Datasets:

griffin

/

ChemSum

like 12

High electrical conductivity and high porosity in a Guest@MOF material: evidence of TCNQ ordering within Cu₃BTC₂ micropores

The host-guest system TCNQ@Cu 3 BTC 2 (TCNQ ¼ 7,7,8,8-tetracyanoquinodimethane, BTC ¼ 1,3,5benzenetricarboxylate) is a striking example of how semiconductivity can be introduced by guest incorporation in an otherwise insulating parent material. Exhibiting both microporosity and semiconducting behavior such materials offer exciting opportunities as next-generation sensor materials.Here, we apply a solvent-free vapor phase loading under rigorous exclusion of moisture, obtaining a series of the general formula xTCNQ@Cu 3 BTC 2 (0 # x # 1.0). By using powder X-ray diffraction, infrared and X-ray absorption spectroscopy together with scanning electron microscopy and porosimetry, we provide the first structural evidence for a systematic preferential arrangement of TCNQ along the (111) lattice plane and the bridging coordination motif to two neighbouring Cu-paddlewheels, as was predicted by theory. For 1.0TCNQ@Cu 3 BTC 2 we find a specific electrical conductivity of up to 1.5 Â 10 À4 S cm À1 whilst maintaining a high BET surface area of 573.7 m 2 g À1 . These values are unmatched by MOFs with equally high electrical conductivity, making the material attractive for applications such as super capacitors and chemiresistors. Our results represent the crucial missing link needed to firmly establish the structure-property relationship revealed in TCNQ@Cu 3 BTC 2 , thereby creating a sound basis for using this as a design principle for electrically conducting MOFs.

high_electrical_conductivity_and_high_porosity_in_a_guest@mof_material:_evidence_of_tcnq_ordering_wi

Introduction<!>Results and discussion<!>Conclusions<!>General information<!>Vapor phase inltration<!>Powder X-ray diffraction<!>Porosimetry measurements<!>Fourier transform infrared spectroscopy<!>Elemental analysis<!>Scanning electron microscopy<!>Auger electron spectroscopy

