showed that the staining run times were significantly lower with PIDT compared to EnVision FLEX (each p < 0.0001 in pairwise comparison).</p><p>To test the reproducibility of the staining, each run included a section of tonsil tissue that was stained with Ki-67 antibody and evaluated for the proportion of cells showing nuclear staining. The mean percentages of nuclear Ki-67-positive cells were compared resulting in a high reproducibility of the stainings without statistically significant differences between the single runs and a standard deviation of only 1.7% regarding the percentage of Ki-67-positive cells.</p><!><p>Representative examples of staining results for selected antibodies in tumor tissues. Bar indicates 200, 100 and 50 µm in the first, second and third line, respectively</p><p>Examples of PIDT and EnVision FLEX staining results of distinctions in signal intensity of different cell populations within a tumor, exemplarily shown is dot-like pan-cytokeratin staining in cells of a small cell lung cancer compared to stronger staining of the local epithelium and stronger CD23 staining of dendritic cells compared to infiltrating CLL cells in a lymph node. Bar indicates 100 µm</p><!><p>The strong signal intensity in combination with the homogenous signal distribution over the tissue section led to a uniform staining result. This property may contribute to more consistent results of quantitative evaluation of immunohistochemical slides, e.g. in case of defining the intrinsic subtypes of breast cancer. Furthermore, computer-assisted evaluation of immunohistochemical stained tumor tissue by image processing may profit from the strong and homogenous signal of PIDT.</p><p>Another point is the yet unknown cause behind the higher signal homogeneity in the PIDT stains. There was no difference between the primary antibodies used. Furthermore, regarding the different quality assessment parameters there is no significant difference between the target retrieval solutions (high vs low ph) used for EnVision FLEX stains, especially not for homogeneity (data not shown). Moreover, the incubation time for the primary antibodies in the PIDT protocol is fairly shorter. Therefore, our findings suggest an improved binding or reduced dissociation of the primary antibody in the PIDT stains. This could be due to the quickly introduced DAB/FITC precipitates coating the primary antibody and preventing dissociation.</p><p>The overall strong signal intensity of PIDT staining led to the question if refined distinctions in staining intensity in some tumor entities are still detectable. Accordingly, tissue sections of small-cell lung cancer (SCLC) and cases of lymph nodes infiltrated by chronic lymphatic leukemia/small lymphocytic lymphoma (CLL/SLL) were stained with Pan-cytokeratin or CD23 antibodies, respectively. Both the attenuated cytokeratin positivity of SCLC cells and the enhanced signal intensity of CD23-positive follicular dendritic cells compared to the CLL/SLL tumor cells could be reliably detected. These results show that the high intensity of PIDT-staining did not impair the detection of signal graduation of different cell types within the tested samples.</p><!><p>Both detection systems showed nearly no slide background and extraordinary low level of tissue background staining without statistically significant differences. Interestingly, despite the higher intensity of PIDT staining only a small increase of background staining could be measured which was not significantly different compared to EnVision FLEX indicating that the specific signal was enhanced by PIDT without enhancing background signal. For PIDT, the acutance was good, however, EnVision FLEX reached a very high score resulting in a statistically significant difference between both detection systems. Finally, tissue slides stained with PIDT showed clearly recognizable, morphological details, however, a better performance of EnVision FLEX could be measured with a statistically significant difference.</p><!><p>Examples of PIDT and EnVision FLEX staining results of bone marrow biopsies with infiltration of follicular lymphoma. Bar indicates 100 µm</p><!><p>Proper immunohistochemical staining of tissue is very challenging for a detection system when tissue sections are sub-optimally fixed. To analyze the robustness of PIDT and EnVision FLEX towards fixation differences some procedure variations were imitated. For this reason, we created a tissue micro array (TMA) with cores of tonsil tissue with over-fixed, under-fixed, and delayed-fixed tonsil tissue cores as well as tonsil tissue fixed after freezing and thawing (analog frozen section). Immunohistochemical stainings with BCL6, CD2, CD10, CD20, CD23, CD68, CK AE1/3, Ki-67, and p63 were conducted. The stainings were evaluated for staining quality parameters as described above. Indeed, frozen sections yielded the lowest staining scores. However, none of the unfavorable fixation conditions impaired the staining quality of either detection system to the extent that staining results were unacceptable for evaluation. Moreover, there was no statistically significant difference regarding the staining results of PIDT and EnVision FLEX stained tissues.</p><p>Overall, both PIDT and EnVision FLEX revealed good to optimal results and were highly able to compensate different unfavorable fixation conditions with average staining scores between 2.4 and 3.0. The only statistically significant difference was seen for signal homogeneity over the tissue section, where PIDT showed better values, and for the acutance of the staining, where EnVision FLEX yielded a sharper signal.</p><p>Both detection systems yielded good to optimal staining results regarding the individual fixation conditions. Statistically significant differences could be measured regarding the homogenous distribution of the signal over the tissue section when the tissue was fixed too shortly, over the weekend, and under standard conditions.</p><!><p>Example of staining results of a TMA containing spots with different fixed tonsil tissue stained for CD20 with PIDT and EnVision FLEX. a Description of the TMA; b Staining results. Bar indicates 100 µm</p><p>Examples for PIDT and EnVision FLEX staining results of poorly fixed tissue (lung adenocarcinoma and breast cancer stained with TTF-1 or estrogen receptor, respectively). Bar indicates 100 µm</p><!><p>In summary, both detection systems PIDT and EnVision FLEX are of comparable high quality regarding the staining results. PIDT is a powerful technology in particular due to its high intensity and homogeneity of the signal distribution over the tissue section especially in bone marrow biopsies. From a laboratory perspective, PIDT enables short run times, which can be particularly useful in pre-therapeutic situations where the challenge is to provide rapid and profound results. Furthermore, laboratories with rather low immunohistochemical throughput would benefit from PIDT as the strength of this technology becomes particularly beneficial when a smaller number of slides per run is stained.</p><!><p>Supplementary file1 (DOCX 14 kb)</p><p>Supplementary file2 (PDF 270 kb)</p><p>Publisher's Note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p>

PubMed Open Access

Photocrosslinking and photopatterning of magneto-optical nanocomposite sol–gel thin film under deep-UV irradiation

This paper is aimed at investigating the process of photocrosslinking under Deep-UV irradiation of nanocomposite thin films doped with cobalt ferrite magnetic nanoparticles (MNPs). This material is composed of a hybrid sol-gel matrix in which MNP can be introduced with high concentrations up to 20 vol%. Deep-UV (193 nm) is not only interesting for high-resolution patterning but we also show an efficient photopolymerization pathway even in the presence of high concentration of MNPs. In this study, we demonstrate that the photocrosslinking is based on the free radical polymerization of the methacrylate functions of the hybrid precursor. This process is initiated by Titanium-oxo clusters. The impact of the nanoparticles on the photopolymerization kinetic and photopatterning is investigated. We finally show that the photosensitive nanocomposite is suitable to obtain micropatterns with submicron resolution, with a simple and versatile process, which opens many opportunities for fabrication of miniaturized magneto-optical devices for photonic applications.Magneto-optic (MO) effects refer to phenomena which modify the light polarization according to an external magnetic field applied to a MO active material 1-3 . One of these MO effects is the Faraday rotation (FR), which is defined as the change produced in the plane of polarization of the light transmitted through a material when a magnetic field is applied: the plane of polarization is rotated. In reflexion mode, this effect is known as the magneto-optic Kerr effect. Amongst the most attractive properties of the magnetic transparent compounds are those related to the magneto-optical (MO) effects and their scientific and industrial applications in areas such as data storage 4 , three-dimensional (3D) imaging 5 , magnonics 6,7 , sensing 8,9 and photonics [10][11][12] . MO active materials are ubiquitous in photonic devices, but they are still lacking in integrated photonic platforms although they are essential components for optical communication systems (optical isolators, optical circulators, optical switches, magneto-optic (MO) modulators) [13][14][15][16][17][18] and high performance magnetic field sensors [19][20][21] .In the past, magneto-optical materials have been mainly developed by physical methods such as sputtering 22,23 , pulse laser deposition 24 , and molecular beam epitaxy [24][25][26] . More recently, nanocomposite approach has been proposed to simplify the synthesis of the material and its shaping into devices 27 . The principle is based on the synthesis of magnetic nanoparticles then their incorporation into a matrix which can be a polymer [28][29][30][31][32][33] , or an inorganic matrix prepared by sol-gel for example 34,35 . The materials can thus be processed by the usual processes adapted to liquid formulations. Solution-based processes appeared with the major advantage of allowing simpler processes without the need of sophisticated equipment 36 . In this context, sol-gel chemistry emerges as a very relevant and versatile solution. The principle of the sol-gel process is based on successive reactions of hydrolysis and condensation of precursors that form networks of metal oxides. One of the interests of this route lies in its compatibility with a large number of precursors. Among numerous commercially available precursors, alkoxides (alkoxysilanes zirconium, titanium, aluminium, etc.) are the most commonly used. Other derivatives are also used (chlorides, nitrates, …). The sol-gel pathway thus offers a number of advantages in terms of energy

photocrosslinking_and_photopatterning_of_magneto-optical_nanocomposite_sol–gel_thin_film_under_deep-

<!>Experimental<!>Sol-gel matrix and nanocomposite solution. 3-methacryloxypropyltrimethoxylsilane (MAPTMS),<!>Results and discussion

<p>harvesting, processing versatility and a wide range of final properties [37][38][39] . The solutions can be deposited as thin films by simple means such as spin-coating, dip-coating or spray-coating. Moreover, the final material can reach good transparency, good mechanical properties and high refractive index. These advantages account for a wide use of sol-gel coating for optical applications.</p><p>We recently introduced a process that relies on the synthesis of a photocurable sol-gel matrix in which magnetic nanoparticles (MNP) can be introduced 40 . Cobalt ferrite (CoFe2O4) MNPs were chosen because they exhibit large Faraday rotation in the 1400-1550 nm spectral range 41 , an important criterion for potential applications in telecommunication devices and photonic integrated circuits. The major advantage of using MNPs incorporated in a non-magnetic host matrix is that the magneto-optical properties are then obtained in the final material without the need for additional thermal post-treatment. The main challenge is to manage and avert the NPs aggregation during the different steps of the process in order to preserve the magnetic properties of the individual MNPs and to avoid light scattering by aggregates in the nanocomposite. The photocurable sol-gel matrix was based on a hybrid precursor that can be crosslinked by light (namely 3-methacryloxypropyltrimethoxylsilane MAPTMS). Photocrosslinking of such matrix can be obtained by Deep-UV laser irradiation (193 nm), which opens the doors towards micro and nanopatterning. Indeed, solubility switch can be induced by laser irradiation which allows defining by laser direct writing the patterns. Other materials and patterning strategies have been proposed. In 42 , Lai et al. prepared a photopatternable material based on a commercial SU8 photoresist doped with magnetite nanoparticles and achieved high resolution patterns and 3D structures by laser direct writing.</p><p>In the present paper, we aim to investigate the photocrosslinking mechanism involved in the nanocomposite and further exemplify the application for photopatterning in the sol-gel matrix that we developed. The optimized synthesis of CoFe 2 O 4 nanoparticles and hybrid sol-gel host matrix is first described. Conditions are defined to reach high concentration of MNPs. The kinetic of photopolymerization is then studied by Fourier transform infrared (FTIR) spectroscopy, in order to investigate the photoinduced mechanisms allowing the photocrosslinking of the material. In particular, we highlight an original photo-induced mechanism based on the excitation of Ti complexes. On this basis, a mechanism can be proposed. In a second part, DUV photopatterning is demonstrated and the effect of the composition on the final results is discussed.</p><!><p>Synthesis of magnetic nanoparticles. The magnetic nanoparticles embedded in the matrix are cobalt ferrite nanoparticles (CoFe 2 O 4 ). The preparation of these nanoparticles was performed by precipitation of cobalt chloride and ferric chloride, based on the method of Massart and Tourinho 43 . The different stages of ferrofluid synthesis can be summarized as follows: First, iron chloride (FeCl 3 ) and cobalt chloride (CoCl 2 ) are mixed in aqueous solution, with a molar ratio Fe/Co equal to 2. Concentrated NaOH (10 mol/L) is then added to the mixture to form the hydroxides of each metal (Fe(OH) 3 and Co(OH) 2 ). The solution is then heated to 100 °C for two hours to convert the hydroxides into cobalt ferrite. After several washing steps in water, an acid treatment is applied (overnight) by adding a 2 mol/L solution of nitric acid. This acid treatment removes hydroxides that would not have been converted into cobalt ferrite. It also makes it possible to shift from a negatively charged surface (charge due to Ogroups on the surface with Na + counter-ions) to a positively charged surface. As cobalt ferrite particles are not stable in an acidic environment, it is necessary to protect them by a surface treatment. This treatment is carried out by adding an almost boiling solution of ferric nitrate (concentration 0.3 mol/L) to the solution containing MNP. Finally, to obtain the ferrofluid, washing steps with acetone and then ether were done and the MNP of cobalt ferrite are dispersed in water. The resulting ferrofluid is an acidic ferrofluid: the MNPs are positively charged at the surface with NO 3 − . The concentration of iron and cobalt ions was determined by atomic absorption spectroscopy method after degradation of the MNPs in a highly concentrated acidic medium. The concentration was confirmed by recording the magnetization curve of the ferrofluids, measured with a vibrating-sample magnetometer (VSM, Quantum Design PPMS).</p><p>The size dispersion and morphology of the particles were determined by analysis of images obtained by transmission electron microscopy (TEM). Transmission electron microscopy (TEM) was performed using a JEOL ARM-200F microscope operating at the 200 keV accelerating voltage. The chemical analysis was performed using a JEOL Centurio detector. The samples were prepared for observation using a Leica ultramicrotome, model EM-UC7, operating at RT. To observe the film cross-section, it was deposited on the surface of a thermanox substrate. The slide thickness was ~ 100 nm.</p><p>Highest concentration in MNPs in the final materials (> 2 vol%) were reached by using a concentrated ferrofluid, obtained by dialysis. This technique consists of concentrating the ferrofluid by reverse osmosis, by immersing a porous dialysis tube filled with the ferrofluid to be concentrated in an aqueous solution of polyethylene glycol (PEG) with a mass concentration of 20,000 g/mol. Typically, dialysis of 20 mL of ferrofluid initially concentrated at 1.45 vol% for 24 h with agitation results in a highly viscous ferrofluid with an MNPs concentration of about 10 vol% (corresponding to a concentration of a factor of ~ 7). By introducing dialyzed ferrofluids into a matrix, it has been possible to achieve doping levels of up to 20 vol%, which is comparable to the highest level of MNPs in nanocomposites described in the literature 44 . The advantage of using a dialyzed ferrofluid is to introduce more magnetic nanoparticles without adding more water to the mixture.</p><!><p>titanium isopropoxide (TTIP), methacrylic acid (MAA), Hydrochloric acid (HCl), 1-propanol, and cyclohexanone were purchased from Sigma Aldrich. Deionized water was used throughout the reactions.</p><p>The main stages of material preparation are shown in Fig. 1. It consists in 5 main steps:</p><p>1. 0.01 mol/L concentrated hydrochloric acid is added to MAPTMS (molar ratio MAPTMS/ H 2 O of 1.3:1). The solution is placed under magnetic stirring for one hour to give a colorless emulsion. The objective of this step is to pre-hydrolyze the MAPTMS 45 and favor a homogeneous introduction of Ti precursor. 2. Methacrylic acid MAA is added to Ti precursor in a molar ratio MAA/Ti = 2.2. This molar ratio was chosen to have a large excess of MAA to ensure a full complexation of Ti precursor. Indeed, according to the literature, a minimum ratio of 1.2 is necessary for total complexation of the titanium precursor 46,47 . The mixture is magnetically stirred for five minutes. This reaction is exothermic. Subsequently, 1-propanol is added to the solution in a ratio of 0.9 molar MAA/1-propanol. The solution is homogenized with a magnetic stirrer for 10 min. A clear, bright yellow solution is obtained. 3. The solutions of MAPTMS and complexed Ti precursor are mixed together. Water is then added with a molar ratio MAPTMS/H 2 O = 0.4. This third step completes the hydrolysis-condensation steps of the sol-gel chemistry. 4. A given volume of ferrofluid containing the MNPs is added to the sol-gel solution. The doped material is homogenized by ultrasonic treatment. The stirring time varies from ten minutes to one hour depending on the amount of MNP introduced. The introduction of MNP has been considered at different stages of the process. The most stable formulations are obtained when the introduction is made at the end of the sol-gel formulation preparation steps and this process was retained (Fig. 1). 5. The doped solution is diluted with 1-propanol. The purpose of this dilution is twofold. On the one hand, the viscosity of the material must be adjusted, which will make it possible to change the thickness of the thin film during spin-coating. On the other hand, dilution ensures the stability and homogeneous dispersion of MNP in the sol. Dilution is particularly important in the case of heavily doped matrices.</p><p>The molar ratio of Si/Ti can be varied in the range 3/1 to 20/1. The reference matrix corresponds to a composition of Si/Ti in a molar ratio of 6/1. Solutions prepared are stable for several months and can be stored at room temperature. www.nature.com/scientificreports/ Thin films preparation and characterization. Substrates (Silicon wafer or glass slides) were first cleaned by rinsing with ethanol and then placed in an UV-ozone cleaner to remove the organic pollutant and increase the polarity of the substrate for good adhesion of the thin film. The formulation was filtered through 0.2 μm PTFE filters. Homogeneous films were obtained by spin coating, with typical thicknesses between 200 and 500 nm, depending on the dilution factor and rotation speed during deposition. The photopolymerization kinetics were followed by real time-FTIR with a Thermo Scientific Nicolet 8700, coupled with a Hamamatsu high intensity mercury-xenon lamp equipped with a light guide (Lightnincure series LC8 lamp). Si wafers (thickness = 0.25 mm) were used as substrates and the irradiance was fixed to 70 mW/cm 2 . With this configuration, the sample can be irradiated in situ, which is more convenient than the ex situ laser irradiation and justifies that we used the DUV lamp for polymerization kinetics. Absorption measurements were performed with a Lambda 950 UV/Vis (Perkin Elmer).</p><p>The Faraday spectra were acquired in the wavelength range 600-1700 nm with a homemade polarimetric optical bench based on the modulation technique combined with an ellipsometric-type calibration method (see 48 for details). The sample (nanocomposite film or ferrofluid cell) is placed perpendicular to the incident beam in the air gap of an electromagnet. The magnetic field can be varied in the ± 0.8 T range. The light from a xenon white light source combined with a monochromator passes through a polarizer, the sample, a photoelastic modulator, an analyzer, a detector, and a lock-in amplifier (LIA). This optical arrangement is suitable for analyzing the polarization state by means of the first (ellipticity) and second harmonic (rotation) signals of the LIA 49 . The calibration method 50,51 allows to measure the absolute value of the polarization rotation with the detection limit of 0.001°.</p><p>Photopatterning. The photopatterning setup relies on a Braggstar (Coherent) nanosecond ArF Excimer laser emitting at 193 nm 40 . The beam section measures 3 × 6 mm 2 . An attenuator located after the laser is used to tune the power. A shutter allows controlling the exposure time. A beam expander makes it possible to enlarge the beam spot by a factor of 5 and thus increases the exposed material surface. Its role is also to homogenize the beam, reduce its divergence and thus increase spatial coherence. At the exit of the expander, the beam reaches a semi-reflecting blade allowing 25% of the power to pass through and returning the remaining 75% of the power to the sample at 90°. The 25% passing through the blade allows the laser power to be measured in real time using a power meter. The sample is placed on a motorized stage in x, y and z. Displacements in x and y position the sample under the beam and the z stage adjusts the sample-interferometer distance (i.e. the focal point).</p><p>Measurements of the films thickness were done by ellipsometric spectroscopy. The measurements were performed on a UVISEL ellipsometer from Horiba-Jobin-Yvon (spectral range 190-830 nm). Data were fitted with the software from the UVISEL ellipsometer. The photopatterned films were characterized by Atomic Force Microscopy (AFM), in tapping mode, with a PicoPlus 5500 System model from Agilent.</p><!><p>Structural, magnetic and magneto-optical properties of Cobalt ferrite CoFe 2 O 4 nanoparticles were first studied. Nanoparticles were characterized by TEM. A typical TEM image is presented in Fig. 2a. The NPs size distribution fitted by a log-normal distribution gives a NPs average size of 8.4 nm with a standard deviation σ = 0.6 (Fig. 2b). The VSM hysteresis curve of the ferrofluid (given in Fig. 2c) shows the classical behavior of superparamagnetic nanoparticles. The magnetization is 800A/m and the concentration of Fe and Co ions measured by flame spectrophotometry is [Fe + Co] = 1.8 mol/L. Ferrofluids prepared under these conditions have excellent stability (several months).</p><p>Cobalt ferrite nanoparticles have been selected for their interesting magneto-optical properties. The nanocomposite material is intended for use in MO devices operating at the wavelength 1550 nm. For this reason, the spectral response of the Faraday rotation of ferrofluid has been investigated. Figure 2d shows the Faraday rotational spectral behavior of the ferrofluid synthesized according to the protocol described above. The Faraday rotation of the CoFe 2 O 4 MNPs is particularly important around 750-850 nm, as well as in the 1500-1600 nm region, where it reaches values higher than 200°/cm. These CoFe 2 O 4 MNPs are therefore good candidates for obtaining MO properties in these two operating windows, at especially at the 3rd telecom wavelength (1.525-1.625 nm), interesting for telecom applications.</p><p>The magneto-optical response of the thin-film material after Deep-UV curing is also shown in Fig. 2d to be compared to the response of the ferrofluid used to incorporate the MNPs. The evolution of the Faraday rotation is measured at normal incidence of a thin layer (Si/Ti: 6/1), doped to 2.2 vol% in nanoparticles. The sample thickness was 1.9 μm, deposited on a glass substrate. It was obtained by deposition of 4 layers of 480 nm, deposited and subsequently irradiated with an energy of 1.5 J/cm 2 by the Excimer laser, which is enough to ensure that the film is dry after exposure. Crosslinking molecular phenomena will be discussed below. The thickness of MO material allows to improve the signal-to-noise ratio. As shown in Fig. 2d, the Faraday rotation of the composite material has the same spectral behavior as that of NPs ferrofluide. This result confirms that the integrity of the nanoparticles is preserved during their incorporation into the sol-gel matrix and the UV cross-linking of the thin film. The DUV cured material presents thus interesting MO properties. The structural characterization of the nanocomposite material was completed by a TEM and EDX analysis (Fig. 2e). The TEM image illustrates the good repartition of the MNPs (bright spots) within the sol-gel matrix. Si and Ti are well distributed within the film thickness, showing that the synthesis strategy is suitable to obtain an homogeneous material, which is needed for optical applications. The next sections are aimed at investigating the Deep-UV induced modification of the material leading to the crosslinking of the material and then to show how this molecular behavior can be used to direct laser write micro-and nanostructures with MO properties.</p><p>As mentioned before, we observed that the film deposited with spin-coating from the sol-gel solution doped with CoFe 2 O 4 MNPs could be crosslinked by laser DUV irradiation. After laser irradiation, the film is tack-free www.nature.com/scientificreports/ and resistant to etching by organic solvents such as alcohols or cyclohexanone. As stated in the introduction, there is no study of the photoinduced phenomena in such nanocomposite materials, which is shown hereafter. Figure 3a displays the typical evolution of the FTIR spectrum of a nanocomposite thin film (450 nm thickness, Si/Ti = 6/1, 0.4 vol% of MNP) under DUV irradiation. Before irradiation, the absorption bands were assigned according to previous works 52 :</p><p>• The 1638 cm −1 band corresponds to the vibration modes of the sp2 carbons (C=C double bonds) of the material. Its presence shows that the C=C bands are not affected by the sol-gel reaction and thus available for forming the polymer network by photopolymerization. • C=O from carboxylic acids and methacrylic functions appears at several wavenumber, according to their environment: free C=O from the methacrylate are visible at 1740-1700 cm −1 . This position is expected for a methacrylate function, which confirms that the methacrylate can be polymerized. A second band located between 1500 and 1550 cm −1 corresponds to the vibrations of the C=O (methacrylic acid) complexed with titanium. This band shows the presence of the complex, formed in the early stages of the sol-gel synthesis, in the thin film after spin-coating. • The sol-gel reaction is also confirmed by the presence of bands in the region 800-1250 cm −1 that can be assigned to Ti-O, Si-O and combinations thereof.</p><p>Figure 3a also shows the evolution of the FTIR spectrum during DUV irradiation. Several modifications were recorded during irradiation. The most obvious change appears at 1638 cm −1 (enlarged in Fig. 3b). This band gradually disappears during the irradiation, showing the consumption of the C=C double bounds. This demonstrates the polymerization of the methacrylate functions and explains the crosslinking of the material under DUV irradiation.</p><p>The conversion of the C=C can be plotted as a percentage of the C=C consumed in reference to the initial quantity of C=C bounds (Fig. 3c). The shape of the curve is classical with a maximum polymerization rate (defined as the slope of the curve) at the beginning of the irradiation and a progressive decrease of the polymerization rate with time up to a maximum conversion ratio. It confirms the very good yield of polymerization achievable with this system, despite the presence of the MNP, with a total final conversion close to 100%. The excellent final conversion is important to guarantee good mechanical and optical properties to the thin film. www.nature.com/scientificreports/ Additionally, we confirmed that the decrease of the C=C double bounds (1638 cm −1 ) is not due to the loss of volatile compounds (free methacrylic acid for example). For this purpose, we followed also the evolution of the C=O bound. We observed a shift of the position of the C=O. This can be explained by the loss of the conjugation between the C=O and the C=C as the C=C is consumed by polymerization. If the area of the corresponding band is plotted versus time, the value keeps constant during polymerization, showing that there is no significant loss of material during photopolymerization.</p><p>One of the reasons explaining the excellent conversion yield of the C=C bounds in the nanocomposite is linked to the limited contribution of the MNPs to the light absorption through the film at the irradiation wavelength. Figure 4a illustrates the value of absorbance and transmittance that were determined by UV spectroscopy at a wavelength (210 nm) close to the irradiation wavelength (193 nm), for several concentrations of MNPs. The data were collected from samples with different thicknesses and the optical properties were calculated for a film thickness of 100 nm. The increase of the concentration of the MNPs only slightly increases the absorption at the irradiation wavelength, the absorption being mainly linked to the host matrix. For the highest load of MNP (20%), the contribution of the MNPs to the total absorption is about 30%. Interestingly, absorption is not linear towards MNP concentration, which can be explained by the partial aggregation of the MNPs when the concentration is increasing. The impact of the MNP on the polymerization kinetic was evaluated for several film thickness (Fig. 4b,c).</p><p>Interestingly, for the lowest MNP concentration (0.4 vol%), the photopolymerization kinetics is only slightly dependent on the film thickness and the polymerization rate and final conversion are very good for thicknesses up to 530 nm. At higher MNP concentration (10 vol%), the polymerization rate is decreased but the final conversion (80%) is high enough to insure good adhesion and mechanical properties of the thin film directly after irradiation, without any further curing. This result confirms that DUV irradiation is effective to trigger the photopolymerization of the nanocomposite. For the maximum MNP concentration achievable (20 vol%), the final conversion was 75% (for 70 J/cm 2 ).</p><p>As mentioned before, the photopolymerization process under deep-UV irradiation (193 nm) relies on the crosslinking of the organic part of the hybrid nanocomposite material. However, since there is no organic photoinitiator added in the matrix to start the polymerization, there are questions arising about the light induced mechanism accounting for the photocrosslinking. As proposed in previous studies 46,53 , metal alkoxides, when exposed to DUV light, can decompose to produce free radical species that are able to start the free radical polymerization of the hybrid matrix. We would like to confirm this mechanism for the nanocomposite material. Figure 5 displays the influence of several parameters involving the Ti complexes in order to demonstrate their central role in the photopolymerization mechanism. We first investigate the influence of the concentration of metal alkoxide on the photopolymerization kinetic (Fig. 5a).</p><p>In Fig. 5a the photopolymerization kinetic was followed by FTIR spectroscopy, with the same method as before (conversion calculated from the decrease of the C=C band at 1638 cm −1 ). This graph clearly shows that the photopolymerization is accelerated when the concentration of metal alkoxide is increased. This confirms the central role of the metal alkoxide complexed with methacrylic acid in the photopolymerization process. We www.nature.com/scientificreports/ observed that there is no significant improvement of the polymerization kinetics between 6/1 and 3/1 for Si/Ti ratio, which justifies the atomic ratio Si/Ti = 6/1 used in this study. Moreover, we observed that the stability of the formulation was not guaranteed after 10 days at highest load of Ti. One reason may be due to ligand exchange on the MNP surface by free methacrylic acid that is used to stabilize the metal. Also, we recorded similar kinetics after adding the MNP at low concentration (0.4 vol%), for all Si/Ti ratio, which means that there are no significant interactions between the MNP and the Ti complex acting as a photoinitiator. Zirconium was also evaluated as a metal for photocatalyst, instead of Ti. (Fig. 5b). With Zr as a metal, the polymerization proceeds with a rate equivalent to the formulation with a very low load of Ti, which means that the photoinitiating efficiency of the Zr complex is much lower than the one of the Ti complex. Since the absorption of both Ti and Zr complexes were found to be close in the DUV range, we concluded that this difference in reactivity can be explained by a difference in redox power between the two species. The interest of Ti as a metal precursor to induce the free radical polymerization of the hybrid sol-gel is thus demonstrated.</p><p>Finally, the nature of the ligand used to complex the titanium alkoxide precursor was also investigated. Figure 5c presents the FTIR kinetic study of the polymerization for different ligands in Ti complexes. Four different ligands were used to complex TTIP: three carboxylic acids (acrylic acid, isobutyric acid and methacrylic acid) and a β-diketone (acetylacetone). In each case, the ligand/Ti ratio was maintained at 2.2 molar % (without MNPs). No significant difference in polymerization kinetics was observed for the different complexing agent, with comparable polymerization rate and final conversion. The ligand chosen to complex the titanium therefore has little influence on the polymerization of the composite formulation. In Fig. 5d, we propose a simplified mechanism to summarize the role of the Ti-complexes in the elaboration of the nanocomposite material. It is admitted that the chosen experimental conditions lead to the incorporation of the Ti complexes as Ti-oxo clusters, as schematized in Fig. 5d. 42 The decrease of the band located between 1500 and 1550 cm −1 that corresponds to the vibrations of the C=O (methacrylic acid) complexed with titanium demonstrates the photolysis of the Ti-oxo clusters under DUV. This result is relevant with the results proposed in previous study, for comparable cluster, but in different conditions. The reactive species are able to trigger the free polymerization of the acrylate functions. The very good polymerization yield suggests a free radical mechanism. Note that the free radical formation of such clusters under DUV irradiation was already proposed in previous studies 46 . Such a property of the Ti complexes suggests that there is no interest to add any organic photoinitiator into the formulation to improve the photopolymerization efficiency. Several commercial photoinitiator known for their efficiency in free radical photopolymerization were added with a concentration of 2 wt% (Irgacure 184, 369 et 819 from Ciba). The corresponding polymerization kinetics revealed only a slight increase of the polymerization rate. This improvement is minor and thus the addition of an organic photoinitiator inside the formulation is not justified.</p><p>In this final part, we discuss the potential of this formulation to be used as a negative tone photoresist to produce sub-micrometric patterns at room temperature with magneto-optical properties. Indeed, as shown before, a very efficient Photocrosslinking can be obtained by DUV irradiation, which can be used for photopatterning the MO material. For this purpose, two home-made photolithography setups were used, as depicted in Fig. 6, in order to show the performance and versatility of this material for photolithography applications: for the lowest resolutions (typical feature lateral size superior to 1000 nm), a proximity printing setup was used, consisting in binary masks (chromium patterned deposited on fused silica substrates) placed close to the sol-gel film (Fig. 6a). In this configuration, the metal lines cut the DUV lights and prevent the Photocrosslinking reaction to occur. For highest resolutions, in order to overcome diffraction problems, an interferometric lithography setup was used (Fig. 6b). With this setup, the period of the patterns can be varied accordingly to the phase mask used. In the present study, we focused on patterns with period of 500 nm generated by a phase mask having a period of 1000 nm. The interference approach allows higher resolutions but, in this case, the light pattern is sinusoidal, since the contrast is generated by the interference between the two diffracted beams. In this configuration, there is thus no 0-light area, which can have some consequences on the shape of the structures, as shown later.</p><p>The coatings were prepared by spin-coating on a substrate cleaned with UV-ozone cleaner. The spin-coating rotation speed and dilution factor of the solution were adjusted to obtain the desired thickness. After irradiation with one of the two configurations described above, the sample is directly developed in a solvent to dissolve the non-irradiated parts. The material behaves like a negative resin, with irradiated parts becoming insoluble. The nature of the solvent as well as the development time have been optimized. Water cannot dissolve the unexposed parts thus cannot be used as a developer for this material. Alcohols (ethanol, methanol) and acetic acid are too strong developers and dissolve the exposed parts and are thus not suitable neither. Cyclohexanone was proved to constitute a good candidate and was chosen in the following as a solvent for development. Well-defined structures could be obtained after 10 s. development in cyclohexanone. No thermal annealing is required after development for stabilization of the sample as a post-treatment. We observed that the time between sample preparation and irradiation shall not exceed 10 min, otherwise, the development is more difficult to carry out. This is due to the condensation reaction that can occur at room temperature, because of atmosphere moisture. This condition is not limiting since the typical irradiation times are shorter than this value (few sec. to few tens of sec.).</p><p>Figure 6c,d show typical patterns obtained in both photopatterning configurations. In both cases, photopatterning could be demonstrated. For the proximity printing lithography (period 1600 nm with line width of 800 nm), well-defined patterned with free substrate between lines and low line edge roughness could be demonstrated (Fig. 6c). The patterns height was 125 nm for a deposited film thickness of 150 nm. We attributed the loss of height to the shrinkage occurring in the material upon DUV irradiation (partial loss of organic moieties). These results illustrate that patterns with width of 1000 nm or more can be obtained.</p><p>In interference lithography, patterns were obtained with higher resolutions but as shown in example in Fig. 6d, the pattern height was lower. Indeed, in this example, though the film thickness was decreased to 80 nm, patterns height was only 50 nm. This result was interpreted as a residual layer remaining between written lines. In order to further investigate the behavior of the material in these conditions, a systematic study of the DUV photopatterning was conducted. Results are exposed in Fig. 7.</p><p>The period of the patterns in Fig. 7 was 500 nm (corresponding to a line width of 250 nm with a space of 250 nm). The heights of the structures were measured by AFM. They are plotted in Fig. 7e. Figure 7a-e show the AFM images of surfaces irradiated with respectively 2.5, 5, 7.5 and 15 mJ/cm 2 . Figure 7f gives a schematic interpretation of the evolution of the pattern structure with the irradiation dose. For the lowest doses (less than 2.5 mJ/cm 2 ), no pattern was observed. We explain this response by a too low conversion within the irradiated parts and thus the crosslinking of the matrix is not enough to promote the adhesion of the material on the substrate during development (case i) in Fig. 7f). This behavior corresponds to a too low conversion of the C=C bond in the organic part of the hybrid matrix, especially at the resin-substrate interface due to the internal filter effect. At 2.5 mJ/cm 2 , the conversion of the organic matrix is sufficient to give rise to a very thin layer of crosslinked material at the substrate surface. It explains why in Fig. 7a, patterns are observable but with a height much smaller (a few nm) than the initial film thickness (80 nm). From 2.5 mJ/cm 2 to 5 mJ/cm 2 , the measured height increases rapidly with the dose as crosslinking proceeds more and more efficiently in the photoresist. However, the case depicted in Fig. 7f-iii is never reached since the maximum height (50 nm, Fig. 7b) was always significantly lower than the initial film thickness (80 nm). This explains the apparition of a residual layer between lines (Fig. 7f-iv) due to the irradiation in dark fringes of the interference pattern. This assumption is confirmed by the gradual decrease of the pattern height with dose (Fig. 7c,d) that corresponds to the increase of the thickness of the layers between lines. Such behavior is partially explained by the interference pattern irradiation configuration. Indeed, one of the drawbacks of this configuration is that the light intensity is sinusoidal so it is not null in the dark fringes.</p><p>As shown in a previous study 40 , the concentration of the MNP has an impact on the photopatterning. Patterns with various concentrations of MNP (between 0 and 20 vol%) were prepared using interference lithography (period 600 nm). Photopatterning can be obtained in this wide range of MNP concentration but there is a strong impact of the MNP load on the quality of the patterns and their height. In particular for the highest loads of MNPs we observed the apparition of roughness at the sample surface after development that can be linked to partial aggregation of the MNPS that occurs at the surface of the patterns. These aggregated nanoparticles may create bridges between close structures, which may account for the difficulty to conduct development in these conditions, which results in remaining material between lines. In Fig. 8, we plotted the typical maximal height and optimal dose for the different MNP concentrations. Interestingly, Fig. 8 reveals that the optimal dose is increased, as expected, with the content of MNP, but only slightly in a 80 nm thin film. In conclusion, photopatterning with submicron resolution is achievable with MNP concentrations as high as 20 vol% but in this case, www.nature.com/scientificreports/ a significant decrease of the pattern modulation, due to the presence of a residual layer between written lines is observed. However, for many applications as gratings or in guided optics, such residual layer is not a problem for practical applications since it can be taken into account in the design of the optical design to produce a given optical function.</p><p>Finally, in Fig. 9, we show the impact of the Ti complex concentration to confirm that the patterning is indeed triggered by the Ti complex, as suggested by the polymerization kinetic studies shown previously, and to evaluate the impact of the Ti complex concentration on the patterns.</p><p>Two concentrations of Ti complexes were used (Si/Ti = 3/1 and 6/1), with the same concentration of MNP (0.4 vol%). In both cases, a residual layer between lines is obtained after photolithography. As expected, the dose needed to achieve the photopatterning of the material is lower for the higher content of Ti, which confirms the role of Ti complex as a photoinitiator of the crosslinking reaction within the material. However, the maximum pattern height was obtained for the lower Ti content, which confirms the interest to use a molar ratio Si/Ti = 6/1, as mentioned previously. Increasing the content of Ti allows decreasing the exposure time for a given power but finally, no significant improvement of the maximum height was observed.</p><p>In conclusion, we have shown in this paper that DUV photolithography (193 nm) is an extremely interesting tool for the micro and nanostructuring of thin films with magneto-optical properties. Starting from solutions whose composition can be easily adapted to modulate the properties, the DUV photolithography step allows to cross-link the material and to structure it at submicrometer scales. No additional step (in particular no thermal annealing) is required to obtain the magneto-optical properties, which opens perspectives for the integration of these materials in devices, on glass, on silicon, but also on plastic.</p>

Scientific Reports - Nature

Tissue-type plasminogen activator requires a co-receptor to enhance N-Methyl-D-Aspartate receptor function

Glutamate is the main excitatory neurotransmitter of the central nervous system. Tissue-type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA-induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the Low-Density Lipoprotein Receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell-surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA-induced events via LDLR engagement.

tissue-type_plasminogen_activator_requires_a_co-receptor_to_enhance_n-methyl-d-aspartate_receptor_fu

INTRODUCTION<!>Animals<!>Materials<!>Preparation of primary cortical neuron cultures<!>Measurement of \xce\x94[Ca2+]i<!>Tissue protein extracts<!>Cell-free cleavage assay<!>Cell-based cleavage assay<!>Immunoblot analyses<!>Electrophysiology<!>Potentiation of NMDA-induced [Ca2+]i is dependent upon proteolytic activity<!>Plasmin, but not tPA, can cleave the NR1 subunit<!>Potentiation of NMDA-induced \xce\x94[Ca2+]i is a function of culture age<!>tPA alone does not alter NMDAR currents<!>Potentiation of NMDA-induced \xce\x94[Ca2+]i is plasmin-independent<!>Expression of PN-1 correlates with culture maturity<!>Potentiation of NMDA-induced \xce\x94[Ca2+]i by tPA requires LDLR engagement<!>DISCUSSION

<p>The serine protease, tPA, is known for its ability to cleave the pro-enzyme plasminogen into the potent protease plasmin, which can in turn lyse blood clots via the digestion of fibrin. In addition to this vascular role, tPA is now recognized to perform important roles within the brain (Samson & Medcalf 2006, Melchor & Strickland 2005). Neurons and glia secrete tPA upon appropriate stimulation (Lochner et al. 2006, Fernandez-Monreal et al. 2004b, Vincent et al. 1998, Polavarapu et al. 2007). Extracellular tPA activity is balanced by inhibitors and spatially targeted by association with cell-surface receptors. tPA−/− mice display cognitive deficits, alterations in addiction and stress, and shifts in the response to pathological situations including seizure and ischemia (Yepes et al. 2002, Wang et al. 1998). Notably, recombinant tPA is used as a thrombolytic agent in patients with ischemic stroke (NINDS 1995). Altogether, a multitude of physiological and pathological roles have been ascribed to tPA. Despite the functional diversity of tPA within the brain, two recurrent themes exist: (i) the physiological actions of tPA are contextual with synaptic plasticity processes; (ii) pathologically, tPA operates as an injurious excitotoxic factor. The paradigms of synaptic plasticity and excitotoxicity are both reliant upon the NMDA receptor (NMDAR). As a result, several recent studies which show interplay between tPA and the NMDAR have received considerable attention (Kvajo et al. 2004, Pawlak et al. 2005a, Nicole et al. 2001, Park et al. 2008, Benchenane et al. 2007, Norris & Strickland 2007, Pawlak et al. 2005b, Martin et al. 2008, Medina et al. 2005).</p><p>A persuasive body of evidence suggests that tPA can directly (i.e. in a plasmin-independent manner) cleave the NR1 subunit and thereby increase the Ca2+-permeability of the NMDAR (Nicole et al. 2001, Fernandez-Monreal et al. 2004a, Benchenane et al. 2007). Our previous analyses have demonstrated that tPA does indeed augment NMDA-induced calcium flux (Δ[Ca2+]i; Reddrop et al. 2005). Indicative of a novel effector substrate, here we show that the potentiating effect of tPA is dependent upon its proteolytic activity and independent of plasmin(ogen). Moreover, we find that tPA does not directly cleave the NR1 subunit. Hence, the mechanism by which tPA increases NMDAR signaling appears to be more complicated that first hypothesized. In line with this notion, we show that the ability of tPA to augment NMDA-induced Δ[Ca2+]i requires a member of the Low-Density Lipoprotein Receptor (LDLR) family. Thus, the potentiating effect of tPA on NMDAR signaling requires a co-receptor and an unknown substrate. In support of a multi-factorial mechanism, we find that tPA does not alter NMDAR-mediated currents in an expressed heterologous Xenopus oocyte system, suggesting that additional cellular factors present in neuronal cultures are necessary for tPA to modulate NMDAR function. Lastly, distinct from the potentiation of NMDAR function, our analyses uncover the unique capacity of plasmin to discretely cleave the NR1 subunit. Thus, tPA has a dual influence on the NMDAR: one being the indirect potentiation of calcium flux via LDLR engagement, and the other being the plasmin-dependent proteolysis of NR1.</p><!><p>C57Black/6 mice between 2-6 months old were used in this study. Experiments adhered to NH&MRC of Australia guidelines for live animal use. Experiments were approved by the appropriate Institutional Animal Ethics Committee.</p><!><p>Unless stipulated all reagents were from Invitrogen. Recombinant human tPA used was Actilyse® (Boehringer Ingelheim). For Fig.1, 2B, 5 and 7 the Actilyse® had been dialysed against 0.35M HEPES-KOH (pH7.4). No difference in the effect of dialysed versus undialysed tPA was seen in this study. Aprotinin, human plasmin, NMDA and MK-801 were obtained from Sigma. Cyanogen bromide-digested human fibrinogen was from American Diagnostica. Human thrombin was from Calbiochem. ctPA was kindly provided by PAION Deutschland GmbH. ctPA was generated from Actilyse® by covalently coupling a 10-fold molar excess of D-phenyl-prolyl-arginine chloromethyl ketone into the reactive centre of the tPA molecule. RAP and anti-human LRP-1 antibody were kind gifts from Prof. Dudley Strickland (University of Maryland, USA). Human LRP-1 was provided by Prof. Phil Hogg (University of New South Wales, Australia). NeuN and GFAP antibodies were donated by Dr Gabriel Liberatore (University of Melbourne, Australia).</p><!><p>Cultures were prepared from E15-16 mice (Reddrop et al. 2005). In brief, cortices were removed in ice-cold HBSS+: Hanks' Balanced Salt Solution with 1mM Na pyruvate, 10mM Hepes-KOH (pH 7.3), 3g/L BSA and 1.2mM MgSO4. The isolated cortices were centrifuged (900×g, 5min, 4°C), supernatant discarded, and tissue pellet incubated in HBSS+ with 0.2g/L trypsin and 80U/ml DNase I (5min, 37°C with agitation). Trypsinization was stopped by the addition of HBSS+ with 0.5g/L trypsin inhibitor, centrifuged (900×g, 5min, 4°C), the supernatant discarded and 10ml of HBSS+ with 0.5g/L trypsin inhibitor and 2.1mM MgSO4 was added. The pellet was triturated through an 18-gauge blunt-ended needle. The resultant single cell suspension was centrifuged (900×g, 5min, 4°C) and the pellet resuspended in Neurobasal media with 1xB27, 10% dialysed fetal calf serum, 0.5mM L-glutamine and 50U/ml penicillin/streptomycin (P/S). The cell suspension was seeded onto poly-D-lysine (BD Bioscience) coated 24- or 12-well plates (±glass coverslips) at 150,000 cells/cm2 and maintained in a humidified 37°C incubator under 5% CO2 and 8% O2. 24 hours after seeding (DIV1; "days in vitro"), the serum-containing media was aspirated and replaced with NBM+ media (Neurobasal media with 1.25xB27, 0.5mM L-glutamine and 50U/ml P/S). At DIV5, an equal volume of NBM+ media was added. All experiments were performed with either DIV5 or DIV12-13 cultures (as indicated) in a humidified 37°C incubator under 5% CO2, 20% O2. Suppl. Fig.S5 demonstrates the cellular constituents of these cultures.</p><!><p>Neurons cultured on coverslips were incubated in phenol red-free NBM+ media containing 1μM Oregon Green® 488 BAPTA-1 AM, for 45min at 37°C. The media was replaced with fluorophore-free NBM+ media and incubated for a further 45min. The coverslips were then assembled into a perfusion chamber (Warner Instruments Model RC-20H) on the stage of a Leica DM-IRBE confocal microscope which was encased in a Perspex incubator and held at 37°C by an electric air heater. A single field of neurons (typically 15-30 neurons) was selected and Flow Buffer (phenol red-free HBSS with 2mM CaCl2 and 0.6mM MgCl2) perfused over the cells at 0.5ml/min. For the assessment of the modulation of NMDA-induced Δ[Ca2+]i, a perfusion protocol involving three stimulations was employed. Δ[Ca2+]i was monitored in 1.8 second intervals. The first and second stimulations involved two identical 45 second exposures to either 25 or 50μM NMDA (separated by 10min), whilst the third stimulation was a 75mM KCl exposure (third stimulation data not shown). Vehicle/Buffer alone (control) or various treatments (tPA, ctPA, RAP, plasmin) were perfused for 5min over the cells in between the two transient NMDA exposures. A thorough example of this procedure has been published (Weiss et al. 2006). The data was analyzed using Leica physiology software with regions of interest (ROI) corresponding to the cell body being selected. Each ROI was assigned a N=1 value and only ROI that displayed a sharp, definitive rise in fluorescence from all three stimulations were analyzed. For each ROI, the Δ[Ca2+]i (i.e. area under the curve) above baseline (i.e. median value of the unstimulated periods) was measured and the second NMDA-induced Δ[Ca2+]i was expressed relative to the first NMDA-induced Δ[Ca2+]i. This value was averaged across all ROI within the same treatment group (i.e. % modulation). The % modulation for each "treated" group was normalized to that of the "control" group (i.e. % modulation relative to control). Each independently seeded culture was assigned an n=1 value. Differences between treatment groups were tested by one-way ANOVA and post-hoc correction for multiple comparisons with p<0.05 being considered as statistically significant. Note, none of the modulatory agents were found to discernibly alter basal calcium flux (suppl. Fig.S6).</p><!><p>Unless indicated, all buffers/manipulations were at 4°C. An adult mouse brain was removed and rinsed with PBS (0.137M NaCl, 2.68mM KCl, 10mM Na2HPO4, 1.76mM KH2PO4 pH 7.4). The cortices were dissected, rinsed again in PBS, then homogenized in RIPA buffer (50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 1mM PMSF, 5mg/L aprotinin, 5mg/L leupeptin, 2mM imidazole, 1mM NaF, 1mM Na3VO4). The homogenate was centrifuged (400×g, 5min) and the supernatant stored at −80°C. Protein extracts of the liver were prepared in the same manner.</p><!><p>An adult mouse brain was removed and washed with PBS. The cortices were dissected, rinsed again in PBS, then homogenized in PBS containing 2.5% Triton X-100. The resulting homogenate was pelleted by centrifugation (400×g, 5min), and the protein concentration of the supernatant quantified and adjusted to 3mg/ml. Proteases (plasmin, tPA, thrombin; 4-25μl) were added to 0.5ml aliquots of the cortical lysates and incubated at 37°C for 10 minutes to 2 hours. 50μg of each sample was then subjected to immunoblot.</p><!><p>DIV12 culture media (in 24-well plates) was replaced with phenol red-free and B27-free Minimum Essential Medium with Earle's salts (400μl per well) and incubated for 1 hour in a humidified 37°C incubator. Proteases (tPA, plasmin, thrombin; 2-10μl) were added to the media and incubated for 10 min or 1 hour, after which the media was aspirated and RIPA buffer added to each well. The lysates were collected, quantified and stored at −80°C.</p><!><p>Samples were boiled in SDS-loading buffer with DTT, subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were probed with primary antibodies [mouse anti-NR1 N-terminal (Chemicon Int., 1:300-1000), goat anti-NR1 C-terminal (Santa Cruz Biotechnology Inc., 1:100-1000), goat anti-tPA (Santa Cruz Biotechnology Inc., 1:1000), sheep anti-plasminogen (Serotec), rabbit anti-GFAP (DakoCytomation, 1:1000), mouse anti-GAPDH (Chemicon Int., 1:5000), 4B3 mouse monoclonal anti-rat PN-1 (Meier et al. 1989)] followed by appropriate HRP-conjugated secondary antibody [sheep anti-mouse IgG (Chemicon Int., 1:5000), rabbit anti-goat IgG (Sigma, 1:5000), rabbit anti-sheep IgG (Chemicon Int., 1:5000)]. Signals were revealed by chemiluminescence (Supersignal, Thermo Scientific).</p><!><p>RNA preparation, oocyte preparation and expression of NMDAR subunits in Xenopus oocytes were performed as described previously (Kloda & Adams 2005). Plasmids with cDNA encoding the rat NR1a and NR2A NMDAR subunits were kindly provided by Dr. J. Boulter (UCLA, Los Angeles, CA, USA). All oocytes were injected with a 1:3 ratio of 5ng NR1a and 15ng NR2A cRNA, respectively and kept at 18°C in ND96 buffer (96mM NaCl, 2mM KCl, 1.8mM CaCl2, 1mM MgCl2 and 5mM HEPES pH 7.4) supplemented with 50mg/L gentamycin and 5mM pyruvic acid 2-5 days before recording. Membrane currents were recorded from Xenopus oocytes using a two electrode voltage virtual ground circuit on a Gene clamp 500B amplifier or an OpusXpress™ 6000A workstation, (Molecular Devices, Sunnyvale, CA), as previously described (Clark et al. 2006). Electrodes were filled with 3M KCl and had resistances of 0.2-1.3Mohm. All recordings were conducted at 20-23°C using a Ca2+ and Mg2+-free solution (115mM NaCl, 2.5mM KCl, 1.8mM BaCl2 and 10mM HEPES at pH 7.3). Current amplitudes were determined by the steady-state plateau response elicited by 30μM glutamate and 10μM glycine, in the absence and presence of 1μM tPA and 250nM Plasmin at a holding potential of −70 mV. Membrane currents were sampled at 500Hz and filtered at 200Hz.</p><!><p>tPA has previously been shown to potentiate NMDA-induced Δ[Ca2+]i (Nicole et al. 2001, Reddrop et al. 2005, Park et al. 2008). To ascertain whether the ability of tPA to augment NMDA-induced Δ[Ca2+]i was dependent on its proteolytic capacity, we utilized a novel inactive variant of tPA termed "ctPA" (PAION GmbH, Germany). Relative to tPA, ctPA retained <0.01% activity by amidolytic assay (data not shown), was 100-1000 fold less active by fibrin zymography (Fig.1A), and was equivalent in molecular weight (suppl. FigS1) and receptor binding (suppl. Table S2). As shown in Fig.1B-C, while 500nM tPA enhanced NMDA-induced Δ[Ca2+]i, ctPA failed to elicit any potentiation. Rather, 500nM ctPA appeared to slightly suppress this calcium response. The basis of this suppression is unknown. Notably, 1μM ctPA also failed to augment NMDA-evoked Δ[Ca2+]i (data not shown). Thus, tPA potentiates NMDA-triggered Δ[Ca2+]i in a manner dependent on its proteolytic activity.</p><!><p>The requirement for proteolytic capacity implies the existence of a tPA-sensitive substrate. Previous studies have identified the NR1 subunit of the NMDAR as the operative substrate in this setting (Nicole et al. 2001, Fernandez-Monreal et al. 2004a, Benchenane et al. 2007). To determine whether tPA can cleave NR1 we performed "cell-based" cleavage assays. In these assays, 500nM tPA was added to the media of neuronal cultures for 10 minutes. As a control, cultures were also treated with 250nM plasmin or 500nM thrombin – two other trypsin-like serine proteases. Cell lysates were then prepared and NR1 content assessed by immunoblot analysis. As shown in Fig.2A, only plasmin was able to cleave the NR1 subunit producing a 90kDa fragment. Incubation of cultures with the indicated proteases for 1 hour resulted in no additional effects (data not shown). This finding suggests that the plasmin-mediated cleavage of full-length NR1 to the 90kDa fragment utilizes portions of the NMDAR that are appropriately exposed on the cell-surface. To account for the possibility that cleavage of the NMDAR by tPA required NMDA engagement, similar cleavage experiments were also performed in the presence of NMDA. Under these conditions, tPA still failed to cleave NR1 (data not shown).</p><p>Our cell-based cleavage assays also show that plasmin treatment does not deplete full-length NR1. This finding reflects the fact that bath-applied proteases cannot access the full complement of cellular NMDARs (as a proportion of NMDARs exist intracellularly (Xia et al. 2001)). To circumvent this, we utilized a "cell-free" cleavage assay, whereby protein lysates of the adult tPA−/− mouse cortex were incubated with plasminogen, tPA, and aprotinin (a reversible plasmin inhibitor). To ensure that tPA was fully active, we also supplemented lysates with cyanogen-digested fibrinogen (CNBr-F); a co-factor that potently enhances tPA activity towards certain substrates (Schaefer et al. 2006, Verheijen et al. 1982). Lysates were then incubated for 15 minutes at 37°C and the NR1 content assessed by immunoblotting. To accommodate for the possibility that NR1 cleavage products contain epitopes that are not recognized by a single antibody, we used two different anti-NR1 antibodies – one directed against the extracellular N-terminal domain and one directed against the intracellular C-terminal domain of NR1 (Fig.2C). As shown in Fig.2B, immunoblotting with either anti-NR1 antibody revealed that whilst tPA or tPA+CNBr-F were unable to proteolyse NR1, co-incubation with tPA+plasminogen resulted in complete NR1 cleavage (see suppl. Fig.S2 for quantitation of Fig.2B). Aprotinin inhibited NR1 degradation caused by tPA+plasminogen incubation. As expected, under these cell-free conditions, a loss of full-length NR1 coincided with the appearance of three NR1 fragments: a ~120, 90 and 60kDa species. The 90kDa fragment is presumably the same as that detected in our cell-based assays, whilst the 60kDa is most likely a consequence of the cell-free conditions whereby regions of NR1 which are spatially hidden become available for ectopic proteolysis. Notably, aprotinin blocked the conversion of single-chain to two-chain tPA (a plasmin-dependent process), but did not inhibit conversion of plasminogen to plasmin (a tPA-dependent process). Thus, plasmin inhibition, rather than tPA inhibition, was responsible for the blockage of NR1 proteolysis by aprotinin. Fig.2B also shows that the anti-NR1 C-terminal antibody failed to detect any cleaved NR1 products. Interestingly, whilst immunoblotting with the anti-NR1 N-terminal antibody revealed that aprotinin potently inhibited plasmin-mediated NR1 degradation, immunoblotting with the anti-NR1 C-terminal antibody revealed only slight inhibition of NR1 degradation. This discrepancy suggests that NR1 has at least two plasmin-sensitive cleavage sites: one that is highly plasmin-sensitive (poorly inhibited by aprotinin) and another that is moderately plasmin-sensitive (effectively inhibited by aprotinin).</p><p>Our cleavage data best fits a model where a highly plasmin-sensitive cleavage site resides within the short intracellular C-terminal tail. Cleavage at this C-terminal site produces the 120kDa fragment. Removal of the intracellular C-terminal tail would explain why all cleavage fragments are not detected by the anti-NR1 C-terminal antibody (Fig.2C). Cleavage at two separate sites yields the 90 and 60kDa fragments</p><p>Given the results obtained from the cell-based cleavage assays, the 90kDa plasmin-generated fragment is the only species that can occur under physiological conditions. Fig.2C provides a schematic of how this 90kDa fragment can be generated from full length NR1 by plasmin.</p><p>To confirm the identity of the NR1 cleavage fragments, immunoprecipitations with the anti-NR1 C-terminal antibody were performed from neuronal culture lysates. The immunoprecipitated material was then plasmin-digested and subjected to anti-NR1 N-terminal immunoblot. The 120, 90 and 60kDa species appeared following plasmin-digestion (suppl. Fig.S3). Thus, plasmin can generate the appropriate NR1 cleavage fragments from immunoprecipitated full-length native NR1. Bioinformatic analysis highlights either Arg704-His705 or Lys316-Tyr317 as the putative plasmin-sensitive cleavage site responsible for the 90kDa fragment (Fig.2C).</p><p>Additional cell-free cleavage assays using wild-type mouse cortical lysates incubated with 1μM tPA, 250nM plasmin or 500nM thrombin for 10 minutes and 2 hours support our conclusion that plasmin, but not tPA, can cleave NR1 (data not shown). Thrombin, albeit with markedly lower efficiency, was also capable of producing the 90 and 60kDa NR1 fragments under cell-free conditions, suggesting utilization of the same cleavage sites (data not shown) (Gingrich et al. 2000).</p><!><p>NR1 is an obligatory subunit of the NMDAR. Therefore, if NMDAR potentiation involved a direct association between tPA and NR1, then tPA should impact on any neuron with functional cell-surface NMDARs. But, despite NMDA eliciting a classical Δ[Ca2+]i in both DIV5 and DIV12 cultures (suppl. Fig.S4), tPA only potentiated NMDA-induced Δ[Ca2+]i in DIV12 cultures (Fig.3). Hence, the ability of tPA to modulate NMDA-induced Δ[Ca2+]i is a function of in vitro culture age. That tPA cannot influence NMDA-induced Δ[Ca2+]i in early (DIV5) cultures denotes a requirement for additional cellular factors besides tPA and the NMDAR.</p><!><p>To confirm that additional cellular factors were required for the potentiation of NMDAR function by tPA, we measured NMDAR-mediated currents in a heterologous non-neuronal system via two-electrode voltage clamp. Expression of heteromeric NR1a/2A NMDARs in Xenopus oocytes generated functional glutamate-activated channels, which were activated by 30μM glutamate and 10μM glycine. The addition of 1μM tPA for 30 seconds to the activated NMDAR revealed no change to the NMDAR-mediated current amplitude (101.3 ±2.5%; p>0.05 by t-test, n=17; Fig.4A). The addition of 250nM plasmin in the open state of the NMDAR also revealed no change to the NMDAR-mediated current (100.5 ±2.5 %; n=4; Fig.4B). As tPA was incapable of altering NMDAR-mediated current in this isolated non-neuronal system, we conclude that tPA requires additional cellular factors to potentiate NMDAR function. This finding, in conjunction with our evidence showing that NR1 is not a tPA-sensitive substrate, contradicts the postulate that tPA directly alters NMDAR function via NR1 cleavage (Nicole et al. 2001, Fernandez-Monreal et al. 2004a).</p><!><p>Having gained strong evidence against NR1 being a tPA-sensitive substrate, we next considered the prototypical substrate for tPA; namely plasminogen. Consequently, we monitored the modulatory effect of 25nM plasmin on NMDA-induced Δ[Ca2+]i. As shown in Fig.5, unlike tPA, the perfusion of 25nM plasmin resulted in no enhancement of NMDA-induced Δ[Ca2+]i. Therefore, we conclude that tPA potentiates NMDA-induced Δ[Ca2+]i in a manner independent of plasmin(ogen). A concentration of 25nM plasmin was chosen on the basis that treatment of neuronal cultures with 25nM plasmin resulted in NR1 cleavage (data not shown), whereas treatment of cultures with tPA failed to result in NR1 cleavage (Fig.2A). Hence, the concentration of plasminogen in our cultures must be less than 25nM.</p><!><p>Besides plasminogen, tPA also displays high proteolytic activity towards several serpins, notably PAI-1, neuroserpin and protease nexin-1 (PN-1) (Hastings et al. 1997, Rossignol et al. 2004, Lawrence et al. 1995). A prior study has shown that PN-1 and tPA together regulate NMDAR function (Kvajo et al. 2004). Consequently, we determined whether our neuronal cultures expressed PN-1. As shown in Fig.6, PN-1 was virtually absent from DIV5 cultures, but was abundant in DIV12 cultures. Whilst we cannot exclude the role of other tPA sensitive serpins, this strong correlation between PN-1 expression and the ability of tPA to enhance NMDA-induced Δ[Ca2+]i led us to postulate that PN-1 may be an additional cellular factor required for the modulation of NMDAR function by tPA.</p><!><p>PN-1 rapidly forms a complex with tPA, which in turn avidly binds to cell-surface LDLRs, initiating intracellular signaling cascades and tPA:serpin complex internalization (Herz & Strickland 2001). Indeed, LDLRs are the sole recognized receptor for tPA:serpin complexes. Accordingly, we assessed whether LDLR engagement was required for tPA to potentiate NMDA-induced Δ[Ca2+]i. For this, we utilized the LDLR pan-ligand blocker, Receptor-Associated Protein (RAP). As shown in Fig.7, we observed that whilst the application of RAP alone produced no significant change in NMDA-induced Δ[Ca2+]i, it fully ablated the ability of tPA to enhance NMDA-induced Δ[Ca2+]i. This observation suggests that tPA-mediated potentiation of NMDA-induced Δ[Ca2+]i is dependent upon LDLR engagement. This finding is consistent with the hypothesis that potentiation of NMDA-induced Δ[Ca2+]i involves tPA:serpin complex formation and subsequent LDLR engagement.</p><!><p>Several studies document that tPA potentiates NMDA-induced Δ[Ca2+]i (Nicole et al. 2001, Reddrop et al. 2005, Park et al. 2008). The currently proposed mechanism involves direct cleavage of NR1 by tPA, which in turn increases the Ca2+-permeability of the NMDAR (Nicole et al. 2001). In support of a proteolytic event, the reversible tPA inhibitor, tPA-STOP, has been shown to diminish the influence of exogenous tPA on NMDA-induced Δ[Ca2+]i (Liot et al. 2004). The low concentration of tPA-STOP (10nM) relative to exogenous tPA (~300nM), however, queries this interpretation. Consequently, our finding that an inactive tPA fails to enhance NMDA-induced Δ[Ca2+]i conclusively demonstrates that the enhancement of NMDA-induced Δ[Ca2+]i relies upon the proteolytic capacity of tPA.</p><p>Implicit in the requirement for proteolytic activity is the existence of an effector substrate. Published evidence from one laboratory defines NR1 as the pertinent substrate (Fernandez-Monreal et al. 2004a, Benchenane et al. 2007). In an attempt to detect proteolysis of NR1, both cell-free and cell-based cleavage assays were performed. Cleavage times were varied from 10 min to 2 hours, and tPA concentrations from 50nM to 1μM were tested. Yet, despite trialing these different conditions, no evidence for the proteolysis of NR1 by tPA was found. Cell-based experiments in the presence of NMDA were also conducted and still no tPA-mediated NR1 proteolysis was observed (data not shown). Therefore, we conclude that NR1 is not a tPA-sensitive substrate. This conclusion extends the findings of others (Liu et al. 2004, Matys & Strickland 2003, Kvajo et al. 2004).</p><p>If not NR1, then what is the operative tPA-sensitive substrate? Platelet-derived growth factor-C (PDGF-C) represented a logical candidate (Fredriksson et al. 2004, Boucher et al. 2003, Su et al. 2008). However, we found that PDGF-C expression remained unchanged during neuronal culture development (data not shown) and thus PDGF-C represents an unlikely effector of tPA-mediated NMDAR modulation.</p><p>Our data, in conjunction with published data (Nicole et al. 2001), also suggests that plasminogen is not the tPA-sensitive substrate in question. Our findings do however, indicate that LDLR engagement is vital for tPA to influence NMDAR function. A direct interaction between tPA and a LDLR could explain how RAP ablates the enhancement of NMDA-induced Δ[Ca2+]i by tPA. Several lines of evidence point against this possibility. First, our surface plasmon resonance experiments (suppl. Table S2), together with other studies (Hu et al. 2006, Orth et al. 1994, Zhuo et al. 2000, Martin et al. 2008), suggest that tPA cannot proteolyse LDLRs. Second, despite having differential effects on NMDA-induced Δ[Ca2+]i, both ctPA and tPA bind to LDLRs with high nanomolar Kd (~330nM; suppl. Table S2). Thus, the potentiation of NMDA-induced Δ[Ca2+]i is unlikely to be explained by the direct association/proteolysis of a LDLR by tPA. Lastly, none of the tested LDLR family members (LRP-1, LRP-1B, ApoER2, Megalin and VLDLR) exhibited differences in expression between DIV5 and DIV12 cultures (data not shown). On the other hand, tPA displays potent and specific proteolytic activity towards several serpins, with the resultant tPA:serpin complex strongly binding to numerous LDLRs with low nanomolar Kd (Horn et al. 1997, Makarova et al. 2003). Additionally, we have observed that PN-1 expression in our cultures increases dramatically from DIV5 to DIV12. Therefore, PN-1 likely represents the tPA-sensitive substrate responsible for the potentiation of NMDA-induced Δ[Ca2+]i. We propose a model whereby tPA first complexes with PN-1 or another differentially expressed serpin, then binds to a LDLR and signals for an enhancement of NMDA-induced Δ[Ca2+]i. Both the requirement for tPA to be proteolytically active and the ability of RAP to block the influence of tPA on NMDA-induced Δ[Ca2+]i are in keeping this model. It will be interesting to determine whether addition of PN-1 to DIV5 cultures restores the ability of tPA to potentiate NMDA-induced Δ[Ca2+]i.</p><p>Further support for a tPA:PN-1 complex being a modulator of NMDAR function stems from the observation that both tPA−/− and PN-1−/− mice have reduced NR1 availability (D. Monard, unpublished data). Other tPA-reactive serpins may also elicit similar effects on NMDAR function. For example, it has been hypothesized that tPA, via complex formation with PAI-1, mediates NMDAR-dependent hyperemia (Park et al. 2008).</p><p>The links between tPA, LDLRs and the NMDAR are compelling. For instance, tPA facilitates NMDAR-dependent synaptic plasticity via engagement of the prototypical LDLR, LRP-1 (Zhuo et al. 2000). And similar to the influence of tPA described here-in, numerous LRP-1 ligands alter NMDA-induced Δ[Ca2+]i (Qiu et al. 2002, Qiu et al. 2003). LRP-1 also physically associates with the NMDAR (May et al. 2004). Lastly, a recent study has demonstrated that tPA may elicit NMDAR activation in a LRP-1-dependent manner (Martin et al. 2008). Given these ties, it is noteworthy that NMDAR- and tPA-dependent LTP remains RAP-blockable despite the absence of neuronal LRP-1 (May et al. 2004). One possible explanation for this is that glial LRP-1 expression is critical for tPA to alter NMDAR function. Astrocytes are key mediators of neurotransmission that facilitate LTP (Yang et al. 2003). Furthermore, astrocytes are a significant component of our cultures (suppl. Fig.S5). Thus, we cannot exclude the involvement of astrocytes in our observations. In fact, a peri-cellular communication mechanism between neurons and astrocytes merits consideration, particularly as astrocytic uptake of tPA is blocked by RAP (Fernandez-Monreal et al. 2004b), as tPA triggers LRP-1 shedding from astrocytes (Polavarapu et al. 2007), and as astrocytes are known NMDAR modulators (Wolosker et al. 2002). Alternatively, it is possible that other LDLRs besides LRP-1 are central to the potentiation of NMDA-triggered events by tPA.</p><p>Distinct from the potentiation of NMDA-induced Δ[Ca2+]i, our experiments reveal the novel ability of plasmin to discretely proteolyse NR1. We propose that plasmin can efficiently remove the very distal C-terminal portion of NR1. As a result, antibodies raised against the C-terminal portion of NR1 do not detect the ~120kDa N-terminal fragment of NR1 (or the subsequent 90 and 60kDa fragments). This model likely explains why previous cell-free experiments have shown that plasmin, instead of discretely cleaving NR1, can completely degrade NR1 (Matys & Strickland 2003). Notably, whilst no effect on rudimentary NMDAR-mediated ion conductance was observed (Fig.4), the impact of plasmin-mediated NR1 cleavage on other NMDAR properties such as allosteric modulation, cell-surface location and internalization rate remains unknown. That plasmin could efficiently proteolyse the extracellular domain of NR1 in the context of native cell-surface NMDARs intimates biological significance. Indeed, cleavage of NR1 by plasmin most likely occurs under chronic stress, a condition where plasmin has been shown to drastically decrease hippocampal NR1 levels (Pawlak et al. 2005b).</p><p>In conclusion, our investigations establish the plasmin-independent potentiation of NMDA-induced Δ[Ca2+]i by tPA. Even though tPA needs to be proteolytically active, we find no evidence that tPA can directly cleave the NR1 subunit. Furthermore, our data suggests that the enhancement of NMDA-induced Δ[Ca2+]i by tPA is mediated by a LDLR co-receptor. A similar set of experimental criteria has been previously described, whereby tPA increases blood-brain barrier permeability in a manner dependent upon proteolysis, independent of plasminogen and reliant upon LRP-1 (Yepes et al. 2003). As such, we hypothesize that tPA acts on a non-plasminogen substrate. Subsequent to this cleavage event, a LDLR is engaged, which in turn augments calcium flux downstream of the NMDAR. Given the dramatic increase in PN-1 between DIV5 and DIV12 cultures, PN-1 presents as the operative non-plasminogen substrate in this setting. This multi-factorial mechanism may underlie some of the proteolytic, yet plasmin-independent roles of tPA (Schaefer et al. 2007, Yepes et al. 2002, Yepes et al. 2003, Pawlak et al. 2002, Kumada et al. 2005, Park et al. 2008). Finally, adding to the ways in which the plasminogen activator system can modulate the NMDAR, our analyses uncover the capacity of plasmin to discretely cleave the NR1 subunit of the NMDAR.</p>

PubMed Author Manuscript

Leaded Aviation Fuel May Present Long-Term Effects on Campus Life from the Adjacent Albert Whitted Airport

Propeller planes and small engine aircraft around the United States, legally utilize leaded aviation gasoline. The purpose of this experiment was to collect suspended particulate matter from a university campus, directly below an airport's arriving flight path's descent line, and to analyze lead content suspended in the air. Two collection sets of three separate samples were collected on six separate days, one set in July of 2018 and the second set in January 2019. The collection procedure began in the morning and continued into the afternoon. Samples were collected with an air abatement monitor, borosilicate glass fiber filters. The negative and positive control samples were collected in sterile conditions; the negative being devoid of trace metal particles and the positive saturated in In and Zn. The fiber filters were digested in in a 2.06M nitric acid and extracted through sonication in an 80°C water bath. They samples were measured on an Induced-Coupled Plasma Mass Spectrometer, utilizing a linear standard. The experiment showed that levels of lead all exceeded the Environmental Protection Agency's federal regulated standards of 0.15µm/m³. Lead levels exceeded 3.2µm/m³ within a 6-hour collection time and reached as high as 6.3µm/m³.

leaded_aviation_fuel_may_present_long-term_effects_on_campus_life_from_the_adjacent_albert_whitted_a

INTRODUCTION<!>Sample Collection<!>Digestion and Extraction<!>Analysis<!>Collection Set I<!>Table 1.<!>DISCUSSION

<p>The University of South Florida, St. Petersburg is placed in an ideal location. Located right on the southside edge of downtown St. Petersburg area and crested on a small inlet of Tampa Bay. The USFSP campus is located next to Albert Whitted Airport (AWA), a private airfield that focuses on charter planes, small jets, single and double piston engines and medical emergency flights. A study in Michigan found that child blood levels declined in the months following 9/11. 3 This independent study was undertaken to check the amount of Pb that is in suspended particulate matter (PM) in air and compare to the EPA's regulated PM of Pb, 0.15µg/m³, according to the 2008 National Ambient Air Quality Standards. The EPA has noted that airports have potential to exceed the ambient Pb concentration set in the NAAQS. 1 The proximity of a small airport and a university campus is not something many realize until hearing an older plane or large engine moving overhead while on its decent. Jet engine aircraft utilize jet fuel for flight, though small engine aircraft use gasoline which still contains lead (Pb), called Tetraethyl lead (TEL). The EPA phased out leaded gasoline from vehicles by the end of 1995. Though piston engines for propeller planes and small aircraft have legally continued use of leaded gas for flight. The Clean Air Act banned the sale of leaded fuel still available in some parts of the country as of January 1 st , 1996, for use in on-road vehicles. 5 Though in 2011, piston engine aircraft used 225 million gallons. 1 AWA landing runway is less than 100 yards from the east side of DAV building, the south side of DAV opens to large grass covered areas, walkways to/from Tampa Bay shoreline, and several locations for students to congregate. It is this area that p lanes arriving at AWA descend over to touch down. Since the EPA regulates that ambient air should not have more than 0.15µg/m³ Pb, it needs to be determined if this small airfield exceed the NAAQS. Dependent on the number of arrivals per given day, the detection of Pb should show different levels. The potential health risk to a university population surely warrants investigation. While forming the experimental procedure, it was predicated that related to the number of arrivals would be directly correlat ed with levels of lead detected on any set day, therefore it is believed that these levels fluctuate.</p><!><p>Samples were obtained using a BDX II Abatement Air Sampler, including polyurethane collection tubing and glass funnel . Two collection sets of three samples were taken. The two sets were separated by six months, each provided three individual days for each set's collection of SPMs. The BDX II pumps monitor for asbestos and lead. 8 Using Whatman Binderless borosilicate glass fiber filters, 31mm diameter and 0.45µm pore size, each sample was taken over a 6-hour interval. The BDX II was placed on a two-story rooftop at the University of South Florida, St. Petersburg's Davis (DAV) building. The experimental collection set had two controls, a negative control, consisting of a blank filter and a positive control, provided with an excess amount of trace metal grade Zn and In. All four controls ran for a 6-hour interval under sterile hood conditions. To provide a significant signature, two clean glass stir rods were used to administer the positive controls saturation of Zn and In delivery at hours 2 and 3, respectively for each collection set. Each of the sample's and control's filter paper were removed following standard laboratory safety, under a sterile hood, using Personal Protective Equipment (PPE) and folded in half, placed in polyurethane bags and stored at ambient temperature until digestion and extraction.</p><!><p>Graduated cylinders were first rinsed with 1% HNO₃, followed by DI water. Using 50mL conical tubes, each folded sample from the polyurethane storage bag was removed by inverting the bag and setting the filter into its corresponding tube. Tubes were filled with 15mL of 2.06 M HNO₃ and placed in a rack, then submerged in an 80°C water bath and ultrasonicated for 60 minutes. After sonication, an additional 15mL of DI water was added for total volume of 30mL. Sample tubes were then centrifuged at 8,000 rpm for 5 minutes. Tubes were then decanted into new corresponding 50mL conical tubes and stored at 4°C until ICP-MS analysis.</p><!><p>Allowed samples to reach ambient temperature and then a second centrifugation of 10,000 rpm for 10 minutes. Using an Agilent 7500 series Inductive Coupled Plasma -Mass Spectrometer, generated a linear gradient using industry standards, Germanium (Ge) and Bismuth (Bi). Analyses was performed on the controls and samples, as well as the two standards Ge and Bi. The linear gradient began at 6.25ppb, increasing two-fold at each interval standard until 200ppb.</p><!><p>The two standards that were used with the analysis was Germanium (72) and Bismuth (209), kept at a controlled 50 ppm. The linear standard that was generated began at 6.25ppm and doubled thereafter up to 200 ppm. Control A showed the absence of In and had 1459 ppb of Zn. Pb reflected 2.821ppm in control A. Control B showed In at 217.5 ppb and Zn at 1711 ppb, while Pb was 2.90 ppb. Samples 1, 2, 3 showed no trace In, suggesting that environmental In is undetectable. The samples showed that Pb levels were 6.328ppm, 4.76ppm, 4.51ppm, respectively. This corresponds to the number of arrivals that landing for each of the three samples collected at 19+, 13+ and 10+ respectively. Control A and B had an average of 2.864 ppb of Pb, the samples had an average of 5.20 ppb. Experimental samples were saturated in Zn. It was determined that not only was there an abundance of Zn suspended particulate in the experimental environment but that the zinc plated screws to hold the glass fiber filter paper was adding considerable amounts of trace particles.</p><!><p>Collection Set II Again, two controls were used, administered in the same manner as Collection Set I. Control A was void of In and had 9331 ppm of Zn. Control B showed 56.92 ppb of In and over 10, 000 ppb of Zn. All three samples also showed levels of Zn exceeding 10,000 ppb. This is most likely due to the removal and replacing the four zinc plated screws when changing the filter between samples and controls. In was again not present in the samples collected, affirming a lack of SPM within the vicinity. The lead measured in the three samples collected for Collection Set II was 3.216, 3.76. and 3.35 ppb respectively. While the arrivals on the</p><!><p>When the EPA selected a new standard for suspended Pb in 2008 which was ten times more rigorous, monitoring data showed that out of all US counties only 18 would violate the new standard. 7 St. Petersburg is within Pinellas county, which was not one of the eighteen the EPA noted. Though within this general area of USFSP, it seems that five to six thousand students, staff, and university employees are exposed to levels that are even above the prior standard that the EPA changed in 2008. Information on arrival flights were obtained through flightaware.com, which provides data on arrival and departure flights for small airports across the US. 10</p><p>The BDX II's filter externally mounted in place at the front of the equipment. It is held by a molded, hard plastic that allows the tubing to inject the air flow. To remove the filter and place a new one inside, four small metal screws must be removed from the plastic that attaches it to the equipment. These screws are small black zinc-plated, as are the other screws used on the equipment. There are mainly two types of screws; zinc plated or galvanized, which is an outer coating used to combat rust from the natur al oxidation of steel and stainless-steel, which is rust proof but more expensive. Kolle describes galvanizing as a Tootsie pop and that the zinc does eventually give way to the steel screw, though there are variations of thickness. 9 The Zn levels are most likely do to fine particulate matter released when removing and replacing these screws, as well as the naturally high levels of Zn in the atmosphere.</p><p>During Collection Set I, samples one and three experienced a random error of significant mention just prior to analyses. The second centrifugation process was performed in equipment that had been solely operated for microbiologic procedures, the 10, 000 rpm was too severe on sample one and three. This led to a crack in the conical tubes from the 15mL level down to the top of the coned bottom. The exterior tubes that held the conical tubes were leaked into. Upon completion of the 10-minute run, these exterior tubes were immediately removed and poured into two new sterile 50mL conical tubes and labeled samples one and two.</p><p>After investigating what potential contaminants may be present from the exterior tubes, the information obtained was that basic microbiological materials may be present. This ranged from EtOH, DI water, NaCl solution, to Taq-polymerase, live bacteria, and DNA. None of the materials associated with the potential contaminants would directly apply to the specific trace metals that were analyzed. Nevertheless, due to this reason the data provided by sample two and controls A and B are free of experimental error. Even still, therefore a secondary experiment, Collection Set 2, was done and measured, as to support the findings of the first.</p><p>The data provided is supportive of the original hypothesis, increased arrival flights of small engine/propeller planes show increased lead levels of suspend particulate matter. It is also assisted by the high Zn levels that were detected, which did not fluctuate from the random error. However, since the random error is potentially a source of contamination the experiment was repeated for conformational data. Aside from the error, the levels of lead that are present in sample two of CS1 is 4.76 ppb. This is 17.86 times greater than the EPA regulation and warrants an in-depth investigation of SPM of lead levels around the USFSP campus. While the average amount of Collection Set 2, which experienced no random errors, was 3.62 times greater than the EPA regulation. Having small samples sizes limits the reproducibility at that point, though if rigorously approached in the same manner as CS I and CS II, further experimentation indicates significant results. Though the EPA website notates that levels of Pb maybe higher in areas of immediate surroundings of airports with small aircraft, having a university campus located direct at the start of the arriving flights runway could be negatively impacting thousands of individuals on an annual basis. It is suggested that t he airport and the city that regulates it, at a minimum, change the approach pattern of arriving flights. This should at least decrease the levels that directly fall over the campus.</p>

ChemRxiv

Divergent modulation of normal and neoplastic stem cells by thrombospondin-1 and CD47 signaling