<p>The development of electrically (semi-)conductive metalorganic frameworks (MOFs) is of great scientic and technological interest, offering the opportunity of making electronic devices with permanent microporosity. 1,2 MOFs are supramolecular coordination complexes composed of metal ions or clusters that are linked by polydentate organic ligands to form 2D or 3D frameworks with accessible porosity. 3 By combining both long-range order found in inorganic semiconductors and high chemical tunability found in organics, semiconducting MOFs are unique among the different classes of conducting materials. In principle, the deep understanding of coordination chemistry and crystal engineering enables rational design of MOF systems at a level that is difficult to achieve in other (particularly non-crystalline) materials. 4,5 However, most MOFs are insulators, originating from the rather ionic coordination bonds of metal carboxylates that tend to suppresses charge transfer between the metal node and linker. 6 Typically, design strategies for electrically conductive MOFs focus on the use of organic linkers that form coordination bonds with improved orbital delocalization between metal and ligand to facilitate charge transfer, and on the use of metal ions with high-energy valance electrons such as Cu 2+ and Fe 2+ . 6,7 In 2014, some of us applied a radically new approach to impart electrical conductivity to MOFs by incorporating the redox-active molecule TCNQ (7,7,8,8-tetracyanoquinodimethane) in the pores of Cu 3 BTC 2 (also known as HKUST-1; BTC ¼ 1,3,5-benzenetricarboxylate). 8 Since then, this striking observation has been conrmed by other groups; 9,10 however, the detailed nature of the host-guest complex and the conductivity mechanism are mainly unsolved. It is proposed that TCNQ bridges two Cu dimer units via two geminal nitrile groups, which is strongly supported by both theory and spectroscopic data. 8,11 Initial measurements of the Seebeck coefficient revealed that holes are the majority charge carriers, 12 and an underlying super-exchange mechanism was proposed. 13 Neumann et al. reported calculations supporting this hypothesis, but required the majority carrier to be electrons, which is in apparent conict with the abovementioned Seebeck measurements. 10 Furthermore, detailed and clear structural evidence for the bridging binding motif has not been obtained, despite intense research efforts. A factor confounding interpretation of prior results is the use of solution-phase techniques for the inltration of TCNQ into the host framework, during which solvent molecules and TCNQ compete for free Cu(II) coordination sites. This likely impedes long-range ordering of TCNQ molecules in the framework and disrupts diffusion pathways for the guest molecule. Moreover, TCNQ is easily reduced (redox potential of +0.2 V vs. Ag/AgCl), 14,15 and in turn can react with the organic solvent and/or water molecules, preventing full control of the oxidation state of TCNQ molecules adsorbed in the Cu 3 BTC 2 framework. In other words, the material obtained via liquid phase inltration lacks rigorous experimental evidence possibly needed for a profound understanding of the conductivity mechanism due to a wider parameter space of compositional and structural characteristics. Is there a single type of TCNQ guest or is there a range of guest species including neutral and charged and how relevant is a homogenous long range ordering vs. short range (nanodomains) or even random and disordered distribution of TCNQ? Is the material phase pure or are their impurities to be considered? What is the role of water and solvent coordinated to Cu centers that are not occupied by TCNQ and do traces of physisorbed water or other protic impurities (e.g. methanol, ethanol) inuence the conductivity of the material? Pristine Cu 3 BTC 2 samples (bulk as well as thin lm) are known to exhibit signicant amounts of Cu(I) defect sites. Do these sites play a role when the material is loaded with redox non-innocent guests such as TCNQ? This more complex scenario of questions prompted us to investigate preparative concepts for loading Cu 3 BTC 2 with TCNQ.</p><p>Recently, D'Alessandro and co-workers applied a vacuum vapor-phase inltration (VPI) for the incorporation of TCNQ into Cu 3 BTC 2 . 16 In their approach, however, inltrated samples were subsequently washed with solvent to remove excess, uninltrated TCNQ. Furthermore, samples were treated under ambient conditions, compromising efforts to keep the hostguest system free from solvent and moisture. Our study builds on their important work, applying an optimized VPI method with rigorous exclusion of moisture and solvent contaminations. Following this new protocol, we obtain samples with precise stoichiometry, having the general formula xTCNQ@Cu 3 BTC 2 (0 # x # 1), that are devoid of complicating factors associated with liquid-phase inltration. The materials were characterized by powder X-ray diffraction (PXRD), infrared (IR) and X-ray photoelectron spectroscopy (XPS) and electrical conductivity measurements, with all measurements strictly conducted under inert conditions. Strikingly, we nd evidence of long-range order for TCNQ@Cu 3 BTC 2 samples with highloadings. Despite the observation that minor amounts of Cu(TCNQ) form as byproduct on the surface of Cu 3 BTC 2 crystals, we nd a relatively high electrical conductivity of these samples. Moreover, by avoiding the use of solvent and careful sample handling, our VPI method yields high permanent porosity as wellmore than ve times higher than achieved previously using liquid-phase inltration 8demonstrating that high electrical conductivity and large pore volume in MOFs can be compatible.</p><!><p>The physical mixture of activated, desolvated Cu 3 BTC 2 and TCNQ (ground together in a mortar) was annealed under vacuum in a glass ampule and heated for 3 days at 180 C to yield the host-guest complex. Using the VPI approach, we produced a series of samples of the general formula xTCNQ@Cu 3 BTC 2 (x ¼ n(TCNQ)/n(Cu 3 BTC 2 ) with x ¼ 0.1, 0.2,., 1.0) with precisely dened guest loadings by varying the amount of TCNQ with respect to a xed amount of Cu 3 BTC 2 . Even though a loading of x ¼ 1.5 would be necessary to saturate all open Cu sites, unreacted, crystalline TCNQ was observed by powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM) for samples with x > 1.0. This is in accordance with the maximum loading capacity of two TCNQ per large pore (one TCNQ per formula unit Cu 3 BTC 2 , x ¼ 1) predicted by theory. 8 The PXRD data of the loaded series of xTCNQ@Cu 3 -BTC 2 (Fig. 1) show that the overall crystal structure of Cu 3 BTC 2 is retained upon TCNQ inltration. The largest change observed is in the intensity of the (111) reection at 2q ¼ 5.82 , which increases monotonically with the loading amount of TCNQ (highlighted in Fig. 1). Additionally, new reections appear at higher angles, of which only the weak reection at 15.77 matches with the principal reection of a potential byproduct, i.e. Cu(TCNQ) phase I (cf. Fig. S1 †). The increase of the (111) reection has previously been interpreted as an indicator for chemisorption of guests at the open Cu sites, 17 and is here indicative of the incorporation of TCNQ into Cu 3 BTC 2 . Since the modied VPI method excludes any intensity changes due to solvent incorporation, this nding is consistent with the suggested bridging binding mode of TCNQ, as the Cu atoms of the paddlewheel nodes direct the planar TCNQ molecule into the (111) lattice plane (Fig. 2). Nevertheless, there will be disordered TCNQ inside of the pores considering that there are two types of large pores in Cu 3 BTC 2 and only one features open Cu sites pointing to the center of the pore, i.e. are available for coordination of TCNQ. Furthermore, we observed weak reections with increasing x in xTCNQ@Cu 3 BTC 2 that are not in agreement with the parent face-centered cubic cell of Cu 3 BTC 2 , e.g. at 10.65 , 15.44 and 18.20 . However, they are consistent with a primitive unit cell of parent Cu 3 BTC 2 having similar lattice parameters, therefore pointing to decreased lattice symmetry for the inltrated MOF. For example, a Pawley prole t with reduced symmetry (Pn 3m) and maintaining the same lattice parameters as the parent Cu 3 BTC 2 can account for all additional reections (Fig. S4 †). A doubling of the unit cell parameters while keeping the face-centered symmetry could similarly lead to a good prole t, which might be in better chemical agreement, reecting the different pores with and without available Cu sites. Determining the origin of these reections requires additional structural analysis that is complex and extends signicantly beyond the scope of the present report. However, this nding is intriguing, pointing to an ordering phenomenon related to TCNQ molecules and a subsequent symmetry reduction of the host-guest system that has not been previously observed. Based on the PXRD measurement, we therefore conclude that our modied VPI method leads to incorporation of TCNQ into Cu 3 BTC 2 with the potential formation of a supercell.</p><p>In order to probe the porosity as function of x in xTCNQ@Cu 3 BTC 2 , we performed nitrogen adsorption experiments. As expected, a decrease of the Brunauer-Emmett-Teller (BET) surface area is observed, from 1833.0 m 2 g À1 for pristine Cu 3 BTC 2 to 573.7 m 2 g À1 for 1.0TCNQ@Cu 3 BTC 2 (Fig. 3), about two thirds of the initial BET surface area. In addition, the decrease is linear with TCNQ loading, providing additional evidence for the incorporation of the guest molecule into the framework. Importantly, the decrease cannot be explained by pore blocking or a simple adsorption of TCNQ at the crystal surface. For instance, the BET surface area of a physical mixture of both components for 1.0TCNQ@Cu 3 BTC 2 would still amount to 1370.4 m 2 g À1 (see detailed discussion in ESI †). It is further important to note that our measured surface areas considerably exceed those for TCNQ-loaded Cu 3 BTC 2 samples reported in the literature. For example, the VPI 0.5TCNQ@Cu 3 BTC 2 sample exhibits a surface area of 1145 m 2 g À1 , whereas the surface area of the material with the same loading synthesized via liquid phase inltration is reported as 214 m 2 g À1 , which is likely due to residual adsorbed water or solvent. 8 The microstructure and phase homogeneity of the TCNQloaded samples was further investigated using SEM (Fig. 4). Nanowire-like structures were discovered on the external surface of the TCNQ-loaded MOF crystallites; the amount and dimensions of these increase with the amount of TCNQ employed during the VPI. To determine the chemical nature of the nanowires, scanning Auger electron microscopy was performed. These data show a homogenous distribution of Cu, C, and N (Fig. S13 †). In combination with the characteristic morphology 18 and the reection at 15.77 in the diffraction pattern, the nanowires were identied as phase I of Cu(TCNQ). For the formation of Cu(TCNQ) to occur, both TCNQ and Cu(II) must be reduced to TCNQ À and Cu(I), presumably involving an oxidation (decarboxylation) of the BTC linker under the conditions of the loading experiment. In the elemental analysis data of the concentration series (Table S2 †), slightly decreased carbon contents compared to the calculated values are found, whereas the nitrogen contents are slightly increased, supporting a decomposition of some BTC molecules. Moreover, Cu 3 BTC 2 is known to contain small quantities of Cu(I) species, which have been related to intrinsic defects and thermal treatment during activation. 19,20 Surface sensitive XPS measurements (Fig. S19 †) reveal a relatively high abundance of Cu(I) species at the surface of Cu 3 BTC 2 crystallites. This Cu(I) concentration is further increased by the inevitable prolonged thermal treatment during the VPI process. The Cu(I) species, in turn, are able to reduce neutral TCNQ (via Cu(I) / Cu(II) + e À ) and thus enable the formation of the surface impurity phase Cu(TCNQ). The presence of several TCNQ species (i.e. coordinated, uncoordinated, radical anion) is suggested by the broadening of the N 1s signature in XPS (Fig. S20 †). The formation of the Cu(TCNQ) byproduct could be reduced, but not fully inhibited, by using lower reaction temperatures (Fig. S15-S17 †). However, reduced temperature also compromise the ordering of TCNQ, as evidenced by the absence of the pronounced (111) reection in PXRD (Fig. S2 †).</p><p>The loading and coordination of TCNQ to open Cu sites can also be tracked by vibrational spectroscopy. The infrared (IR) spectra of pristine and TCNQ-loaded Cu 3 BTC 2 are shown in Fig. 5. Whereas in pristine Cu 3 BTC 2 a band in the CN vibration region is absent, a CN vibration mode at 2222 cm À1 appears in TCNQ-loaded samples and becomes more pronounced with higher TCNQ loading. Notably, a second vibrational mode at 2200 cm À1 and a shoulder at 2170 cm À1 appear for samples with high TCNQ loading (cf. Fig. S6 †). A red shi of the CN vibration was previously attributed to coordinated TCNQ involving a partial charge transfer. 8,21 Due to the presence of the CuTCNQ byproduct in the samples, we here also assign some contribution to the signal at 2000 cm À1 and the shoulder at 2170 cm À1 to Cu(TCNQ), whose IR signature is known from the literature. 22 The indication of multiple TCNQ species in the IR spectra matches well with the XPS signature in the N 1s regime.</p><p>Thermogravimetric analysis (TGA) of 1.0TCNQ@Cu 3 BTC 2 (Fig. S7 †) shows a decomposition that proceeds in one step with an onset at 300 C. This temperature is slightly lower than observed for pristine Cu 3 BTC 2 , which starts to decompose at 330 C. The corresponding features in the differential scanning calorimetry (DSC) data are at 349.7 C and 338.3 C for the pristine and the loaded MOF, respectively. The 1.0TCNQ@Cu 3 BTC 2 sample shows an additional smaller peak at 314.2 C, indicating a step-wise decomposition.</p><p>Electrical transport measurements were performed on pressed pellets using an air-tight two-point-probe setup. I-V curves were recorded from À5 V to 5 V (Fig. S9 †) and the conductivity s was calculated using eqn (1):</p><p>in which I, V, d and A are the measured electrical current, the applied voltage, the thickness of the pellet and the area of the pellet, respectively. The calculated room-temperature conductivities are plotted against the TCNQ loading (Fig. 6). We observe an exponential increase of the electrical conductivity upon TCNQ loading, starting from immeasurably small values for pristine Cu 3 BTC 2 to 1.5 Â 10 À4 S cm À1 for 1.0TCNQ@Cu 3 -BTC 2 . This value is lower than the electrical conductivity reported for thin lm samples inltrated via the liquid phase (0.07 S cm À1 ), 8 pointing at additional inuence of grain boundaries and particle sizes as well as at the different measurement techniques used (pressed powder vs. thin lm). The observed exponential increase of the electrical conductivity can be described by classical percolation theory. 24,25 At low TCNQ loadings only few of the copper paddlewheel units are bridged by TCNQ and form localized conducting regions. With increasing TCNQ loading these bridged domains become interconnected and give rise to charge transport through the framework. Given the fact that TCNQ@Cu 3 BTC 2 samples in the literature showed high conductivities but no pronounced (111) reection, [8][9][10] disordered TCNQ might also contribute to the charge transport through the material.</p><p>The impact of the Cu(TCNQ) byproduct on the electrical transport, however, is intrinsically difficult to assess because Cu(TCNQ) can crystallize in two different phases. Phase II is a poor conductor, whereas phase I is an electrical semiconductor with a room temperature conductivity of 0.25 S cm À1 . 18 Mechanical removal of the nanowires by sonication was unsuccessful and led to a disordering of the TCNQ molecules and a signicant decrease in the conductivity (Fig. S3, S11 and S18 †). A physical mixture of 1% CuTCNQ/ Cu 3 BTC 2 (estimated amount from SEM images) did not show any conductivity. In combination with previous ndings in the literature for TCNQ@HKUST-1 in which Cu(TCNQ) as an impurity phase was not observed, we ascribe the increasing electrical conductivity as function of TCNQ loading to the formation of our host-guest system. Moreover, 1.0TCNQ@Cu 3 BTC 2 exhibits one of the highest-reported electrical conductivities paired with permanent porosity reported to date, comparing with other conductive MOFs of similar porosities, such as Cd 2 (TTFTB). 