Thrombospondin-1 is a secreted matricellular protein that regulates the differentiation and function of many cell types. Thrombospondin-1 is not required for embryonic development, but studies using lineage-committed adult stem cells have identified positive and negative effects of thrombospondin-1 on stem cell differentiation and self-renewal and identified several thrombospondin-1 receptors that mediate these responses. Genetic studies in mice reveal a broad inhibitory role of thrombospondin-1 mediated by its receptor CD47. Cells and tissues lacking thrombospondin-1 or CD47 exhibit an increased capacity for self-renewal associated with increased expression of the stem cell transcription factors c-Myc, Sox2, Klf4, and Oct4. Thrombospondin-1 inhibits expression of these transcription factors in a CD47-dependent manner. However, this regulation differs in some neoplastic cells. Tumor initiating/cancer stem cells express high levels of CD47, but in contrast to nontransformed stem cells CD47 signaling supports cancer stem cells. Suppression of CD47 expression in cancer stem cells or ligation of CD47 by function blocking antibodies or thrombospondin-1 results in loss of self-renewal. Therefore, the therapeutic CD47 antagonists that are in clinical development for stimulating innate anti-tumor immunity may also inhibit tumor growth by suppressing cancer stem cells. These and other therapeutic modulators of thrombospondin-1 and CD47 signaling may also have applications in regenerative medicine to enhance the function of normal stem cells.

divergent_modulation_of_normal_and_neoplastic_stem_cells_by_thrombospondin-1_and_cd47_signaling

1. Introduction<!>2. Stem cells and extracellular matrix<!>3. TSP1 in hematopoietic stem cell differentiation and self-renewal<!>4. Thrombospondins in other adult stem cells<!>5. CD47 is a hematopoietic stem cell marker<!>6.1 Function of stem cell CD47 as a SIRP\xce\xb1 counter-receptor<!>6.2 CD47 as a signaling receptor for TSP1 in stem cells<!>7. TSP1-CD47-SIRP\xce\xb1 regulation in bone development<!>8. CD47 in cancer stem cells<!>8.1 Increased CD47 expression in cancer stem cells<!>8.2 Modulation of cancer stem cells by altering CD47 expression<!>8.3 Modulation of cancer stem cells using CD47 ligands<!>9. Future Prospects<!>

<p>The concept that stem cells can give rise to both normal and malignant tissues has attracted growing interest in developing therapeutics to target these cells. Pluripotent cells were first identified in mouse embryos in 1981 (Evans and Kaufman, 1981), and a mouse teratocarcinoma stem cell line was established the same year (Martin, 1981). Pluripotent stem cell lines were first generated from pigs and sheep (Notarianni et al., 1991). Blastocysts produced by in vitro fertilization for clinical purposes were used to develop the first human human embryonic stem cell (ESC) line (Thomson et al., 1998). One approach to circumvent the ethical issues surrounding the use of human embryos for regenerative medicine was to create induced pluripotent stem (iPS) cells using somatic cells isolated from adult tissues. Routine creation of iPS cells was enabled by identification of the four critical stem cell transcription factors cMyc, Sox2, Oct3/4 and Klf4 (Takahashi and Yamanaka, 2006). Forced expression of these four proteins is sufficient to convert various somatic cells into iPS cells. However, the potential of transplanted iPS cells to form teratomas or teratocarcinomas in patients spurred efforts to develop lineage-committed adult stem cells that lack this potential.</p><p>Stem cells are also of growing interest in cancer research. Cancers may arise by transformation of tissue stem cells, or transformed somatic cells may activate the self-renewal program of stem cells (Pardal et al., 2003). Regardless of their origin, it is clear that many tumors are sustained by a minor population of tumor initiating cells that share many properties of stem cells. In this review, we examine the role of the matricellular protein thrombospondin-1 (TSP1) and its receptors in stem cell biology, focusing on how TSP1 interactions with its receptors, including CD47, differentially modulate stem cell physiology in normal and neoplastic cells.</p><!><p>The concept of a stem cell niche encompasses supporting cells, which provide specific cues needed to regulate quiescence of stem cells and maintain their asymmetric division, and a specialized niche extracellular matrix (ECM) that releases growth factors and engages specific ECM receptors on stem cells to control their fate. Signals provided by these ECM receptors mediate dynamic communication between ESCs and their niche (Gattazzo et al., 2014). This ECM is composed of soluble and bound macromolecules that facilitate three-dimensional assembly of stem cells and supporting cells that provides organizing and signaling cues. Changes in stem cell-ECM interactions provide environmental cues that guide wound healing, homeostasis, aging, and stem cell maintenance (Discher et al., 2009; Watt and Fujiwara, 2011).</p><p>Thrombospondins are a family of five secreted proteins in vertebrates (Adams and Lawler, 2011). Some thrombospondins are constitutive elements of ECM, but TSP1 and thrombospondin-2 (TSP2) are matricellular proteins that are not constitutive in ECM but are expressed transiently under specific conditions, where they alter cell behavior and ECM remodeling via their interactions with growth factors in the ECM and by engaging specific transmembrane receptors including proteoglycans, integrins, and the nonintegrin receptors CD36, CD47, and CD148 (Calabro et al., 2014; Roberts et al., 2012; Takahashi et al., 2012).</p><p>CD47 is a signaling receptor for TSP1 that is ubiquitously expressed, but at a higher level on some stem cells. CD47 consists of an extracellular IgV domain followed by five membrane-spanning segments and a short variably spliced C-terminal cytoplasmic tail (Soto Pantoja, 2013.). TSP1 binding to CD47 results in cell type-specific signaling that can alter cell adhesion, motility, growth, differentiation, and survival (Oldenborg, 2013; Soto-Pantoja et al., 2015) (Fig. 1). Some of these signals are mediated by lateral interactions of CD47 with specific integrins and other signaling receptors in the plasma membrane. CD47 also serves as the counter-receptor for signal regulatory protein-α (SIRPα) and SIRPγ (Barclay and Van den Berg, 2014; Matozaki et al., 2009). SIRPα is highly expressed on phagocytes, and binding of the IgV domain of CD47 on a target cell to the IgV domain of SIRPα on macrophages elicits signaling mediated by the phosphatase SHP1 that blocks phagocytosis of the target cell (Fig. 1) (Oldenborg et al., 2000). Accumulating evidence indicates that CD47 expressed by stem cells serves both as a cell-autonomous signaling receptor and as a SIRPα counter-receptor to limit stem cell clearance. Although less explored in stem cells, SIRPα binding could potentially elicit signaling though CD47 that may be distinct from that produced by TSP1 binding.</p><!><p>A role for TSP1 in hematopoietic stem cells (HSCs) was first reported in 1990 (Long and Dixit, 1990). Nonadherent low density human bone marrow cells were shown to adhere specifically on immobilized TSP1. This was true for mixed-lineage progenitors as well as those from colonies containing erythroid burst-forming cells (BFU-E), and erythroid, granulocyte/macrophage, and megakaryocyte colony-forming cells (CFU-GEMM). This activity was mapped to a large C-terminal region of TSP1 and shown to be inhibited by specific monoclonal antibodies that bind to this domain of TSP1. A subsequent report in 1992 reproduced these results using sorted CD34+DR−CD15− human bone marrow cells that exhibited characteristics of HSCs including self-renewal and the ability to differentiate into multiple hematopoietic lineages (Long et al., 1992). The CD34+DR−CD15− hematopoietic progenitor cells attached on TSP1 but not on immobilized fibronectin. In combination with c-Kit, TSP1 was shown to function as a colony-stimulating factor for CD34+DR−CD15− progenitors. In contrast, TSP1 inhibited colony formation driven by the cytokine IL-3. This suggested that TSP1 is a context-dependent positive and negative regulator of HSC differentiation. Another early study using murine progenitor cells showed that TSP1 significantly inhibited murine megakaryocytopoiesis at a concentration of 1 µg/ml (2.2 nM) (Chen et al., 1997). In contrast to the stimulatory activity described above, this inhibitory activity was reproduced by a recombinant N-terminal domain of TSP1. TSP1 was further shown to inhibit the growth of multipotent hematopoietic colony-forming units (CFU-GEMM) but not those committed to granulocyte/macrophage (CFU-GM) or erythroid (BFU-E) lineages. Studies using a CD36 antibody that inhibits TSP1 binding suggested that the TSP1-induced inhibition of megakaryocytopoiesis is mediated in part by the binding of TSP1 to its receptor CD36 expressed on the megakaryocytic progenitors (Yang et al., 2003). Notably, the N-terminal domain of TSP1 does not contain its CD36-binding site, so it is unclear how the results of these two studies can be rationalized.</p><!><p>Recombinant human TSP1 had a negative effect on the angiogenic potential of human endothelial colony-forming stem cells (Smadja et al., 2011). Suppression of either TSP1 expression or CD47 expression in the endothelial colony-forming cells using siRNA enhanced their angiogenic potential, implicating CD47 as the inhibitory TSP1 receptor in these stem cells. Consistent with the known role of TSP1/CD47 signaling in limiting vascular cell nitric oxide (NO)/cGMP signaling (Soto-Pantoja, 2015), cGMP signaling protected endothelial progenitor cells by suppressing oxidative stress and the expression of TSP1 in a murine salt-sensitive hypertension model (Xie et al., 2010). Salt-induced hypertension was associated with elevated TSP1 expression in this model.</p><p>TSP1 promotes the neuronal differentiation of neural progenitor cells (Lu and Kipnis, 2010). Conditioned medium from cultured wild type (WT) astrocytes but not thbs1−/− astrocytes promoted neurogenesis in WT neural progenitor cells. TSP1 also promotes the differentiation of bronchioalveolar stem cells derived from mouse lung into multiple lineages (Lee et al., 2014a). BMP4 signaling induced endothelial cell TSP1 expression via calcineurin/NFATc1 in a 3-dimensional coculture system with bronchioalveolar stem cells. Thbs1−/− endothelial cells were defective in inducing bronchioalveolar stem cell differentiation despite supporting increased colony numbers when cocultured with bronchiolar or alveolar cells. Thbs1−/− stem cells also had no intrinsic differentiation defect. Combined with results from transplantation studies, these data indicate that TSP1 produced by endothelial cells in the lung stem cell niche selectively promotes the alveolar differentiation of bronchioalveolar stem cells in vivo.</p><p>Regulation of stem cell differentiation extends to at least one other member of the thrombospondin family. Increased colony-forming activity was found for bone marrow stromal cells isolated from TSP2 null mice (Hankenson et al., 2000). In another study TSP2 secreted by mesenchymal stem cells was reported to promote chondrogenic differentiation in a paracrine manner via PKCα, ERK, p38/MAPK, and Notch signaling pathways (Jeong et al., 2013). TSP1 and TSP2 share several receptors including some integrins, proteoglycans, and CD36 (Calzada and Roberts, 2005), suggesting that both may regulate stem cells via common mechanisms. On the other hand, TSP2 lacks the activities of TSP1 to activate latent TGF-β or bind to α3β1 integrin (Calzada and Roberts, 2005; Schultz-Cherry et al., 1995), so stem cell responses mediated by these pathways may not be shared by TSP2.</p><!><p>Mouse thoracic duct lymph contains HSCs with multilineage potential (Massberg et al., 2007). These cells originate from bone marrow cells that enter into the bloodstream and then enter into peripheral lymphatics and return to bone marrow via the bloodstream. This suggests that a highly adaptive pool of HSCs continuous recirculates and, by differentiating locally, can rapidly generate immune effector cells. CD47 is highly expressed on HSCs and has several proposed functions on these cells. One hypothesis is that CD47 expression on HSCs protects these circulating cells from clearance by macrophages that express SIRPα (van den Berg and van der Schoot, 2008). This is supported by observations that engraftment of human hematopoietic cells into immune-compromised mice is supported by the nonobese diabetic (NOD) SIRPα mutation, which enables high affinity recognition of human CD47 (Kwong et al., 2014; Takenaka et al., 2007; Yamauchi et al., 2013). CD47 binding to SIRPα is generally species-specific (Kwong, 2014; Subramanian et al., 2006; Takenaka, 2007), and CD47 in the parental C57Bl/6 mice used by Yamauchi is known to bind only weakly to human CD47 (Kwong, 2014). Conversely, down-regulation of CD47 expression on HSCs from hemophagocytic lymphohistiocytosis patients correlated with their increased engulfment by macrophages (Kuriyama et al., 2012). Thus, increasing CD47/SIRPα interactions can increase hematopoiesis, and decreasing the interaction can limit hematopoiesis. However, other studies have clearly shown that some tissues lacking CD47 can engraft in a WT host (Wang et al., 2010), and the successful hematopoiesis in cd47−/− and sirpacytΔ mice clearly indicates that HSCs can form and mediate hematopoiesis in the complete absence of CD47/SIRPα signaling. Presumably other don't eat me signals compensate for the missing CD47 in these models (Oldenborg, 2000).</p><p>In addition to highly expressing CD47, HSCs have been reported to express SIRPα (Seiffert et al., 2001). This suggests that CD47/SIRP interactions may modulate intercellular signaling between HSCs, but this idea remains to be explored.</p><p>In the context of hematopoietic cell recirculation, evidence that CD47 regulates transmigration of monocytes and T cells through endothelial or epithelial monolayers suggested an additional role for CD47 in the trafficking of bone marrow-derived HSCs (Cooper et al., 1995; de Vries et al., 2002; Liu et al., 2002; Liu et al., 2001). However, another study concluded that cd47−/− polymorphonuclear neutrophils have no defect in transmigration. Rather, the lower number of neutrophils at a site of inflammation resulted from a CD47-dependent defect in granulopoiesis (Bian et al., 2013). This was associated with a deficiency in IL-17 levels, suggesting that CD47 indirectly regulates the differentiation of specific hematopoietic lineages. Activity of the CD47 antibody MAb100.1, which inhibited stroma-supported erythropoiesis in vitro of erythroid progenitor cells co-cultured on stromal cells (Furusawa et al., 1998), further implicates CD47 in the lineage-specific differentiation of HSCs.</p><!><p>As shown in Figure 1, one proposed function of CD47 expressed on stem cells is to protect the stem cells from clearance by phagocytic cells. This is a passive function of CD47, where the active signaling is mediated by SIRPα on the phagocytes. Changes in CD47 expression during HSC mobilization are consistent with this function. Experimental mobilization of HSCs using cyclophosphamide/G-CSF showed increased cell surface CD47 levels on cKit+ cells at day 2 (Jaiswal et al., 2009). The highest expression of CD47 on progenitors occurred just prior to entry into the bloodstream, and was maintained during migration to splenic, liver and bone marrow sinusoids. By day 5 when egress stopped, levels returned to near baseline. HSCs in cord blood and peripheral blood similarly showed elevated cell surface CD47 levels. Parallel experiments in WT versus cd47−/− mice indicated that CD47 is not necessary for the migration of HSCs during mobilization. Consistent with other studies, HSCs from cd47−/− mice did not engraft in irradiated WT recipients, and cd47−/− cells failed to colonize WT bone marrow in a cd47−/−/WT parabiosis model. All of these results are consistent with a protective role of CD47 for inhibiting phagocytic clearance of stem cells.</p><p>The increased clearance of HSCs with reduced CD47 expression in patients with hemophagocytic lymphohistiocytosis provides additional correlative evidence for a protective function of CD47 in human stem cells (Kuriyama, 2012). This parallels the reduced colonization of hemizygous cd47+/− bone marrow cells relative to WT bone marrow cells when transplanted into an irradiated WT recipient (Jaiswal, 2009). This clearly establishes that reduced CD47 expression on HSC creates a fitness deficit, and this deficit correlates with their increased sensitivity to macrophage phagocytosis. Direct evidence was obtained for preferential macrophage clearance of transplanted cd47+/− cells when macrophages were activated in the recipient mice using lipopolysaccharide. Therefore, under these experimental conditions increased cell surface expression of CD47 on HSCs provides a significant protection against macrophage-mediated clearance.</p><!><p>Mammalian stem cells resemble the blastomeres of planktonic and benthic organisms with small eggs, and their persistence in adult organisms may contribute to the growth and maintenance of tissues via proliferation and the regulation of organ size via cell loss (Weissman, 2015). Adult stem cells are maintained in a quiescent state but are able to exit quiescence and rapidly expand and differentiate in response to stress or injury to support tissue repair. TSP1 expression is induced in response to certain acute and chronic injuries, and mice lacking TSP1 or CD47 show an increased capacity to recover from such injuries (Isenberg et al., 2007a; Isenberg et al., 2007b; Isenberg et al., 2008a; Isenberg et al., 2008b). CD47 and/or TSP1 inhibition protects tissues in WT mice in the same injury models (Isenberg et al., 2007c; Maxhimer et al., 2009a). Some of these protective responses involve increased NO/cGMP signaling, and other studies demonstrated that TSP1 signaling through CD47 inhibits NO/cGMP signaling (Roberts, 2012). The NO/cGMP pathway also regulates differentiation of stem cells (Mujoo et al., 2011), suggesting that enhanced NO signaling in tissue stem cells may account for some effects of CD47 blockade on tissue regeneration. However, other studies revealed that TSP1 signaling through CD47 regulates stem cell self-renewal by controlling expression of the transcription factors c-Myc, Oct4, Sox2 and Klf4, and blockade of CD47 signaling increases stem cell self-renewal by increasing expression of these four transcription factors (Kaur et al., 2013). Consistent with an inhibitory role of TSP1/CD47 signaling in stem cell maintenance, NO donors were previously shown to dose-dependently induce expression of Oct4 expression in multipotent progenitors isolated from mouse bone marrow (Chu et al., 2008). However, this effect of NO appeared to be independent of cGMP signaling.</p><p>thbs1−/− mice have more circulating CD13+/VEGFR-2+/CD45−/CD117+ endothelial progenitor stem cells (EPCs) relative to WT mice (Shaked et al., 2005). The increase in EPCs was suppressed by using a drug targeting CD36, suggesting that this activity of TSP1 is mediated via CD36. On the other hand, EPCs express high levels of CD47, and knockdown of CD47 expression enhanced their cell proliferation and angiogenic potential (Smadja, 2011). Recombinant human TSP1 inhibited the angiogenic potential of EPCs in vitro, which was mediated by CD47 binding. Ischemia and granulocyte macrophage-colony stimulating factor induced mobilization of EPCs for neovascularization (Takahashi et al., 1999). Enhancement of EPCs is a potential strategy for treating ischemic vascular diseases as well as for tissue regeneration, and these studies suggest that therapeutics inhibiting TSP1/CD47 signaling could achieve this goal.</p><p>Primary human umbilical vein endothelial cell cultures can be converted to iPS cells by forced expression of the transcription factors Klf4, Oct4, Sox2, and c-Myc (Lagarkova et al., 2010; Panopoulos et al., 2011). We found that primary murine lung endothelial cells isolated from thbs1−/− and cd47−/− mice spontaneously undergo a similar reprogramming to a multipotent stem cell state without requiring transfection or exogenous stem cell growth factors (Kaur, 2013) (Fig. 2). Expression of the four stem cell transcription factors is higher in cd47−/− and thbs1−/− primary lung endothelial cells. WT primary endothelial cells become senescent and stop proliferating when deprived of serum, but the null cells continue growing and spontaneously form multicellular structures consistent with embryoid bodies (EBs). Several pluripotency markers are expressed in these EBs including Nanog, stage-specific embryonic antigen-1 (SSEA1), and cKit. As expected for multipotent stem cells, asymmetric cell division is up-regulated in the induced cd47−/− EBs. The multipotency of these EBs was established by demonstrating their ability to differentiate into mesodermal, ectodermal, and endodermal lineages when exposed to appropriate differentiating growth factors. The four stem cell transcription factors are also expressed at higher levels in some tissues of cd47−/− and thbs1−/− mice, and immunohistochemical analysis indicated that Sox2+ stem cells are more abundant in lung and spleen tissues of these mice.</p><p>These studies suggest that suppression CD47 or TSP1 expression could be a therapeutic approach to enhance self-renewal for tissue regeneration. Our previous studies established that therapeutics designed to suppress CD47 expression or TSP1 binding to CD47 enhance the recovery of tissues subjected to ischemic injuries or ionizing radiation in rodents and miniature pigs (Isenberg, 2007c; Isenberg et al., 2008c; Maxhimer, 2009a; Maxhimer et al., 2009b; Soto-Pantoja et al., 2013). Antisense knockdown of CD47 acutely elevated cMyc, Sox2, and Oct4 expression in WT cells (Kaur, 2013). Conversely, re-expression of CD47 in CD47-deficient cells dose-dependently decreased cMyc expression. Dose–dependent suppression of cMyc mRNA by treatment with TSP1 in WT but not in CD47-deficient cells established that TSP1 is the relevant ligand that induces CD47 signaling to suppress stem cell character.</p><!><p>TSP1 is a major regulator of latent transforming growth factor-β1 (TGF-β) activation, and TSP1 control of latent TGF-β activation is critical for regulation of TGF-β activity in some diseases (Nor et al., 2005; Sweetwyne and Murphy-Ullrich, 2012). Following activation from its latent form, TGF-β engages its receptors and induces signaling to regulate bone development and remodeling. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and contribute to bone remodeling following injury and during tumor metastasis. Treatment of growing human bone marrow-derived MSCs with TSP1 increases the level of active TGF-β (Bailey Dubose et al., 2012). The MSCs express TSP1, and both TSP1 expression and TGF-β activity decrease during osteoblast differentiation. Exposure to TSP1 and active TGF-β blocks the osteoblastic differentiation of MSCs grown in osteogenic media as measured by decreased expression of Runx2, a key transcription factor associated with osteoblast differentiation, and alkaline phosphatase. The inhibitory effect of TSP1 on osteoblast differentiation results from its ability to activate latent TGF-β. A peptide that blocks TGF-β activation by TSP1 restored osteoblast differentiation assessed by increased Runx2 and alkaline phosphatase expression. A TGF-β neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies demonstrate that TSP1 can regulate osteoblast differentiation by activating latent TGF-β. (Bailey Dubose, 2012).</p><p>Similarly, CD47 has been implicated in the differentiation of osteoclasts. Parathyroid hormone-stimulated bone marrow cultures derived from cd47−/− mice showed a significant reduction in multinuclear osteoclasts expressing tartrate-resistant acid phosphatase (TRAP+) and production of M-CSF and RANKL as compared with WT bone marrow cultures (Lundberg et al., 2007). Tyrosine phosphorylation of SIRPα was reduced in cd47−/− bone marrow stromal cells. Stromal cells lacking the cytoplasmic signaling domain of SIRPα (sirpacytΔ) also showed defective osteogenic differentiation, and both the cd47−/− and non-signaling sirpacytΔ stromal cells showed reduced ability to support osteoclastogenesis by WT bone marrow macrophages. Thus, CD47-induced SIRPα signaling is critical for stromal cell support of osteoclast formation. These findings were supported by in vivo evidence that femoral bones of 18- or 28-week-old cd47−/− mice contained significantly reduced osteoclast and osteoblast numbers and exhibited an osteopenic bone phenotype. Thus, a lack of CD47 strongly impairs SIRPα-dependent osteoblast differentiation, resulting in impaired bone formation and reduced formation of osteoclasts (Lundberg, 2007). Blocking antibodies against CD47 and SIRPα also reduced the numbers of TRAP+ osteoclasts formed in cultures of murine hematopoietic cells stimulated by M-CSF and RANKL. Further study identified reduced expression of the osteoclastogenic genes nfatc1, Oscar, Trap/Acp, ctr, catK, and dc-stamp in bone marrow cultures from cd47−/− mice stimulated with parathyroid hormone or 1α,25(OH)2-vitamin D3 (Koskinen et al., 2013). Stromal cells lacking either CD47 or the cytoplasmic tail of SIRPα were defective in supporting osteoclastogenesis in WT bone marrow-derived macrophages. Therefore, CD47/SIRPα signaling in stromal cells is necessary for supporting the osteoclast differentiation of bone marrow stem cells.</p><p>Proliferation and differentiation of osteoclasts depends upon cell-cell fusion. CD47/ SIRPα interactions have been implicated for macrophage fusion (Han et al., 2000), which plays an important role for bone development and differentiation of osteoclasts (Hobolt-Pedersen et al., 2014; Yagi et al., 2006). Further studies are needed to define how TSP1/CD47 interactions and CD47/SIRPα interaction alter osteoclast survival, differentiation, and regeneration.</p><!><p>Although decreased expression of CD47 can be advantageous for nontransformed stem cells, data from a number of laboratories has established that cancer stem cells (CSCs) frequently express higher levels of CD47. Furthermore, clinical data for a number of malignancies indicates that high CD47 expression is a negative prognostic factor (Majeti et al., 2009; Willingham et al., 2012; Yoshida et al., 2015; Zhao et al., 2011). Finally, murine xenograft and syngeneic murine tumor models show favorable responses to CD47 knockdown or treatment with function-blocking CD47 antibodies (Lee et al., 2014b; Majeti, 2009; Maxhimer, 2009b; Soto-Pantoja et al., 2014; Zhao, 2011). One fundamental question raised by these data is why CSCs appear to respond differently than normal stem cells to modulation of CD47 expression or signaling. Currently, there are two major hypotheses to explain a selective advantage of high CD47 expression in CSCs (Fig. 3). The "don't eat me" hypothesis proposes that resistance to macrophage-mediated phagocytic clearance provides a selective pressure for CSCs to maintain high CD47 expression. According to this hypothesis, CD47 serves a passive role as a counter-receptor for SIRPα, resulting in a myeloid-specific immune checkpoint (Fig. 3A). The second hypothesis posits that CD47 has a cell-autonomous function in CSCs that promotes their maintenance (Fig. 3B). The "don't eat me" hypothesis is analogous to that discussed above for adult stem cells and in the context of cancer has been reviewed elsewhere (Chao et al., 2012a; Chao et al., 2012b; Weiskopf and Weissman, 2015). Here we will focus on the second hypothesis, which has gained experimental support from several recent publications.</p><!><p>Cell surface CD47 expression is elevated on human and mouse myeloid leukemia cells (Jaiswal, 2009) and on a subset of self-renewing leukemia stem cells (Majeti, 2009). This increased CD47 expression predicted decreased overall survival in three independent cohorts of adult acute myeloid leukemia patients. CD47 was also identified in an unbiased proteomics screen of plasma membrane proteins on CD34+ acute myeloid leukemias from two patients (Bonardi et al., 2013). This finding was extended to tumor-initiating cells from primary human bladder carcinomas defined as Lineage-CD44+CK5+CK20− (Chan et al., 2009). These cells showed higher CD47 expression than the bulk tumor cells. Similar up-regulation of CD47 was found in the granulocyte-macrophage progenitor population of patients with myelodysplastic syndrome at the high-risk refractory anemia with excess blasts (RAEB) stage (Pang et al., 2013). Another study found that elevated CD47 on erythroblasts of myelodysplastic syndrome patients positively correlated with their peripheral red blood cell count, consistent with HSC function (Jiang et al., 2013). Association of high CD47 expression with stem cell/tumor-initiating populations has also been reported in gastric cancer (Yoshida, 2015), hepatocellular carcinoma (Lee, 2014b), and pancreatic ductal adenocarcinoma (Cioffi et al., 2015).</p><p>Elevated CD47 expression has been associated with a metastasis-initiating population of human luminal breast cancers (Baccelli et al., 2013). The metastasis-initiating cells expressed EPCAM, CD44, CD47 and MET. In patients with metastases, the number of EPCAM+CD44+CD47+MET+ circulating tumor cells, but not of bulk EPCAM+ circulating tumor cells, correlated with decreased overall survival and an increased number of metastasic sites. Basal breast cancers are known to have a higher percentage of tumor initiating cells and to express a higher level of CD47 than luminal breast cancers (Zhao, 2011). Recently we reported that CD47 is expressed higher on breast CSCs than differentiated cells derived from the triple negative MDA-MB-231 cell line (Kaur et al., 2016). Analysis of TCGA data for patients with basal triple-negative breast carcinoma showed higher CD47 expression relative to tumors from patients with HER2+ and ER+ luminal breast cancers, but CD47 expression within the triple negative group did not correlate significantly with overall survival.</p><!><p>Microarray analysis of tumor initiating cells from hepatocellular carcinoma identified multiple genes with altered expression when CD47 expression was suppressed using shRNA (Lee, 2014b). A number of these genes showed parallel changes in expression when the tumor initiating cells were induced to differentiate. Among these, cathepsin-S mRNA in the tumor initiating cells was decreased when the cells differentiated and when CD47 expression was suppressed. CD47 expression correlated with that of cathepsin-S in hepatocellular carcinoma patient specimens, and CD47 regulated cathepsin-S expression in tumor initiating cells through activation of NFκB. Cathepsin-S in turn controlled tumor initiating cell migration and invasion through activation of protease-activated receptor-2, which is a substrate for this protease. Protease-activated receptor-2 is a G protein-coupled receptor that regulates cell survival, proliferation, and motility (Gieseler et al., 2013) . Consistent with these in vitro results, antisense morpholino suppression of CD47 suppressed the growth and metastasis of hepatocellular carcinoma xenografts in NOD/SCID mice, and combining morpholino knockdown of CD47 with doxorubicin treatment enhanced tumor growth inhibition. This study established that decreasing CD47 expression can cell-autonomously suppress CSCs.</p><p>The mechanisms that elevate CD47 expression in stem cells remain poorly understood. Elements in the CD47 promoter have been defined that respond to aPal/NRF1 and NFκB (Chang and Huang, 2004; Lo et al., 2015), and miR-141 targets the 3'-UTR of CD47 mRNA to inhibit CD47 expression (Tang et al., 2013). High CD47 expression was recently associated with high Hif expression in breast cancers and correlated with stem cell character (Zhang et al., 2015). CD47 mRNA and cell surface protein expression were induced by hypoxia. Chromatin immunoprecipitation demonstrated hypoxia-induced binding of Hif1α and Hif1β to the CD47 promoter. Hif-deficient cells were subject to increased phagocytosis by mouse bone marrow-derived macrophages. Notably, SUM159 breast cancer cells cultured as nonadherent spheroids (mammospheres), which enriches for CSCs, expressed twofold higher CD47 mRNA levels than control adherent cultures. Conversely, shRNA knockdown of CD47 in SUM159 cells reduced their formation of mammospheres and reduced expression of the CSC marker aldehyde dehydrogenase. These data indicate that elevated CD47 expression promotes the specification and survival of breast CSCs in a cell-autonomous manner, independent of CD47 interactions with SIRPα on phagocytes.</p><!><p>Repeated passage in immune-competent mice to select resistant cells from Lewis lung carcinoma resulted in the isolation of cells with increased CSC characteristics as well as increased CD47 expression (Zheng et al., 2015). Notably, the selected cells also had very low TSP1 expression. Treatment of the selected cells with recombinant TSP1 reduced cell proliferation and was associated with increased expression of the cell cycle inhibitor p21 and decreased expression of cMyc, Klf4, Sox2 and Oct4. TSP1 also increased levels of cleaved caspase-3. Notably, knockdown of CD47 using a shRNA vector blocked these responses to TSP1. TSP1 treatment also inhibited proliferation and suppressed sphere formation in human colon cancer (HCT116), non-small cell lung cancer (A549), and cervical cancer (HeLa) cell lines (Zheng, 2015). These data further support a cell-autonomous function of CD47 signaling in CSCs and implicate TSP1 signaling through CD47 in regulating CSC fate.</p><p>CD47 is highly expressed by pancreatic ductal adenocarcinomas and their metastases as compared to normal pancreatic tissues, but CD47 protein expression in the cancers was not significantly correlated with clinical outcome (Cioffi, 2015). However, CD47 expression was significantly elevated when pancreatic adenocarcinoma cells were induced to form nonadherent spheres, relative to the same cell lines grown as adherent cultures. CD47+ and CD133+ stem-like cells exhibited more sphere formation than CD47− and CD133− cells. Consistent with the "don't eat me" hypothesis, treating the pancreatic CSCs with a CD47 antibody that blocks SIRPα binding (B6H12) specifically induced phagocytosis by macrophages. However, the CD47 antibody induced death of pancreatic CSCs that was independent of macrophages. Pancreatic CSCs treated with the CD47 blocking antibody B6H12 exhibited higher annexin-V binding, suggesting that the antibody cell-autonomously induces apoptosis, although other forms of programmed cell death were not excluded. Finally, treatment of mice bearing pancreatic tumor xenografts with B6H12 either as a single agent or in combination with chemotherapy significantly reduced the percentage of tumor cells expressing the CSC surface markers CD133 and SSEA1. These data suggest that, in addition to enhancing innate immune clearance, this CD47 blocking antibody can directly eliminate pancreatic CSCs in vitro and in vivo (Cioffi, 2015).</p><p>We recently found that the CD47 blocking antibody B6H12 directly alters the expression of many genes in human breast CSCs (CD44hi/CD24low) derived from the MDA-MB-231 cell line (Kaur, 2016). B6H12 inhibited asymmetric cell division and cell proliferation of breast CSCs, which is consistent with the pancreatic CSC data (Cioffi, 2015). Treatment with the B6H12 antibody down-regulated the expression of Klf4 mRNA and protein, which contrasts with the elevated Klf4 expression in endothelial cells and tissues of cd47−/− mice (Kaur, 2013). This indicates that CD47 induces different signaling pathways to regulate Klf4 in cancer cells versus normal cells. B6H12 also inhibited proliferation of T47D breast carcinoma cells, but proliferation of the ER+ line MCF7 and the nontransformed breast line MCF10A was enhanced rather than inhibited by B6H12. The latter cell lines contain very low percentages of stem cells, suggesting that this CD47 antibody may be selectively inhibitor for more malignant breast cancers that have a high percentage of CSCs. The positive proliferative responses of MCF10A mammary epithelial cells to the blocking antibody B6H12 is consistent with our previous findings that nontransformed cd47−/− cells proliferate faster than WT cells (Kaur, 2013). The MCF7 data indicates that there may be exceptions to the rule that CD47 signaling triggers opposing responses in nontransformed stem cells versus CSCs.</p><p>The changes in gene expression induced in triple negative breast CSCs following B6H12 treatment also provided some insight into the molecular mechanism by which CD47 signaling regulates breast CSC differentiation. B6H12 strongly suppressed mRNA levels of EGF and EGFR and acutely inhibited EGFR phosphorylation in breast CSCs (Kaur, 2016). EGFR and Klf4 are known targets of the micro-RNA miR-7 (Okuda et al., 2013; Webster et al., 2009), and we further observed that B6H12 increased the expression of miR-7-5P in breast CSCs but not in differentiated cells from the same cancer cell line. Other studies have identified miR-7 as a suppressor of CSC and normal stem cells (Cui et al., 2014; Okuda, 2013; Pek et al., 2009; Zhang, 2015). Therefore, regulation of this noncoding RNA may mediate some of the effects of CD47 signaling on CSCs.</p><p>We also compared genes exhibiting altered expression in bCSC following B6H12 treatment with a list of genes that correlate with CD47 mRNA expression in the Cancer Genome Atlas RNA-Seq data for triple-negative breast cancers (Kaur, 2016). Sixty genes achieved significant correlation with CD47 in both bCSC and triple-negative breast cancer tumor data. Consistent with our in vitro data, EGFR mRNA expression positively correlated with CD47 mRNA expression in triple-negative breast cancer primary tumors. Several known stem cell markers and regulators correlated with CD47 mRNA expression in the TCGA breast carcinoma dataset including a positive correlation with cKit protein expression, negative correlation with phosphorylation of PDK1 at Ser241, and negative correlation with mRNA expression of the transcription factor PATZ1, which maintains stem cells by its regulation of Pou5f1, Nanog, cMyc, and global changes in histone modification (Ma et al., 2014; Ow et al., 2014). Significant negative correlations between PATZ1 and CD47 mRNA expression were also found in TCGA datasets for melanoma, head and neck squamous cell carcinoma, and bladder carcinoma, suggesting a more general regulation of CSCs by CD47.</p><!><p>Specific signaling pathways that are regulated by CD47 have been identified in normal stem cells and in several types of neoplastic stem cells (Figs. 2 & 4). However, it is not clear which of these signaling pathways account for the differential responses of normal and neoplastic stem cells. cMyc is one obvious candidate in that Myc signaling is dysregulated in many cancers. We have shown that Burkitt's lymphoma cells, where the 5' promoter region of cMyc is replaced with Ig heavy chain promoter sequence, are resistant to growth inhibition by CD47 over-expression, and cMyc expression is unperturbed by CD47 in these cells (Kaur, 2013). Others have identified NFκB dependent regulation of cystatin-S/protease activated receptor-2, p21, Gβγ-dependent activation of the PI3-kinase/Akt pathway, and ubiquitin-like with PHD and ring finger domains 1 (UHRF1)-dependent down-regulation of p16INK4A as targets of CD47 signaling in specific cancer cell lines (Boukhari et al., 2015; Lee, 2014b; Sick et al., 2011; Zheng, 2015). Further study is needed to determine which of these CD47 target pathways generalize to other cancer types, and which may differ in neoplastic versus nontransformed stem cells.</p><p>Quiescence may preserve the self-renewal of normal stem cells and is a critical factor in the resistance of CSCs to chemotherapy that leads to relapse of cancer. Improved knowledge of quiescence mechanisms is needed to target drug-resistant quiescent CSCs. For example, quiescent CD34+ cells from CML patients are resistant to the tyrosine kinase inhibitor Imatinib mesylate (Bhatia et al., 2003; Cheung and Rando, 2013). Thus, an improved understanding of the molecular mechanisms of quiescence in adult stem cells is critical for success of molecular targeted therapies (Li, 2011; Maugeri-Sacca et al., 2011). Although TSP1/CD47 signaling clearly plays roles in both normal and neoplastic stem cells, no studies have examined its potential role in stem cell quiescence. Considering that at least some organs in cd47−/− mice have a higher abundance of stem cells, the remarkable radioresistance of cd47−/− and thbs1−/− tissues could result in part from higher stem cell numbers. Validating this hypothesis will require evidence that the null stem cells are more quiescent or intrinsically more radioresistant.</p><p>Studies are beginning to identify the intrinsic and extrinsic regulatory mechanisms that control stem cell quiescence (Hittelman et al., 2010). p53 plays an important role in self-renewal for regulating stem cell quiescence (Lin et al., 2005; Meletis et al., 2006) and is known to regulate TSP1 expression (Dameron et al., 1994; Watnick et al., 2015). Other intrinsic regulatory mechanisms include Hif-1α (Takubo et al., 2010) and negative regulators of the mTOR pathway (Ito et al., 2008; Thompson et al., 2008; Yilmaz et al., 2006). As previously discussed, Hif1α regulates CD47 expression, and other studies have shown that CD47 signaling controls autophagy (Soto-Pantoja et al., 2012), which is downstream of mTOR. Many extrinsic stimuli from the ECM in the stem cell niche also regulate stem cell quiescence including TGF-β and integrin signaling (Li and Bhatia, 2011), activation of which is controlled by TSP1 and CD47, respectively. These studies may guide future efforts to define signaling controlled by TSP1, CD47, and other TSP1 receptors in the stem cell niche.</p><p>Although the origin of CSCs from transformation of normal stem cells rather than de-differentiation of tumor cells into stem-like cells remains controversial, solid tumors clearly contain mixed populations of terminally differentiated cells with a minor population of cancer cells that can initiate tumor growth (Pignalosa and Durante, 2012). It is important to understand the signaling pathways that control self-renewal and survival of CSCs to develop novel therapies to prevent regrowth of tumors following radiation therapy. CD47 is a promising candidate that can act differentially in normal versus CSCs. For this therapeutic strategy to succeed, we need to better understand how the TSP1/CD47 axis regulates stem cells in normal tissues verses tumors.</p><p>It is also important to further investigate how CD47 signaling in CSC regulates their responses to cytotoxic chemotherapy and targeted therapeutics. Published studies have reported synergism between CD47 blockade and doxorubicin (Lee, 2014b) and synergism with some therapeutic antibodies including those targeting CD20 and HER2/Neu (Chao et al., 2010; Zhao, 2011). The latter synergism has been attributed to enhancement of ADCC responses, but another possibility is that altered CD47 signaling in CSC increases their sensitivity to these targeted therapies in a cell-autonomous manner.</p><!><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.</p>

PubMed Author Manuscript

A new crystal form of human acetylcholinesterase for exploratory room-temperature crystallography\nstudies

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 \xc3\x85 resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature.

a_new_crystal_form_of_human_acetylcholinesterase_for_exploratory_room-temperature_crystallography\ns

Introduction<!>Protein expression and purification.<!>Crystallization.<!>X-ray data collection.<!>New crystal form of hAChE<!>Low temperature complexes of P31 hAChE with reversible ligands<!>Donepezil*hAChE low temperature complex.<!>9-Aminoacridine*hAChE low temperature complex.<!>BW284c51*hAChE low temperature complex.<!>Room temperature structure of the BW284c51*hAChE complex<!>Comparison of structures at the tertiary and quaternary levels<!>Conclusions