6,26 The synthesis of a reference sample of xTCNQ@Cu 3 BTC 2 (x ¼ 0.4 as determined by EA) via liquid phase inltration revealed no indication of TCNQ ordering, but a conductivity in the order of 10 À1 S cm À1 , which is signicantly higher than for samples with comparable TCNQ loading prepared via VPI. However, this sample shows a non-ohmic behavior (deviation from the linear I-V curve) at high potentials that we assign to electrochemical processes of water/ solvent inside the pores (Fig. S21-S24 †). This nding emphasizes the importance of solvent exclusion during the guest inltration step and highlights the need for in-depth structural and spectroscopic studies of Guest@MOF systems to develop and validate structure-property relationships.</p><!><p>We developed an optimized VPI strategy under thermodynamic control and used it to introduce stoichiometric amounts of TCNQ to obtain a concentration series of xTCNQ@Cu 3 BTC 2 with 0 # x # 1.0 under strict inert conditions. No washing step is needed to remove excessive TCNQ, which is advantageous over the kinetically controlled loading procedure presented in previous studies. [8][9][10]16 High reaction temperatures and long exposure times evidently promote an ordered, periodic arrangement of TCNQ within the (111) crystal lattice plane and the bridging coordination motif of TCNQ and the Cu atoms of two neighbouring paddle-wheel units. To our knowledge this is the rst crystallographic evidence for the integration of TCNQ into the framework of Cu 3 BTC 2 , and further demonstrates that the introduction of a non-innocent guest molecule can be regarded as a new element of MOF property design. Additional evidence supporting the accommodation of TCNQ within the pores of the framework is the systematic decrease in the BET surface area. SEM images of the inltrated samples show the formation of nanowires on the MOF crystal surface during VPI that were identied as Cu(TCNQ). Their formation can be reduced but not entirely suppressed at lower temperatures. IR data recorded during this work shows two nitrile vibration modes that can be attributed to TCNQ molecules inside the Cu 3 BTC 2 framework and to the TCNQ anion in Cu(TCNQ). The electrical conductivity of the concentration series increases exponentially with the amount of TCNQ used in the VPI. Even though, a quantitative analysis of the contribution of the hostguest complex and the Cu(TCNQ) byproduct to the electrical conductivity of the material is difficult, the obtained material shows high conductivity values accompanied by high permanent porosity. In fact, the BET surface area of 1.0TCNQ@Cu 3 BTC 2 is among the highest for electrically conductive MOFs. 6,26 The combination of these two properties creates a high potential for using this material in sensing or electronic device fabrication where access to the pores provides an essential new function. In this respect, well dened, oriented Cu 3 BTC 2 thin lms deposited and inltrated with TCNQ using solvent-free vapor phase techniques represent the ultimate goal. Pointing to the complexity of this system, further detailed analysis is required, beyond standard characterization techniques, to fully understand the ordering of TCNQ and the conductivity mechanism.</p><!><p>All chemicals were purchased from commercial suppliers (ABCR, Acros Organics, Alfa Aesar, Sigma Aldrich) and used without further purication unless otherwise stated. Solvents used for the synthesis and washing steps were reagent grade or higher.</p><!><p>Cu 3 BTC 2 was synthesized following the literature procedure. 27 The tile crystalline product was continuously washed with ethanol and dichloromethane in a Soxhlet apparatus for one week, respectively, to rigorously remove all reactants and highboiling solvents. The washing solution was replaced by the respective fresh solvent once. Subsequently, the blue powder was desolvated under high vacuum ($10 À6 mbar) at 180 C to yield pristine Cu 3 BTC 2 . TCNQ was recrystallized three times from acetonitrile under inert conditions to yield molecular, crystalline TCNQ in high purity.</p><p>Inside an argon glovebox, 200 mg of Cu 3 BTC 2 and distinct amounts of TCNQ were physically mixed and lled into borosilicate glass tubes to prepare a concentration series of xTCNQ@Cu 3 BTC 2 (x ¼ n(TCNQ)/n(Cu 3 BTC 2 ) with x ¼ 0.1, 0.2,., 1.0). The glass tube was then evacuated ($10 À3 mbar) and ame sealed to give a closed system for the VPI process. The sealed ampules were placed inside a convection oven at 180 C for 72 hours allowing the TCNQ to sublime and diffuse into the MOF. Aer cooling down to room temperature the ampules were transferred into the glovebox and stored for further characterization.</p><!><p>Powder X-ray diffraction (PXRD) data was collected in a 2q range of 5-50 in steps of 0.0016413 (2q) on a PANalytical Empyrean equipped with a Cu X-ray tube operated at 45 kV and 40 mA. The samples were lled into borosilicate capillaries of 0.7 mm diameter and mounted onto a capillary spinner. The radiation was focused onto the sample through a focusing X-ray beam mirror equipped with a 1/8 divergence slit and 0.02 radian soller slits. The diffracted beam was detected by a PIXcel1D detector in receiving slit mode equipped with a 1/8 anti-scatter slit and 0.02 radian soller slits.</p><p>Thermogravimetric analysis/differential scanning calorimetry TGA/DSC measurements were carried out on a Mettler Toledo TGA/DSC 1 equipped with an auto sampler unit. Aluminum crucibles (100 mL) were lled with 5-10 mg of MOF powder inside the glovebox and tightly capped with an aluminum lit. The crucible was transferred into the TGA chamber and a hole was pinched into the lit while the stream of Ar (40 mL min À1 ) was already on. The temperature was ramped from 30 C to 550 C at a rate of 5 C min À1 . The instrument was calibrated to a blank sample prior to the measurements.</p><!><p>Porosimetry measurements of pristine and guest-loaded MOF powders were performed using a Micromeritics 3ex to determine their BET surface area. Therefore, approximately 60 mg of a sample was lled into a BET tube and evacuated for 3 h at room temperature prior to the measurement. Nitrogen isotherms were recorded at 77 K. The BET surface area was calculated from data points in the relative pressure range of 0.01 to 0.1.</p><!><p>Fourier transform infrared spectroscopy (FTIR) of powder samples was done in argon atmosphere on an ALPHA FTIR spectrometer (Bruker) equipped with a Pt attenuated total reectance (ATR) unit at room temperature in the range of 400-4000 cm À1 with a resolution of 2 cm À1 . 64 scans were recorded per measurement.</p><!><p>Elemental analysis (EA) for the elements C, H, N, and S was carried out on a Hekatech EuroEA Elementaranalysator. Cu contents were determined by atom absorption spectroscopy (AAS) aer decomposing the sample in a mixture of sulfuric acid and nitric acid in a CEM microwave.</p><!><p>Top-view micrographs of MOF powders were conducted on a Merlin high-resolution scanning electron microscope (Carl Zeiss) using an acceleration voltage of 5 kV. The samples were transferred from a glove box into the analysis chamber under argon atmosphere using a transfer vessel (Leica EM VC500). To avoid charging, the sample was coated with 5 nm of Pt prior to the measurement using a Leica EM ACE600. Energy dispersive X-ray spectroscopy (EDX) analysis was conducted using an XMAX EXTREME EDX detector (Oxford Instruments). Measurements were carried out by application of an acceleration voltage of 5 kV and a probing current of 100 pA for SEM and 1000 pA for EDX.</p><!><p>AES measurements were performed at room temperature with a scanning Auger electron spectroscope (JEOL Ltd. JAMP-9500F eld emission scanning Auger microprobe) system. Samples were prepared by spreading powder particles over a gold-coated surface. AES spectra were acquired with a primary beam of 10 keV. The take-off angle of the instrument was 0 . The differential energy spectrum was used to subtract background from the direct Auger spectrum for calculating the band-to-band intensity. The rst differential d(N(E))/d(E) Auger spectra were obtained by numerical derivation of the direct N(E) integrated Auger data displaying an absolute scale with counts/second units by a universal Savitzky-Golay (SG) quadratic differential lter using seven points and used to calculate the band-to-band intensity of Auger electrons and derive the elemental compositions. The differential spectrum is simply the differential of the direct spectrum with respect to energy. The spectra were calibrated with the carbon band at 263.0 eV. For Auger elemental analysis an 8 nm probe diameter was used. Elemental mapping was analysed by AES. Elemental images were acquired with a primary beam of 10 keV. The take-off angle of the instrument was 0 . The coloured images are elemental</p>