<p>Structural investigations of acetylcholinesterase (AChE; EC 3.1.1.7) catalytic subunit flourished after its amino acid sequence was deduced from cDNA cloning [1], beginning with the X-ray structure determination of electric ray AChE (Torpedo californica, TcAChE) [2] in 1991. In the following decades more than two hundred AChE structures have been deposited into the Protein Data Bank (PDB). The first solved TcAChE structure represented a breakthrough in understanding the basis of the enzyme's highly evolved catalytic mechanism and provided a 3D template for design of therapeutically important inhibitors, such as donepezil (Aricept, E2020) [3], for partial symptomatic treatment of Alzheimer's disease. This structure also informed the design of oxime antidotes for reactivation of AChE covalently inhibited by organophosphate pesticides and chemical warfare agents. Nearly 95% of all PDB X-ray depositions (~152,500 as of June 2019), including practically all AChE structures, have been obtained from the diffraction experiments run at cryogenic temperatures, 210 °C below human body temperature. Among the AChE structures, less than 20% are structures of therapeutically appropriate, human AChE (hAChE) target.</p><p>The benefits of cryogenic X-ray data collection arise from reduced radiation damage of crystal allowing for high dataset completeness, improved resolution of the diffraction image and good accuracy of resulting atomic coordinates. Our goal is to explore the feasibility of hAChE structural analysis under conditions closer to physiological conditions (37 °C or 310 K) and to use room temperature X-ray data collection (at 22 °C or 295 K), instead of cryogenic temperatures (−173 °C or 100 K). While potentially sacrificing higher resolutions typically obtained at cryogenic temperature our hope is to gain better insight of a range of physiologically more relevant protein conformations indicative of structural flexibility, while also avoiding protein crystal flash freezing and use of cryoprotectants which are known to harm X-ray diffraction quality and interfere with small molecule ligand binding [4]. One way of counteracting crystal damage associated with X-ray exposure at room temperature is to perform diffraction using large, high-quality crystals capable of producing complete diffraction datasets before the radiation causes substantial degradation. Using a hAChE construct lacking one of the protein's three glycosylation sites, we have been able to grow larger than usual crystals of human AChE in the P31 space group. These larger crystals are able to better withstand radiation damage at room temperature and allow for the collection of reasonably complete datasets with acceptable resolution of the final X-ray structure. Unlike previously characterized hAChE crystals (most belonging to P3121 space group and a few to H32, P6, P61 or P21 space groups) the unit cell dimensions of the P31 hAChE crystals appear similar (a = b ≅ c). These crystals can grow ~250 times larger than the previously reported crystals and diffract well at room temperature. In the asymmetric subunit of the P31 form two kinds of hAChE homodimers are observed: the commonly found four-helix bundle (4HB) dimer and a "face-to-face" (FF) dimer. Due to the absence of one of the three hAChE glycosylation sites, the two monomers can establish a dimerization interface close to the opening of the hAChE active center gorge in the FF dimer. This dimer formation does not interfere with ligand access to the active center gorge, thus allowing entrance of small molecules during soaking experiments. We present here the structures of hAChE in space group P31 at room temperature in complex with the specific reversible AChE inhibitor BW284c51, and at low temperature in complexes with the drug donepezil (Aricept, E2020), with BW284c51 and with 9-aminoacridine, a congener of the drug tacrine (Cognex). Comparison of these structures with published structures reveals nearly identical positions of those ligands in the active center gorges of TcAChE and in different crystal forms of hAChE, thus confirming that the P31 hAChE form represents a valid structural template for low temperature studies, and can serve to explore X-ray analysis of hAChE structure under conditions approaching physiological temperature.</p><!><p>Human AChE was expressed in monomeric form truncated at C-terminal amino acid 547 and with a FLAG tag placed at the N-terminal end [5]. Stably transfected Gnt1− HEK293 mammalian cell culture deficient in complex N-glycans was used for expression. Cells were grown at 37 °C and 10% CO2 in Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum. The enzyme was purified on an anti-FLAG affinity chromatography column (Sigma-Aldrich) using proteolytic elution by specific Recombinant HRV 3C protease (Sinobiological.com) at the engineered Prescission protease recognition site. Profinity IMAC Resin (Bio Rad) was used to remove the HRV 3C protease. The N-terminal sequence of the purified, eluted hAChE was G-P-L-E-G-R-. The amino acid sequence of the mature hAChE protein starts at E-G-R- and ends at the truncated C-terminus [5] with the sequence -S-A-T-D-T-L-D547.</p><!><p>Ligands, donepezil, 9-aminoacridine and BW284c51 were obtained from Sigma-Aldrich and 30–60 mM stock solutions were prepared in DMSO. In preparation for co-crystallization, samples of hAChE were dialyzed in 10 mM NaCl, 10 mM HEPES, pH 7, and concentrated to 6–10 mg/mL. About 1 h prior to crystallization, the solution of hAChE was combined with stock solutions of ligands in a molar ratio of 1:5 or 1:10 to obtain binary complexes. Co-crystals were grown by vapor diffusion at 10 °C in sitting drop microbridges or 9-well glass plates (Hampton Research, Aliso Viejo, CA). Well solutions containing 10 mM sodium citrate, 100 mM HEPES, pH 7.5, 7–12% PEG6000 were used in crystallization experiments with donepezil and BW284c51 and 200 mM potassium nitrate, 100 mM HEPES pH 7.5, 9% PEG 3350 were used to produce crystals with 9-aminoacridine. Crystals typically appeared within 2-4 weeks (~0.1-0.2 mm in the longest dimension) and over 2-4 months, depending on the ligand, slowly grew to full size (> 1 mm). Largest crystals were observed in drops that contained 1-3 crystals.</p><!><p>X-ray crystallographic data were collected at room temperature (~22 °C; RT) and from frozen crystals at 100 K. Prior to data collection at 100 K crystals were subjected to two very brief consecutive soaks in the cryoprotectant solutions, first in 12% glycerol followed by 25% glycerol, and then flash cooled by plunging into liquid nitrogen. For the RT data collection, crystals were mounted on Litholoops (Molecular Dimensions, Maumee, OH) directly from the crystallization or soaking drops and kept hydrated using the MiTeGen (Ithaca, NY) room temperature setup. Diffraction data were collected on the ID19 (100 K) and BM19 (293 K) beamlines at SBC-CAT using Pilatus3 X 6 M (ID19) and MAR CCD 165 mm (BM19) detectors at the Advanced Photon Source (APS). X-ray diffraction data were integrated and scaled using the HKL3000 software suite [6]. The structures were solved by molecular replacement using the CCP4 suite [7]. The structure of the apo-hAChE (PDB ID 4EY4) [8] was used as a starting model with all waters and the N-linked glycosylated saccharides removed. Refinement was performed using the phenix.refine program in the PHENIX [9] suite and the resulting structure analyzed with molprobity [10]. The structures were built and manipulated with the program Coot [11]. Figures were generated using Coot, Biovia Discovery Visualizer (Dassault Systems) and PyMol molecular graphics software (Schrödlnger LLC). A summary of the crystallographic data and refinements is given in Table 1. Crystallographic data have been deposited to PDB under codes 6O4W (donepezil*hAChE), 6O4X (9-aminoacridine*hAChE), 6O50 (LT: BW284c51*hAChE) and 6O52 (RT: BW284c51c*hAChE).</p><!><p>Human AChE truncated at the amino acid residue 547 was expressed in a HEK-293 cell line as a monomeric form [5] and is devoid of the N-linked oligosaccharide chain at position 350. While none of the three glycosylation sites (265, 350 and 464) appeared clear enough in the electron density maps to be unequivocally modelled, the absence of an expected oligosaccharide at 350 was obvious from both complete absence of electron density and as inferred from the immediate proximity of the neighboring chain of the homodimer which would be incompatible with oligosaccharide at Asn350 (Fig. 1).</p><p>Glycosylation of Asn350 is clearly visible in published hAChE structures that crystallize in a P3121 space group, for example in 4EY7 (Fig. 1) [8]. Our construct thus forms two distinct dimers in the crystal, one stabilized by the C terminal 4HB dimer interaction and the other one by "face-to-face" interaction of the two subunits near Asn350 in the general vicinity of the two active center gorge openings. Areas and solvation energies of dimer interfaces calculated by the PISA server [12,13] show clear dominance of 4HB dimer interactions, but also significant contribution of the face-to-face interface in the P31 dimer (Table 2).</p><p>It is interesting that in the P31 packing the energetically most stabilizing 4HB dimer interactions (red helices in Fig. 2) are distributed both horizontally and vertically in a 3D network unlike that seen in P3121 packing where only 2D, horizontal layers of hydrophobic "sticky" interactions are formed.</p><p>As a result of this homodimer packing, our P31 crystals are more compact with unit cell dimensions a = 125.18 Å, b = 125.18 Å, c = 130.28 Å, in contrast to hAChE 4EY7 P3121 crystals which have more elongated needle-like appearance (a = 105.15 Å, b = 105.15 Å, c = 322.98 Å) [8]. The influence of glycosylation on crystal packing is further seen in the complex of donepezil with fish TcAChE (PDB code 1EVE) where a different distribution of four glycosylation sites prevents formation of FF dimers, while 4HB dimer interaction remains (Table 2). Instead of FF dimers, a new, less stabilizing face-to-back (FB) dimer is formed (Fig. 1; Table 2) resulting in the 3D distribution of most hydrophobic "sticky" interactions (Fig. 2), but a less compact crystal (a = 111.93 Å, b = 111.93 Å, c = 136.90 Å) [3], The compact P31 hAChE packing is amenable to forming large, more than 1 mm long stick-like crystals (Image 1) with good quality X-ray diffraction properties critical for data collection under conditions of high radiation damage in a room temperature X-ray diffraction experiment.</p><!><p>All three reversible ligands, donepezil (E2020), BW28451c (BW) and 9-aminoacridine (9AA) classify as low nanomolar AChE inhibitors (Table 3) and co-crystalize with hAChE in the same P31 space group with a nearly identical relative orientation of monomers in a 4HB dimer (Fig. 3). A barely distinguishable monomer tilt observed in 9AA and BW complexes compared to E2020 complex (Fig. 3A) seems to indicate only slightly more compact dimers for the donepezil*hAChE complex (black and blue graphs, Fig. 3B) as indicated by differential distance analysis between corresponding amino acid Cα atoms of individual homodimers [18]. This is also reflected in the Cα-based rigid body dimer overlay (Table 4).</p><!><p>The positioning of the S enantiomer of donepezil in our co-crystals is identical to the one found in hAChE co-crystals in 4EY7 [8] (RMSD = 0.3 Å for heavy atoms of the donepezil molecules), based on Cα atom overlays of four hAChE monomers (two monomer chains from each structure) (Fig. 4). Bound donepezil, fully resolved by electron density maps is stabilized in the P31 hAChE (6O4W) active center gorge by precisely the same interactions (Fig. 4) as observed in 4EY7 [8]. This is despite noticeable monomer tilt in the 4EY7 dimers compared to our structure (revealed by mismatch in yellow and teal colored dimer ribbons and associated green graph of the differential distance analysis in Fig. 3) and the presence of the FF dimers in the P31 structure in the general vicinity of the bound donepezil. The observed quaternary structure differences, thus, did not influence binding of the slender donepezil molecule which spans the base and the opening of the hAChE active center gorge in precisely the same manner in all structures. Donepezil is stabilized (Fig. 4) by stacking interactions with aromatic rings of Trp 86, Trp 286, Tyr337 and Phe338, hydrophobic interactions with Tyr341, and hydrogen bonds with backbone carbonyls of Ser293 and Phe295, and hydroxyl of Tyr72. It also hydrogen bonds to five water molecules in the immediate vicinity, H2O water molecules 34, 88, 155, 157 and 260. Consistent with previous observations [8] the binding is somewhat different in the donepezil*TcAChE complex (1EVE) obtained by soaking, with a slight tilt of the plane of the peripheral 5,6-dimethoxy-2,3dihydroinden-1-one ring and a flip of the central piperidine ring. Neither of the two structures are affected by the observed quaternary structure level differences (Fig. 1) between the P31 donepezil*hAChE complex 6O4W and the 4EY7 and 1EVE structures of the same complex.</p><!><p>This is the first structure of 9AA, a congener of the initial Alzheimer's disease drug tacrine (Cognex) in complex with an AChE. Complexes of tacrine with TcAChE (1ACJ) and human BChE (4BDS) have been previously published [19,20]. The only difference between 9AA and tacrine is that in tacrine one of the benzene rings of 9AA is fully saturated. Their binding affinities for AChE are less than two-fold different (Table 3) [15], as is also the case for BChE [14]. Fully resolved by electron density maps 9AA binds at the base of the hAChE active center gorge establishing close stacking interactions between the tricyclic aromatic ring and Trp86 on one side and with Tyr337 on the other side forming a nearly equidistant π orbital sandwich (Fig. 5). Additional weaker stacking stabilization is provided by the 5.8 Å distant imidazole of the catalytic triad His447. Tacrine is stabilized in TcAChE and human BChE (1ACJ and 4BDS structures) in virtually the same position by stacking interactions with Trp86 but the His447 stacking is missing due to ring saturation in tacrine (Fig. 5). A slight ~0.6 Å shift of 9AA towards Ser203 compared to tacrine is consequently observed (Fig. 5). In all three structures, amino groups of both 9AA and tacrine form hydrogen bonds with two adjacent water molecules.</p><!><p>The AChE-selective inhibitor BW284c51, similarly to donepezil, spans the active center gorge of hAChE between Trp86 at the base and Trp286 at the gorge opening. The two inhibitors are similar in size and have similar inhibition constants in the low nanomolar range, yet they are stabilized differently in the gorge of different AChEs. In the hAChE structure 6O50, BW284c51as a symmetric, charged bis-quaternary compound, primarily forms cation-π and π-π interactions between its terminal exocyclic quaternary nitrogens and aromatic rings of Trp86 and Trp286 (Fig. 6), a Coulombic attraction to Glu202, and aromatic hydrophobic π-π stacking of its lower aromatic ring with Tyr337 and upper aromatic ring with Trp286. The terminal alkenes of BW284c51 are additionally stabilized by Tyr337 and Tyr449 at the gorge base and by Leu76 at the gorge opening. In the published complex of BW284c51 with TcAChE (PDB:1E3Q) [21] the lower half of the ligand is nearly identically stabilized at the aromatic gorge base, but the upper benzene ring is drawn by 1–1.5 Å deeper into the gorge to curl (Fig. 6) and establish an additional weak, hydrophobic π-π sandwich between ~5 π distant rings of Trp279 and Tyr334 at the peripheral site (homologous to Trp 286 and Tyr341 in hAChE).</p><p>In our BW284c51*hAChE co-crystalized complex the ligand, fully resolved by electron density maps, extended by ~1 Å farther outside of the gorge than in the BW284c51*TcAChE complex obtained by soaking. This is even ~1.7 Å farther out than the extension of donepezil in its hAChE or TcAChE complexes. In TcAChE both BW284c51 and donepezil extend similarly far outside of the TcAChE gorge, both significantly less than BW284c51 in our hAChE complex. This may reflect steric occlusion caused by the symmetry-related TcAChE molecule in the crystal packing, restricting access at the active center gorge opening by the associated FB dimer. Indeed, the closest residue of the symmetry-associated FB monomer to the bound BW284c51 of 1E3Q is His181, only 3.9 Å away, serving as a steric and possibly also electrostatic obstacle to relaxed ligand binding for the P3121 TcAChE complexes. In our P31 hAChE complexes with 4HB and FF dimers, we do not observe this kind of limitation imposed by crystal packing. In fact, we note very fast ligand soaking into this P31 crystal form of hAChE (Gerlits et al., J. Biol. Chem. 2019, in press).</p><!><p>The unique properties of the novel P31 crystal packing of hAChE allowed us to grow about 1.5 × 0.2 × 0.2 mm3 crystals suitable for X-ray data collection at room temperature (Image 1), without the need to use cryoprotectants and flash freezing of crystals both of which can introduce structural artifacts [4]. We were able to collect a sufficiently complete room temperature X-ray diffraction data set and solve an RT structure of the BW284c51*hAChE complex at the 3.2 Å resolution (6O52; Table 1). The resulting electron density of the RT structure looks surprisingly well-defined and similar to the higher resolution 2.4 Å structure of the same complex collected under LT conditions (6O50; Figs. 7 and 8), perhaps due to the high binding affinity of BW284c51, allowing for their comparison. Ligand conformation and position, and hAChE backbone and sidechain conformations, in both LT and RT structures appear nearly identical, particularly within the active center gorge (Fig. 7). The exception is a relatively small local surface loop difference (with up to 2.4 Å deviations for residues 261–265). In the more detailed differential distance analysis using Ser203 as a frame of reference [18] the overall difference in Cα positions between RT and LT structures appeared slightly larger than the difference between the two monomers from homodimers in the same LT structure (red vs grey traces, Fig. 7C). In fact, the difference between the two LT structures, the one of 9AA*hAChE (6O4X) compared to the BW284c51*hAChE (6O50) complex (black vs grey traces in Fig. 7 graph), appears slightly larger. This may not be surprising for a tight binding of a low nM inhibitor such as BW284c51, which fills the hAChE active center gorge fully, thus stabilizing both ligand and protein conformations, at both LT and RT. The two largest peaks of difference between 9AA and BW284c51 LT complexes correspond to the Ω loop (residues 69–96) and α-helical residue stretch between 337 and 350 (Fig. 7C) that structurally represent the active center gorge lid and the upper gorge "floor". Both of these structural elements participate in stabilization of bound BW284c51 (Fig. 6), but not of the much smaller 9AA that occupies only the base of the gorge (Fig. 5). Hence, the upper part of the gorge opens up slightly even at LT in the 9AA complex, when compared to the BW284c51 LT complex.</p><!><p>Using protein backbone α-carbon-based overlays and resulting RMSD values as criteria, we compared effects of data collection temperature and unit cell symmetry for hAChEs as well as primary sequence differences between hAChE and TcAChE, on the similarity of their monomeric tertiary structure folds and similarity in organization of monomers into 4HB dimeric quaternary structure (Tables 4 and 5). Total of nine structures were included in the comparison. Four of them were P31 hAChEs from this study and four were previously published P3121 complexes of donepezil, tacrine and BW284c51 with TcAChE and hAChE. The human BChE*tacrine complex was also included in the comparison because of the similarity in the BChE backbone fold with AChEs and because of the similar, yet distinct 4HB quaternary organization in the two enzymes. While the resolution of the structures may not allow for a detailed statistical analysis, several obvious "out of range" value comparisons are readily noticeable.</p><p>The effect of diffraction data collection temperature seemed slightly larger on the hAChE tertiary structure than on the quaternary organization of its 4HB homodimer. The 4HB dimer of the BW284c51 RT complex (6O52) overlaid with the LT complex (6O50) had a relatively small RMSD of 0.38 Å (Table 4). When compared to other P31 hAChE LT complexes from Table 4 (numbers in the yellow box), the RMSD ranged from 0.38 Å to 0.63 Å. This was only slightly more than the 0.37 Å to 0.49 Å RMSD range determined for comparison of the same structures with the 4HB dimer of the BW284c51 LT complex. When quaternary comparison was extended to the P3121 dimer of the 4EY7 hAChE and P3121 TcAChE dimers, no difference can be observed between LT and RT complexes (magenta box in Table 4). Temperature-dependent tertiary structural differences revealed from comparison of monomers were, however, larger (Table 5). The comparison of BW P31 hAChE monomers to all other P31 monomers (within the yellow box in Table 5) shows a RMSD range of 0.34 Å to 0.42 Å for the RT complex but a noticeably smaller 0.24 Å - 0.30 Å range for the LT complex. An extension of the tertiary structural comparison to P3121 monomers of the 4EY7 hAChE and P3121 TcAChE monomers, revealed no difference between LT and RT complexes (magenta box in Table 5). Effectively, while RMSD numbers in the two rows in Table 5 corresponding to RT structures were noticeably larger than other numbers within the yellow box of Table 5 (0.34 Å - 0.43 Å vs. 0.26 Å - 0.31 Å; excluding a to b chain comparisons of the same structure), the same did not hold for RMSD values within the magenta box of Table 4 (0.48 Å – 0.92 Å vs. 0.46 Å - 0.94 Å). In conclusion, the effect of temperature was observed only for small RMSD values (≤ 3 Å) when comparing monomer folds within the same P31 space group, but the quaternary structures were not noticeably affected by temperature.</p><p>The effect of unit cell symmetry change from P31 to P3121 for hAChE is emphasized at the quaternary level where 4EY7 hAChE dimers differ from 4HB dimers of P31 hAChE structures by 1.45–1.52 Å RMSD whereas comparison of monomers yields only 0.46–0.53 Å RMSD. It is interesting that the P3121 dimers of the tacrine*TcAChE complex (1ACJ) show noticeably greater similarity with P31 dimers (0.90–1.06 Å RMSD) than the other two P3121 TcAChE complexes containing much longer ligands, donepezil (1EVE, 1.48–1.56 Å RMSD) and BW284c51 (1E3Q, 1.46–1.62 Å RMSD). In addition to binding in the active center gorge these longer ligands also occupy peripheral site at the mouth of the TcAChE gorge. Thus the greater effect of unit cell symmetry might be a consequence of the pressure of peripherally bound ligands on tight packing of FB TcAChE dimers (Table 2; Fig. 1) that also had an effect on the more "curled" conformation of BW284c51 in the P3121 TcAChE complex (1E3Q) compared to its P31 hAChE complex (6O50) shown in Fig. 6C.</p><p>The effect of primary sequence differences on quaternary and tertiary AChE structure is illustrated by cross-species comparisons between hAChE and TcAChE within the same P3121 space group (teal boxes in Tables 4 and 5). A larger magnitude RMSD difference is observed in quaternary structure comparisons between species relative to those within species (0.96–1.45 Å between hAChE and TcAChE dimers vs. 0.32–0.59 Å within TcAChE dimers). The difference was also significant for the tertiary structure comparisons (0.93–0.97 Å between hAChE and TcAChE dimers vs. 0.32–0.39 Å within TcAChE dimers). Thus, both monomers and dimers are different between AChEs from different species although they crystalize in the same space group.</p><p>Comparisons of AChEs with the human BChE*tacrine complex (4BDS) reveal two interesting points. The BChE tertiary backbone structure was similarly distinct to that of both TcAChE and hAChE structures with ~1.1 Å RMSDs (Table 5) which is relatively close to comparisons of TcAChE vs. hAChE (~0.9 Å RMSDs). The BChE quaternary structure was, however, clearly distinct from those observed in AChEs (~3 Å RMSDs; Table 4). Still, it appears that 4HB dimers of BChE were more similar to 4HB dimers of P31 hAChE (~2.7 Å RMSD) than to P3121 4HB dimers (3.23 Å – 3.57 Å RMSD). The exception is the P3121 TcAChE*tacrine dimer (1ACJ) which had a 2.75 Å RMSD (Table 4). It seems that the size of a ligand (small tacrine or 9-aminoacridine vs. large donepezil (E2020) or BW284c51) and its binding site within the active center gorge of either AChE or BChE could influence, at least modestly, the quaternary structures of these enzymes.</p><!><p>Crystallogenesis from a glycosylation-deficient form of hAChE yields relatively large (1.5 mm × 0.2 mm × 0.2 mm) crystals of space group P31 and a new unit cell, not previously obtained for AChE from any species. Specific arrangement of monomers in the P31 hAChE crystals simultaneously includes two kinds of homodimers, the previously observed 4HB dimer and a novel FF dimer, thus creating a compact 3D packing amenable to formation of large, well-diffracting crystals suitable for RT X-ray crystallography.</p><p>Co-crystallization of hAChE-ligand complexes in the P31 unit cell with ligands binding to either the base of the active center gorge or with those spanning its full length were not affected by FF dimer formation and revealed LT X-ray structures nearly identical to those obtained in previous LT X-ray structures. The P31 hAChE crystals are readily amenable to soaking by ligands of the shape and size used in this study.</p><p>Successful determination of a RT hAChE structure at 3.2 Å from large P31 crystals of BW284c51*hAChE complex lays the groundwork for future comprehensive studies of RT structures of lower affinity complexes, such as oxime reactivators associated with hAChE, where more temperature-related diversity could be expected in both oxime and hAChE conformations (Gerlits et al., J. Biol. Chem. 2019, in press) to better inform structure-based design of novel reactivators of hAChE under conditions approaching physiological temperature.</p>

PubMed Author Manuscript

Natural gas at thermodynamic equilibrium Implications for the origin of natural gas

It is broadly accepted that so-called 'thermal' gas is the product of thermal cracking, 'primary' thermal gas from kerogen cracking, and 'secondary' thermal gas from oil cracking. Since thermal cracking of hydrocarbons does not generate products at equilibrium and thermal stress should not bring them to equilibrium over geologic time, we would not expect methane, ethane, and propane to be at equilibrium in subsurface deposits. Here we report compelling evidence of natural gas at thermodynamic equilibrium. Molecular compositions are constrained to equilibrium,and isotopic compositions are also under equilibrium constraints:The functions [(CH4)*(C3H8)] and [(C2H6)2] exhibit a strong nonlinear correlation (R2 = 0.84) in which the quotient Q progresses to K as wet gas progresses to dry gas. There are striking similarities between natural gas and catalytic gas generated from marine shales. A Devonian/Mississippian New Albany shale generates gas with Q converging on K over time as wet gas progresses to dry gas at 200°C.The position that thermal cracking is the primary source of natural gas is no longer tenable. It is challenged by its inability to explain the composition of natural gas, natural gases at thermodynamic equilibrium, and by the existence of a catalytic path to gas that better explains gas compositions.

natural_gas_at_thermodynamic_equilibrium_implications_for_the_origin_of_natural_gas

Background<!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!>Conclusion<!>Competing interests<!>Authors' contributions<!>Acknowledgements

<p>The hydrocarbons in natural gas are believed to come from two sources, one biological ('biogenic gas'), and the other from thermal cracking, 'primary thermal gas' from kerogen cracking and 'secondary thermal gas' from oil cracking [1,2]. Although there is general agreement on the source of biogenic gas, disagreement persists over the origin of thermal gas. One point of controversy is that thermal cracking does not produce a gas resembling natural gas. Oil and kerogen pyrolysis typically give between 10 and 60% wt methane (C1–C4) [3-9] while natural gas contains between 60 and 95+% methane [10]. None of the explanations that have been offered to explain this [11-15] are satisfactory [16]. Catalysis by reduced transition metals can, in theory, explain high-methane in natural gas [17], and this hypothesis is supported by experimental results. Crude oils and n-alkanes decomposed over reduced nickel and cobalt oxides produce gas resembling natural gas in molecular and isotopic compositions [18]. There is, however, no evidence of metal activity in sedimentary rocks and therefore no compelling reason to question thermal cracking theory. Moreover, recent hydrous pyrolysis experiments with metal-rich Permian Kupferschiefer shale showed little evidence of catalytic activity, seemingly dismissing the possibility of a catalytic path to natural gas [19].</p><p>This changed with the recent disclosure of natural catalytic activity in marine shales at temperatures 300° below thermal cracking temperatures [20]. Shales generated gas under anoxic gas flow at 50°C, nearly five times more gas than the same shale would generate at 350°C through thermal cracking. Although there was only indirect evidence for transition metals as the active catalysts, it nevertheless established natural catalytic activity in source rocks believed to be major sources of natural gas. There are, therefore, two possible paths to natural gas, a thermogenic path operating almost exclusively at high temperatures, and a catalytic path operating at much lower temperatures. The latter redefines the time-temperature dimensions of gas habitats opening the possibility of gas generation at subsurface temperatures previously thought impossible.</p><p>Thermodynamic equilibrium could shed light on which of these paths might dominate in nature. Thermal reactions are generally under kinetic control and their products removed from thermodynamic equilibrium while catalytic reactions are often under equilibrium control and their products near equilibrium. Hydrocarbons can achieve equilibrium through metathesis where homologues interconvert (Reaction 1).(1)</p><p>Olefin metathesis [21,22] is a well-known catalytic reaction shown in Reaction 2 for propylene. It is an extraordinary catalytic process because it breaks and makes carbon-carbon double bonds and reshuffles olefinic carbons distributing them randomly to new partners. Catalyzed by a variety of transition metals, it proceeds to equilibrium at low temperatures with conservation of π and σ bonds [23]. Metathesis of methane, ethane, and propane is illustrated in Reaction 3, referred to here as 'gas metathesis'. Although hypothetical, it bears a strong resemblance to olefin metathesis in stoichiometry (1) and to low-temperature gas generation in marine shales [20]. Hydrocarbons decompose over reduced nickel and cobalt oxides to C1–C3 compositions near equilibrium, possibly through catalytic gas metathesis [18]. Equilibrium between hydrocarbons in natural environments is not limited to metathesis, however. Metastable equilibria have been reported from the interaction of hydrocarbons, water, and authigenic mineral assemblages [24-26].(2)(3)</p><p>Gas metathesis without the aid of a catalytic agent is highly unlikely. Hydrocarbon cracking generates methane, ethane, and propane removed from thermodynamic equilibrium [3,27], and their extraordinary thermal stabilities [28,29] preclude equilibrium over geologic time. Thus natural gas at metathetic equilibrium (3) would implicate catalytic assistance.</p><p>Here we address metathetic equilibrium in shale gas generation and in natural gas deposits, and discuss the genetic implications.</p><!><p>Methane or propane tends to dominate the hydrocarbons emerging from marine shales under anoxic isothermal gas flow [20]. Fig. 1 illustrates two examples, a methane-rich gas from a high-maturity Mississippian Barnett shale (a) and a propane-rich gas from a lower-maturity Devonian/Mississippian New Albany shale (b).</p><!><p>Shale decomposition products under anoxic conditions, 200°C isothermal helium flow. The figure shows hydrocarbon concentrations (ppm vol) in the effluent gas coming off the indicated shales over time. The experimental procedure and product analysis are described elsewhere [20]. (a) Barnett Shale is Mississippian from the Delaware Basin, TX (Reeves County, well cuttings, 3500 m). Yield = 0.04 mg gas/g rock (C1–C5). Rock-Eval: TOC = 8.1% wt; S1 = 0.95 mg/g; S2 = 1.1 mg/g; S3 = 0.25 mg/g; Tmax = 548. (b) Upper Dev/L Miss New Albany Shale from the Illinois Basin, KY (side wall core, 1025 m). Rock-Eval: TOC = 6.2% wt; S1 = 2.2 mg/g; S2 = 17 mg/g; S3 = 0.3 mg/g; Tmax = 448. Yield = 1.2 mg gas/g rock (C1–C5).</p><!><p>The possibility that these are preexisting hydrocarbons desorbed under isothermal gas flow is inconsistent with the order in which hydrocarbons are released over time. Desorption under isothermal gas flow is a first order process where light hydrocarbons (Cx) will desorb before heavy hydrocarbons (Cy) with concentrations [Cx] and [Cy] in the effluent gas stream falling exponentially over time [30]. In our analysis of exponential desorption, ratios ([Cx]/[Cy]) will also fall exponentially over time irrespective of the concentrations adsorbed on surfaces or in solution. Thus, the relative amounts of heavy and light hydrocarbons adsorbed will effect the ratio ([Cx]/[Cy]), but not its exponential fall over time with isothermal gas flow. It would not be possible for ([Cx]/[Cy]) to remain constant or increase over time, for example.</p><p>There is no evidence of desorption in Fig. 1. [C1]/[C3] in (a) rises from 20 to over 100 in the first 30 minutes of gas flow, then falls to 60 at the end of data collection. The ratio in (b) is nearly constant over 35 minutes of gas flow (~0.3), then rises sharply to 2.5 at the end of data collection. The remarkable proportionality between ethane and propane sustained throughout both reactions and its independence of methane concentrations are also inconsistent with desorption. Since desorption under isothermal gas flow should not produce [C2]/[C3] ratios remaining constant over time and [C1]/[C3] ratios increasing over time, it must be dismissed as a possible source of the gases produced in these experiments.</p><p>The gases are distinct in one other respect. Barnett gas is near thermodynamic equilibrium while New Albany gas is far removed from equilibrium. Equation 4 shows the equilibrium constant K for methane, ethane, and propane (3) (where C1 = CH4; C2 = C2H6; C3 = C3H8).(4)</p><p>Log K for equilibrium at a reaction temperature of 200°C is 0.90 at one atmosphere [31]. The average composition for the gases in Fig. 1 place the quotient Q, Q = (C1 *C3)/(C2)2, at log Q = 0.40 for Barnett gas and log Q = -1.30 for New Albany gas, where C1, C2, & C3 are % vol.</p><p>A catalytic reaction can be metathetic, under equilibrium control, and still yield these hydrocarbons removed from equilibrium. All catalytic reactions tend to equilibrium over time in hydrocarbons that interconvert. Product compositions will therefore change over time, removed from equilibrium initially (short residence times), but approaching equilibrium with time. Thus, in flow reactors where residence times are short, gas metathesis could generate these hydrocarbons removed from equilibrium.</p><p>The New Albany reaction was repeated under closed conditions for evidence of gas metathesis over longer residence times. Gas flow was continued for 20 minutes to insure active gas generation, then the reactor was closed and its contents allowed to stand for 200 hours at 200°C. Fig. 2 shows the changes in gas composition over time and Fig. 3 shows the approach to equilibrium that attends these changes.</p><!><p>Gas compositions over time, closed reactor, anoxic procedure, New Albany shale (Fig. 1), 200°C, Helium. After anoxic helium flow for 20 min., the reactor was closed, and the gas was analyzed (GC) at the indicated times by opening the reactor briefly to allow gas from the reactor to pass into a six-way valve for GC analysis [20].</p><p>Gas composition over time and thermodynamic equilibrium. Gas compositions are shown in Fig. 2. Q is the quotient [(C1)(C3)]/[(C2)2], where concentrations are % vol. The horizontal line is the equilibrium constant K (K = 10) for the reaction conditions [31]. The solid line through the data is from the equation Q(t) = 10*(1-e-αt), where Q(t) is the quotient at time t (hours), 10 is Q at infinite time, and the constant α was set to 0.0094 to fit the data.</p><!><p>The New Albany shale generates gas removed from equilibrium under flow conditions (Fig. 1b) and approaching equilibrium under closed conditions (Fig. 3). Equilibrium over time is a signature of catalysis. In this instance, it progresses to equilibrium and gas dryness in concert (Fig. 2). Natural gases might also progress to equilibrium and dryness in concert if the natural process is similarly catalytic.</p><p>Two methods were used to approximate the equilibrium constant K (eq. 4) in the subsurface. One approximates Gibbs free energies as a function of temperature and pressure [32] and the other log K values at various temperatures for ideal gases at one atmosphere [31]. For temperatures between 325 K and 575 K, and pressures between 3 MPa and 150 MPa, log K falls between 0.9 and 1.3 [32]. The second approximation [31] also places equilibrium limits within the same region: log K = 0.73 (575 K) and 1.4 (325 K). If natural gas compositions are constrained by equilibrium forces, they should have log Q values near these limits relative to the log Q limits for unaltered thermogenic gases.</p><p>Figure 4 is a histogram of log Q for offshore Gulf of Mexico gases [33]. These gases were chosen because they are mostly free gases not associated with crude oils or other materials that might compromise their thermodynamic properties. The gases are divided into non-microbial and microbial according to dryness (% wt C1 in C1–C4). The non-microbial gases are largely within the approximated equilibrium limits, while the microbial gases are clearly removed from those limits.</p><!><p>Histogram log Q, 87 Offshore Gulf of Mexico Gases [29]. % vol was used to calculate Q, the quotient [(C1)(C3)]/[(C2)2]. Microbial gases have % wt C1 (C1–C4) > 99% and average log Q = 3.1 ± 0.53; δ13C1 average -61.7 ± 7.1, a signature considered biogenic. Only one had δ13C1 below – 50. Non-microbial gases have % wt C1 (C1–C4) < 99% and average log Q = 1.2 ± 0.38. The dark vertical lines indicate approximate thermodynamic equilibrium limits for subsurface conditions [31,31]. The log Q region marked 'Thermal Cracking' represents the products of thermal cracking based on laboratory experiments [3,27,34-36].</p><!><p>Gas products from thermal cracking experiments fall within the region marked 'Thermal Cracking' in Fig. 4[3,27,34-36]. We would expect 'primary thermal gas' from kerogen cracking and 'secondary thermal gas' from oil cracking to fall within this region as well. The displacement of natural gases to the right of this region by two log unit is therefore significant.</p><p>Figure 5 shows a similar displacement in 1600 gases from various basins in North America.</p><!><p>Natural gas compositions and their relationship to thermodynamic equilibrium. Histogram of log Q (Q = [(C1)*(C3)/(C2)2]) for 1600 gas compositions obtained from the U.S. Department of Interior; mean log Q = 0.90 ± 0.43 [10]. Hydrocarbon concentrations used to calculate log Q were % vol C1–C5. These gases do not include compositions with C2 or C3 < 0.5% vol. Since concentrations were rounded off to the nearest tenth in the DOI database, values in that range introduced substantial errors in calculating Q. The vertical dark lines and the horizontal bar are defined in Fig. 4.</p><!><p>Figs. 4 &5 challenge the notion that thermal cracking is the source of natural gas irrespective of thermodynamic equilibrium. How do we explain log Q values displaced from where they should be by two log units? The fact that they are displaced towards thermodynamic equilibrium, in this case metathetic equilibrium, raises the possibility that they were generated catalytically under equilibrium constraints, not thermally under kinetic constraint. It is possible, in other words, that there was no displacement; they were generated where they are.</p><p>Figure 6 supports this supposition. It shows a strong correlation between [(CH4)*(C3H8)] and [(C2H6)2] consistent with gas compositions constrained to equilibrium. It follows a power function (the solid line) rather than a linear function (lines parallel to the dashed line). The ratio Q ([(CH4)*(C3H8)]/[(C2H6)2]) thus varies systematically with concentrations, displaced from equilibrium at high concentrations of C2 and C3 (wet gas) and at equilibrium at low concentrations C2 and C3 (dry gas). Fig. 7 shows the approach to equilibrium with gas dryness. The line through the data is an equilibrium curve for a reversible reaction approaching equilibrium over time (t): Q = Kequi(1-e-αt). Time (t) has been replaced with C1/(C2+C3) in Fig. 7, consistent with the generally accepted notion that wet gas converts to dry gas over geologic time [1,2].</p><!><p>Equilibrium plot of C1–C3 (eq. 1) in 1600 natural gases (Fig. 5). Hydrocarbon concentrations are % vol in C1 – C5. These gases do not include compositions with C2 or C3 < 0.5% vol since the data, rounded off to the nearest tenth %, injects unacceptable error into the x and y functions. The dark line through the data is the regression line for the power equation y = 0.0282 x1.308, where y = (C2H6)2, x = (CH4)*(C3H8), and R2 = 0.840. The dashed line is for x/y = 12.0, thermodynamic equilibrium at 400 K [31]. Gas compositions were obtained from the U.S. Department of Interior [10]. The mean log Q (Q = (x/y) for the data = 0.90 ± 0.43.</p><p>Thermodynamic equilibrium and gas dryness. Q = (C1)*(C3)/(C2)2. The data is taken from Fig. 6. The black line passing through the data is the equilibrium curve, where Q approaches the equilibrium constant K (10.4) with gas dryness: Q = 10.4(1-e-α(C1/C2+C3))), α was set to 0.1 to fit the data.</p><!><p>Isotope ratios (13C/12C) in petroleum hydrocarbons are believed to be functions of primary biological inputs and isotope effects in gas generation and decomposition [2]. Isotopic equilibrium is another factor that can alter primary biological isotope ratios. Replacing 12C with 13C lowers the free energy of hydrocarbons because the carbon and hydrogen bonds to 13C are stronger than the same bonds to 12C. Bond energy enhancement increases with carbon number. Replacing 12C with 13C in ethane yields more additional bond energy than it does in methane, for example. Thus, at isotopic equilibrium, 13C will be distributed preferentially in the higher hydrocarbons according to their respective lower free energies [37]. Original 13C input will control the amount of 13C shared between hydrocarbons at equilibrium, but their respective free energies will control how 13C is distributed between them. The distribution of 13C at equilibrium will therefore be independent of original input, rates of gas generation and rates of gas decomposition.</p><p>The isotopic equilibrium reaction for methane and ethane is shown in Reaction 5 and for methane and propane in Reaction 6 (the position of 13C in C3H8 is unspecified).(5)(6)</p><p>Eqs. 7 and 8 are the isotopic equilibrium equations with K1,2 (7) the carbon isotopic equilibrium constant for methane and ethane, and K1,3 (8) the carbon isotopic equilibrium constant for methane and propane: 12Cn and 13Cn are fugacities; 13C2 = 13C12CH6, and 13C3 = 13C12C2H8.(7)(8)</p><p>Table 1 shows the molar isotope ratios and the quotients Q for methane, ethane, and propane in 285 natural gases and it includes data for catalytic gases [18] for comparison, to be discussed below. The quotients Q1,2 and Q1,3 are very close to theoretical equilibrium values at 400 K: K1,2 = 2.039; K1,3 = 3.101 [37]. All ratios show substantial invariance. The variance in Q1,2 and Q1,3 is one half that in the molar isotope ratios within them reflecting the correlations between molar isotope ratios shown in Fig. 8. Table 1 also displays the extraordinary match between catalytic gases and natural gases.</p><!><p>The correlations between molar isotope ratios in methane, ethane, and propane in 285 gases (USGS, Table 1). Ratios are molar, calculated as described in Table 1. The lines are linear regression lines with a coefficients of correlation R2 = 0.638 for 13C2/12C2 with slope (zero intercept) = 2.028, and R2 = 0.47 for 13C3/12C3 with slope (zero intercept) = 3.055.</p><p>Statistical properties of molar isotope ratios and isotopic equilibrium constants in 285 natural gases and 5 catalytic gases.</p><p>Natural gas data was taken from USGS Energy Resource Organic Geochemistry Data Base, http://energy.cr.usgs.gov/prov/og/. Catalytic gases are from octadecene decomposition over reduced nickel oxide (180 – 210°C) [18]. C1–C3 compositions were normalized to % wt carbon. δ13C values were converted to mass ratios which were used to calculate wt 13C at each carbon number: x1 at C1, x2 at C2, and x3 at C3. Wt 13C1 = x1; Wt 13C2 is wt C2 with composition 13C12C = x2+((12/13.00335)x2); wt 13C3 is wt C3 with composition 13C12C2 = x3+((24/13.00335)x3). Wt 12C at C2 was calculated as the total wt 12C at C2 minus the wt 12C in 13C2. Wt 12C at C3 was also the total wt 12C at C3 minus the wt 12C in 13C3. Weights (per 100 g) were converted to moles/(100 g C1–C3) which were used throughout our analysis. The quotient Q1,2 = (13C2)*(12C1)/(13C1)*(12C2) and Q1,3 = (13C3)*(12C1)/(13C1)*(12C3), where concentrations are moles/100 g. Variance (v) is (sd)2 for log (ratio).</p><!><p>Fig. 9 shows the proximities of natural gases and catalytic gases to isotopic thermodynamic equilibrium on a log K scale.</p><!><p>Histogram of isotopic quotients (log Q1,2 and log Q1,3) for 285 natural gases (Table 1). All values of Q1,2 and Q1,3 were calculated as described in Table 1. The arrows beneath the chart (catalytic gas) mark the positions of log Q1,2 and log Q1,3 for Catalytic Gases in Table 1. The vertical lines mark isotopic equilibrium constants at 300 K, log K1,2 = 0.31259; log K1,3 = 0.49602, and at 500 K, log K1,2 = 0.30786; log K1,3 = 0.49142 [37].</p><!><p>The approach to equilibrium with dryness (Figs. 6 & 7) mirrors the experimental results shown in Figs. 2 &3. The isotopic results (Figs. 8 &9) reinforce the position that hydrocarbons in natural gas are generated under equilibrium constraints. It is a metathetic equilibrium and therefore a catalytic equilibrium.</p><p>Other explanations are less satisfactory. It is difficult to explain dry gas generation through thermal cracking [28,29] and harder to explain gas metathesis through thermal stress. Equilibrium requires the facile exchange of carbon atoms between methane, ethane, and propane. Carbon-carbon and carbon-hydrogen σ bonds are broken and reformed with overall bond conservation. This is unprecedented in thermal hydrocarbon reactions and inconceivable without catalytic assistance.</p><!><p>The following results support our position that natural gases are at or near thermodynamic equilibrium:</p><p>1) Gas compositions are significantly displaced from thermogenic compositions to equilibrium compositions (Figs. 5 &6).</p><p>2) There is a strong nonlinear correlation between [(C1)*C3)] and [(C2)2] in which the quotient Q converges on equilibrium as wet gas progresses to dry gas (Figs. 6) consistent with an approach to equilibrium over geologic time (Fig. 7).</p><p>3) The isotopic compositions of methane, ethane, and propane are also constrained to equilibrium compositions (Figs 8 &9).</p><p>We propose catalytic gas metathesis as the source of equilibrium in natural gas. The natural catalytic activity in marine shales [20], or some similar activity in other sedimentary rocks, is probably the source of equilibrium in natural gas deposits. This view is supported by the New Albany shale experiment in which Q progressed to metathetic equilibrium over time as wet gas progressed to dry gas (Figs. 2 &3). A mechanistic connection between degradation to methane [20] and metathesis is suggested.</p><p>Catalysis by low valent transition metals [16-18] may be the source of gas metathesis and degradation in the origin of natural gas. The match in isotope ratios between catalytic gases and natural gases (Table 1) supports this position and the sensitivity of marine shales to oxygen poisoning [20] supports it as well.</p><p>The position that thermal cracking adequately explains the origin of natural gas [1,2] is no longer tenable. It cannot explain the high concentrations of methane in natural gas [16], the distribution of light hydrocarbons [38,39], and the associations with thermodynamic equilibrium reported here. Of the two possible pathways to natural gas, the catalytic path [20] appears the more attractive. It is simple, economic, and does not suffer the now mounting deficiencies challenging thermal cracking theory.</p><!><p>The authors declare that they have no competing interests.</p><!><p>FDM formulated theory, experimental design and supervised the experimental work. DJ contributed to the experimental work and helped in shaping the paper. EH was instrumental in early strategy and in writing the ms.</p><!><p>We thank Stephen Garcia for experimental assistance.</p>