Royal Society of Chemistry (RSC)

Studying a Cell Division Amidase Using Defined Peptidoglycan Substrates

Three periplasmic N-acetylmuramoyl-L-alanine amidases are critical for hydrolysis of septal peptidoglycan, which enables cell separation. The amidases cleave the amide bond between the lactyl group of muramic acid and the amino group of L-alanine to release a peptide moiety. Cell division amidases remain largely uncharacterized because suitable substrates to study them have not been available. Here, we use synthetic peptidoglycan fragments of defined composition to characterize the catalytic activity and substrate specificity of the important E. coli cell division amidase, AmiA. We show that AmiA is a zinc metalloprotease that requires at least a tetrasaccharide glycopeptide substrate for cleavage. The approach outlined here can be applied to many other cell wall hydrolases and should enable more detailed studies of accessory proteins proposed to regulate amidase activity in cells.

studying_a_cell_division_amidase_using_defined_peptidoglycan_substrates

<p>The bacterial cell wall (murein sacculus) is an essential cellular component that maintains cell shape and protects against lysis due to high internal pressure. It is composed of crosslinked strands of peptidoglycan (PG) that form a mesh-like structure around the inner membrane (Figure 1a).1 Although it provides stability, the cell wall is a dynamic structure; it is continuously modified by a number of synthetic and degradative enzymes that are finely tuned to allow the sacculus to grow and divide without lysing. The action of degradative enzymes known as murein hydrolases is especially critical during the late stages of cell division. In Escherichia coli, three periplasmic N-acetylmuramoyl-L-alanine amidases, AmiA, AmiB, and AmiC, have been shown to be particularly important for hydrolysis of septal PG, which enables cell separation.2 The amidases cleave the amide bond between the lactyl group of muramic acid and the amino group of L-alanine to release a peptide moiety (Figure 1b). Cell division amidases remain largely uncharacterized because suitable substrates to study them have not been available.3 Existing assays to monitor amidase activity are mainly based on in-gel degradation of isolated murein sacculi, which are heterogeneous, insoluble, and contain peptide chains of varying length, composition, and degree of crosslinking.1 This variability complicates any attempts to analyze amidase substrate preferences and possible modes of activation.</p><p>We have developed methods to synthesize the PG precursors Lipid II (1)4a–b and Lipid IV (2)4c, and have established enzymatic methods to convert these substrates to longer glycan strands (nPG, 3) that vary only in chain length.4c–e Here, we use these defined PG substrates to characterize the E. coli cell division amidase, AmiA.5 The approach described, which is applicable to many other amidases and cell wall hydrolases, enables a more detailed understanding of the activity and regulation of this important class of enzymes.</p><p>AmiA is a member of the zinc-dependent amidase 3 family, and one member, B. polymyxa va. colistinus CwlV, has been crystallized (PDB ID 1JWQ), allowing us to predict the active site residues involved in metal binding (Figure 2a).6 We overexpressed and purified C-terminally His-tagged AmiA7 and incubated it with nPG containing a radiolabel on the pentapeptide (3) in the presence and absence of zinc. Reactions were analyzed using paper chromatography.8 Unreacted nPG remains at the baseline, cleaved peptides migrate, and conversion is quantified by scintillation counting. The peptide product was verified using an authentic standard. Control experiments using lysozyme confirmed that nPG (3) could be cleaved by a PG hydrolase and that the reaction could be analyzed by paper chromatography (Figure S1). Without added zinc, AmiA exhibited no initial activity; in the presence of Zn2+, the radiolabeled peptide was cleaved from nPG (3) in a concentration-dependent manner (Figure 2a, right, wt rates). Adding zinc chelators such as EDTA and 1,10-phenanthroline prevented hydrolysis, but the reaction could be rescued by supplying an excess of zinc (Figure S2).</p><p>We also examined the initial rates of several mutants made based on comparisons of amidase family 3 catalytic domains. E242 is predicted to act as a general base catalyst, and H65, E80 and H133 are proposed to coordinate zinc to form the active site (Figure 2a). Mutation of any of these residues results in a full loss of activity, except for E80A, which suffered a 100-fold decrease in rate compared to wt (Figure S3). Altering residues D69 and D109, which are also highly conserved but are not predicted to be essential for activity, produced more modest decreases in reaction rate (about 10-fold less than wt). We have concluded that AmiA is a zinc-dependent metalloprotease with an active site configuration similar to CwlV.</p><p>The substrate requirements of CwlV, AmiA, or related autolytic amidases have not been determined: until now, the murein sacculus was the only reported substrate for these enzymes. In order to assess the substrate requirements of AmiA, we tested defined PG fragments of different glycan chain lengths that represent various stages of PG synthesis in the periplasm (Figure 2b). The substrates were incubated with purified enzyme under similar conditions and the reactions were analyzed by gel electrophoresis (Figure 2c).9 AmiA cleaves substrates 2 and 3 to produce a common low molecular weight band, which was identified as the released pentapeptide (4) by correlation to an authentic standard using HPLC and electrophoretic mobility (Figure S4). AmiA does not cleave the peptide from 1 (compare lanes 2 and 4 to lane 6), and a maximum of 50% of the radiolabeled peptide can be cleaved from 2 (data not shown). These results show that Lipid II (1) is not a substrate and suggest that AmiA contains an extended binding pocket that recognizes sugars on either side of the glycopeptide substrate. Consistent with this idea, disaccharide-peptide fragments obtained by treating nPG (3) with lytic transglycosylases are also not cleaved by AmiA (Figure S5). Hence, our results show that AmiA requires at least a tetrasaccharide as a substrate.</p><p>The use of compositionally well-defined PG polymer substrates has allowed us to characterize a cell wall amidase, E. coli AmiA, involved in PG degradation during division. The turnover number for cleavage of the polymer is 0.05 min−1 (Figure S3). Since cleavage rates from sacculi by similar purified enzymes have not been reported, there is no in vitro data for comparison.3d Although this rate is slower than estimates required to support bacterial growth,10 slow rates are typically observed in vitro for the enzymes that synthesize glycan strands.11 A possible explanation is that cell wall synthetic and degradative enzymes are proposed to operate as components of multi-protein machines with tightly-coordinated activities. Faster in vitro rates may be observed when other important components of the system are reconstituted. Some protein candidates for amidase regulation have been suggested, but it is unclear if they simply recruit amidases to the appropriate cellular location or if they influence activity through direct interaction with the amidase or its substrate.1,12 Access to homogeneous substrates rather than crude cell wall fractions provides the capability to evaluate amidase kinetics in response to prospective activators and could, in turn, help illuminate the nature of these complex interactions.</p><p>E. coli amidases cleave peptide crosslinks from peptidoglycan (PG): (a) schematic representation of an E. coli cell. Amidase activity is required for cell separation to occur during division; (b) chemical structures of synthetic labeled PG fragments Lipid II (1), Lipid IV (2) and uncrosslinked (nascent) PG (3).1c</p><p>Analysis of AmiA substrate preferences and catalytic features: (a) predicted zinc-binding site of AmiA based on alignment with B. polymyxa var. colistinus CwlV (left); comparison of relative cleavage rates of 3 (7.2 μM) by mutants of AmiA (4.0 μM) (right); (b) reaction scheme for AmiA cleavage of [14C]-pentapeptide (4) from potential PG substrates that differ in length; (c) gel electrophoresis of 3 (lanes 1, 2), 2 (lanes 3, 4), and 1 (lanes 5, 6) without and with AmiA addition, respectively, under similar reaction conditions. AmiA cleaves 3 and 2, but not 1, to produce a new band that represents 4.</p>

PubMed Author Manuscript

Reconstructing Reactivity in Dynamic Host-Guest Systems at Atomistic Resolution: Amide Hydrolysis Under Confinement in the Cavity of a Coordination Cage

Spatial confinement is widely employed by Nature to attain unique efficiency in controlling chemical reactions. Notable examples are enzymes, which selectively bind reactants and exquisitely regulate their conversion into products. In the attempt to mimic natural catalytic systems, supramolecular metal-organic cages capable of encapsulating guests in their cavity and of controlling/accelerating chemical reactions under confinement are attracting increasing interest. However, the complex nature of these systems, where reactants/products continuously exchange in-and-out the host, makes it often difficult to elucidate the factors controlling the reactivity in dynamic regimes.As a case study, here we focus on a coordination cage that can encapsulate amide guests and enhance their hydrolysis by favoring their mechanical twisting towards reactive molecular configurations under confinement. We designed an advanced multiscale simulation approach that allows us to reconstruct the reactivity in such host-guest systems in dynamic regimes. In this way, we can characterize the amide encapsulation/expulsion in/out the cage cavity (thermodynamics and kinetics), coupling such host-guest dynamic equilibrium with the characteristic hydrolysis reaction constants.All computed kinetic/thermodynamic data are then combined, obtaining a statistical estimation of reaction acceleration in the host-guest system that is found in optimal agreement with the available experimental trends. This shows how, to understand the key factors controlling accelerations/variations in the reaction under confinement, it is necessary to take into account all dynamic processes that occur as intimately entangled in such host-guest systems. This also provides us with a flexible computational framework, useful to build structure-dynamics-property relationships for a variety of reactive host-guest systems.

reconstructing_reactivity_in_dynamic_host-guest_systems_at_atomistic_resolution:_amide_hydrolysis_un

Introduction<!>Results and Discussion<!>Relationship between amide conformations and reactivity<!>Amide encapsulation/expulsion in-and-out the cage cavity<!>Molecular determinants of reactivity in dynamic host-guest systems<!>Conclusions<!>Computational methods and supplementary figures<!>S2<!>Preface on reconstructing dynamics from metadynamics<!>Ab initio metadynamics simulations<!>S10<!>Encapsulations/expulsion metadynamics simulations