PubMed Open Access

Involvement of hnRNP A1 in the matrix metalloprotease-3-dependent regulation of Rac1 pre-mRNA splicing

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57-nucleotide exon 3b. Rac1b was originally identified through its over-expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signalling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase-3 (MMP-3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP-3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP-3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer.

involvement_of_hnrnp_a1_in_the_matrix_metalloprotease-3-dependent_regulation_of_rac1_pre-mrna_splici

INTRODUCTION<!>Cell culture and MMP-3 treatment<!>Cell transfections<!>Cellular fractionation<!>In vitro transcription<!>RNA-binding assays<!>Rac1 minigene construction<!>RNA extraction, RT, PCR, and qPCR<!>Mass spectrometry<!>Histology and Immunohistochemistry<!>Statistical analysis<!>hnRNP A1 binds to Rac1 alternative exon 3b<!>hnRNP A1 is a repressor of Rac1 exon 3b inclusion<!>MMP-3 treatment disassembles an hnRNP A1-containing complex from Rac1 exon 3b<!>MMP-3-triggered Rac1 splicing regulation is modulated by hnRNP A1<!>hnRNP A1 and Rac1b show inverse regulation in vivo<!>DISCUSSION

<p>Pre-messenger RNA (pre-mRNA) alternative splicing can generate multiple protein variants from a single gene. It is a highly controlled process and represents the major source of protein diversity [Nilsen and Graveley, 2010]. One way alternative splicing is regulated is by the interaction of splicing factors such as serine-arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNP proteins) with their target sequences in the pre-mRNA [Black, 2003]. Typically, SR proteins recognize intronic or exonic splicing enhancers (ISEs and ESEs, respectively) and hnRNP proteins recognize intronic or exonic splicing silencers (ISSs and ESSs, respectively) [Matlin et al., 2005]. In addition, the regulation of alternative splicing by signalling pathways is an important mechanism required for maintenance of cellular homeostasis and deregulation of alternative splicing is associated with pathological conditions and progression to cancer [Blaustein et al., 2007; David and Manley, 2010; Dutertre et al., 2010; Srebrow and Kornblihtt, 2006]. There has been particular interest in the association between tumor progression and alternative splicing [Skotheim and Nees, 2007; Srebrow and Kornblihtt, 2006; Venables, 2004], though our knowledge about how splicing regulation is controlled by pro-tumorigenic stimuli is limited [Cheng and Sharp, 2006; Cheng et al., 2006; Karni et al., 2007; Valacca et al., 2010].</p><p>hnRNPs play important roles in every step of mRNA metabolism. The human hnRNP family consists of at least 24 members, which are among the most abundant nuclear proteins. hnRNP A1, a member of the hnRNP A/B subfamily, has been studied extensively and participates in the regulation of splicing, mRNA export, translation, telomere regulation and micro RNA (miRNA) processing [Cammas et al., 2007; Chabot et al., 2003; Dreyfuss et al., 2002; Guil and Caceres, 2007; Izaurralde et al., 1997; LaBranche et al., 1998; Lewis et al., 2007; Zhang et al., 2006]. Although hnRNP A1 localizes predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm [Pinol-Roma and Dreyfuss, 1992], and this shuttling can be regulated by signalling cascades [van der Houven van Oordt et al., 2000].</p><p>Rac1b, an alternatively spliced isoform of the small GTPase Rac1, was initially identified as expressed in breast and colorectal cancer cells [Jordan et al., 1999; Schnelzer et al., 2000]. Subsequent investigations of purified recombinant Rac1b revealed that it possessed limited GTPase activity, decreased affinity for nucleotides, and differential association with effector proteins [Matos et al., 2003; Orlichenko et al., 2010]. Functional investigation has revealed that Rac1b transforms NIH3T3 cells [Singh et al., 2004], promotes colorectal cancer cell survival [Matos and Jordan, 2008; Matos et al., 2008], and is an essential intermediate in the induction of epithelial-mesenchymal transition (EMT) in mouse mammary epithelial cells exposed to matrix metalloproteinase-3 [Radisky et al., 2005]. EMT is a highly regulated process in which epithelial cells acquire the invasive, motile mesenchymal phenotype [Thiery and Sleeman, 2006]. EMT has been best characterized for its role in development, but recent studies have shown that EMT processes are activated during tumor progression. MMPs can trigger EMT both in vivo and in cell culture [Egeblad and Werb, 2002; Stallings-Mann and Radisky, 2007; Thiery and Sleeman, 2006]. Treatment of functionally normal mammary epithelial cells with MMP-3 promotes cytoskeletal modifications, loss of cell adhesion, acquisition of an invasive phenotype, down-regulation of epithelial markers and up-regulation of mesenchymal markers [Lochter et al., 1997; Lochter et al., 1998; Sternlicht et al., 2000], and was shown to require increased expression of Rac1b [Nelson et al., 2008; Radisky et al., 2005]. However, the processes by which exposure of cells to MMP-3 led to increased expression of Rac1b were unclear.</p><p>In the present study we identify a key regulator of exon 3b inclusion into Rac1 mRNA. Using a proteomic approach, we find that hnRNP A1 binds to the alternative exon 3b and has a strong repressor activity on this exon inclusion. We show that MMP-3 treatment alleviates exon 3b from splicing inhibition by the disassembly of a repressor complex composed of (at least) hnRNP A1 and A2, leading to an enhanced inclusion of this oncogenic exon. We also compare hnRNP A1 and Rac1b expression in normal breast lobules and in breast cancer tissue, finding that progression to cancer is associated with reduced expression of nuclear hnRNP A1 and increased expression of Rac1b, consistent with the loss of a hnRNP A1 repressive role in breast cancer. These results shed light on how a transforming stimulus such as the disturbed expression of MMPs leads to alternative splicing regulation and ultimately to cellular transformation.</p><!><p>SCp2 and EpH4 stable cell lines bearing a tetracycline-regulated MMP-3 transgene were maintained as described previously [Radisky et al., 2005]. For transgene repression, a 5 mg/ml stock solution of tetracycline in ethanol was diluted 1:1,000 into culture medium and changed daily. To stimulate cells with MMP-3, we used medium that had been conditioned by cells grown in the absence of tetracycline. Conditioned medium from cells grown in the presence of tetracycline was used as control. These conditioned media were analyzed by zymography to verify that MMP-3 was only expressed upon removal of tetracycline (Supplementary Figure 1; Lochter et al 1997), and that EMT was induced by extracellular proteolytic activity (not shown). Cells were incubated for 3–5 days in the presence of conditioned media.</p><!><p>For experiments requiring higher transfection efficiency in order to analyze endogenous mRNAs, SCp2 cells were used. DNA transfections were carried out in 6-well plates using 6 ml of Lipofectamine 2000 (Invitrogen) and 2 μg of plasmid DNA in 2.5 ml Opti-MEM. RNAi transfections were performed with 5 μl of Lipofectamine 2000 (Invitrogen) and siRNA concentration was 40 nM in a final volume of 2.5 ml Opti-MEM. Cells were plated 24 h before transfection (3.5×105 cells for DNA and 1.5×105 for RNA). In both cases the medium was changed 4 hours after transfection.</p><!><p>Nuclear and cytoplasmic fractions from either SCp2 or EpH4 cells were obtained either by the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, for small scale) according to the manufacturer instructions or by a commonly used laboratory procedure (for large scale). In the latter case, cells were washed and scraped from 10 10-cm plates in ice-cold PBS, spun, and resuspended in 10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF. After incubation for 10 min on ice, cells were dounce-homogenized (15 storkes with pestle B), and centrifuged at 12,000 g for 10 min. The supernatant (cytoplasmic fraction) was placed in pre-cooled eppendorf tubes, adjusted to 10% glycerol and stored at −80°C. The nuclear pellet was resuspended in 25 mM HEPES-KOH pH 7.9, 25% glycerol, 1.5 mM MgCl2, 1 mM EDTA, 0.6 M AcOK, 0.5 mM DTT, and 0.5 mM PMSF, and kept on ice for 40 min vortexing every 5 min. After a centrifugation step at 12,000 g for 30 min, the supernatant (nucleoplasmic fraction) was placed in a pre-cooled eppendorf tube and stored at −80°C. For RNA-binding studies, the nuclear extract was dialyzed against 20 mM HEPES-KOH pH 7.9, 100 mM AcOK, 0.2 mM EDTA, 20 % glycerol, 0.5 mM DTT, and 0.5 mM PMSF for 2 hours prior to the binding reaction. Protein concentration in the extracts was measured either by the Bradford assay (Biorad) or with the Quant-iT Protein Assay Kit using the Qubit Fluorometer (Invitrogen).</p><!><p>RNAs were synthesized with the Riboprobe in vitro Transcription System (Promega) according to the manufacturer instructions. For radioactively labeled RNAs, [α-32P]-CTP was added to the transcription reactions. Reactions were treated with RQ1-DNase (Promega) and run on a 10% acrylamide/8M urea gel. Bands were cut, eluted overnight, quantified with a scintillation counter and subsequently used for binding reactions.</p><!><p>RNA-affinity purification, UV crosslinking/Immunoprecipitation and RNA EMSA assays were performed as described [Rothrock et al., 2005]. Biotinylated RNA oligos were from Dharmacon. The wt exon 3b oligonucleotide sequence was (5′ to 3′): GTTGGAAGACACATGTGGTAAAGATAGACCCTCCAGGGGCAAAGACAAGCCGATTGCC. For the binding reaction, 50 pmol of 5′-biointylated RNA (Dharmacon) was incubated with 100 μg of SCp2 nuclear extract in a 500 μl binding reaction (3.2 mM MgCl2, 20 mM phosphocreatine, 1 mM ATP, 1.3% polyvinyl alcohol, 25 ng of yeast tRNA, 1 mM DTT, 0.1 μl RNasin (Invitrogen, 40 U/μl), 75 mM KCl, 10 mM Tris, pH 7.5, 0.1 mM EDTA). Binding reactions were incubated with gentle agitation for 30 min at 30°C. Streptavidin agarose beads (Pierce) were pre-blocked in GFB100 (100 mM KCl, 20 mM Tris–Cl, pH 7.5, and 0.2 mM EDTA, pH 8.0), plus 1 μg/μl heparin and washed with GFB100. Pre-blocked strepavidin beads (25 μl) were added to the RNA-binding reactions, with additional heparin, to a final concentration of 0.5 μg/μl, 1.6 mM MgCl2, 100 μl BC400, in a final volume of 1 ml, and incubated for 60 min at 4°C with gentle agitation. RNA–protein-bead complexes were washed with GFB100, resuspended in 2X SDS loading buffer, denatured for 5 min at 95°C, resolved by SDS-PAGE (Acrylamide/Bis 37.5:1, BioRad), and detected by coomassie staining. The "B mut" oligonucleotide was identical to the wt 3b oligo except for nucleotides at possitions16 to 25 that were replaced by a linker containing the Mlu I restriction enzyme sequence as already described [Konig et al., 1998].</p><!><p>Two fragments of the murine Rac1 gene were PCR-amplified using mouse genomic DNA as template and Platinum HiFi DNA Polymerase (Invitrogen). One fragment contained Rac1 exon 3 and 5′ portion of intron 3. The other fragment contained a 3′ portion of intron 3, exon 3b, the whole intron downstream, and part of exon 4. Fragments were joined by overlapping PCR and cloned into the pcDNA3.1(+) vector (Invitrogen). The minigene was sequenced and confirmed not to have any mutations within the exons, intron/exon boundaries, or intron sequences at a distance of 600 nucleotides from exons. Mutations were performed using standard PCR-mediated mutagenesis using the wild type minigene as a template.</p><!><p>RNA was isolated with TRIZOL reagent (Invitrogen) or TRI reagent (Molecular Research Center) and cDNA was synthesized using MMLV-RT (Invitrogen). For quantitative real-time PCR analysis of Rac1 splicing, oligonucleotide primers specific for the exon 3-containig Rac1 splice isoform and for exon 3-exluding Rac1 isoform were used. PCR was carried out using Taq Platinum (Invitrogen) and SYBR green dye. Regular (end point) RT-PCR of Rac1 minigene-derived transcripts was performed by extracting RNA and synthesizing cDNA with a minigene specific primer. PCR was performed with Taq Platinum and minigene specific primers. The ratios of the splicing isoforms were shown not to change between 26 and 32 cycles of PCR, and 28 cycles were routinely carried out. Primer sequences are available upon request.</p><!><p>Proteomic analysis was performed as described previously [Orlichenko et al., 2010]. Briefly, isolated gel bands were excised, destained, reduced and alkylated with dithiothreitol and iodoacetamide. Proteins were digested for 4 h with 0.6 μg trypsin (Promega) in digestion buffer (20 mM Tris pH 8.1/0.0002% Zwittergent 3–16, at 37°C) followed by peptide extraction with 60 μl of 2% trifluoroacetic acid, then 60 μl of acetonitrile. Pooled extracts were concentrated and then brought up in 0.1% formic acid for protein identification by nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analysis using a ThermoFinnigan LTQ Orbitrap Hybrid Mass Spectrometer (ThermoElectron Bremen) coupled to an Eksigent nanoLC-2D HPLC system (Eksigent, Dublin, CA). The MS/MS raw data were converted to DTA files using ThermoElectron Bioworks 3.2 and correlated to theoretical fragmentation patterns of tryptic peptide sequences from the NCBI nr database using both SEQUEST™ (ThermoElectron, San Jose, CA) and the Mascot™ 3 (Matrix Sciences London, UK) search algorithms running on a 10 node cluster. The mass spectrometry analysis was done at Mayo Proteomics Research center (Mayo Clinic College of Medicine, Rochester, MN).</p><!><p>Non-malignant breast (cancer-free prophylactic mastectomy tissue) and breast cancer biopsies were derived from waste surgical material from different deidentified patients, and were formalin-fixed and paraffin-embedded. Sections from four non-malignant and four breast cancer samples were evaluated. For immunohistochemistry, tissue sections were deparaffinized by placing them into three changes of xylene and rehydrated in a graded ethanol series. The rehydrated tissue samples were rinsed in water and sections were subjected to heat antigen retrieval as described by the manufacturer (Dako, Carpinteria, CA). Slices were incubated with the appropriate primary antibody (Rac1b, Millipore #09-271; hnRNP A1, Abcam #ab5832) for 30 min at room temperature. Sections were then rinsed with Tris-buffered saline/Triton-X100 (TBST) wash buffer, and secondary incubation was with DAKO DUAL+, horseradish peroxidase (HRP) for 15 minutes. Tissue slices were rinsed with TBST wash buffer and then incubated in 3,3′-diaminobenzidine (DAB+) (K3467, DAKO), and counterstained with modified Schmidt's hematoxylin. Stained slices were scanned and the stain intensity of hnRNP A1 and Rac1b was assessed using the Aperio positive pixel count algorithm in the Imagescope software (Aperio, Vista, CA). The algorithm used is based on spectral differentiation between brown (positive) and blue (counter) staining.</p><!><p>Statistical significance in the splicing assays, both for the minigene and endogenous transcripts, was tested with a two-tailed t-test. Whenever a difference is said to be observed between two conditions, a p-value<0.05 was obtained except where otherwise indicated.</p><!><p>Rac1b, a splicing variant of Rac1, was shown to be necessary and sufficient for mammary epithelial cell transformation [Radisky et al., 2005]. Exposure of functionally normal mouse mammary epithelial cells lines (SCp2 or EpH4) to MMP-3 activates a signalling pathway that leads to the inclusion of exon 3b into Rac1 mRNA, consequently rendering a constitutively active Rac1 protein. In order to gain insight into the mechanisms regulating Rac1 alternative splicing, we investigated which nuclear proteins have the ability to bind the Rac1 mRNA. Comparing the human and murine exon 3b sequence and its flanking introns, there are two conserved regions, one being the exon itself and the other a region in the downstream intron. Taking this into account, we performed RNA-affinity purification using nuclear extracts from SCp2 cells and a 5′-biotinylated murine exon 3b RNA probe. Upon incubation with nuclear extract, proteins bound to the 3b RNA were pulled-down with streptavidin-agarose beads and subjected to SDS-PAGE and coomassie staining. A doublet of approximately 35 KDa was consistently observed (Fig. 1A) and these bands were isolated, subjected to in-gel trypsin digestion, and analyzed by nanoLC-tandem mass spectrometry, which identified the proteins as hnRNP A1 and hnRNP A2 (Supplementary Figure 2). To confirm this finding, we performed western blot analysis of the samples subjected to mass spectrometry analysis with different antibodies: anti-hnRNP A1, anti-hnRNP A2, and anti-SRSF1 (as a control). We found that both hnRNP A1 and hnRNP A2 were pulled down with the exon 3b RNA but not with a control oligo of the same length, and that the splicing factor SRSF1 (previously known as SF2/ASF) did not bind to the Rac1 exon 3b RNA under the same conditions (Fig. 1B). The interaction between these hnRNPs and Rac1 exon 3b was confirmed by RNA electrophoretic mobility shift assay (EMSA)/supershift (data not shown). Similar results were obtained with the EpH4 cell line. To characterize the hnRNP A1 binding sequence within Rac1 exon 3b RNA, we first searched along the 57-nt sequence for known binding sequences. While no canonical hnRNP A1 consensus sequences were found [Burd and Dreyfuss, 1994], we did identify a 6-nt sequence (UAAAGA) that closely resembles the consensus reported by Chabot and colleagues, matching 5 out of 6 nucleotides [Blanchette and Chabot, 1999] (Supplementary Figure 3). We found that replacing a 10-nt sequence (hereafter termed B element) containing this putative hnRNP A1 binding site by an unrelated sequence ("B mut") substantially decreased hnRNP A1 binding to exon 3b as measured by UV crosslinking followed by hnRNP A1 immunoprecipitation (Fig. 1C). To confirm this result, we performed RNA affinity purification followed by western blot as in figure 1B, but competed the binding reactions with either non-biotinylated wt or non-biotinylated B mut exon 3b RNA oligos. We found that a 30X molar excess of the wt exon 3b RNA greatly diminished hnRNP A1 binding whereas the same molar excess of the B mut exon 3b RNA had no effect (Fig. 1D). These results show that the sequence UAAAGA within exon 3b functions as a bona fide hnRNP A1 binding site.</p><!><p>Since hnRNPs are known to have a strong splicing inhibitory activity, we tested whether hnRNP A1 could regulate Rac1 pre-mRNA splicing. As observed in figure 2A, over-expression of hnRNP A1 diminished exon 3b inclusion into endogenous Rac1 mRNA. In agreement with the identification of hnRNP A2 by our initial RNA affinity-purification experiments, over-expression of hnRNP A2 also decreased exon 3b inclusion levels. We then constructed a Rac1 splicing reporter minigene consisting of exon 3b, part of its flanking exons (3 and 4), its complete downstream intron and a shortened version of its upstream intron (Fig. 2B left panel). We found that over-expression of hnRNP A1 decreased Rac1 exon 3b inclusion in transcripts derived from the co-transfected minigene by 10-fold as assessed by radioactive RT-PCR (Fig. 2B right panel; Fig. 2C). To further define the role of hnRNP A1 in Rac1 pre-mRNA splicing, we created a core hnRNP A1 binding consensus (UAGG, [Burd and Dreyfuss, 1994; Del Gatto-Konczak et al., 1999] within exon 3b. The UAGG-containing exon 3b (termed "UAGG 3b" when referring to RNA and "TAGG 3b" when referring to DNA) binds hnRNP A1 with greater affinity than the wild type exon as determined by EMSA/supershift (Fig. 2D, compare lanes 4 and 8) and the protein-UAGG 3b complex is more resistant to disruption by an anti-hnRNP A1/A2 antibody that recognizes an epitope within one of the RNA recognition motifs (RRMs) of hnRNP A1 and A2 (Fig. 2D, compare lanes 3 and 7). Consistent with a repressive role for hnRNP A1, creation of this hnRNP A1 binding consensus completely inhibited exon 3b inclusion into Rac1 minigene-derived mRNA (Fig. 2E). Importantly, different mutations in the same region had no effect on Rac1 alternative splicing, ruling out the possibility that the splicing effect of the TAGG motif is due to disruption of a positive regulatory sequence within the exon (Supplementary Figure 4). These results show that hnRNP A1 functions as a repressor of Rac1 exon 3b splicing, as either its over-expression or its enhanced binding diminish exon 3b inclusion into Rac1 mRNA.</p><!><p>We then tested whether MMP-3-mediated signalling was acting through an hnRNP A1-dependent pathway to regulate Rac1 pre-mRNA splicing. We reasoned that MMP-3 treatment could be altering the amount of the hnRNPA1-containing complex assembled on Rac1 exon 3b. We assayed for protein complex binding to exon 3b and found that nuclear extracts prepared from MMP-3-treated cells displayed lower exon 3b-protein complex formation than the nuclear extracts prepared from untreated cells (Fig. 3A wt 3b probe; Fig. 3B). This MMP-3 regulatory effect was lost when using the UAGG 3b as a probe (Fig. 3A, UAGG 3b probe; Fig. 3B). Consistent with these findings, UV crosslinking followed by immunoprecipitation with an anti-hnRNP A1 antibody showed decreased binding to exon 3b upon MMP-3 treatment (Fig. 3C), negatively correlating hnRNP A1 binding with exon 3b inclusion. We also found that inclusion of the 3b exon into minigene-derived mRNA was increased in SCp2 cells treated with MMP-3 (Fig 3D, wt 3b minigene) as already observed for endogenous Rac1 transcripts (Radisky 2005), but that this effect was lost with the TAGG mutant minigene (Fig. 3D, TAGG 3b minigene). Similar results were obtained with EpH4 cells (data not shown). hnRNP A1 mRNA and protein levels showed a slight decrease upon MMP-3 treatment (Fig. 3E; Fig. 3F). These results provide a link between MMP-3-regulated Rac1 exon 3b splicing and hnRNP A1 binding. Treatment with MMP-3 significantly reduces its binding to exon 3b and inclusion of this exon is triggered.</p><!><p>Considering that our results pointed to hnRNP A1 as a repressor of exon 3b splicing, we investigated the effect of its knockdown on exon 3b inclusion levels. To this end, a specific siRNA duplex targeting the murine hnRNP A1 mRNA sequence was designed, which caused a reduction of hnRNP A1 protein levels of approximately 50% (Fig. 4A). Endogenous Rac1 pre-mRNA alternative splicing was measured by quantitative real-time PCR using specific primers for exon 3b-containing (3b+) and exon 3b-lacking (3b−) mRNA isoforms (Fig. 4B), and the results were expressed as 3b+ over 3b− ratio. As observed in figure 4C, cells transfected with a control siRNA were still responsive to the MMP-3 treatment showing an increase in the 3b+/3b− ratio (2.5 fold effect, siRNA Ctl.). However, when cells were transfected with the hnRNP A1-targeting siRNA, MMP-3 treatment exerted a greater effect (4.5 fold, siRNA hnRNP A1). It is noteworthy that diminishing hnRNP A1 and/or hnRNP A2 protein levels in untreated cells did not relieve exon 3b from repression, suggesting that in their absence other unidentified factor(s) are responsible for maintaining splicing repression (Fig. 4C black bars and data not shown). Importantly, over-expression of hnRNP A1 decreases exon 3b inclusion into endogenous Rac1 mRNA in a dose dependent manner as assessed by the same quantitative real time-PCR protocol (Fig 4D). These results suggest that Rac1 exon 3b inclusion is repressed under normal conditions by several factors, including hnRNP A1. While a 50% reduction in hnRNP A1 protein levels does not affect the repressed state of exon 3b splicing, it apparently sensitizes the cells to MMP-3 treatment.</p><!><p>Rac1b was previously identified as overexpressed in breast cancer tissue [Schnelzer et al., 2000]. Since we had identified an inhibitory role for hnRNP A1 as a regulator of Rac1b, we assessed whether progression from non-malignant breast to breast cancer was associated with decreased expression of hnRNP A1. Immunohistochemical analysis of non-malignant breast (obtained from prophylactic mastectomy tissue and determined by pathologic analysis to be cancer-free) and of breast cancer tissue revealed that normal breast tissue showed high nuclear levels of hnRNP A1 and diffuse/punctate cytoplasmic expression of Rac1b in the lobular epithelial cells, while breast cancer tissue showed decreased levels of nuclear hnRNP A1 and increased expression of Rac1b (Fig. 5). These results are consistent with a repressive role for hnRNP A1 on Rac1b expression in vivo.</p><!><p>We show here that hnRNP A1 is involved in Rac1 alternative splicing regulation in mouse mammary epithelial cells. Moreover, our experiments provide insight into the mechanism by which MMP-3 stimulates exon 3b inclusion into Rac1 mRNA. We find that hnRNP A1 is bound to the alternative exon 3b under normal conditions and is able to repress exon 3b splicing. Upon treatment with MMP-3, hnRNP A1 is displaced from exon 3b, relieving it from the splicing-repressive effect and leading to an enhanced inclusion of the exon. As suggested by RNAi experiments, it is expected that other proteins playing a repressive function cooperate with hnRNP A1 to maintain low inclusion levels of 3b exon under normal conditions. In addition, it is possible to speculate that not only the displacement of hnRNP A1 but also the binding of a positive regulator is required for MMP-3-triggered increase in 3b exon inclusion (Fig. 6). This remains to be elucidated.</p><p>When exploring the link between cancer and splicing, two kinds of cancer-associated alterations in splicing patterns can be distinguished. One group is composed by alterations that are attributable to mutations that create or disrupt splice sites or splicing enhancers and silencers. However, in only few cases has a causal relationship been determined [Srebrow and Kornblihtt, 2006; Venables, 2004]. In the second group, no mutations have been observed in cis-acting splicing elements within the involved genes, suggesting that the alterations could be due to changes in the activity of trans-acting splicing regulators [Ghigna et al., 1998; Stickeler et al., 1999; Venables, 2006]. Rac1 belongs to this second group, together with Ron, CD44, prolactin receptor, fibroblast growth factor receptor, and MDM2 [Ghigna et al., 2008; Srebrow and Kornblihtt, 2006]. In this respect, a strong connection between expression levels of a splicing factor, alternative splicing and changes in cellular behavior related to tumorigenesis has been described for Ron pre-mRNA. Over-expression of SRSF1 has been postulated to regulate malignant transformation by inducing production of ΔRon, a constitutively active isoform of the Ron receptor tyrosine kinase, in turn leading to the loss of epithelial phenotype and acquisition of migratory capacity [Ghigna et al., 2005]. SRSF1 is up-regulated in many human tumors, partially due to an amplification of its gene SFRS1. By ectopic expression of SRSF1 in culture cell lines, this factor has been defined as an oncoprotein that functions in establishment and maintenance of cellular transformation by altering the splicing pattern of several cancer-related genes [Karni et al., 2007]. Recently, SRSF1 and SRSF3 (previously known as SRp20) levels have been shown to regulate Rac1 splicing in human colorectal tumor cells in a PI3K- and catenin/TCF4-dependent manner. It is noteworthy that although the authors of this latter study did not assess hnRNP A1 involvement in Rac1 splicing in these cells, a mutation that disrupts the hnRNP A1 consensus binding site we report in the present work was found to greatly increase exon 3b inclusion [Goncalves et al., 2009].</p><p>Culture and animal models in which de-regulation of MMPs expression is associated with tumorigenesis have been well described [Lochter et al., 1997; Sternlicht et al., 1999; Sympson et al., 1994]. In particular, expression of an MMP-3 transgene targeted to mouse mammary epithelial cells was shown to induce spontaneous neoplastic progression in the mammary gland [Sternlicht et al., 1999]; and aberrant MMP expression has been linked to activation of EMT in human breast cancer progression as well [Radisky and Radisky, 2010]. Here, we show an inverse pattern of expression for hnRNP A1 and Rac1b in nonmalignant breast tissue and in breast cancer; further studies will be necessary to define a potential role for MMPs in this process.</p><p>During the last few years, the involvement of alternative splicing regulation in EMT has been the focus of gene-specific as well as high throughput studies. Not only the expression of epithelial specific splicing factors but also alternative splicing switches have been proposed as crucial regulatory points for this cellular process and for early steps of metastatic progression [Brown et al., 2011; Ghigna et al., 2005; Valacca et al., 2010; Warzecha et al., 2009]. Recently, an EMT-associated splicing signature controlled by different splicing factors including members of the hnRNP family has been proposed [Shapiro et al., 2011].</p><p>A vast body of work links hnRNP A1 activity with different aspects of tumorigenesis. This protein functions as an auxiliary factor for the processing of mir18-a, a microRNA that belongs to a miRNA cluster with oncogenic potential [Guil and Caceres, 2007; Michlewski et al., 2011]. hnRNP A1 is highly expressed in proliferating and transformed cells [Biamonti et al., 1993] as well as in breast, colon and lung cancers, among others [Zerbe et al., 2004]. Silencing of hnRNP A1 and A2 promotes apoptosis in a variety of human and mouse cancer cell lines, while has no effect on normal epithelial and fibroblastic cell lines [Patry et al., 2003], and hnRNP A2 controls invasive cell migration of ovarian carcinoma cells through alternative splicing of TP53INP2 [Moran-Jones et al., 2009]. Our work provides another physio-pathological context in which hnRNP A1 activity affects the malignant phenotype; identification of the pathways that regulate hnRNP A1 function may provide new insight towards early indicators of cancer development as well as novel approaches to anticancer therapies.</p>

PubMed Author Manuscript

Self-Assembled Biocompatible Fluorescent Nanoparticles for Bioimaging

Fluorescence is a powerful tool for mapping biological events in real-time with high spatial resolution. Ultra-bright probes are needed in order to achieve high sensitivity: these probes are typically obtained by gathering a huge number of fluorophores in a single nanoparticle (NP). Unfortunately this assembly produces quenching of the fluorescence because of short-range intermolecular interactions. Here we demonstrate that rational structural modification of a well-known molecular fluorophore N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD) produces fluorophores that self-assemble in nanoparticles in the biocompatible environment without any dramatic decrease of the fluorescence quantum yield. Most importantly, the resulting NP show, in an aqueous environment, a brightness which is more than six orders of magnitude higher than the molecular component in the organic solvent. Moreover, the NP are prepared by nanoprecipitation and they are stabilized only via non-covalent interaction, they are surprisingly stable and can be observed as individual bright spots freely diffusing in solution at a concentration as low as 1 nM. The suitability of the NP as biocompatible fluorescent probes was demonstrated in the case of HeLa cells by fluorescence confocal microscopy and MTS assays.

self-assembled_biocompatible_fluorescent_nanoparticles_for_bioimaging

Introduction<!>Photophysical Properties of the Molecules in Solution<!><!>Photophysical Properties of the Molecules in Solution<!><!>Photophysical Properties of the Molecules in Solution<!>Preparation and Characterization of the NPs<!><!>Preparation and Characterization of the NPs<!><!>Preparation and Characterization of the NPs<!>Size Characterization of 2NP by Fluorescence Optical Tracking<!><!>Size Characterization of 2NP by Fluorescence Optical Tracking<!>Biological Experiments<!><!>Conclusions<!>Experimental Section<!>Synthesis of 4-Azido-7-Nitrobenzofurazan (NBD-N3)<!>Synthesis of 4-Triarylphosphazo-7-Nitrobenzofurazan (1-3)<!>4-Triphenylphosphazo-7-Nitrobenzofurazan (1)<!>4-Tri-(2-Methylphenyl)Phosphazo-7-Nitrobenzofurazan (2)<!>4-Tri-(2,4,6-Triemthoxyphenyl)Phosphazo-7-Nitrobenzofurazan (3)<!>Absorption and Fluorescence Spectra<!>Steady State Fluorescence Spectra<!>Excited State Lifetimes<!>Fluorescence Anisotropy Spectra<!>Epifluorescence Microscopy<!>Particle Tracking<!>Dynamic Light Scattering<!>Transmission Electron Microscopy<!>Cellular Experiments<!>Data Availability<!>Author Contributions<!>Conflict of Interest Statement