<p>In billion of years of evolution, Nature has evolved systems (or materials) to control chemical reactivity with unique specificity, efficiency and fidelity. 1,2 Enzymes, capable of catalyzing reactions in their substrate-binding cavities, are a notable example. 3,4 In these systems, the reactants are dynamically encapsulated in the enzymes binding site, where the reaction occurs, and the products are then expelled leaving the reaction site free for hosting another reaction. 5 Such a machinery is controlled by a delicate modulation of host-guest interactions, which control key factors, such as, e.g., the residence time of the reactants/products (guest) in the reaction site of the enzymes (host), conformational changes in the guests favoring the reaction, etc., controlling de facto, the reactivity in the system. [6][7][8] In the attempt to mimic natural catalytic systems, chemists have designed synthetic cavities capable of encapsulating reactants with high selectivity. [9][10][11] In particular, supramolecular coordination cages have been designed, which can host catalytic reactions in their internal cavities. 10,[12][13][14][15] Reactivity, regio-selectivity and enantio-selectivity can be manipulated in such systems, e.g. by engineering the structural and electronic properties of the cage frameworks or by changing the guest structure. 9,16 Noteworthy, mimicking what happens in some proteolytic enzymes, 17 in a recent work it has been shown that the encapsulation of amide guests in a coordination cage may result in a considerable acceleration of amide hydrolysis.</p><p>In particular, the molecular crowding in the cavity of the cage was found to favor the mechanical twisting of the amides towards reactive (cis isomers) configurations. 10 The rational design of similar synthetic molecular systems requires a detailed comprehension of (i) the molecular and chemical-physical factors that control their reactivity in space and time, and (ii) how to master them in order to produce new classes of catalytic systems. However, in such supramolecular host-guest systems, the reactivity is coupled with a dynamic equilibrium where the guests exchange in/out the cage hosts, which makes them difficult to rationalize. 16 In general, the reactivity in such systems is controlled by several factors, such as the guests binding/unbinding, the solubility of the guests in the outer solution, the character-istic timescale for the reactions, possible entrapment in metastable states, and molecular concentrations in the system. In presence of coordination-cages, a major role in the reactivity is played by the non-covalent cavity-guest interactions. 16 Despite notable efforts, 9,15,[18][19][20] , reaching a detailed understanding in terms of the molecular factors that control the reactivity in systems in which the molecular species are in continuous exchange is typically difficult at experimental level. Computer simulations are extremely useful to this end. Quantum mechanical (QM) approaches, 21 or semi-empirical approaches such as Density Functional Tight Binding methods (DF-TB) 22 have been employed to study chemical reactivity and reactive pathways with notable precision. Ab initio molecular dynamics (MD) [23][24][25] and metadynamics [26][27][28] simulations have been also widely adopted to study chemical reactions of reactants in reactive configuration, in cases where the reactions require crossing free-energy barriers. However, the study of such systems on timescales that allows accounting also for their supramolecular host-guest dynamics (i.e., dynamic exchange of guests in-and-out the host) is not trivial. For this reason, a comprehensive description of such complex dynamic systems has been until now difficult to attain. Recent computational approaches based on enhanced sampling methods, such as, e.g., metadynamics (MetaD), holds great potential in this sense. 16,[29][30][31] Recently employed, e.g., to study the isomerization of photochromic switches (azobenzenes) encapsulated in coordination cages, such approaches allowed to demonstrate the tight interplay between isomerization dynamics, molecular crowding, and host-guest exchange dynamics. 16 This approach is versatile and holds a high potential for studying chemical reactions in host-guest systems in dynamic regimes in general.</p><p>As a case study, here we focus on the neat host-guest system recently reported by the group of Fujita, 10 where the encapsulation of amide guests within a coordination cage was found to considerably enhance the amide hydrolysis. This is an interesting test case not only because one amide functional group (the peptide bond) is the structural foundation of proteins, but also because the hydrolysis of amides is a classic example of a spontaneous reaction hindered by very high kinetic barriers, which can be lowered by physical means. 32 X-ray crystallography and NMR measurements were used in this system to characterize the encapsulation of a number of electron-rich diaryl amides into different octahedral coordination cages, differing in their metal corners, having electron-deficient walls. In particular, here we focus on the host-guest system of Figure 1, for which an acceleration in the amide hydrolysis of ⇠ 14x (compared to the same amide free in solution) has been experimentally reported when amide 2 is co-encapsulated with co-guest cage 3 inside cage 1 (Figure 1b: red vs. blue curves). This has been imputed to the fact that reactive conformations of the amide guest (cis-twisted) are stabilized within the cage host. 10 Such evidence found consistency with single-crystal X-ray diffraction analyses, showing that the amide can twist when encapsulated within the cage cavity. To obtain a submolecular-resolution insight into the behavior of such system in dynamic regime, here we combine all-atom (AA) MD and MetaD simulations that allow us to reconstruct the structural and dynamical features of this host-guest system. Ab initio MetaD simulations 25 allow us to study the chemical reactivity of the amide guest in the conformations that are most favored within the cage cavity. Coupled with the AA-MetaD thermodynamic/kinetic study of the amide (and co-guest) exchange in-and-out the cage, we can formulate a general scheme revealing the key factors controlling the reaction enhancement in this system. We obtain results that are found in excellent agreement with the available experimental trends, as well as a flexible computational framework, which can be used, in principle, to study a variety of dynamically reactive host-guest systems and to draw structure-dynamics-reactivity relationships useful for rational design.</p><!><p>Atomistic modeling of the host-guest system As a representative example of a supramolecular host, here we focus on the coordination cage 1 reported in Figure 1a recently employed by Takezawa et al. to host the hydrolysis of encapsulated amide guests. 10 This is an octahedral coordination cage composed of four self-assembled electron-deficient panel ligands (2,4,6-tris(4pyridyl)-1,3,5-triazine) and six metal-based corners (cis-endcapped Pd(ii) complexes). 33 As a first step, starting from the X-ray crystal structure reported in the literature, 10 we built an AA model for cage 1 which was then preliminary minimized and equilibrated in explicit water solvent and in standard (room) conditions of temperature and pressure via a classical MD simulation. In particular, all the AA models used herein have been parametrized based on the General Amber Force Field (GAFF) 34 and all simulations have been run with the GROMACS-2020.2 software 35 patched with Plumed-2.7. 36 (details available in the Computational Methods section in the Supporting Information, SI). We analyzed the equilibrium MD trajectories to study the degree of flexibility of the cage in realistic conditions. Analysis of the evolution along the MD of, e.g., the internal volume of the cage cavity, Root Mean Square Deviation (RMSD) of the atomic positions, and of two variables (d1 and d2) estimating respectively the height and equatorial width of the octahedral cage revealed that the cage structure is rather rigid in experimentally relevant conditions (see FigureS2 in the SI). This is due to the tetrahedral T d symmetry of this cage. Differently from other examples of flexible cage hosts, 16 cage 1 thus shows a large volume (⇠1.2 nm 3 ), open, with a rather persistent hydrophobic cavity that allows encapsulating one or multiple guest molecules in its interior (the electron-acceptor ⇡ planes of the triazine-based ligands of 1 are particularly apt at interacting with electron rich guests). 10,33 As the main guest, here we focus on guest 2, an N-(2,4-Dimethoxyphenyl)thiophene-2-carboxamid, an electron-rich diaryl amide (Figure 1a) that was experimentally shown to produce the considerable reaction acceleration following to encapsulation in cage 1 (Figure 1b). 10 The central bond in amide guest 2 can undergo hydrolysis according to the reaction schematized in Figure 1b (top). Guest 2 is mildly apolar (logP = 2.77). 37 Experimentally-obtained crystal structures show that cage 1 can encapsulate at the same time up to two 2 molecules in its internal cavity. 10 An AA model was developed for guest 2, paying particular attention to the accuracy in the force field parametrization of the central amide bond dihedral, which defines the trans and cis conformers of the amide, how much one is energetically favored respect to the other, and the related transition barrier (see Figure S1 and details in the Computational Methods section in the SI).</p><p>We built inclusion complexes where guest 2 is encapsulated within cage 1 in different stoichiometries: i.e., 2 ⇢ 1 and 2 2 ⇢ 1, where respectively one or two 2 amide guests are encapsulated inside cage 1. In particular, the AA model for the 2 2 ⇢ 1 complex was built starting form the available experimental crystal structure for this complex, 10 while that for the 2 ⇢ 1 was obtained by deletion of one of the 2 guests. Aromatic amides, like 2, exist mainly in a trans-planar conformation in solution. However, experimental evidences demonstrated that 2 may adopt a cis-twisted conformation within the T d symmetric cavity (forming a pseudo-S 4 symmetric dimer) in cage 1. 10 In particular, X-ray and NMR measurements showed signals corresponding to a cis:trans 1:1 2 dimer in the 2 2 ⇢ 1 complex, and to a cis-twisted conformation in the 2 • 3 ⇢ 1 complex.