<p>Fluorescence imaging is a not invasive, highly sensitive, technique that allows to investigate biological organisms with high tridimensional resolution in real time, by making use of suitable fluorescent contrast agents (Rio-Echevarria et al., 2011; Cauzzi et al., 2012; Chen et al., 2015; Grimm et al., 2015; Lee et al., 2015; Mei et al., 2015; Tang et al., 2015; Antaris et al., 2016; Proetto et al., 2016; Xu et al., 2016). Tailored fluorescent nanoparticles (NP) (Jiang et al., 2015; Ma et al., 2015; Pyo et al., 2015; Wolfbeis, 2015; Muller et al., 2018), promise to surpass conventional molecular probes as fluorescent markers especially as far as sensitivity is concerned: in fact, NPs potentially emit a much brighter signal, with respect to molecules, in the same excitation conditions. Nevertheless, achieving such an enhanced brightness (defined as B = εQY, where ε is the molar absorption coefficient of the NP and QY is fluorescence quantum yield of the NP) is still a challenge (Ow et al., 2005; Wu et al., 2006; Sun et al., 2010; Cho et al., 2011; Volkov et al., 2011; Trofymchuk et al., 2017; Melnychuk and Klymchenko, 2018).</p><p>Most of the NP proposed and applied for bio-imaging, in fact, result from the assembly of molecular fluorophores (MF) in an organized nanostructure. These assemblies may be stabilized: (i) via covalent bonds, hence by modifying the MF chemical structure with reactive terminal groups [e.g., alkoxysilanes (Rio-Echevarria et al., 2010; Rampazzo et al., 2011; Selvestrel et al., 2013), acrylates (Chen et al., 2009), or thiolates (Battistini et al., 2008; Bonacchi et al., 2016)] to form polymer/copolymer or by (ii) non-covalent interactions, that involve either the MF or additional groups specifically introduced in the structure to achieve supramolecular polymerization (Genin et al., 2014; Montalti et al., 2014; Reisch and Klymchenko, 2016; Faucon et al., 2017; Boucard et al., 2018). Control of size and size distribution is a critical issue in NP design and it can be achieved by exploiting surfactants or stabilizers as templates, hence molecules that are physically or chemically incorporated in the NP typically giving a compartmentalized structure (e.g., core-shell).</p><p>From the point of view of fluorescence brightness, the ability of NP to generate an intense fluorescence signal, even in the low intensity excitation regime, results from the co-presence of a high number of MF in each NP. Here, we report the preparation in a bio-compatible environment of NP with a diameter of about 90 nm containing as much as about 1 × 106 MF/NP obtained by the self-assembling of new molecular fluorophores specifically designed to achieve highly bright NP.</p><p>We would like to stress that without a rational design, MF normally aggregate in highly densely packed NP undergoing strong fluorescence quenching, a process which reduces their QY to almost zero. This phenomenon known as aggregation caused quenching (ACQ) (Genovese et al., 2013), typically occurs in self-assembled multi-fluorophoric systems and it is the result of short-range interactions between MF in the NP.</p><p>ACQ is particularly severe in the case of actual contact between the MF, as it occurs when aggregates are formed, and it can be prevented, at least in part, by spacing the MF (Mak et al., 1999) by incorporating them in an inactive matrix (e.g., silica) as in typical dye-doped NP. A major drawback of this strategy, that becomes effective for NP with a dye/matrix ratio below ~1%, is a drastic reduction of the density of emitting molecules in the NP and hence of the ε of the NP with respect to matrix-free NP.</p><p>Here we describe the synthesis and the properties of a new family of MF, which are water insoluble and do not suffer from ACQ, and we demonstrate that a rational design of the molecular structure allows us to achieve a molecular unit that self-organizes in highly bright, and fluorescent NPs which are very stable in PBS (phosphate buffered saline) solution.</p><p>In designing the new MF, our attention was attracted by the N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD) dye derivatives (Fery-Forgues et al., 1993; Mukherjee et al., 1994). This family of dyes has very interesting properties for imaging applications. In particular the synthetic precursor NBD chloride is quite inexpensive and it is easily conjugated with amine derivatives yielding fluorescent dyes featuring absorption and emission in the visible range, large Stokes shift and good quantum yields.</p><p>Unfortunately, emission of NBD derivative in the aggregated form is often very low and it is almost completely quenched in aqueous environment (Fery-Forgues et al., 1993). We reasoned that such a limitation could be overcome by the introduction of a triphenylphosphazene group in the NBD structure. Indeed such chemical modification, even if scarcely studied, has been reported to result, in the case of fluorescent dyes, into a red shift of both the absorption and luminescence maxima, an increase of the Stokes shift and of the luminescence intensity (Bodige et al., 1999; Nifant'ev et al., 2008; Joshi et al., 2014; Xu et al., 2015;Ragab et al., 2016).</p><p>Three different NBD-triarylphosphazene derivatives were prepared in high yields with straightforward procedures. The NP were then prepared via nanoprecipitation in the presence of Pluronic F127 as a stabilizer and they were characterized, from the photo-physical point of view, via UV-Vis absorption spectroscopy and steady-state and time resolved fluorescence spectroscopy. Although different surfactants have been proposed as stabilizer for nanoprecipitation we chose Pluronic F127 in virtue of its well-known biocompatibility (Pitto-Barry and Barry, 2014). The formation and stability of the NPs were demonstrated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Moreover, wide field fluorescence microscopy proved that these NP are stable at a concentration as low as 1 nM.</p><p>By comparing the NP to the molecular precursors, an increase of the brightness of about five order of magnitude, with respect to the fluorophore in organic solvent, could be estimated as a result of the self-assembly. A direct comparison with the fluorophore in aqueous medium was not possible because of the lack of solubility but making reference to an NBD water soluble derivative, we could appraise for the NP a brightness of more than six orders of magnitude higher. In order to demonstrate that these NPs were suitable for bio-imaging, they were incubated with living HeLa cells and their ability to label the cells was demonstrated via confocal scanning fluorescence microscopy. Finally, toxicity assays demonstrated the high biocompatibility of the NPs. We believe that these highly bright, functionalizable NPs are very promising platform for the design of new versatile multifunctional nanoprobes.</p><!><p>Molecular fluorophores 1-3 are shown in Figure 1 and they were synthesized following the general reaction reported in Figure 1. Photophysical properties of molecules 1-3 where investigated in CH2Cl2 solution at the concentration 5 × 10−5 M and they are summarized in Table 1. In particular, the effect of the substituent on the phenyl ring was investigated. We would like to stress that fluorophores 1-3 are insoluble in water but that they can be dispersed in water in the form of NPs as discussed in the next section.</p><!><p>Chemical formula of compounds 1, 2, 3 and scheme of the synthetic reactions for their preparation.</p><p>Photophysical data of compounds 1, 2, 3, 4 and of 2NP and 3NP.</p><p>PBS.</p><p>Calculated from TEM.</p><p>From Fery-Forgues et al. (1993).</p><!><p>UV-Vis electronic absorption spectra are shown in Figure 2 (continuous lines) together with the normalized fluorescence spectra. The absorption spectrum of compound 1 shows a maximum at λ = 482 nm (ε = 34,300 M−1cm−1) while the fluorescence band presents a peak at 534 nm. Both absorption and fluorescence maxima are very close to those reported for the same molecule in acetonitrile (Ragab et al., 2016), and they are very similar to those reported for the parent compound, diethylamino-NBD (4) (Fery-Forgues et al., 1993). The photophysical properties of NBD derivatives in different solvents have been investigated by Lopez and coworkers who reported for 4 in dichloromethane an absorption and fluorescence band with maxima at 482 and 534 nm, respectively (Fery-Forgues et al., 1993). These bands are attributed to an electronic transition with charge transfer (CT) character, the amino group acting as the electron donor and the nitro group as the acceptor. Because of its nature, the CT bands are affected both by the polarity of the environment (e.g., the solvent) and of substituents. Considering the similarity between NBD-amine and 1, we can conclude that the triphenylphosphazene group and the diethylamino group have a very similar electronic effect on the nitro-aromatic system. Moreover, the fluorescence QY = 0.50 and the excited state lifetime τ = 5.7 ns of 1 in CH2Cl2 are very similar to those of NBD.</p><!><p>Absorption (continuous lines) and fluorescence spectra (dashed lines) of molecules 1 (black, λexc = 450 nm) 2 (green, λexc = 450 nm) and 3 (red, λexc = 490 nm) in dichloromethane (concentration 5 × 10−5 M).</p><!><p>The introduction of the methyl group on the phenyl substituent to give 2 produces a shift of the absorption maximum to 488 nm and of the fluorescence to 524 nm. It is interesting observing that going from 1 to 2, only a very small decrease of the energy of the lowest singlet excited state is observed, from 2.44 eV for 1 to 2.45 eV for 2, but a relevant decrease of the Stokes shift from 0.26 to 0.18 eV occurs. This indicates that the effect of the methylation is to increase the hindrance of the bulky triphenylphosphazene substituents on the phosphorous atom and hence it reduces the degree of conformational reorganization of the excited states which produces the Stokes shift. The electronic effect of the weak electron donor methyl group, on the other hand, is marginal and since the electronic transition has a strong charge-transfer character, no relevant difference in the electronic transition is observed. The rigidification effect due to the bulkier substituents also affects the fluorescence QY and the excited state lifetime that rises, with respect to 1, to QY = 0.65 and τ = 7.7 ns in 2.</p><p>On the contrary, the substitution of each of the three phenyl rings with three strong electron donor methoxy groups to give 3 produces a large hypsochromic shift both of the absorption band, λ = 524 nm (ε = 26,200 M−1cm−1) and of the fluorescence maximum λem = 555 nm, corresponding to a decrease of the energy of the transition to 2.30 eV. The further increase of the hindrance of the phosphazene substituents in 3 with respect to 2 leads to a decrease of the Stokes shift to 0.13 eV. On the other hand the presence of the electron rich trimethoxyphenyl group causes a decrease of the fluorescence QY to 0.09 and of the excited state lifetime to τ = 4.2 ns</p><!><p>Dye molecules 1, 2, and 3 are water insoluble and nanoparticles (NPs) were prepared by nanoprecipitation (Reisch and Klymchenko, 2016). A small volume (10 μL) of a THF solution of 1, 2 or 3 (2 mg/ml) and the surfactant Pluronic F127 (20 mg/mL) was rapidly injected into 2.5 mL of Millipore water under vigorous stirring. The reaction vial was kept open to atmospheric air in order to allow complete evaporation of the organic solvent. After 2 h of stirring, a precipitate was formed in the case of compound 1 while transparent, colored suspensions were obtained for samples containing 2 and 3.</p><p>The formation of the NP constituted by 2 and 3 was demonstrated by dynamic light scattering (DLS), transmission electron microscopy (TEM) and fluorescence microscopy (FM). In particular, DLS measurements showed the presence of a quite monodisperse single population of NPs both in the sample containing 2 (2NP, d = 128 nm, PdI = 0.11) and 3 (3NP, d = 140, PdI = 0.06). The size distribution of the two samples is shown in Figures 3a,b, respectively. DLS measurements were performed after dilution of the NPs suspension in phosphate-buffered saline (PBS) solution (1:50, vol:vol). After such a dilution, the concentration of the surfactant Pluronic F127 was 0.13 μM, hence more than three order of magnitude below the critical micelles concentration (cmc = 0.3 mM at r.t.) (Rampazzo et al., 2011). In order to exclude the formation of NPs constituted by the surfactant (micelles that, on the other hand, have been reported to show size of tens of nm), a blank sample was prepared following the same procedure used for 2NP and 3NP. No relevant scattering signal was detected in the case of the blank samples, confirming the absence of NPs.</p><!><p>Top: size distribution obtained by DLS analysis of 2NP (a) and 3NP (b). Center: representative TEM images of 2NP (c) and 3NP (d). Bottom: size distribution resulting from the analysis of the TEM images of 2NP (e) and 3NP (f).</p><!><p>The size distribution of 2NP and 3NP was also investigated by TEM: representative images of the two samples are shown in Figures 3c,d, respectively while the histogram obtained by measuring the size of the NP in the images with the software Image J are shown in Figures 3e,f. Fitting the data with a Gaussian model, we obtained the NP size: 91 ± 13 and 54 ± 9 for 2NP and 3NP, respectively. While the diameter measured for 2NP by TEM was consistent with the hydrodynamic diameter measured by DLS, a significant difference was observed in the case of 3NP. The larger hydrodynamic diameter measured by DLS for this latter sample revealed a partial aggregation of 3NP in water.</p><p>The photophysical characterization of 2NP and 3NP was performed in PBS, results are summarized in Table 1. Since it was not possible to compare the properties of 2NP and 3NP with the molecular components 2 and 3 in aqueous medium because of their insolubility, we compared them to the parent compound diethylamino-NBD (4), as shown in Table 1.</p><p>The UV vis absorption spectra of 2NP and 3NP are shown in Figures 4, 5, respectively. The molar absorption coefficient (ε) was calculated for the molecules 2 and 3 in the NP considering their average concentration. Figures 4, 5 clearly show that the aggregation in the NP has only a minor effect on the absorption properties of the dye molecules that undergo only a modest hypsochromic shift and a moderate decrease of ε.</p><!><p>Absorption (continuous lines) and fluorescence spectra (dashed lines, λexc = 450 nm) of compound 2 (black, dichloromethane) and of 2NP (red, PBS).</p><p>Absorption (continuous lines) and fluorescence spectra (dashed lines, λexc = 490 nm) of compound 3 (black, dichloromethane) and of 3NP (red, PBS).</p><!><p>On the contrary, NP formation had a very different effect on the fluorescence properties of 2 and 3. In particular, 2 maintains in the NPs an acceptable quantum yield (QY = 0.31) while 3 undergoes strong aggregation induced quenching (QY = 0.01). This observation suggests that the introduction of methyl on the bulky tri(phenyl)phosphazene group is suitable to decrease the intermolecular electronic interactions in the NPs by reducing the overlap of the molecular orbitals of the fluorescent NBD units. Nevertheless, the presence of electron donating tri-methoxyphenyl groups is known to cause fluorescence quenching because of the formation of charge-transfer non-fluorescent excited states (Shukla and Wan, 1993). Fluorescence anisotropy measurements demonstrated that the quenching effect is enhanced by excitation energy migration inside the NPs (Bonacchi et al., 2008; Jiang and McNeill, 2017). Both 2 and 3 in fact showed in a high viscosity medium like propylene glycol, quite a high value of fluorescence anisotropy r (r = 0.21 for 2 and r = 0.23 for 3 at r.t.). On the contrary, the two fluorophore immobilized in the NPs showed a fluorescence anisotropy which is zero both for 2NP and 3NP. The complete depolarization observed in the NP is in contrast with the lack of rotational freedom of the fluorophores in the nanostructures and can be explained only by considering a fast depolarization involving the homo-energy transfer processes (Genovese et al., 2013).</p><p>Time resolved fluorescence measurements (time correlated single photon counting, TCSPC) showed that the spectral changes observed upon NP formation were due to the presence, in the NPs, of populations of fluorophores experiencing different environments. Tri-exponential decays were observed both in the case of 2NP (τ1 = 0.46 ns, B1 = 3,196, τ2 = 1.45, B2 = 1,085, τ3 = 6.16, B3 = 44) and 3NP (τ1 = 0.62 ns, B1 = 4,132, τ2 = 2.44, B2 = 3,724, τ3 = 7.81, B3 = 1,559). From these data the average excited lifetime was calculated to be < τ> = 2.53 ns and < τ> = 0.77 ns for 2NP and 3NP, respectively. This result is in agreement with the low fluorescence QY measured for 3NP.</p><p>In order to evaluate the order of magnitude of the fluorescence brightness of 2NP and 3NP, the number of molecules per particles was estimated as reported in Table 1. This number was calculated considering the molar volume of 2 and 3 (molecular volume of 1, 2, and 3 were calculated to be 338.0, 380.9, and 533.6 Å3, respectively) and the hydrodynamic diameter of the NPs.1</p><p>Molar volume for 2NP and 3NP were 2.4 × 105 and 5.0 × 104 L mol−1 while molar volume for 2 and 3 were 0.23 Lmol−1 and 0.32 L mol−1, respectively. Hence 2NP and 3NP contain about 1.1 × 106 molecules and 1.6 × 105 dye molecules, respectively. Considering these values, the brightness of 2NP results to be as high as ~1010M−1cm−1 while 3NP show a brightness which is almost two orders of magnitude lower than 2NP.</p><!><p>Thanks to their outstanding brightness, 2NP could be detected, in suspension, as single bright spots in a conventional fluorescence microscope. Using an acquisition time as short as 10 ms, the NP appeared motionless as shown in Figure 6 (inset). Thermal motions of the NP were clearly observed by time lapsed acquisition (4,000 frames). The resulting movies were analyzed to measure the linear displacements of the NPs using the plugin MOSAIC for Image J (Sbalzarini and Koumoutsakos, 2005; Chenouard et al., 2014). The displacements were then plotted in a histogram as shown in Figure 6. We would like to stress that an analogous experiment was performed for a 3NP sample and no emissive spots attributable to NP diffusion could be observed. This demonstrated that these NPs were not bright enough to be detectable as individual objects by fluorescence microscopy. Moreover, by comparing two samples of 2NP and 3NP with the same concentration (8 μg/ml), the average intensity measured within a frame in the case of 2NP was more than 2 orders of magnitude higher than the one measured for 3NP.</p><!><p>Histogram of the linear displacements measured for 2NP in PBS by fluorescence microscopy for a time interval Δt = 10 ms. Images were acquired in time-lapse mode (4,000 frames) with an EMCCD Camera and processed with Image J (Plug-in MOSAIC). Each pixel corresponds to 0.16 μm. A representative image of the NP (bright white spots) is shown in the inset.</p><!><p>The diffusion coefficient of 2NP was calculated by tracking the fluorescent NP via fluorescence microscopy considering the following equations:</p><p>Where P is the probability of observing a displacement of an NP from the position x0 to the position × after a time delay t and D is the diffusion coefficient of the NP that, in a spherical approximation, is dependent on the diameter of the NP according to the Stokes-Einstein equation:</p><p>Where k is the Boltzmann constant, T the temperature and η is the viscosity of the medium. Image sequences were processed to acquire the positions of the NP in each frame and to identify individual NP movements. The trajectories of the NPs were used to get the displacement (in pixels, where a pixel corresponds to 0.16 μm) of the NP in the frame acquisition time interval (t = 1.0 × 10−2 s). Data represented in the histogram were fitted with a Gaussian model, as shown in Figure 6, to obtain the diffusion coefficient D = 2.65 × 10−12 m2s−1; a value that corresponds to NPs with an average diameter of 160 nm in good agreement with the DLS analysis.</p><!><p>To demonstrate their efficacy as a fluorescent probe, 2NP and 3NP were incubated with HeLa cells at the relatively low dose of 80 ng/ml at 37°C. After confocal microscopy analysis of 20 h, cells (Figure 7) revealed an intense structured 2NP signal within the cell cytoplasm, suggesting endosomal NP internalization, although cytoplasmatic internalization cannot be completely ruled out. As expected, based on their weak intrinsic fluorescence, 3NP cellular signal was much weaker. MTS assays showed that both 2NP and 3NP are devoid of the toxic effects on HeLa cells of up to 1 μg/ml (Figure 8).</p><!><p>Fluorescence signal of NPs in cells. HeLa cells grown on glass cover-slips were incubated for 20 h with no NPs (medium alone), 2NP or 3NP, as indicated and analyzed by fluorescence confocal microscopy with the same instrumental setting for comparison. Representative images are shown. In the case of 2NP cell signal distribution details are shown at higher magnification. Bars are the standard deviations of the means.</p><p>Cytotoxicity of 2NPs and 3NPs. HeLa cells were incubated for 20 h with the indicated doses of NPs in DMEM at 37°C and subjected to MTS assay. Data, expressed as % of control (no NP) samples, are mean ± SE (N=3).</p><!><p>Summarizing the results so far discussed, we found that molecule 1 does not form stable NP upon nanoprecipitation in our experimental conditions. On the contrary, nanoprecipitation of molecules 2 and 3 leads to NP formation. However, the quantum yield of molecule 3, which is already low in the non-aggregated form, further decreases upon assembly in the NP. As a result, the brightness of 3NP is about two orders of magnitude lower than the one of 2NP. Such a difference is so relevant that while 2NP can be clearly tracked at the single NP level in solution at very low concentration by fluorescence microscopy, 3NP cannot be observed with the same technique. Most interestingly, the brightness of 2NP could be estimated to be about six orders of magnitude higher than an NBD water soluble derivative used as reference. These results demonstrate that rational design of the molecular precursor is fundamental for producing stable and strongly bright nanoparticles by self-assembly. Cellular experiments proved that 2NPs are suitable to be used as fluorescent contrast agents for bioimaging, also thanks to their good biocompatibility. We believe that our approach can be extended to other molecules and surfactants in order to tune the excitation/emission wavelength as well as the NP size.</p><!><p>General: Solvents were purified by standard methods. All the reagents used were purchased by Sigma-Aldrich and used as received.</p><p>TLC analyses were performed using Merck 60 F254 precoated silica gel glass plates. Column chromatography was carried out on Macherey-Nagel silica gel 60 (70–230 mesh).</p><p>NMR spectra were recorded using a Bruker AV 500 spectrometer operating at 500 MHz for 1H, 125.8 MHz for 13C. Chemical shifts are reported relative to internal Me4Si. Multiplicity is given as follow: s = singlet, d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet, br = broad peak.</p><p>ESI-MS mass spectra were obtained with an Agilent Technologies LC/MSD Trap SL mass spectrometer. EI/MS spectra were obtained with an Agilent Technologies 6850-5973 GS/MS.</p><!><p>Into a flask, covered with an aluminum foil and containing NaN3 (0.350 g, 5.39 mM) dissolved in a EtOH/H2O mixture (1:1 v/v, 20 mL), a solution of NBD-Cl (0.5 g, 2.505 mM) in EtOH (40 mL) was added dropwise within a 1 h period. The reaction was left stirring for 6 h at RT. Subsequently, the solvent was removed under reduced pressure and crude was purified via column chromatography on silica gel using petroleum ether/AcOEt 4:6 as eluent. The product 1 was obtained as an orange solid in 90% yield.</p><p>1H NMR (300 MHz, d6-DMSO), δ: 8.70 (d, J =5.3 Hz, 1H), 6.40 (d, J = 5.3 Hz, 1H).</p><p>ESI/MS, m/z: 207.8 (15%, M+H+), 178.8 (100%, M-N2+H+).</p><p>EI/MS, m/z: 207 (100%, M+), 180, 150, 133, 120, 104, 92, 77, 64, 52.</p><!><p>Into a flask, covered with aluminum foil and containing 1 (0.1 g, 0.485 mM) and the desired phosphine (3 eq.) dissolved in THF (8 mL), H2O (2.6 mL, 145.553 mM) was added at once and left to react overnight under stirring. Subsequently, the formed solid was filtered-off, washed with cold THF and dried giving the final product as an orange solid with quantitative yield.</p><!><p>1H NMR (300 MHz, CDCl3) δ 8.19 (d, J = 8.3 Hz, 1H), 7.74 (m, J = 13.0, 10.7, 4.7 Hz, 6H), 7.65–7.56 (m, 3H), 7.54–7.37 (m, 7H), 6.56 (d, J = 8.5 Hz, 1H).</p><p>31P NMR (202 MHz, CDCl3) δ 14.01.</p><p>13C NMR (126 MHz, CDCl3) δ 137.04, 134.06, 134.02, 133.06, 132.92, 130.21, 130.04, 126.84, 125.51, 40.86, 40.58, 40.30, 40.02, 39.74, 39.47, 39.19.</p><p>ESI-MS, m/z: 425.2 (100%, M+H+).</p><!><p>1H NMR (500 MHz, DMSO-d6) δ 8.20 (d, J = 8.8 Hz, 1H), 7.71 (t, J = 7.4 Hz, 3H), 7.58–7.46 (m, 10H), 5.50 (d, J = 8.9 Hz, 1H), 2.20 (s, 9H).</p><p>31P NMR (202 MHz, DMSO-d6) δ 21.31.</p><p>13C NMR (126 MHz, DMSO-d6) δ 154.93, 150.91, 143.15, 143.08, 137.24, 134.40, 134.32, 134.09, 133.98, 133.49, 133.40, 127.61, 127.51, 123.92, 123.15, 121.57, 117.25, 109.16, 109.06, 31.16, 30.06.</p><p>ESI-MS, m/z: 483.2 (100%, M+H+), 505.1 (10%, M+Na+).</p><!><p>1H NMR (500 MHz, Acetone-d6) δ 8.54 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 9.4 Hz, 1H), 7.90 (s, 1H), 6.25 (d, J = 4.7 Hz, 6H), 5.83 (d, J = 8.4 Hz, 1H), 3.88 (s, 10H), 3.60 (s, 27H).</p><p>31P NMR (202 MHz, Acetone-d6) δ 3.03.</p><p>13C NMR (126 MHz, Acetone-d6) δ 167.49, 163.92, 142.36, 140.66, 140.53, 135.71, 129.25, 125.86, 118.39, 96.10, 56.14, 55.65.</p><p>ESI-MS, m/z: 711.2 (100%, M+H+).</p><!><p>UV-VIS absorption spectra were recorded at 25°C by means of Cary 300 UV-Vis spectrophotometer (Agilent Technologies).</p><!><p>The fluorescence spectra were recorded with a Horiba Fluoromax-4 spectrofluorimeter and with an Edinburgh FLS920 fluorimeter equipped with a photomultiplier Hamamatsu R928P. Quartz cuvettes with optical path length of 1 cm were used for both absorbance and emission measurements.</p><!><p>Excited state lifetime was measured with an Edinburgh FLS920 fluorimeter equipped with an electronic card for time correlated single photon counting TCSPC900. The kinetic tracks were fitted with a tri-exponential model: I(t)=A+B1e-t/τ1+B2e-t/τ2+ B3e-t/τ3 with the software package FAST.</p><!><p>All fluorescence anisotropy measurements were performed on an Edinburgh FLS920 equipped with Glan-Thompson polarizers. Anisotropy measurements were collected using an L-format configuration, and all data were corrected for polarization bias using the G-factor.</p><p>In particular four different spectra were acquired for each sample combining different orientations of the excitation and emission polarizers: IVV, IVH, IHH, IHV (where V stands for vertical and H for horizontal with respect to the plane including the excitation beam and the detection direction; and the first subscript refers to the excitation and the second subscript refers to the emission). The spectra were used to calculate the G-factor and the anisotropy r: G = IHV/IHH and r = IVV-GIVH/IVV+2GIVH.</p><!><p>The fluorescence images were obtained with an inverted microscope (Olympus IX71) equipped with a Xenon lamp for excitation. Excitation, dichroic and emission filters were purchased from Chroma and Thorlabs. Excitation filter: 475 ± 17.5 nm; emission filter: 530 ± 21.5 nm; dichroic Reflection/Transmission): 470–490/508–675 nm. Fluorescence images were acquired with an Electron Multiplying Charge Coupled Device EMCCD Camera (Princeton Instruments, Photon Max 512). Acquisition time was 30 ms per frame at the maximum amplification gain using a 100x oil immersion objective for fluorescence (Olympus UPLFLN100XO2).</p><!><p>Trajectories were tracked by analyzing sequences of images acquired with an integration time τ of 10 ms per frame. The particles were localized and tracked by using the plug-in MOSAIC for the software ImageJ (Sbalzarini and Koumoutsakos, 2005; Chenouard et al., 2014). The displacement distribution was processed with the software Sigmaplot to obtain histograms that were fitted with Gaussian peaks. The Stokes-Einstein equation was used to obtain the particle diameter d.</p><!><p>Light Scattering measurements were performed using a Malvern Nano ZS instrument equipped with a 633 nm laser diode. Samples were housed in disposable polystyrene cuvettes of 1 cm optical path length. DLS measurements were performed after dilution of the NPs suspension in phosphate-buffered saline (PBS) solution (1:50, vol:vol).</p><!><p>A Philips CM 100 transmission electron microscope operating at 80 kV was used. For TEM investigations, a 3.05 mm copper grid (400 mesh) covered by a Formvar support film was dried up under vacuum after deposition of a drop of nanoparticles solution.</p><!><p>HeLa cells were maintained in a DMEM medium (Gibco) supplemented with 10% FCS (Euroclone) and antibiotics (penicillin and streptomycin, Invitrogen) at 37°C in a humidified atmosphere containing 5% (v/v) CO2; cells were split every 2–3 days. For MTS cytotoxicity assay, cells (1 × 104 cells) were plated onto a 96-well culture plate the day before the experiment. Cells were then incubated for 20 h with NPs at different concentrations in DMEM, added with 10% FCS. Cellular mitochondrial activity (indicator of cellular viability) was evaluated by MTS assay (Promega) according to the instruction manual. For the MTS test N = 3 independent experiments were run in triplicate. t test (significativity p < 0, 05) were performed, but differences compared to control (no particles) were always not significant (p > 0, 05). For the assessment of intracellular distribution of NPs, cells (1 × 105) were seeded on cover glasses and after 24 h they were incubated for 20 h at 37°C with NPs, washed with PBS and directly analyzed by confocal microscopy (Leica SP2). Images were processed using ImageJ software.</p><!><p>All datasets generated for this study are included in the manuscript and/or the supplementary files.</p><!><p>MM designed the synthesis of the NPs and supervised their preparation and characterization. FM and JT synthesized molecules 1, 2, and 3. VC prepared and characterized the NP. AC performed the photophysical characterization of molecules. RT and EP performed the cellular experiments.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

A dynamic structural model of expanded RNA CAG repeats: A refined X-ray structure and computational investigations using molecular dynamics and umbrella sampling simulations

One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded r(CAG) repeats can cause Huntington\xe2\x80\x99s and other disease through translation of toxic proteins. Herein, crystal structure of r[5\xca\xb9UUGGGC(CAG)3GUCC]2, a model of CAG expanded transcripts, refined to 1.65 \xc3\x85 resolution is disclosed that show both anti-anti and syn-anti orientations for 1\xc3\x971 nucleotide AA internal loops. Molecular dynamics (MD) simulations using Amber force field in explicit solvent were run for over 500 ns on model systems r(5\xca\xb9GCGCAGCGC)2 (MS1) and r(5\xca\xb9CCGCAGCGG)2 (MS2). In these MD simulations, both anti-anti and syn-anti AA base pairs appear to be stable. While anti-anti AA base pairs were dynamic and sampled multiple anti-anti conformations, no syn-anti\xe2\x86\x94anti-anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, over 800 ns explicit solvent MD simulation was run on r[5\xca\xb9GGGC(CAG)3GUCC]2, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn-anti conformation), transformed to anti-anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn-anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1\xc3\x971 nucleotide AA internal loops in r(CAG) repeats is anti-anti but can adopt syn-anti depending on the environment. These results are important to understand RNA dynamic-function relationships and develop small molecules that target RNA dynamic ensembles.

a_dynamic_structural_model_of_expanded_rna_cag_repeats:_a_refined_x-ray_structure_and_computational_

INTRODUCTION<!>Crystallization and structure refinements<!>Preparation of model systems for MD simulations<!>Molecular dynamics simulations<!>Umbrella sampling simulations<!>Minimization<!>Equilibration<!>Production runs<!>Analysis<!>Structure of r(3xCAG)X-RAY (a model of r(CAG)exp<!>Pathways for syn-anti\xe2\x86\x94anti-anti AA transformation<!>Stability of syn-anti and anti-anti AA base pairs<!>Dynamics of model 3xCAG system<!>RNA CAG loops in literature<!>SUMMARY and CONCLUSIONS

<p>Many diseases are caused by transcripts that contain expanded repeats. The physiological consequences of these expansions, and thus the mechanism of disease, are varied. For example, r(CUG) expansions (r(CUG)exp) in the 3ʹ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA cause myotonic dystrophy Type 1 while myotonic dystrophy type 2 is caused by an expansion of r(CCUG) (r(CCUG)exp) in intron 1 of the zinc-finger 9 (ZNF9) protein mRNA. These diseases are characterized by muscle weakness and slow relaxation of muscles after contraction.1,2 Fragile X-associated tremor ataxia syndrome (FXTAS) is caused by 50-200 CGG repeats (r(CGG)exp) in the 5ʹ UTR of the fragile X mental retardation 1 (FMR1) gene.3-5 In each of these cases, the RNAs are in a non-coding region and are not translated into protein. Thus, disease is caused by an RNA gain-of-function.1,6,7 In this mechanism, expanded repeats sequester proteins, typically ones involved in pre-mRNA splicing regulation, affecting their function. Muscleblind-like 1 protein (MBNL1) is sequestered and inactivated by r(CUG)exp and r(CCUG)exp while Sam68 is inactivated by r(CGG)exp.</p><p>In contrast to the above cases, expansions of CAG repeats (r(CAG)exp) that cause disease are typically found in the coding regions of mRNAs. For example, r(CAG)exp have been found in Huntingtin (HTT), androgen receptor (AR), spinocerebellar ataxia (SCA), and atrophin-1 (ATN1) genes and cause Huntington disease (HD), Spinal and Bulbar Muscular Atrophy (SBMA), Spinocerebellar Ataxia Type 1 (SCA1), and Dentatorubral-pallidoluysian atrophy (DRPLA), respectively. Since r(CAG)exp is in the coding region of these transcripts, it is translated into toxic polyglutamine (polyQ) proteins that cause disease.1,8-12</p><p>More recently it has also been shown that r(CAG)exp in coding regions can also sequester proteins such as MBNL1 and contribute to neurodegeneration.13-15 Moreover, expanded repeats in UTRs can be translated via a Repeat Associated Non-ATG Translation.16 Thus, although the disease mechanisms described above for each type of repeat are the most well established, it is not as clear-cut as it once appeared and the interplay of RNA and protein toxicities may ultimately cause disease.</p><p>In order to gain insights into the role of RNA structure in these diseases, various approaches have been used to gain structural information. For example, chemical and enzymatic probing has shown that triplet repeat expansions form expanded hairpin structures with 1×1 nucleotide internal loops closed by GC pairs.17 For several of these repeats, high-resolution structural information has been obtained through refinement of NMR spectral or X-ray crystallographic data.18-23</p><p>The structure of a model of r(CAG)exp has been reported by Kiliszek et al,23 who studied the structure of the RNA duplex (GGCAGCAGCC)2. They reported that the adenosines are in the anti conformations with a single C2-H2…N2 hydrogen bond, with significant distortions compared to a standard Watson-Crick paired duplex. This work did not identify fluxional behavior characteristic of large amplitude motions that might be expected for a 1×1 nucleotide internal loop. Information about such motions could improve our understanding of the conformations that toxic repeating transcripts adopt and how these conformations affect the binding of protein or small molecules.24,25 Thus, the conformational flexibility of r(CAG) repeat-containing duplexes is of significant interest.26</p><p>Computational technology is an emerging scientific tool that can make useful predictions of the atomic details of structure as well as of RNA dynamics. Such studies are described herein and utilized to study the conformational heterogeneity of r(CAG) repeats. Methods such as residue-centered force fields (coarse-grained),27 atom-centered force fields (AMBER,28 CHARMM,29,30 and GROMOS31), approximate quantum mechanics,32,33 and mixed quantum-mechanics/molecular mechanics (QM/MM),34-41 are typically used for predicting the structure and dynamics of RNA. Molecular mechanics force fields are computationally economical such that molecular dynamics (MD) simulations on the microsecond or faster time scales are possible.42-46 Application of these methods allows for comparing the structural and thermodynamic properties of nucleic acids with experiments.47-52 Free energy calculations are particularly important because free energies control a reaction coordinate. Combined with the weighted histogram analysis method (WHAM),53,54 umbrella sampling55-58 is a powerful computational approach to extract the free energy landscape along multiple reaction coordinates such as base opening59 and base flipping60-62 that can occur in DNA and RNA.</p><p>In this paper, we present the crystal structure of a self-complementary RNA duplex, r[5ʹUUGGGC(CAG)3GUCC]2, (a model of r(CAG)exp) refined to 1.65 (r(3xCAG)X-RAY) Å resolution. The crystal structure shows multiple conformations for 1×1 nucleotide AA internal loops (anti-anti and syn-anti) that suggest fluxional behavior. To provide insight into these results and probe more deeply into the structure of the AA internal loops, MD simulations using the Amber force field in explicit solvent were run on the model systems r(5ʹGCGCAGCGC)2 (MS1), r(5ʹCCGCAGCGG)2 (MS2), and r[5ʹGGGC(CAG)3GUCC]2 (3xCAG) including a detailed analysis of the dynamics of 1×1 nucleotide AA internal loops. After choosing two reaction coordinates, which mimic base flipping and base orientation with respect to sugar moieties for the 1×1 nucleotide AA internal loops, a 2D free energy landscape for syn-anti↔anti-anti transformation was generated by combining WHAM with umbrella sampling MD simulations. It was found that the AA base pairs are dynamic and can form stable structures both in anti-anti and syn-anti conformations with one hydrogen bond. The results indicate that the anti-anti and syn-anti 1×1 nucleotide AA internal loops are major and minor conformations, respectively. Multiple pathways are located for the syn-anti↔anti-anti transformation. Moreover, an electronegative binding pocket was determined around syn-anti AA base pairs where individual Na+ ions can bind for as long as 50 ns. These results are important to better understand how r(CAG)exp contributes to disease, as well as to assist in the design of small molecules that bind r(CAG)exp and ameliorate disease.</p><!><p>The RNA duplex, r[5UUGGGC(CAG)3GUCC]2 (r(3xCAG)X-RAY), at 1.2 mM concentration was dissolved in DEPC-water and folded by heating to 60 °C for 5 min and cooling to room temperature. Crystal of r(3xCAG)X-RAY was grown by the sitting drop vapor diffusion method using 0.2 μL of 1.2 mM RNA and equal volume of precipitants. The precipitants for r(3xCAG)X-RAY were 15 mM Mg(CH3COO)2, 50 mM Na cacodylate, pH 6.0, 1.7 M (NH4)2SO4. A diffraction data set with Bragg spacings to 1.65 Å for r(3xCAG)X-RAY was collected on a MAR325 CCD detector at beam line 12-2 of the Stanford Synchrotron Radiation Laboratory. The datasets were integrated and scaled using HKL2000.63 Phases for RNA structure was obtained by molecular replacement using Phaser 64 in Phenix program interface 65 with PDB entry 3SYW as a search model. Crystallographic refinement was performed using Phenix 65 and multiple rounds of manual model fittings were performed using Coot. Data collection and the crystallographic refinement statistics are summarized in Table 1.</p><!><p>Model systems MS1, MS2, and 3xCAG (Figure 1) were prepared for MD simulations. MS1 and MS2 were modeled using the nucgen module of AMBER966 with AA base pairs in anti-anti and syn-anti conformations. 3xCAG (Figure 1) was modeled from crystal structure r(3xCAG)X-RAY by trimming down the flanking uridines. In the model 3xCAG structure, one of the terminal AA base pair was modified to be in anti-anti conformation. This was done because the crystal structure of r(3xCAG)X-RAY is symmetric and has both the terminal AA base pairs in syn-anti conformations that puts redundancy to the analysis of potential syn-anti↔anti-anti transformations. The systems were neutralized with Na+ ions67 and solvated with TIP3P water molecules68 in a truncated octahedral box. The MS1/MS2 and 3xCAG systems had 4025 and 8995 water molecules, respectively. The Amber force field28 with revised χ69 and α/γ70 torsional parameters were used in molecular dynamics and umbrella sampling simulations.</p><p>The main reason why the flanking uridine bases were removed from the structure was to mimic the in vivo r(CAG)exp. In the crystallization process, flanking uridines were included in order to crystallize the structure, which resulted in terminal AA base pairs in syn-anti conformations. The observation of this conformation, which was not seen before in RNA CAG repeats, motivated us to delve into the mechanism behind it. By trimming down the uridine bases, we disrupted the interaction between the flanking uridines and the terminal AA base pairs, giving us the opportunity to investigate the dynamics of AA base pairs when starting with syn-anti conformations in 3xCAG.</p><!><p>The systems were first minimized and equilibrated in two steps as described previously.71,72 In the production runs, constant pressure dynamics with isotropic positional scaling was used. The reference pressure was 1 atm with a pressure relaxation time of 2 ps. SHAKE73 was turned on for bonds involving hydrogen atoms. An atom-based long-range hard cutoff of 8.0 Å was used in all simulations. A total of 854 ns and ~500 ns of MD were run at 300 K with a 1 fs time step for 3xCAG and MS1/MS2, respectively. Simulations were carried out with the PMEMD module in AMBER9.66 Trajectory files were written at each 5000 fs time step. For all these calculations, over 210K CPU hours were used.</p><!><p>In order to predict the potential pathways for the syn-anti↔anti-anti transformation, a 2D potential of mean force (PMF) surface was calculated for the model system MS2. Rotation around the glycosidic bond is responsible for the syn and anti base orientations of adenosine with respect to the adjacent sugar. As a result, the χ torsional angle was chosen as one of the reaction coordinates to mimic the base orientation (Figure 2a). A second reaction coordinate, called center-of-mass pseudo-dihedral angle (θ), was chosen to mimic unstacking of adenosine as shown in Figure 2b. Similar reaction coordinates for base flipping studies of oligonucleotides were used previously.60,61,74 Each reaction coordinate was rotated with an increment of 10°, yielding 36×36=1296 conformations. Initial structures with different (χ, θ) combinations were created for the model system MS2.</p><!><p>Each structure was minimized in two steps: 1) All residues except the adenosines were held fixed with a restraint force of 10 kcal/mol-Å2. χ and θ dihedrals were held fixed in a particular combination using a square bottom well with parabolic sides that had force constants of 50000 kcal/mol-rad2. Steepest descent minimization of 2000 steps was followed by a conjugate gradient minimization of 2000 steps. 2) Similar to the first step, the χ and θ dihedrals were held fixed in their initial combination. Watson-Crick base pairing restraints were imposed on the GC base pairs neighboring the 1×1 nucleotide AA internal loop. Steepest descent minimization of 25000 steps was followed by a conjugate gradient minimization of 25000 steps (Tables S1 and S2).</p><!><p>After minimization, two steps of pressure equilibration were completed: 1) the RNA molecule was held fixed with a restraint force of 10 kcal/mol-Å2. Constant volume dynamics with an atom-based long range hard cutoff of 8.0 Å was used. SHAKE73 was turned on for bonds involving hydrogen atoms. The temperature was raised from 0 to 300 K in 20 ps. Langevin dynamics with a collision frequency of 1 ps−1 was used. A total of 20 ps of MD were run with a 2 fs time step. 2) The above conditions were chosen, except the following: χ and θ dihedrals were held fixed in their initial combination with a force constant of 500 kcal/mol-rad2. Watson-Crick base pairing restraints were imposed on GC base pairs neighboring 1×1 nucleotide AA internal loops. Constant pressure dynamics with isotropic positional scaling was turned on. The reference pressure was 1 atm with a pressure relaxation time of 2 ps. A total of 100 ps of MD were run with a 2 fs time step. Particle Mesh Ewald (PME)75,76 was used in all calculations.</p><!><p>Each production run was similar to the second step of the pressure equilibration described above. No restraints except the ones imposed on χ and θ dihedrals were used in the simulations. A square bottom well with parabolic sides was used to restrain χ and θ dihedrals with force constants of 50 kcal/mol-rad2. For each simulation, a total of 2 ns of MD were run with a 2 fs time step. (χ, θ) data were written at intervals of 10 fs. Simulations were carried out with the sander.MPI module in AMBER11. For umbrella sampling calculations, over 267K CPU hours were used.</p><!><p>Dihedral and root-mean-square deviation (rmsd) analyses were completed using the ptraj module in the AMBER package.66 Weighted Histogram Analysis Method77,78 (WHAM) was used to calculate the PMF surface using WHAM ver.2.0.4 written by Alan Grossfield (http://membrane.urmc.rochester.edu/content/wham). In the WHAM analysis, the last 1.5 ns data of each umbrella sampling simulation was used, and periodicity was turned on in both dimensions.</p><!><p>Figure 3A shows the secondary structure of an RNA construct that has three copies of the 5ʹCAG/GAC motif. Crystal structure was refined to 1.65 Å resolution (r(3xCAG)X-RAY). The central 5ʹCAG/GAC region of r(3xCAG)X-RAY is in the anti-anti 1×1 nucleotide AA internal loop conformation while the terminal AA base pairs are in the syn-anti conformations (Figures 3B and 4). The duplex crystallized in double-stranded helical structure and made pseudoinfinite helices with the neighboring RNAs in the crystal lattice (Figures 3C). The duplex has A-form conformation and the overlay of r(3xCAG)X-RAY and A-form RNA shown in Figure 3D displays almost identical backbone structures except the 5ʹUU dangling ends, which interact with the terminal 1×1 nucleotide AA internal loops (Figure 3E). Amino and 2ʹOH groups of A12 and A29 are in close contact with U−17 and U−2, respectively, which cause A12 and A29 to adopt syn conformations (Figures 3E and 4). Almost all riboses are C3ʹ-endo conformation (sugar pucker) (Table S3). Dihedral angle values for α, β, γ, δ, ε, and ζ can be found in Table S3. Parallel to Figure 3D, global helical parameters calculated for r(3xCAG)X-RAY show characteristic of A-form conformation with a minor groove width of 15.5 Å (Tables S4-S8).</p><p>Electron density maps of individual 1×1 AA internal loops for r(3xCAG)X-RAY are in line with syn-anti and anti-anti conformations for terminal and central 1×1 nucleotide AA internal loops, respectively (Figure 4). Temperature factor comparisons of the central anti-anti AA loop have higher average values in r(3xCAG)X-RAY than base paired regions (Figure 4 and S1). This is evidenced by the well-defined electron density for base paired regions while the central 1×1 nucleotide AA internal loops have less well-defined electron density, indicating dynamic nature (Figures 4 and S1). Electron densities for the CG closing base pairs are consistent with three hydrogen bonds (Figures S1 and S2). Interestingly, a similar dynamic character has been observed in a refined X-ray structure of the 1×1 nucleotide UU internal loops in r(CUG)exp models, suggesting that many repeats can sample multiple conformations.19 In contrast, a refined X-ray structure of r(CGG) repeats shows that this RNA adopts a rather rigid structure, and each 1×1 nucleotide GG internal loop has a syn-anti conformation.18</p><!><p>Umbrella sampling MD simulations were used to construct the 2D potential mean force (PMF) surface in order to visualize the potential pathways for the syn-anti↔anti-anti AA transformation (Figure 5). The x- and y-axes in Figure 5 are the reaction coordinates for unstacking (θ) and base orientation with respect to the sugar moiety (χ) of A5 in the 1×1 nucleotide AA internal loop (Figure 2). Three pathways are found for syn-anti↔anti-anti AA transformation (Figure 5). In path P1, A5 first unstacks from the helical axis through the minor groove direction (motion in x-axis). This motion relaxes the state and gives A5 enough space to make the syn↔anti transformation through a rotation around the glycosidic bond, χ (motion in y-axis). A5 then stacks back into the helical axis through the major groove direction to form the AA base pair. In P2, almost a direct syn↔anti transformation occurs in the system without A5 unstacking from the helical axis. In P3, a transformation similar to P1 is seen except that unstacking occurs through the major groove direction. P1 has two energy barriers in the syn-anti transformation pathway with a stable local minimum at around (−75, 60) (Figure 5). In addition, P1 has a broader space in its transformation pathway compared to P2 and P3, which makes the transformation more probable. To explicitly show the differences between the pathways, 1D free energy profiles along the most likely direction for each path were extracted (Figures S3 and S4). In Figure 5, the sampling range of the syn-anti region is very tight compared to the range of the anti-anti region. This means that the anti-anti region is dynamic and sample more than one conformation while syn-anti is much more rigid in structure compared to anti-anti.</p><p>In general, RNA loops are flexible, which gives them functional roles in the cell such as when AA base pairs form in RNA CAG repeats. This property, however, makes it hard to determine how small molecules bind to these flexible RNA regions, which was recently discussed by Al-Hashimi group at the University of Michigan at Ann Arbor.79 While Figure 5 discloses different potential pathways for the syn↔anti transformation, there are some results that should not be overlooked. RNA residues have three significant motions that affect the global RNA structure: 1) Base opening/flipping, 2) Base orientation with respect to sugar, and 3) Sugar puckering. The first two motions will affect the RNA structure the most. In Figure 5, the x-axis represents base opening/flipping while the y-axis represents χ rotation. Even though the lowest energy regions are around (50,50) (adenosine stacked on the helical axis in a syn conformation) and (60,180) (adenosine stacked on the helical axis in an anti conformation) Figure 5 shows several other local minima regions in unstacked states such as around (−125,50), (−125,190), and (−75, 300). When AA base pairs are not interacting with anything, the structure prefers the anti-anti form while upon interacting with something such as the flanking uridine bases they can adopt the syn-anti form. This interaction-driven conformational preference process can be applied to other local minima regions, too, as shown in Figure 5. It is possible that the local minima regions could be dominated by AA base pairs if a small molecule was designed to capitalize on these regions in binding to RNA.</p><!><p>Model systems MS1 and MS2 (Figures 1a and 1b) were designed in both syn-anti and anti-anti 1×1 nucleotide AA internal loop conformations in order to investigate the dynamics. For each case, at least 500 ns MD simulations were run. Each system has a total of 16 Na+ ions that neutralize the systems. Na+ binding and rmsd analysis show unique properties for each conformation (see Figure S5).</p><p>Rms devations (rmsd) of 1×1 nucleotide AA internal loops (with respect to initial conformations) and distances of each Na+ ion from a loop as a function of time for each MD simulation were plotted (Figure S5). In the MD simulations, different 1×1 nucleotide AA internal loop types were observed (Figures 6 and S5). Different colors were used in Figure 6 to distinguish each 1×1 nucleotide AA internal loop type, and this color notation was conjoined with the rmsd analysis in Figure S5 to emphasize the structural transformations observed in the simulations. Additionally, different colors were used for each Na+ ion in the Na+ binding analysis to highlight the time spent by each ion around the 1×1 nucleotide AA internal loops (Figure S5). It was observed that both syn-anti and anti-anti 1×1 nucleotide AA internal loop conformations were stable in these model systems. While the syn-anti 1×1 nucleotide AA internal loop remained almost all the time in the initial configurations, multiple anti-anti→anti-anti 1×1 nucleotide AA internal loop transformations were observed (Figures 6 and S5).</p><p>In the MD simulations that were started with syn-anti 1×1 nucleotide AA internal loop, there was a Na+ binding pocket around the 1×1 nucleotide AA internal loop, which held different Na+ ions throughout the simulations (Figures 7 and S5, panels a1 and b1). Almost no structural change was seen in syn-anti AA base pair in these simulations. When 1×1 nucleotide AA internal loops were in syn-anti conformations, Na+ ions were bound in this pocket for as long as over 50 ns (Figure S5). In this state, Na+ was interacting with the electronegative groups of anti A and neighboring G (Figure 7). On average, distances between Na+ and A-N7, G-N7, and G-O6 were 2.4, 2.6, and 3.3 Å, respectively (Figure 7). This Na+-bound state is one reason why 1×1 nucleotide AA internal loop in the syn-anti conformation did not fluctuate too much from its initial state (Figure S5). Indeed, when there were fluctuations in syn-anti 1×1 nucleotide AA loops in the MD simulations, there were no Na+ ions bound to this binding pocket (Figure S5, panels a1 & a2 and b1 & b2). Around 125 and 440 ns in the MD simulation of model system MS1 (Figure S5a) and around 325 ns in the MD simulation of model system MS2 (Figure S5b), no Na+ ions were bound in the pocket. At these times, syn-anti AA transformed to a transition state for a short period of time that corresponds to the orange colored AA state shown in Figure 6d and highlighted with orange in the rmsd analysis (Figure S5, panels a1 & a2 and b1 & b2). Thus, the dynamics of syn-anti 1×1 nucleotide AA internal loops were controlled by the Na+ binding in this pocket.</p><p>In the MD simulations that were started with anti-anti 1×1 nucleotide AA internal loops, no Na+ binding pocket was found (Figure S5, c and d). Na+ ions were spending at most 10 ns around AA base pairs in these systems (Figure S5). On average, a Na+ ion was present within 5.5 Å of AA for 33% and 91% of the time in the anti-anti and syn-anti AA systems, respectively. The anti-anti 1×1 nucleotide AA internal loops, however, were dynamic. Multiple anti-anti↔anti-anti AA conformational transformations displayed in Figure 6 were observed in the simulations (Figure S5, c and d). This result is in line with the umbrella sampling predictions that span a wide range for the anti-anti 1×1 nucleotide AA internal loops in the 2D PMF surface (Figure 5).</p><p>One explanation for not observing the syn-anti↔anti-anti AA transformation in these systems might be due to the energy barrier, which could be too high to overcome. In order to test this hypothesis, ten independent temperature jump MD simulations were completed on model system MS2, which were started both in syn-anti and anti-anti 1×1 nucleotide AA internal loop conformations. The temperature in each MD simulation was gradually increased from 300 to 400 K within 20 ns without any restraints, and final structures were analyzed. Five out of ten MD simulations that started with syn-anti 1×1 nucleotide AA internal loops ended up in anti-anti 1×1 nucleotide AA internal loop conformation while none of the simulations that started with anti-anti 1×1 nucleotide AA internal loops transformed to syn-anti AA. In model systems MS1 and MS2, there are four Watson-Crick GC base pairs on both sides of the 1×1 nucleotide AA internal loops while in the original triplet r(3xCAG)X-RAY system there are only two GC base pairs between each consecutive 1×1 nucleotide AA internal loop. The results, therefore, suggest that model systems MS1 and MS2 have rigid stem regions that increase the energy barrier of the syn-anti↔anti-anti AA transformation.</p><!><p>Model 3xCAG system (Figure 1c) resembles the original triplet RNA, r(3xCAG)X-RAY, (Figure 3a) much more than the model systems MS1 and MS2 (Figures 1a and 1b). As a result, the dynamics of 3xCAG was investigated by doing an 854 ns long MD simulation. There were a total of 32 Na+ ions that neutralized the system.</p><p>A different rmsd and Na+ binding analysis was completed on 3xCAG (Figure 8). As before, each individual Na+ ion is highlighted with different colors (Figure 8, c/f/i). Similar to the analysis completed for MS1 and MS2, the rmsd's of each 1×1 nucleotide AA internal loop with respect to initial conformation were plotted (Figure 8, b/e/h). Colors representing unique 1×1 nucleotide AA internal loop types in Figure 6 were used in the rmsd analysis to emphasize the type of transformations seen in the MD simulation (Figure 8, b/e/h). Moreover, the rms deviations of backbone, and GC/CG stems located between the consecutive 1×1 nucleotide AA internal loops were plotted in order to investigate the rigidity of different regions of 3xCAG with respect to time (Figure 8, a/d/g).</p><p>Similar to the results of MS1 and MS2, it was observed that there was a Na+ binding pocket around the syn-anti type 1×1 nucleotide AA internal loop (see Figure 8i, first 125 ns). The initial conformation of A12-A23 was syn-anti, and this conformation was stable for the first 125 ns (Figure 8h). In this syn-anti state, different Na+ ions were binding to the pocket for as long as 30 ns (Figure 8, h and i). At around 175 ns, however, a full transformation from synA12-antiA23 to antiA12-antiA23 was observed (Figure 8h). This structural transformation was initiated when there were no Na+ ions bound to the pocket (Figure 8i). Without a Na+ ion in the pocket, synA12 first unstacked through the minor groove side to transform itself into a transition state with two hydrogen bonds (Figure 6d and highlighted with orange color in Figure 8h). This particular transformation from the synA12-antiA23 to the 'transition state' synA12-antiA23 was seen a couple of times in the first 175 ns (highlighted with orange in Figure 8h). At around 175 ns, synA12 transformed fully to antiA12 to form the antiA12-antiA23 base pairing (Figure 8h). Once the A12-A23 base pair was in the anti-anti form, multiple anti-anti→anti-anti transformations were observed until the end of the MD simulation (Figures 6 and 8h).</p><p>In order to explicitly define the synA-antiA↔antiA-antiA transformation seen in the MD simulation, the (χ, θ) states occupied by A12 between 125 and 175 ns were plot (Figure S6). The pathway P1 predicted by umbrella sampling calculations (Figure 5) was followed by A12 in the synA12-antiA23↔antiA12-antiA23 transformation. Note that the prediction of pathway P1 for MS2 by umbrella sampling calculations is almost identical to the 3xCAG MD simulation result (Figures 5 and S6).</p><p>The other two 1×1 nucleotide AA internal loops, A6-A29 and A9-A26, which were started in anti-anti conformations, never transformed to a syn-anti state. Similar to the results of MS1 and MS2, no Na+ binding pocket was found when the 1×1 nucleotide AA internal loop was in the anti-anti conformation (Figure 8, c and f). As before, multiple anti-anti↔anti-anti AA transformations were observed for these internal loops (Figure 8, b and e).</p><p>Model 3xCAG is not as rigid as model systems MS1 and MS2. The two Watson-Crick GC/GC base pairs between each 1×1 nucleotide AA internal loop in 3xCAG were responsive to the environment. Rms deviations of G10C11/G24C25 at around 185 ns (right after the synA→antiA transformation) and 600 ns, and of G7C8/G27C28 at around 750 ns were over 3 Å (Figure 8, d and g). These fluctuations have a direct effect on the RNA backbone, giving an rmsd over 10 Å during these times (Figure 8a). Such flexible neighbors around 1×1 nucleotide AA internal loops were not observed in the model systems MS1 and MS2 that may contribute to lowering the free energy barrier for the synA↔antiA transformation.</p><p>Except for the transition state, all stable 1×1 nucleotide AA internal loops in the MD studies of 3xCAG, MS1, and MS2 have one hydrogen bond (Figure 6). These non-canonical pairs are weak compared to Watson-Crick base pairs, which have two or three hydrogen bonds.80-82 As a result, any slight perturbation of the 1×1 nucleotide AA internal loop will have an effect on its conformation. Adenosine has three electronegative regions (N1, N3, N7) (Figure 2a) that can make non-covalent electrostatic interaction with cations such as Na+ and molecules. In a 1×1 nucleotide AA internal loop with one hydrogen bond, there are a total of five free electronegative groups on the adenosine bases that can interact with Na+ ions. Except for the Na+ ions bound to the pocket in syn-anti AA, the ions spent only short amounts of time near these electronegative regions (Figures 8c/f/i, and S5). It was observed that, depending on the momentum of the ions, they could assist 1×1 nucleotide AA internal loops in the structural transformations represented in Figure 6 (Movies S1-S3).</p><!><p>The 1×1 nucleotide AA internal loop conformations predicted by MD simulations (Figure 6) are also observed in RNA crystal structures. The Cate group at the University of California at Berkeley refined crystal structures of Escherichia coli (E. coli) ribosome bound to different complexes.83-90 In these structures, there are 5ʹCAG/3ʹGAC motifs with syn-anti 1×1 nucleotide AA internal loop conformations. Yet, the ribosomal structure is much more complex compared to the 3xCAG duplex structure that is presented herein. Thus, different long-range interactions can play roles to alter the conformation of the 5ʹCAG/3ʹGAC motif in the ribosome. For example, in the bacterial ribosome the conformation of one of the terminal GC base pairs in a 5ʹCAG/3ʹGAC motif does not form a canonical GC paired structure due to the formation of a pseudoknot. In the crystal structure of the ribosome-bound cricket paralysis virus, the 5ʹCAG/3ʹGAC motif has anti-anti AA conformation that is similar to Figures 6a and 6c.91 The crystal structure of r(GGCAGCAGCC)2 has two 5ʹCAG/3ʹGAC motifs that have both 1×1 nucleotide AA internal loops in anti-anti form similar to the structure presented in Figure 6b.23 This crystal structure, however, has two sulfate (SO42−) molecules that interact with the amino groups of 1×1 nucleotide AA internal loops to stabilize the conformation. This observation further suggests that the interaction of ions or small molecules can affect the structures of the 1×1 nucleotide internal loops in transcripts that contain expanded repeats in significant ways. These ligand-induced structural changes could also affect the binding of protein to these expanded repeats.</p><!><p>Purines and pyrimidines have different electronic structures, and this difference brings different properties to their nucleotides. One such structural difference is seen in the preferred glycosidic dihedral angles, χ, which are responsible for the base orientation with respect to sugar. While pyrimidines prefer mostly the anti conformation, purines have a tendency to be in the syn state.69 This main difference between pyrimidines and purines will have an affect on properties of different RNA triplet repeats. Recently, the χ torsional parameters for the Amber force field were revised, yielding better structural and thermodynamic predictions for unique RNA systems.69,71,72 Backed with the revised χ parameter sets, structural and thermodynamic calculations on other RNA triplet repeats such as CUG, CCUG, CCG, and CGG will produce important results that can be used against the genetic neurological diseases described above.</p><p>In this report, we described a refined crystal structure of r[5ʹUUGGGC(CAG)3GUCC]2 (r(3xCAG)X-RAY), a model of r(CAG)exp, refined to 1.65 Å resolution. In conjunction with MD simulations, the structure showed that the 1×1 nucleotide AA internal loops sample anti-anti and syn-anti conformations. Moreover, the 1×1 nucleotide AA internal loops have structures that are sensitive to their environment. Our MD studies provide key insights to the properties of the AA internal loops. While the 1×1 nucleotide AA internal loops in r(3xCAG)X-RAY repeats prefer the anti-anti conformation the syn-anti form is another stable conformation. The latter conformation has structure that is affected and stabilized by a Na+ binding pocket formed near this internal loop. Parallel to this result, the crystal structure shows dangling uridine bases stabilizing the syn-anti conformations. These studies thus suggest that a small molecule that binds to 1×1 nucleotide AA internal loops can affect conformation. Such information is useful in understanding the potential structural consequences of ligand binding to repeating transcripts and designing compounds that affect biological function.</p><p>For diseases caused by r(CAG)exp, small molecules that can inhibit protein binding thus modulating RNA gain-of-function toxicity and inhibit translation of toxic polyQ proteins would be ideal. Both of these active areas of research24,92-94 can be aided by a more thorough understanding of the structural nature of expanded repeats including information about dynamics.</p>

PubMed Author Manuscript

Exploiting a Global Regulator for Small Molecule Discovery in Photorhabdus luminescens

Bacterially-produced small molecules demonstrate a remarkable range of structural and functional diversity and include some of our most useful biological probes and therapeutic agents. Annotations of bacterial genomes reveal a large gap between the number of known small molecules and the number of biosynthetic genes/loci that could produce such small molecules, a gap that most likely originates from tight regulatory control by the producing organism. This study coupled a global transcriptional regulator, HexA, to secondary metabolite production in Photorhabdus luminescens, a member of the Gammaproteobacteria that participates in a complex symbiosis with nematode worms and insect larvae. HexA is a LysR-type transcriptional repressor, and knocking it out to create a P. luminescens \xce\x94hexA mutant led to dramatic upregulation of biosynthesized small molecules. Use of this mutant expanded a family of stilbene-derived small molecules, which were known to play important roles in the symbiosis, from three members to at least nine members.

exploiting_a_global_regulator_for_small_molecule_discovery_in_photorhabdus_luminescens

<!>UvrY and Lrp do not regulate stilbene production in P. luminescens<!>HexA regulates stilbene virulence factor production in P. luminescens<!>HexA regulates stilbene and anthraquinone production in P. temperata<!>L-proline dose-response effects in P. luminescens \xce\x94hexA<!>Stilbenes possess multipotent activities<!>Conclusion<!>Genetic inactivation of lrp and hexA in P. luminescens<!>P. temperata \xce\x94hexA proline dose-response<!>P. luminescens \xce\x94hexA proline dose-response<!>NMR analysis<!>

<p>Photorhabdus luminescens, a Gammaproteobacterium, uses a functionally diverse suite of secondary metabolites to participate in a complex symbiosis with nematode worms (Heterorhabditis spp.) and insect larvae. The bacteria persist quietly in the guts of infective juvenile (IJ) nematodes that hunt insect larvae. When a worm succeeds in entering its prey's circulatory system (hemolymph), it regurgitates the bacteria, which then proceed to make toxins that kill the larva, proteases and esterases that liquefy the larva's interior, signals that cause the IJ worms to become reproducing adults, molecules that counter insect defense mechanisms, and molecules that protect their prey from competing bacteria and fungi. Some of the small molecules produced by P. luminescens have been identified (1), but despite efforts in many laboratories, these known small molecules represent only a small fraction of the bacteria's metabolic potential. The sequenced P. luminescens genome contains at least 33 genes in 20 loci that encode proteins similar to polyketide synthases, nonribosomal peptide synthetases, and β-lactam-producing enzymes (2). The genomic potential for secondary metabolism seen in P. luminescens rivals members of the Streptomyces genus, the most productive antibiotic-producing bacterial genus (3).</p><p>To access these uncharacterized small molecules, we searched for the molecular signals and their targets that control P. luminescens metabolism. Recently we reported that the bacteria respond to the high concentrations of L-proline in insect hemolymph by initiating a profound upregulation of secondary metabolite production (4). L-proline enhances the production of small molecules known to be involved in antibiosis, insect virulence, and nematode mutualism along with many structurally and functionally uncharacterized molecules. L-proline acts both as an osmoprotectant in the high solute concentrations characteristic of insect hemolymph (5) and more importantly, as a nutrient signal and electron source to enhance the proton motive force believed to regulate downstream pathways involved in antibiotic production and virulence (4). This report concerns the downstream regulation of metabolite production and the discovery of previously undescribed small molecules involved in important aspects of the symbiosis.</p><p>Global regulators, which affect the transcription of gene ensembles via regulatory cascades, typically govern the production of small molecules in bacteria (6). Identification and manipulation of these global regulators could provide a powerful approach to complete sets of biologically important and previously uncharacterized small molecules.</p><!><p>To define a link between the L-proline response and the global regulatory genes involved in controlling the transition from nematode symbiosis to insect pathogenesis, we investigated three candidate genes. The first involved a two-component regulatory system, BarA/UvrY, that regulates a selection of virulence genes. A uvrY-deficient P. luminescens mutant exhibited decreased production of proteases and toxins, as well as decreased bioluminescence (7). UvrY also regulated several genes with suspected roles in antibiotic synthesis and efflux, as well as motility and oxidative stress response (7). However, metabolomic profiling of organic extracts from the uvrY-deficient strain showed no significant changes compared to wild type (WT) in the production of known antibiotics and small molecule virulence factors – anthraquinone polyketides and stilbenes (Figure S1). The second candidate was a leucine-responsive protein (Lrp) regulator that the Goodrich-Blair laboratory had identified as a global regulator of metabolic switching in Xenorhabdus nematophila (8), a bacterium that participates in a symbiosis similar to that of Photorhabdus. When Lrp binds a small molecule ligand, often an amino acid, it becomes a transcriptional activator. Markerless deletion of the homologous lrp gene in P. luminescens by allelic exchange mutagenesis did not result in significant changes to anthraquinone or stilbene production compared to WT (Figure S1). Since earlier work had shown that both Photorhabdus and Xenorhabdus used L-proline to initiate the metabolic switch, this difference in downstream regulation fits a convergent evolution model for the Photorhabdus and Xenorhabdus systems (4).</p><!><p>The third candidate was the LysR-type transcriptional regulator HexA. The Clarke laboratory had shown that the related species Photorhabdus temperata uses HexA to repress general antibiotic activity while dwelling within its nematode host (9). Indeed, disruption of this gene, homologous to the hexA (hyperproduction of exoenzymes) gene of Erwinia carotovora, (10) led to increased (derepressed) antibacterial activity in P. temperata as judged by a larger zone of inhibition phenotype (9). Obtaining a stable hexA knockout in P. luminescens proved challenging in our hands. Insertional inactivation by plasmid integration could be successfully achieved and confirmed by PCR, but the genetic insertion was repeatedly lost in the subculturing attempts needed to obtain a pure mutant strain.</p><p>It seemed likely that upregulation of antibiotics and protein toxins in the hexA knockout of P. luminescens caused the instability, so we reasoned that L-proline might have a protective effect under these conditions, perhaps through the activation of compensatory pathways such as efflux pumps or resistance proteins. By supplementing the medium with 100 mM L-proline, we were able to propagate cultures of the P. luminescens insertional hexA mutants for chemical interrogation. The stabilizing effect of L-proline in these mutants provides further evidence of its important role in the life cycle of P. luminescens.</p><p>Metabolomic profiling of organic extracts from the P. luminescens hexA mutant by high-pressure liquid chromatography (HPLC) revealed upregulation of multiple metabolites compared to the WT strain (Figure 1). Production of known stilbenes (1 – 3) in addition to other compounds with stilbene-like UV absorbance was increased in the ΔhexA mutant (starred peaks in Figure 1), while production of anthraquinone compounds (10 and 11) was marginally downregulated compared to WT. These differential effects complement those obtained from analysis of the P. luminescens proline transporter mutants (ΔproU and ΔputP), in which both the ΔproU and ΔputP strains demonstrated dramatically increased production of anthraquinones and decreased stilbene production (4). The reciprocal effects between the ΔproU/ΔputP and ΔhexA mutants argue that L-proline transport and its subsequent metabolism contributes to derepression of HexA and upregulation of the stilbene class in P. luminescens.</p><p>A number of the compounds upregulated in the ΔhexA strain were isolated and structurally characterized (Figure 2). Analysis of the unknowns by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS) led to the discovery of six new stilbene derivatives (4 – 9). These new compounds mostly share a similar carbon skeleton as the previously identified stilbenes but differ in oxidation states. Because known stilbenes (1 – 3) mediate several critical functions in the symbiosis, their biosynthesis has been carefully studied (11–13). The identification of six new derivatives in the L-proline stabilized ΔhexA mutants illustrates the ability of genetically manipulating global regulators to uncover both the quantitative and qualitative molecular diversity in this important class.</p><!><p>To correlate metabolite production to the larger zone of inhibition phenotype previously observed in the P. temperata ΔhexA mutant (9), we also analyzed organic extracts from the mutant and its WT parent. Differential metabolomic profiling of the mutant compared to WT revealed greatly enhanced production of both stilbenes and anthraquinones in the ΔhexA mutant, which was independent of proline concentration (Figure S2). Production of the stilbene metabolites by ΔhexA far exceeded that of the WT strain even in cultures supplemented with high L-proline concentrations (Figure S2). Regulation of the major metabolites in the P. temperata mutant resulted in a dramatic increase in production of a small number of compounds, including known stilbenes and anthraquinones. The P. luminescens hexA knockout, on the other hand, displays a less dramatic upregulation of a larger number of compounds, as described below. Moreover, while these results confirm hexA's regulatory role as repressor of stilbene biosynthesis in Photorhabdus species, they also illustrate differences in anthraquinone regulation across the species; anthraquinone production is upregulated in the P. temperata ΔhexA mutant, but marginally downregulated in P. luminescens ΔhexA.</p><!><p>To further investigate the effect of increasing L-proline concentration on the production of metabolites by P. luminescens ΔhexA, L-proline dose-response curves were generated for the upregulated stilbenes (Figure 3). Similar to the effects seen in P. temperata ΔhexA (Figure S2), P. luminescens ΔhexA exhibits constitutive production of many of these metabolites compared to WT. Production of the reduced stilbene derivative (4), for example, was upregulated 50-fold in ΔhexA with high concentrations of supplemental L-proline. Under high L-proline concentrations, metabolites 5 – 8 are repressed in both WT and ΔhexA, perhaps due to their conversion to other compounds, suggesting a metabolic shift rather than a general upregulation. Production of 9 is unchanged in the ΔhexA mutant (Figure S3), but this metabolite diverges structurally from the other stilbenes and may serve other biological functions.</p><!><p>Stilbenes are common plant metabolites, but Photorhabdus is the only known producer of stilbenes outside the plant kingdom (14). The stilbenes in Photorhabdus are biosynthesized from the condensation of two β-ketoacyl intermediates (Scheme 1), while plant-derived stilbenes arise through the linear elongation of an activated phenylpropanoid starter unit with malonyl-CoA extender units (11), which indicates that the Photorhabdus biosynthetic pathway evolved independently of plants. In spite of its independent bacterial origin, the pathway's genes have an odd organization. While bacterial biosynthetic pathways for secondary metabolites, including those for many of the annotated pathways in the P. luminescens genome (15), typically have clustered genes, the stilbene biosynthetic genes are not clustered (11). They are organized more like the scattered genes found in plant biosynthetic pathways.</p><p>The newly-identified reduced stilbene (4) is especially interesting from both biosynthetic and physiological perspectives. The biosynthesis of 1 proceeds from phenylalanine, derived from prephenate, and leucine (11). Similar transformations on 2,5-dihydrophenylalanine (DHPA) would lead to the synthesis of 4 rather than 1 (Scheme 1). DHPA, also derived from prephenate (16), is a known antibiotic produced by Streptomyces sp., and functions as a microtubule-disrupting agent (17). Compound 4 and any of its proposed precursors could serve as oxidative sinks to protect the bacterium from the massive levels of oxidative stress it encounters in the insect hemolymph (18). Oxidation would yield the corresponding metabolite along the pathway to 1. DHPA, for example, can spontaneously oxidize to phenylalanine (19). This strategy of producing easily oxidized metabolites resembles the production of redox active pigments by Pseudomonas aeruginosa and Staphylococcus aureus (20–21) and suggests still another biological role for this stilbene family in addition to those described below (11). The biosyntheses of the remaining new metabolites remain unknown, but they are likely to be oxidation products of the major stilbenes 1 or 2.</p><p>The previously described Photorhabdus stilbenes (1 – 3) exhibit a broad range of biological activities that illustrate their critical roles in many aspects of the complex symbiosis. They mediate interactions with competing microbes, with their nematode hosts, and with their insect prey. These stilbenes have been shown to possess antibiotic activity against fungi and Gram-positive bacteria (22), they are potent inhibitors of phenoloxidase, one of the insect's key defenses against microbial pathogens (13), and they are essential in maintaining the bacterium's mutualistic symbiosis with its nematode host by influencing nematode development (11).</p><p>In order to efficiently exit their IJ stage and become reproducing adults, the nematodes respond to molecular signals made by their bacterial symbionts (23). Previous investigations have demonstrated that IJ nematodes grown on a stilbene-deficient strain of P. luminescens, ΔstlA mutants with the first committed step of stilbene biosynthesis knocked out (Scheme 1), have a recovery rate only 5–15% of that achieved with WT P. luminescens (11). Growing IJ nematodes on the ΔstlA strain supplemented with either stilbene 1 or the StlA product cinnamic acid yielded almost full IJ recovery, while no recovery was observed when IJs were supplemented with 1 or cinnamic acid but no bacteria (11). These results indicate that stilbene-derived compounds are essential for nematode recovery, but stilbene 1 cannot induce recovery on its own. It is likely that 1 is converted by P. luminescens (and possibly further elaborated by the nematode) to the biologically active but not yet identified compound.</p><p>The IJ stage of the nematode H. bacteriophora is analogous to the dauer stage of the much better known model nematode Caenorhabditis elegans. In C. elegans, entry into dauer is mediated by the dauer pheromone, which is made by the nematodes. Exit from dauer is governed by an unknown molecular signal called the 'food signal' (24) because it is made by the bacteria that C. elegans consume. While dauer pheromones for C. elegans have been actively studied, the food signal has been ignored, and access to the new stilbenes could provide the molecular tools needed to study this developmental switch. Further biological experiments will be needed to fully explore the roles played by individual members of the stilbene family uncovered in this study.</p><!><p>Identifying the small molecules produced by orphan biosynthetic pathways – pathways that can be identified in sequenced genomes but whose products have not been characterized – represents both a great opportunity and a substantial challenge. The cryptic metabolites produced by these pathways typically outnumber the known metabolites by an order of magnitude, and many would be expected to have potential therapeutic applications. These metabolites are most likely cryptic because the pathways that produce them are tightly regulated, and we currently know very little about what conditions activate them. This study shows that identifying global regulators and manipulating them can lead to the accelerated discovery of cryptic metabolites. The study also illustrates the power of focusing on symbiotic associations both as a way to identify regulatory triggers and as a way to place any metabolites that might be produced into their biological context.</p><!><p>P. luminescens gDNA was isolated as previously described (25). The entire coding sequence from start to stop codons of lrp (locus tag: Plu1600; Protein Accession: NP_928891) was excised by allelic-exchange mutagenesis to generate a markerless deletion mutant. The exchange sequence for lrp consisted of ~1 kB of upstream and downstream genome sequence fused by overlap extension PCR (26) (see Supplemental Methods). The full-length lrp exchange sequence was digested with SacI, inserted into the corresponding site in pDS132 (27), and verified by restriction analysis (pDiLrp). Cloning was carried out in E. coli strain WM3618 lambda pir.</p><p>Because markerless deletion attempts of hexA failed in our hands, an internal hexA (locus tag: Plu3090; Protein Accession NP_930322) gene fragment was amplified, digested with SacI, and inserted into the corresponding site in pDS132 (pDiHexA) for plasmid integration (see Supplemental Methods). Ligation products in both directions were successfully taken forward to insertionally inactivate hexA by pDiHexA plasmid integration containing a chloramphenicol resistance marker.</p><p>Mutants were generated using similar procedures as previously described (4). The pDS132 deletion constructs (pDiLrp or pDiHexA) were transformed into the diaminopimelic acid (dap) auxotroph donor strain, E. coli WM6026 lambda pir (28), by heat-shock transformation (29). The donor E. coli and recipient WT P. luminescens TT01 were filter mated, replated on LB-chloramphenicol, then selected on LB sucrose plates for counterselection (see Supplemental Methods). Positive deletions were identified by colony PCR and sequence verified. For insertional inactivation of hexA, agar plates used for filter mating and all subsequent plating steps were also supplemented with 100 mM L-proline. Successful hexA plasmid integrants were identified by colony PCR and sequence verified (see Supplemental Methods). No SacB counter selection was performed.</p><!><p>Rifampicin-resistant ΔhexA and its rifampicin-resistant parent strain of P. temperata were assessed for metabolite stimulation with increasing concentrations of L-proline (0–100 mM) (9). All experimental conditions were identical to those previously described (4).</p><!><p>The P. luminescens ΔhexA mutant was grown on LB agar + 100 mM L-proline + 25 μg mL−1 chloramphenicol for two days at 30 °C. The WT strain was cultured similarly, but without antibiotic. Single colonies were selected and grown two additional days in 5 mL LB broth (WT) or LB with 25 μg mL−1 chloramphenicol (ΔhexA). Cultures were then centrifuged and resuspended in 5 mL fresh LB (to remove chloramphenicol and ensure medium consistency between mutant and WT). For metabolite stimulation assays, 50 μL of this resuspended culture was used to inoculate 5 mL of a rich tryptone-yeast extract based medium (2 g tryptone, 5 g yeast extract, and 10 g NaCl per L) with increasing amounts of L-proline (0–100 mM). Triplicate cultures were grown to stationary phase over 72 h at 30 °C and 250 rpm. The cultures were vigorously extracted with 6 mL ethyl acetate, then centrifuged and 4 mL of the top organic layer dried. These dried extracts were resuspended in 1 mL methanol, and 50 μL of this mixture was injected for HPLC analysis to quantify metabolite production (see Supplemental Methods for details).</p><p>Due to the tendency of the hexA mutant to revert to WT in the absence of proline, we reasoned that cultures supplemented with little to no proline may similarly have reverted during the 3-day incubation prior to extraction for metabolite analysis, thereby skewing the results. Prior to extraction for assessment of metabolite production, representative cultures with high and low concentrations of proline were therefore sampled and used as template in a PCR assay to evaluate whether reversion to WT was occurring. Results demonstrated only low levels of reversion in cultures with no supplementary proline over the 3-day growth period (Figure S4).</p><!><p>NMR experiments (Varian: 1H, gCOSY, gHSQC, and gHMBC) were performed in deuterated methanol with a symmetrical NMR microtube susceptibility-matched with the solvent (Shigemi, Inc.) on a Varian INOVA 600 MHz NMR. Known metabolites were confirmed by 1H NMR and mass spectrometry.</p><!><p>Representative HPLC trace overlay (210 nm) of organic extracts of WT (black) and ΔhexA (blue) P. luminescens cultures, both grown in 5 mM L-proline. Starred peaks denote stilbene derivatives, upregulated in the ΔhexA strain. Numbers above peaks refer to compounds in Figure 2.</p><p>Structures of new and known stilbene derivatives isolated from P. luminescens. 1 – 3 have been previously isolated from Photorhabdus. 4 – 9 are new natural products. All four stereoisomers of 5 were isolated in a single fraction. 9 was previously described as a side product in a synthetic scheme (30).</p><p>Fold change in production of stilbene metabolites by WT (black •) and ΔhexA (blue ) P. luminescens with increasing concentrations of supplementary L-proline. Numbers above curves refer to structures in Figure 2. Compounds 6 – 8 are overlapped on the HPLC trace and were therefore integrated together.</p><p>Proposed biosynthesis of reduced stilbene 4, based on the established biosynthesis of 1. The pathways to these compounds diverge at prephenate, and oxidation of any intermediate along the route to 4 leads to the convergent production of 1.</p>

PubMed Author Manuscript

Local vs global motions in protein folding

It is of interest to know whether local fluctuations in a polypeptide chain play any role in the mechanism by which the chain folds to the native structure of a protein. This question is addressed by analyzing folding and non-folding trajectories of a protein; as an example, the analysis is applied to the 37-residue triple \xce\xb2-strand WW domain from the Formin binding protein 28 (FBP28) (PDB ID: 1E0L). Molecular dynamics (MD) trajectories were generated with the coarse-grained united-residue force field, and one- and two-dimensional free-energy landscapes (FELs) along the backbone virtual-bond angle \xce\xb8 and backbone virtual-bond-dihedral angle \xce\xb3 of each residue, and principal components, respectively, were analyzed. The key residues involved in the folding of the FBP28 WW domain are elucidated by this analysis. The correlations between local and global motions are found. It is shown that most of the residues in the folding trajectories of the system studied here move in a concerted fashion, following the dynamics of the whole system. This demonstrates how the choice of a pathway has to involve concerted movements in order for this protein to fold. This finding also sheds light on the effectiveness of principal component analysis (PCA) for the description of the folding dynamics of the system studied. It is demonstrated that the FEL along the PCs, computed by considering only several critically-placed residues, can correctly describe the folding dynamics.

local_vs_global_motions_in_protein_folding

Introduction<!>UNRES model and simulation details<!>Principal component analysis in internal coordinates<!>Results and Discussion<!>FEPs along \xce\xb8i and \xce\xb3i angles of the folding trajectory<!>FEPs along \xce\xb8i and \xce\xb3i angles of the non-folding trajectory<!>FEPs along the distances between the residues in folding and non-folding trajectories<!>Hairpin I<!>Hairpin II<!>PCA of the folding trajectory<!>Conclusions<!><!>Conclusions

<p>In spite of long, assiduous experimental and theoretical studies, one of the most important problems of biophysics and biochemistry, how proteins reach their biologically active conformations, still remains unsolved. In order to understand the kinetics and thermodynamics of protein folding, it is important to know why proteins fold properly or do not reach their native state, and what governs the way that proteins fold. From theoretical and conceptual points of view, it is well-known that a study of free-energy landscapes (FELs) holds the key to understand how proteins fold and function.1–3 However, the selection of a correct model for protein folding kinetics and the coordinates along which the intrinsic folding pathways can be identified in order to interpret experimental data still remains challenging. Some progress to treat this problem has been made recently based on the analysis of molecular dynamics (MD) simulations of protein folding,4–12 particularly, in the use of principal component analysis (PCA),7–10 transition disconnectivity graphs (TRDG),4–6 and network analysis.6,12 This involves proper choices for the reaction coordinates and the treatment of progress variables. However, the question as to what governs the way in which proteins fold was not addressed in those studies.</p><p>It is well established that such important questions about protein folding can be answered by examining the behavior of small model proteins and peptides. Therefore, in the present study, in order to find the reasons why a protein may or not fold properly to its native state, we analyzed MD trajectories of a particular 37-residue protein as an example, namely, the triple-β-stranded WW domain from the Formin binding protein 28 (FBP28) (PDB ID: 1E0L)13 (Figure 1), generated with the coarse-grained united-residue Fig. 1 (UNRES) force field.10,14–21</p><p>The FBP28 WW domain is a member of the WW domain family. The WW domains have been the subject of extensive theoretical7,9,10,22–30 and experimental31–36 studies because of their small size, biological importance,37 and interesting fast-folding kinetics. From the folding point of view, the FBP28 WW domain is a very interesting system to study because of its biphasic folding kinetics22,23,33 [i.e., coexistence of slow (three-state) and fast (two-state) phases]. Also, there is an interesting dilemma offered by β-sheets regarding the mechanism controlling the folding rate. Previous experimental and theoretical studies have shown that either local interactions at the turns38,39 or the interstrand interactions of hydrophobic residues control the folding,40,41 although some studies illustrated a close competition between these mechanisms.42</p><p>It should be noted that, in spite of the biological importance of the WW domain, our interest in this work is in the folding problem per se (therefore, a new approach for the analysis of MD trajectories of protein folding is presented), and not specifically in the properties of the WW domain protein. Recently, we studied30 not only the wild type FBP28 WW domain but also its mutants and truncated mutants at different temperatures, and widely discussed the agreements and discrepancies of our results with experiment.31–34 Here, several important aspects of folding (folding time, folding-transition temperature, folding scenario, the behavior of hairpins, and the role of loops in folding) are compared with experiment (see Materials and Methods, and Results and Discussion sections).</p><p>We address the protein folding of the FBP28 WW domain by carrying out 10 canonical UNRES MD trajectories at 330K, which have been grouped in three categories: fast-folding [3 MD trajectories in which this protein folds within the first 40 ns (corresponding to about 40 μs of actual time because the timescale is reduced by about 1000 in UNRES MD simulations43)], slow-folding (4 MD trajectories in which this protein folds after 40 ns), and non-folding (3 MD trajectories in which this protein never folds during the entire MD simulation) (Figure S1 in the Supporting Information). All trajectories start with the same initial (extended) structure but with different velocities. From an analysis as to why different initial velocities of the MD simulations lead to proper or improper folding pathways, light can be shed on the mechanisms leading to folding or misfolding of a protein in solution.</p><p>There is strong evidence44–47 that protein folding is initiated and governed by local motions of the residues which, subsequently, result in global conformational changes. Therefore, in the present study, the folding and non-folding trajectories of the FBP28 WW domain were analyzed in terms of the relation between the local dynamics of each residue and the global dynamics of the entire system. This local and global analysis enabled us to determine which residues play an important role in folding of the FBP28 WW domain. We also answered questions regarding the effectiveness of PCA, and the role of formation of loops and β-strands, for folding and non-folding trajectories of the FBP28 WW domain.</p><p>The paper is organized as follows. The Materials and Methods section contains descriptions of the UNRES model, details of MD simulations, and the PCA method. The Results and Discussion section contains four subsections. In the first two subsections, the free-energy profiles (FEPs) along the internal coordinates of the backbone, for folding and non-folding MD trajectories, respectively, are presented and discussed to identify the key residues participating in successful folding of the FBP28 WW domain. The distances, as a function of time, and the FEPs along the distances between selected pairs of residues for folding and non-folding MD trajectories are illustrated and discussed in the third subsection. The fourth subsection answers the question as to how reaction coordinates for folding the FBP28 WW domain can be built from PCA using a relevant subset of internal coordinates of the polypeptide. A summary and conclusions are given in the Conclusions section.</p><!><p>The UNRES model of polypeptide chains10,17,48 is illustrated in Figure 2. A polypeptide chain is represented as a sequence of α-carbon (Cα) atoms linked by virtual Cα…Cα bonds with united peptide groups halfway between the neighboring Cα's, and united side chains, whose sizes depend on the nature of the amino acid residues, attached to the respective Cα's by virtual Cα…SC bonds. The effective energy is expressed by Eq. 121</p><p> (1)U=wSC∑i<jUSCiSCj+wSCp∑i≠jUSCipj+wppf2(T)∑i<j-1Upipj+wtorf2(T)∑iUtor(γi)+wtordf3(T)∑iUtord(γi,γi+1)+wb∑iUb(θi)+wrot∑iUrot(αSCi,βSCi,θi)+wbond∑iUbond(di)+∑m=36wcorr(m)fm(T)Ucorr(m)+wSS∑iUSS;i with21</p><p> (2)fm(T)=ln(e+e-1)ln{exp[(TT0)m-1]+exp[-(TT0)m-1]};T0=300K where the successive terms represent side chain-side chain, side chain-peptide, peptide-peptide, torsional, double-torsional, bond-angle bending,16 side-chain local (dependent on the angles α and β of Fig. 2), distortion of virtual bonds, multi-body (correlation) interactions, and formation of disulfide bonds, respectively. The w's are the relative weights of each term. The correlation terms arise from a cumulant expansion17,49 of the restricted free energy function of the simplified chain obtained from the all-atom energy surface by integrating out the secondary degrees of freedom. The temperature-dependent factors of Eq. 2, introduced in our recent work21 and discussed further in reference 50, reflect the fact that the UNRES effective energy is an approximate cumulant expansion of the restricted free energy. The virtual-bond vectors are the variables used in molecular dynamics.</p><p>We carried out canonical MD runs at 330 K (10 trajectories), with the force field parameterized51 on the β-strand protein 1E0L and the α-helical protein 1ENH. The Berendsen thermostat52 was used to maintain constant temperature. The trajectories selected for detailed analysis corresponded to the near folding-transition temperature, Tf = 339 K51 for 1E0L (experimental Tf = 337 K33), at which the protein either exhibits quite a stable native state and, once the system folds, it remains in the native state until the end of the simulation or the native state is still stable, but only a few folding/unfolding events are observed. The time step in molecular dynamics simulations was δt = 0.1 mtu (1 mtu = 48.9 fs is the "natural" time unit of molecular dynamics53) and the coupling parameter of the Berendsen thermostat was τ = 1 mtu. A total of 120,000,000 steps (about 0.6 μs of MD time) were run for each trajectory. It should be noted that the experimental folding time of the FBP28 WW domain is ~ 30 μs,33 while the presented fast-folding trajectory folds in ~ 37 ns. However, in order to compare the experimental and UNRES time, the latter should be multiplied by ~ 1000 because the fast degrees of freedom are averaged out.53 After taking this extension of the time scale into account, it can be seen that the experimental and computational folding times are comparable.</p><!><p>In the PCA method in internal coordinates,7–10,54–56 the correlated internal motions of a protein are expressed quantitatively by the covariance matrix Cij [Eq. (3)] of the 2 × (N − 2) variables [Eq. (4a)] representing the coarse-grained angles θ, and of the 2 × (N − 3) variables [Eq. (4b)] representing the coarse-grained angles γ (see Fig. 2 for the definitions of the angles)</p><p> (3)Cij=〈(ui-〈ui〉)(uj-〈uj〉)〉, where <…> denotes the average over all sampled conformations in the trajectory with i and j varying between 2 and 2 × (2N − 5) with N being the number of residues. The variables u in Eq. (3) are defined by</p><p> (4a){u4i-7;u4i-6}={cos[θi(t)],sin[θi(t)]} for i = 2 to N−1 and by</p><p> (4b){u4i-5;u4i-4}={cos[γi(t)],sin[γi(t)]} for i = 2 to N−2.</p><p>The diagonalization of the covariance matrix Cij leads to 2 × (2N − 5) eigenvalues λk, ordered by decreasing value: λ1 > λ2 >… > λ2X(2N−5), and eigenvectors ek = { e1k, e2k, …, e2N−5k}T, where T is the transpose, with eik={eik(1);eik(2)} where eik(n) is the nth component of the projection of the eigenvector ek on the ith internal coordinate. The contribution of each angle i to a mode k is the so-called influence: (5)vik=[(eik(1))2+(eik(2))2]</p><p>The total mean-square fluctuation MSF of the coarse-grained internal coordinates can be decomposed into modes: (6)MSF=∑i〈(ui-〈ui〉)2〉=∑k∑iλkvik</p><p>The eigenmodes with the largest eigenvalues λk correspond to the collective modes contributing the most to the MSF of the protein [see Eq. (6)]. The projection of the trajectory on the eigenvector ek is named the principal component qk.</p><!><p>We studied two types of trajectories of the FBP28 WW domain: folding (i.e., in which the protein folds during the trajectory) and non-folding (i.e., in which the protein never folds during the entire MD simulation). In order to understand the folding mechanism of the FBP28 WW domain protein, we studied the local motions of each residue along the sequence. In particular, we investigated the FEPs along the backbone virtual-bond angle θ and backbone virtual-bond-dihedral angle γ, of each residue (defined in Figure 2). The quantity θi for residue i is the angle formed by the vectors (virtual bonds) joining three successive Cα atoms (i − 1, i, i + 1) along the primary sequence. The first angle along the sequence is θ2 and the last is θN−1 in which N is the total number of residues. The quantity γi for residue n is the dihedral angle formed by the vectors (virtual bonds) joining four successive Cα atoms (i − 1, i, i + 1 and i + 2) along the primary sequence. The first dihedral angle along the sequence is γ2 and the last is γN−2. This approach along the backbone virtual-bond-dihedral angles γi was used previously for describing how a single native protein diffuses on its free-energy landscape.57–59</p><p>It should be noted that, although the FEPs along the θi and γi angles of the entire trajectory [(μ)θ=− kBT ln P(θ), μ(γ)=− kBT ln P(γ), where P, T and kB are the probability distribution function (pdf), the absolute temperature, and the Boltzmann constant, respectively] are very helpful to identify the key residues in the folding process,10 the analysis of the FEPs of the entire trajectory does not provide information about the way in which each residue explores its own FEP in the course of time nor to what extent the motion of each residue is coupled to the global motion of the protein as it proceeds towards its native state. To answer these questions, we have selected one folding trajectory and calculated FEPs along the θi and γi angles for certain periods of time in the non-native state, during which significant structural changes occur before the protein reaches its native state. In order to find out which residues play a crucial role in folding, we compared these FEPs to those computed for one non-folding trajectory in the same time-periods in which the FBP28 WW domain makes noticeable jumps although it never folds. It should be noted that the selection of time periods does not influence the FEPs of the non-folding trajectory significantly, as shown below in the "FEPs along θi and γi angles of the non-folding trajectory" subsection.</p><p>It should be noted that the FEPs presented here are effective FEPs because they are computed from a non-equilibrium probability density and depend on the time duration and on the initial conditions of the trajectory. The effective FEP differs from the actual FEP, which is an equilibrium thermodynamic property and should be computed from the entire sets of trajectories (folding and non-folding). Because of the dependence of the effective FEP on the time duration of the trajectory and on the initial conditions, we use the effective FEP to analyze in detail the MD trajectories and extract the reasons why a protein folds or not in a single MD trajectory.</p><p>Figure 3 shows how the periods of time, over which the FEPs were calculated, were selected. Panel (a) illustrates the RMSD as a function of time of folding (black) and non-folding (red) MD trajectories. The first ~58 ns of both trajectories were selected and expanded [panels (b) and (c)]. In the first 37 ns, the FBP28 WW domain remains unfolded in the folding trajectory, forming unfolded and intermediate states, and subsequently jumps into the native state (t > 37 ns). Based on significant changes in the RMSD of the folding trajectory [panel (b)], three time-periods were selected to calculate the FEPs. These are: ~4.5 ns (blue rectangle), ~21 ns (red rectangle) and ~37 ns (green rectangle).</p><!><p>Figure 4 shows the FEPs along the θi and γi angles computed from the folding [Figures 4(a) and 4(b), respectively] and non-folding [Figures 4(c) and 4(d), respectively] MD trajectories. The blue, red, green and black curves correspond to the FEPs calculated over 4.5 ns, 21 ns, 37 ns and over the entire duration of the trajectories, respectively. It should be noted that, in order to avoid some confusion caused by the deep minimum in the neighborhood of 180°, the range of the fluctuations of the γi angles, which is between [−180°; 180°], was shifted to [0°; 360°] in Figures 4(b) and 4(d).</p><p>Two main (representative) shapes in the FEPs computed over the entire folding trajectory [black lines in Figure 4(a)] can easily be distinguished along the sequence of the θi angles. The first shape, with one deep minimum at ~ 90°, represents FEPs of loop and end residues (FEPs with black numbers); the second shape, with a broad, deep minimum at ~ 120° – 130° and a narrow (less deep) local minimum at ~ 90°, represents FEPs of β–strand residues (FEPs with red numbers); the shapes of the FEPs along the θi angles, which include residues from both loops and β–strands, or ends and β–strands (FEPs with green numbers) either have a "transition" shape between the representative shapes for the loops and β–strands (θi, i = 8, 31), or resemble one of them [i.e., the FEPs along θi, i = 7, 13, 14, 16, 23 – 25, 30 resemble the representative shape of loop residues, and the forms of the FEPs along θi, i = 17, 26 resemble the representative shape of the β–strand residues]. Only a few FEPs along the θi angles (θi, i = 22, 32, 33) have forms that are different from their representative shapes.</p><p>Based on the FEPs along the θi angles of the selected time-periods, during which the whole system remains in the non-native state [blue, red, and green lines in Figure 4 (a)], most of the θ angles within the first and middle β–strands (θi, i = 9 – 12, 18 – 21) completely explore the narrow local minimum at ~ 90°. These θ angles jump to their native minimum, as does the whole system simultaneously, and stay in the deep global minimum at ~ 120° – 130° during the rest of the trajectory [black lines in Figure 4 (a)]. It should be noted that, in the non-native state, these θ angles explore not only the local minimum, but they also spend some time (less than in the local minimum for θi, i = 9, 10, 18; and more than in the local minimum for θi, i = 11, 12, 19–21) in the global minimum [blue, red, and green lines in Figure 4 (a)]. The θ angles of the third β–strand (θi, i = 27 – 29) partially explore the narrow local minimum at ~ 90° while the protein is in the non-native state. After the protein jumps to the native state [shown by the decrease of the RMSD (Figure 3 (b)], the θ angles of the third β–strand start to interconvert between the local (90°) and global (~ 120° – 130°) minima. Like the θ angles of the first and middle β–strands, the θ angles of the third β–strand also start exploring the native minimum in the non-native state [blue, red, and green lines in Figure 4 (a)]. Moreover, the θ angles of the first β–strand edges (θi, i = 7, 13, 14), the N-terminus (θi, i = 5, 6), and some θ angles in the neighborhood of the middle β–strand (θi, i = 17, 23, 26) either completely or partially explore their local minima or "shoulder" before the protein jumpsto the native state. The rest of the θ angles (θi, i = 2 – 4, 8, 15, 16, 22, 24, 25, 30 – 36) gradually explore their own minima during the entire trajectory and, hence, do not follow changes in the dynamics of the whole system.</p><p>A more detailed, quantitative representation of the results illustrated in Figure 4(a) is given in Table S1(a) [see the Supporting Information].</p><p>Unlike the FEPs along the θi angles shown in Figure 4(a), the shapes of the FEPs along the γi angles of the full trajectory [black lines in Figure 4(b)] are diverse. However, the FEPs for the β–strand residues (FEPs with red numbers) and their edges (FEPs with green numbers) are similar to each other. In particular, they exhibit one deep minimum at ~ 180° (γi, i = 8 – 12, 16 – 22, 26 – 29) along with a few shallow minima in the region of 40–100° (γi, i = 8 – 12, 16, 18 – 22, 26 – 29) and in the vicinity of 270° (γi, i = 9, 11, 12, 17, 19, 22, 26 – 29). There is no representative shape for the FEPs at the loop residues connecting the β–strands. This is not surprising because there are not enough loop residues to define a γ angle; consequently, every γ angle in this part of the protein is influenced by the neighboring β–strand. Only a few γi angles at the N- and C-terminal loops have FEPs similar to each other (γi, i = 5, 34, 35). It should be noted that, as in the FEP along the θ33 angle, the shape of the FEP for γ33 is similar to the representative shape of the FEPs of the β–strand residues.</p><p>The FEPs along the γi angles for selected time-periods [blue, red, and green lines in Figure 4 (b)] clearly illustrate that the residues of the first and middle β–strands and their edges (γi, i = 7 – 21) completely explore all possible shallow local minima before jumping to the native state and then remain in a deep global minimum until the end of the trajectory [black lines in Figure 4 (b)]. As in the FEPs along the θ angles [Figure 4 (a)], the residues of the third β–strand and its edges (γi, i = 24 – 30) partially explore the local minima in the non-native state [blue, red, and green lines in Figure 4 (b)] and, based on the fluctuations of the θ and γ angles in the course of time (not shown), start to jump back and forth between the local and global minima when the whole system is in the native state [black lines in Figure 4 (b)].</p><p>As for the θ angles, a more detailed, quantitative representation of the results illustrated in Figure 4(b) is given in Table S1(b) [see the Supporting Information].</p><p>Based on the results of the FEPs along the θi and γi angles (Figure 4) and those of Table S1 (a and b), it can be concluded that, in the folding trajectory, (i) most of the residues of hairpin I (β–strands 1 and 2) completely follow all the changes in the dynamics of the whole system (these residues are called consistent); (ii) the residues of the third β–strand partially follow the changes in the dynamics of the whole system (these residues are called partially consistent), which indicates that the third β–strand is not as conformationally stable as the first two.</p><p>Also, it should be noted that most of the FEPs along the γ angles representing the β-strands and their edges exhibit three minima [one (deepest) corresponds to the native state, and the other two (shallow) correspond to the unfolded and intermediate states, respectively], while the FEPs along the θ angles representing the same parts of the protein have mainly two minima [one (deepest) corresponds to the native state, and the second (shallow) corresponds to the unfolded and intermediate states]; such behavior of θ and γ angles indicates that the γ angles are more sensitive and correlated to the global motions of the protein than are the θ angles.</p><!><p>The FEPs along the θi angles for the loop and end residues (black numbers) computed over the entire non-folding trajectory [black lines in Figure 4(c)] are similar to the FEPs along the θi angles for the loop and end residues computed over the entire folding trajectory [black lines in Figure 4(a)]. However, most of the FEPs along the θi angles pertain to β–strands (red numbers) and some FEPs along the θi angles for the edges of loops and β–strands, which include the motions of residues from both loops and β–strands of the non-folding trajectory (green numbers), differ from the corresponding FEPs of the folding trajectory [black lines in Figure 4 (a, c)]. Indeed, in the non-folding trajectory, most of the FEPs along the θi angles pertain to β–strands (θi, i = 9 – 12, 18, 20 – 22, 27 – 29), and have a deep minimum at ~ 90° and a shallow minimum at ~ 120° – 130°; the reverse is true for these residues in the folding trajectory. It, therefore, turns out that, in the non-folding trajectory, the β–strand residues are trapped in local minima.</p><p>The FEPs along the θi angles computed over the selected time-periods and over the entire duration of the non-folding trajectory [blue, red, green, and black lines in Figure 4(c)] show that almost each angle (except θ32) gradually explores its own FEP.</p><p>By comparing the FEPs along the γi angles computed over the entire duration of the non-folding trajectory [black lines in Figure 4(d)] to the FEPs along the γi angles computed over the entire duration of the folding trajectory [black lines in Figure 4(b)], differences can be observed not only for the angles pertaining to the β–strands (γi, i = 9 – 11, 19 – 21, 27, 28), but also for the angles pertaining to the loop, and the edges between loops and β–strands (γi, i = 4, 6 – 8, 12, 13, 15, 16, 22 – 25, 31, 32). The main difference for the angles pertaining to the β–strands is that the deepest minimum of their FEPs is shifted from 180° (in the folding trajectory) to the region of 0° – 80° (in the non-folding trajectory), which is similar to the shift occurring for the FEPs along the θi angles pertaining to the β–strands, i.e., from 120° – 130° [in the folding trajectory, Figure 4(a)] to 90° [in the non-folding trajectory, Figure 4(c)]. For the non-folding trajectory, the molecule also explores wider ranges of the γi angles, which is manifested in a small number of regions in which the FEPs are undefined [Figure 4 (d)], compared to those in the plots corresponding to the folding trajectory (Figure 4 (b)]. This indicates that the protein explores a larger portion of conformational space when it does not fold, as expected. The regions, which are undefined in the FEPs along the γi angles in the folding trajectory and explored by the γi angles in the non-folding trajectory, are far from the NMR-derived structural data for most of the angles (red circles in Figs. 4b and 4d); therefore, the exploration of these regions either delays the folding or it can be one of the reasons for non-folding. It should be noted that, despite the many differences between the FEPs along the γi angles computed from the folding and non-folding trajectories, there are some similarities, as well, at the flexible N- and C-terminal parts (γi, i = 2, 3, 33, 35) [Figure 4 (b, d)].</p><p>In the non-folding trajectory, almost all θ and γ angles explore their FEPs gradually, and a majority of the angles pertaining to the β–strands and their edges are trapped in their local minima, and do not conform to the global minimum [Figure 4(c, d)]. This is in contrast to the folding trajectory in which the local minima (non-native state) were explored before the global minimum (native state) [Figure 4 (a, b)].</p><p>Based on the analysis of the FEPs described above for the folding and non-folding trajectories, the following can be concluded: (i) most of the residues of all three β–strands (especially the first and middle ones) and their edges and loops connecting the β–strands play a crucial role in folding the FBP28 WW domain; (ii) the residues of the N- and C-termini do not follow the changes in the dynamics of the whole system; hence, their contribution to the folding event is minimal; (iii) the main reason for non-folding is that many residues (especially the residues of the β–strands) are trapped in a metastable conformation (i.e., in local minima of the FEPs computed over the entire folding trajectory), and there is no consistency between the FEPs along the angles involving these residues and the whole system, in contrast to the behavior observed for the folding trajectory; (iv) our finding, that almost all residues (except the residues at the N- and C-termini) in the folding trajectories move more or less in a concerted (correlated) fashion and the angles involving these residues are consistent with the changes of the whole system (monitored by the RMSD), reflects the observation7 that the internal coordinate PCA applied to the θ and γ angles was very efficient for the description of the folding and less efficient for the description of the non-folding trajectories of the FBP28 WW domain. The point is that the collective motion in a protein is any motion that involves a number of atoms moving in a concerted fashion, and PCA appears to be the best method for extracting collective variables from MD simulations.60</p><p>In order to justify and strengthen the aforementioned statements concluded from the analysis of only one folding and one non-folding trajectory, we computed the FEPs along the θ and γ angles for the three fast-folding trajectories [Figure S2 (a, b) in the Supporting Information]. We also computed the Pearson average correlation coefficients Fig. 5 R59,61[Figure 5(a)] between the FEPs along the θ and γ angles of the three fast-folding [Figure S1(a–c)] and the four slow-folding [Figure S1(d–g)] trajectories, and the correlation coefficients [Figure 5(b, c)] between the FEPs along the θ and γ angles of all folding (7 MD trajectories) and the three non-folding trajectories [Figure S1(h–j)]. All FEPs of the three fast-folding trajectories are very similar, hardly-distinguishable from each other [black lines in Figure S2 (a, b)]. Minor differences between the FEPs of the non-native states [red lines in Figure S2 (a, b)] are caused by the time-differences between the non-native states explored by the system in the fast-folding trajectories. The average correlation coefficients between the FEPs along both the θ and γ angles of the fast-folding and slow-folding trajectories [Fig. 5(a)] are very high (R > 0.96 for FEPs along the θ angles, R > 0.87 for FEPs along the γ angles), which indicates the similarity between the folding trajectories. The differences between the average correlation coefficients for the FEPs along the θ and γ angles of all folding and non-folding trajectories are noticeable at β-strands, edges and N- and C-termini [Figure 5 (b, c)]. In particular, for the FEPs along the θ angles, Ravg = 0.86, 0.76, 0.83 for the first, second and third β-strands, respectively, whereas Ravg = 0.98, 0.95 for the N- and C-termini, respectively [Figure 5 (b)]. For the FEPs along the γ angles, Ravg = 0.74, 0.66, 0.65 for the first, second and third β-strands, respectively, whereas Ravg = 0.95, 0.90 for the N- and C-termini, respectively [Figure 5 (c)]. The average correlation coefficients for the edges are Ravg = 0.55, 0.80, 0.12, 0.64 [Figure 5 (c)]. These results indicate that the FEPs of the angles representing the C- and N-termini of the protein in the folding and non-folding trajectories are highly correlated; and the FEPs of the angles representing the β-strands and their edges of the protein in the folding and non-folding trajectories are less correlated. From the above analysis, the conclusions based on one folding and one non-folding trajectory can be extrapolated to other folding and non-folding trajectories of the FBP28 WW domain.</p><p>It should be noted that our recent studies7 of the folding and non-folding trajectories for FBP28 WW domain by PCA also revealed the strong correlations between the first few PCs and the RMSDs in the folding trajectories, and non-correlations between the PCs and the RMSDs of the folding and non-folding trajectories. Similar results have been obtained for the trajectories studied here (not shown).</p><p>Although the FEPs along the θi and γi angles identified the residues that participate in the folding of the FBP28 WW domain (see Figure 4), it is still not clear which parts of the protein form first, and which residues play a crucial role in folding. In order to answer these questions, the FEPs along the distances between selected residues for both folding and non-folding trajectories were examined.</p><!><p>Figures 6 and 7 illustrate the FEPs along the distances between the selected residues [GLU(10) – TYR(20), THR(13) – LYS(17), ALA(14) – GLY(16), TYR(20) – SER(28), ASN(23) – LEU(26)] for folding and non-folding trajectories, respectively. The residues in both trajectories [see panels (c), (f), (i), (l), and (o) in both Figures 6 and 7] were selected with the purpose of trying to identify the consequence of the formation of native contacts in all parts of the protein, and those residues that might be key for folding. The FEPs in Figures 6 and 7 were calculated for the same time-periods as the FEPs in Figure 4; hence, the FEPs in Figures 6 and 7 correspond to the large changes in the RMSD of the whole system illustrated in Figure 3.</p><!><p>The first three pairs of the selected residues represent hairpin I in folding [panels (a) – (i) of Figure 6] and in non-folding [panels (a) – (i) of Figure 7] trajectories. In particular, (1) panels (a) and (b) of Figures 6 and 7 illustrate the distance between residues GLU(10) and TYR(20) as a function of time for the entire trajectory, and for the selected time periods, respectively, and panel (c) of Figures 6 and 7 shows the FEPs along the distances for the selected time-periods (blue, red, green lines) and the entire trajectory (black line); (2) panels (d) – (f) and (g) – (i) of Figures 6 and 7 illustrate the same variables (distances and free energy) for the following pairs of residues: THR(13) – LYS(17), and ALA(14) GLY(16), respectively.</p><p>Residues GLU(10) and TYR(20) are located in the middle of the first and second β–strands, respectively; hence, the distance between them as a function of time describes the behavior of hairpin I. Based on the results shown on panels (a) and (b) of Figures 6 and 7, the changes in the dynamics of the whole system, illustrated by the RMSD vs time in Figure 3, are detected correctly by the distance between residues GLU(10) and TYR(20) for both the folding and non-folding trajectories, which indicates that, regardless of the type of trajectories (folding or non-folding), the middle part of hairpin I follows the dynamics of the whole system. The reasons for folding and non-folding are illustrated in panel (c) of Figures 6 and 7, in which the FEPs computed from the folding and non-folding trajectories differ from each other. In particular, the FEP computed over the entire folding trajectory [black line in panel (c) of Figure 6] exhibits one shallow minimum (at ~ 9.8 Å), which is explored completely before reaching the native state [see blue, red, and green lines in panel (c) of Figure 6], and one narrow deep minimum (at ~ 6.0 Å), formed after jumping into the native state. By contrast, the FEP computed over the entire non-folding trajectory [black line in panel (c) of Figure 7] shows two deep minima [at ~ 8.8 Å (deepest) and ~ 5.9 Å], and both of them are gradually explored over time [see blue, red, and green lines in panel (c) of Figure 7]. Based on these results, it can be concluded that, in the folding trajectory, hairpin I (at least its middle part) is very stable. In other words, formation of hairpin I coincides with the jump of the protein into the native state, and it remains formed until the end of the trajectory. Unlike the folding trajectory, hairpin I in the non-folding trajectory is very unstable, and the amplitude of the fluctuations in the distance of its middle part (between two deep minima) is ~ 3.0 Å [panels (a) – (c) of Figure 7]. It should be noted that the calculated distance between residues GLU(10) and TYR(20) in the folding trajectory (~ 6.0 Å) differs from the experimental value, small red square (~ 4.4 Å).13</p><p>In order to corroborate the conclusions about the stability/instability of hairpin I, a second pair of residues [THR(13) and LYS(17)] located on the edges of the first and middle β–strands and the loop connecting these β–strands was selected [panels (d) – (f) of Figures 6 and 7]. The results shown in panels (d) and (e) of Figures 6 and 7 indicate that the distance between residues THR(13) and LYS(17) is as sensitive to the changes of the whole system (monitored by the RMSD, Figure 3), as was the distance between residues GLU(10) and TYR(20), except for the first large change of the protein RMSD at ~ 4.5 ns [see blue rectangle in panel (e)], which was not as clearly detected by residues THR(13) – LYS(17) as was by residues GLU(10) – TYR(20). However, the FEPs along the distances between the both pairs of residues [GLU(10) – TYR(20) and THR(13) – LYS(17)] computed over the entire folding trajectory are similar to each other [panels (c, f) of Figure 6]; in addition, the deep minimum in panel (f) (~ 5.8 Å), which corresponds to the native basin, is closer to the experimental value, small red square (~ 5.4 Å)13 than the one for residues GLU(10) – TYR(20) [panel (c)]. The FEPs in panels (c) and (f) (black lines) indicate that hairpin I is very stable and follows the dynamics of the whole protein. The FEP along the distance between residues THR(13) – LYS(17), computed over the entire non-folding trajectory [panel (f) of Figure 7] reveals even a more complicated shape with three minima (~ 5.9 Å, 7.9 Å, and 11.3 Å) than the one for residues GLU(10) – TYR(20) [panel (c) of Figure 7], which is additional evidence for the instability of hairpin I in the non-folding trajectory.</p><p>In order to understand the role played by the loop of hairpin I in folding/non-folding, the fluctuations and the FEPs along a third distance, selected between residues ALA(14) and GLY(16), located in the loop connecting the first and middle β–strands, were computed [panels (g) - (i) of Figures 6 and 7]. In both the folding and non-folding trajectories, the fluctuations of the distance ALA(14) – GLY(16) over time [panels (g) and (h) of Figures 6 and 7] do not reflect the large jumps found in the RMSDs (Figure 3) as well as the distances of previous pairs of residues did [panels (a, b) and (d, e) in Figures 6 and 7]. However, the behavior of the distance between residues ALA(14) and GLY(16) over time differs in the folding and in the non-folding trajectories [compare panels (g) and (h) of Figures 6 and 7]. The loop of hairpin I is more flexible at the beginning of the folding trajectory than in the non-folding trajectory [see the amplitude of the fluctuations during the first ~ 4.5 ns (blue rectangle) in panel (h) of Figures 6 and 7]. However, in the folding trajectory, the amplitude of the fluctuations decreases gradually and becomes stable in the intermediate state (between ~ 21 ns and ~ 37 ns). On the contrary, the loop of hairpin I in the non-folding trajectory is quite rigid at the beginning of the trajectory, but becomes more flexible over time, and the amplitude of the fluctuations increases almost twice [panels (g) and (h) of Figure 7].</p><p>The FEPs along the distance between ALA(14) and GLY(16) for both trajectories, in general, exhibit a similar shape with one minimum at ~ 5.5 Å for all time periods; however, the differences observed in panels (g) and (h) regarding the fluctuations are reflected in the shapes of the FEPs [panel (i) of Figures 6 and 7]. Based on the results illustrated in panels (g) – (i) of Figures 6 and 7, it can be concluded that the loop connecting the first and middle β–strands forms in the early stage of the trajectory and remains formed until the end of the trajectory with enhanced stability over time for the folding trajectory and reduced stability over time for the non-folding trajectory. Therefore, formation of the loop of hairpin I seems to play an important role in the early stage of folding and helps the protein to fold if the loop fluctuates slightly in the vicinity of the most probable distance ALA(14) – GLY(16) (5.5 Å), whereas too large fluctuations may impede folding. This finding is in agreement with an earlier experimental study,31 in which the major influence of formation of the loop of hairpin I on the folding rate was observed. It should be noted that, since the angle θ15 is formed by residues ALA(14) – ASP(15) – GLY(16), there is a striking similarity between the FEPs along θ15 [panels (a, c) of Figure 4] and along the distance ALA(14) – GLY(16) [panel (i) of Figures 6 and 7] for both folding and non-folding trajectories.</p><!><p>The two other distances were selected to represent hairpin II in folding [panels (j) – (o) of Figure 6] and non-folding [panels (j) – (o) of Figure 7] trajectories. In particular, panels (j) – (l) and (m) – (o) of Figures 6 and 7 illustrate the same variables (fluctuations of the distances over time and free energy) as the above [panels (a) – (i)] for the distances: TYR(20) – SER(28) and ASN(23) – LEU(26), respectively. It should be noted that, because of the short length of the loop connecting the second and third β–strands, the pair of residues representing this loop was not selected for this analysis.</p><p>Residues TYR(20) and SER(28) are located in the middle of the second and third β–strands, respectively; hence, the time-dependence of the distance between them describes the general behavior of hairpin II. Based on the results shown in panels (j) and (k) of Figures 6 and 7, the changes in the dynamics of the whole system (monitored by the RMSD as shown in Figure 3) are detected correctly in the fluctuations of the distance TYR(20) – SER(28) over time both in the folding and in non-folding trajectories. This indicates that, regardless of the type of trajectory (folding or non-folding), the middle part of hairpin II follows the dynamics of the whole system. However, unlike hairpin I (panels (a) and (c) of Figure 6), after reaching the native state, hairpin II exhibits an unstable behavior with large fluctuations [as reflected by the distance TYR(20)–SER(28) in panel (j) of Figure 6]. Hence, the FEP computed over the entire folding trajectory [black line in panel (l) of Figure 6] exhibits only one deep minimum (~ 6.0 Å), and a shoulder instead of a shallow minimum, which is not explored completely (see blue, red and green lines) before jumping into the native state. It should be noted that the distance TYR(20)–SER(28) starts exploring the native basin much earlier (after 4.5 ns) [red line in panel (l) of Figure 6] than the whole system jumps to the native state and, after that, interconverts between the native basin and a local minimum (shoulder) located around 8–10 Å [panel (l) of Figure 6].</p><p>The FEP along the distance TYR(20) – SER(28) computed over the entire non-folding trajectory [black line in panel (l) of Figure 7] exhibits one broad minimum and resembles the FEP illustrated in panel (c) of Figure 7. Like hairpin I, based on these results, hairpin II in the non-folding trajectory is also very unstable; the distance between the middle parts of the β-strands of hairpin II oscillates between ~ 5 Å and ~ 10 Å [panel (l) in Figure 7].</p><p>The results for the last pair of selected residues, ASN(23) and LEU(26), located at the edges of the middle and third β–strands and the loop connecting these β–strands of the folding trajectory [panels (m) – (o) of Figures 6] also indicate that, despite the consistency with the dynamics of the whole system in the non-native state, after jumping into the native state of the whole system, the distance between residues ASN(23) and LEU(26) still fluctuates with a large amplitude (~ 5.0 Å) from time to time [panel (m) of Figure 6] showing the highest flexibility of hairpin II. The corresponding FEP of the distance ASN(23) – LEU(26) computed over the entire folding trajectory [panel (o) of Figure 6], as expected, is quite similar to the FEP for the previous pair [panel (l) of Figure 6]. Unlike the folding trajectory, the distance between residues ASN(23) and LEU(26) in the non-folding trajectory does not follow the dynamics of the whole system [panels (m) and (n) of Figure 7], and the corresponding FEPs exhibit a broad, "unstructured" shape [panel (o) of Figure 7].</p><p>It should be noted that this behavior of hairpin II, which is caused mainly by the high flexibility of the third β–strand, leads to biphasic kinetics in the folding of the FBP28 WW domain.22,23,33 The conclusions drawn in these studies22,23,33 regarding the biphasic kinetics coincide with ours. In particular, the misregistered structure in hairpin II is the cause of the biphasic kinetics, which was traced to the mobility of the third β-strand within the context of otherwise folded conformations.</p><p>Moreover, the behavior of hairpins I and II and the role of loops in folding, observed in this study, is in harmony with earlier experimental31,62 and theoretical26 results.</p><!><p>Since most of the residues move in a concerted fashion in the folding trajectory of the FBP28 WW domain, as was observed above [Figure 4 (a) and (b)], it is interesting to examine whether the FEL along the corresponding PCs computed with the involvement of several θi and γi angles, can correctly describe protein folding. Therefore, three different analyses of the folding trajectory were carried out by PCA: (i) PCs were computed by considering the whole protein; (ii) PCs were computed by considering only five sets of θ and γ angles (9 – 11, 26, 29); (iii) PCs were computed by considering only six sets of θ and γ angles (2 – 4, 33 – 35).</p><p>The angles in analyses (ii) and (iii) were not selected randomly. Indeed, they correspond to the angles with the largest and smallest contributions, respectively, to the first two PCA modes (defined in Materials and Methods); i.e., the principal modes (with the largest eigenvalues λk) contributing the most to the structural fluctuations [mean-square-fluctuations (MSF)] of the protein. Figure 8 illustrates the contributions of the two main principal modes [solid lines with filled (principal mode 1) and empty (principal mode 2) rectangles] to the MSF along the θ [panel (a)] and γ [panel (b)] angles for the folding (black lines) and non-folding (red lines) trajectories.</p><p>Based on the results shown in panel (a), the main contribution to the fluctuations in both trajectories comes from the β–strands; however, some contribution from the loop connecting the first and middle β–strands should also be mentioned. Panel (b) illustrates different results for the folding and non-folding trajectories. In particular, the major contribution to the fluctuations in the folding trajectory comes from the β–strands, especially from the first β–strand; however, the contributions from the loops, especially from the loop connecting the middle and third β–strands, are also noticeable. The major contribution to the fluctuations in the non-folding trajectory comes from the loop connecting the first and middle β–strands; however, the contributions from the β–strands are also noticeable. It should also be noted that the magnitude of the contribution of principal mode 1 at some θ and γ angles for the non-folding trajectory is several times greater than the one for the folding trajectory. This is not surprising because the system in the non-folding trajectory is more flexible and the amplitude of the fluctuations is much larger than in the folding trajectory; consequently, λ1 for the non-folding trajectory is a few times larger than the one for the folding trajectory.</p><p>Comparing the results shown in Figures 4, 6 – 8, we can see the correlation between local and global motions. The angles, whose fluctuations over time are consistent with the dynamics of the whole system (i.e., the fluctuations of the RMSD), are the main contributors to the MSF; hence, they are the main players in folding. In other words, they impel the protein to follow their dynamics.</p><p>The FEPs, μ(qi)=−kBT ln P(qi), constructed along the first three PCs (i = 1 – 3), and the FELs along the first two PCs, μ(q1, q2)=−kBT ln P(q1, q2), for the whole protein [panels (a) and (b)], for only five sets of θ and γ angles (9 – 11, 26, 29) [panels (c) and (d)], and for only six sets of θ and γ angles at the N- and C-termini of protein (2 – 4, 33 – 35) [panels (e) and (f)] for the folding trajectory, are shown in Figure 9. The sets of θ and γ angles (9 – 11, 26, 29 and 2 – 4, 33 – 35) were selected by their maximal and minimal contributions in principal modes 1 and 2.</p><p>Both FEPs and FELs, computed by considering the whole system [panels (a) and (b)] and five sets of θ and γ angles [panels (c) and (d)], are very similar to each other, which is not surprising since all five angles are the main contributors to the MSF, and their local motions are consistent with the dynamics of the whole system. Both FEPs along the first PC and FELs exhibit three-state folding dynamics, which coincide with the results obtained in earlier theoretical7,9,10,25,30 and experimental33 studies. The FEPs and FEL computed by considering six sets of θ and γ angles at the N- and C-termini (panels e and f), do not resemble the FEPs and FELs illustrated in panels a – d, and, therefore, do not describe folding dynamics correctly.</p><!><p>By analyzing the MD trajectories of the FBP28 WW domain, generated with the coarse-grained UNRES force field, in terms of the local motions of each residue and PCA, we have studied the dynamics of the folding and non-folding trajectories and found the following:</p><!><p>The loop connecting the first and middle β–strands forms in the early stage of simulation26 and consequently plays an important role in folding/non-folding of the protein at the beginning of the trajectory.</p><p>The dynamics of the residues of the first and middle β–strands and their edges with the loop connecting these β–strands, in the folding trajectory, are consistent with the dynamics of the whole system and exhibit very stable behavior; hence, their role in folding is crucial. The residues of the third β–strand in the folding trajectory follow the motions of the whole system on some level; however, after reaching the native state their behavior is not as stable as that of the residues of the first two β–strands. None of the residues in the non-folding trajectory exhibits complete consistency with the dynamics of the whole system.</p><p>In the folding trajectory, hairpin I forms first, and remains formed until the end of the trajectory, whereas hairpin II, because of the behavior of the third β–strand, shows unstable behavior, which is the reason for biphasic kinetics in the folding of the FBP28 WW domain. These findings support earlier experimental31,33,36,62 and theoretical22,23,26 results.</p><p>The residues, whose motions are consistent with the dynamics of the whole system in the folding trajectory, are the main contributors to the MSF in principal modes 1 and 2. This indicates the correlations between local and global motions in protein folding, which, to the best of our knowledge, is a new observation.</p><p>Unlike the non-folding trajectory, for the folding trajectory most of the FEPs along the θi and γi angles for loops and β–strand residues can be identified by their shapes, and most of the residues move in a concerted fashion and follow the dynamics of the whole system. The main differences between the FEPs of the folding and non-folding trajectories are along the angles belonging to the β–strands.</p><p>The reason why PCA has proved to be an effective tool for the analysis of protein folding trajectories is that this type of analysis involves concerted motions of many residues, which can be captured by a few PCs with the largest eigenvalues.</p><p>The key residues, involved in the folding/non-folding of the studied trajectories and identified here, are the "main players", in general, in the folding/non-folding of the FBP28 WW domain. Perhaps, this also applies to other β–proteins (for this reason some additional studies are planned in the future).</p><p>Five sets of θ and γ angles are enough to construct the FEP and FEL thereby correctly describing the folding dynamics of the FBP28 WW domain.</p><!><p>Finally, it should be noted that the questions addressed in the present study, i.e., why the FBP28 WW domain protein folds or does not fold, and what governs the FBP28 WW domain to fold, are very important for understanding misfolding and, consequently, the diseases associated with misfolding of the WW domain proteins.</p>

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