</p><p>To the purpose of our investigation, here we decided to model all possible combinations of conformers, mainly aiming to explore the presence of any correlation between confinement, crowding and the rotation of the amide bond. We also parametrized an AA model for coguest 3 (Figure 1a). Starting also in this case from a corresponding experimentally available crystal structure, 10 we use this to built an additional AA model for the 2 • 3 ⇢ 1 complex, a ternary inclusion complex where one 2 guest and one 3 co-guest are simultaneously encapsulated in cage 1. Particularly interesting for this study, the 2 • 3 ⇢ 1 system was observed experimentally to produce the larger hydrolysis acceleration among all explored cases (Figure 1b: ⇠ 14x acceleration compared to the free amide). 10 We then ran classical MD simulations to equilibrate all the considered complexes in explicit water and in standard (room) conditions of temperature and pressure (at least 1 µs for each system -see SI for details). From the equilibrium MD trajectories, we then extracted multiple data indicative of the encapsulation (Figure 2). When in the cage, 2 tends to stay shifted from the geometrical center of the cavity to maximize interactions with the walls, with the 2 trans conformer showing in general a larger shift compared to 2 cis (Figure 2b). This is imputable to the thiophene ring, which tends to partially stick out of the cavity preferring interaction with the solvent (scheme of Figure 2a, top: in cyan). Co-encapsulation with 3 induces a larger decentralization of the amide guests. Noteworthy, the augmented growing in such a case is observed to causes a significant reduction in terms of 2 cis mobility in the cage cavity, as evidenced by the narrower distribution in Figure 2b (solid red curve). This behavior correlates with an augmented number of contacts between 2 and the cage 1 (Figure 2c). In general, the 2 cis guest shows increased contacts than the 2 trans one (augmented guest-cage interaction), while this trends is even increased when co-guest 3 is also present within 1 (higher crowding). We will discuss The Solvent Accessible Surface Area (SASA) of the guests shows that 2 is less exposed to the solvent when this is encapsulated within 1, and even less when this is co-encapsulated together with 3 (Figure 2d). In general, we observe that the 2 cis conformer has a smaller SASA, and is mode compact than 2 trans in all cases. These SASA and contacts data provide information on the different packing of the 2 cis and 2 trans conformers within the cage in increasing crowding conditions. These are consistent with the FESs reconstructed from the histograms extracted from the MD of Figure 2a, where it is evident how 2 cis allows for a tighter packing within the cage (bottom). At the same time, the 2 cis conformer is found less mobile within 1, as it is demonstrated by a narrower FES dark minimum (minimum energy configuration) compared to that of the 2 trans conformer (top).</p><p>As a fist and generally rate-limiting step, the amide hydrolysis reaction requires the nucleophilic attack of water (or of OH , if the reaction occurs as catalyzed in basic conditions) to the carbonyl carbon. 32,38 While the cavity of cage 1 is markedly apolar, and the encapsulation of the guests in the cavity is essentially driven by hydrophobic effects, some accessibility by the solvent is therefore still required for the reaction to take place. We calculated the number of contacts between the solvent molecules and the carbonyl group of the 2 guest in the various case. In general, we can observe that the amide of 2 trans is slightly more accessible to the solvent compared to that of 2 cis when the guests are free in solution (dotted distributions), and a similar depletion is observed upon mono-guest confinement (dashed).</p><p>However, the situation is surprisingly switched in the tightly packed 2•3 ⇢ 1 systems (Figure 2e). In highly confined conditions, the amide of 2 cis is found more exposed to the solvent within cage 1 compared to that of 2 trans (red vs. blue solid curves). This suggests that, while on the one hand the cis conformer of 2 is more tightly packed within the cage, the equilibrium configuration of 2 cis in the 2 • 3 ⇢ 1 complex may allow, at the same time, for an increased propensity to react. We explore at a deeper level the difference in terms of reactivity of the trans vs. cis conformers of 2 in the next section.</p><!><p>The encapsulation of amide 2 within cage 1 was shown to enhance amide hydrolysis in considerable way in mildy basic conditions (experimental results of Figure 1b have been obtained at [NaOH] = 100 mM 10 ). To obtain information on the effect of confinement on the hydrolysis reaction (slow-down, inhibition, acceleration, etc.), here we modeled the hydrolysis reaction when the amide is free in solution vs. when this is encapsulated the the cavity of cage 1. In moderate pH conditions (6 < pH < 13), and in the absence of other catalysts, the hydrolysis of amides occurs via hydroxide attack, forming a tetrahedral intermediate (TI), followed by a second step consisting in the C-N bond rupture. 32 The first step of the reaction is typically considered the rate determining step of the process 38 (although in some cases the subsequent bond rupture can contribute to, and also control, the rate of hydrolysis). 32 For this reason, for the study of the hydrolysis of amide 2, here we focused our investigation only on the formation of the TI via nucleophilic attack by a solvated hydroxide (Figure 3a).</p><p>Previously used, e.g., to investigate the hydrolysis of formamide in basic conditions, 38 here we relied on ab initio well-tempered metadynamics (WT-MetaD) simulations 39 to study the reactivity of 2 as free in solution vs. confined in the cage cavity. Considered the computational cost of these simulations, and the complexity of our systems, we employed a semi-empirical density-functional tight-binding (DF-TB) method, 40,41 in its self-consistent charge corrected variant SCC-DFTB. 42 Recently shown to provide comparable accuracy to DFT with large basis sets in terms of prediction of barrier heights and reaction energies for organic molecules, 43 SCC-DFTB guarantees satisfactory accuracy in our case at an affordable computational cost. 22 We simplified our models by studying a system with an OH nearby the amide (Figure 3a), and constraining the C-N-C(O)-C dihedral (!) of amide 2 to representative values, in order to simulate attack to different conformers. We compared 2 conformers with ! equal to 0 (cis conformer), ⇡ (trans conformer), and ⇡/4 (a twisted cis The blue profile refers to 2 in trans conformation, the red one refers to the reaction when 2 is cis, while the violet profile refers to the free energy profile of a cis-distorted configuration of 2 with ! = ⇡/4. A relative reactivity score for each amide conformers ( ! ), normalized based on the maximum measured value (i.e., that for !=⇡/4, set to 1), is associated to the simulated conformers of 2 (right secondary y axis). (c) Isomerization of 2 in the cage, co-encapsulated with 3 (left: trans-2, right: cis-2). (d) Free energy profiles for the isomerization of 2 (i) when this is free in solution (dotted curve, cis in pink), (ii) when 2 is encapsulated in 1 (dashed curve, cis in dark pink), (iii) when this is co-encapsulated with another (trans) 2 guest in cage 1 (dot-dashed curve, cis in light red), and (iv) when 2 is encapsulated in 1 together with the co-guest 3 (solid curve, cis state in light red). The data show that an increasing crowding stabilizes more and more the reactive 2 conformations (e.g., cis) in the cage cavity. Right secondary y axis: relative probabilities (P conf ! ) for the different conformations (!) of 2 in the various host-guest systems calculated based on the G values extracted from WT-MetaD simulations. conformer). We employed replica infrequent ab initio WT-MetaD simulations 25 to obtain information on the reaction coefficients (rate of hydroxide attack and TI formation) for the various conformers of 2 (see Methods section in the SI for details). The reaction barrier of ! = ⇡/2 has been also tested, but this conformer was found too unstable to compute meaningful kinetic data. From multiple infrequent WT-MetaD runs activating/biasing the transition (R!TI, and the TI!R processes), we reconstructed the unbiased kinetics for the transition events and could estimate the characteristic transition times, ⌧ of f and ⌧ on . The kinetic constant for hydroxyde release can be calculated as k of f = 1/⌧ of f . The kinetic constant for hydroxyde attack (k on ) can be obtained in similar way from the ⌧ on and accounting for the OH concentration in the system (in our simplified setup, where OH is constrained in close proximity of the amide -perfectly basic conditions -, the theoretical OH concentration to be considered is equal to that of pure water: 55.6 M -see Methods for details).</p><p>This simulation approach allowed us to obtain an throughout thermodynamic and dynamic characterization of the reaction as a function of the amide conformation (for some relevant discrete ! values). The results of Figure 3b show a strong dependency of reactivity on the ! dihedral. As expected, the lowest reactivity against hydroxyde attack was observed for the trans conformer, while the cis and the twisted cis amide conformers were found as more reactive. In particular, the latter is the only conformer with a K reac = k on /k of f >1 (Figure 3b: R is higher in free energy than TI -see complete data in Table S2). In particular, from the K data, we can obtain relative reactivity scores ( ! ) useful to compare the reactivity between the different amide conformers. The ! scores of Figure 3b (right secondary y axis) clearly show how, compared to the twisted cis amide conformer (!=⇡/4), the cis amide (!=0) is ⇠ 1000 times less reactive, while the reactivity of the trans amide conformer (⇡) is basically negligible (⇠ 10 9 times less reactive than the ⇡/4 conformer).</p><p>To our purpose, the reactivity of the different conformers of 2 has to be put into context, namely, considering the actual probability for finding such conformers in solution vs. in the different encapsulated systems (i.e., under different crowding conditions). In realistic conditions, amide bonds are usually found in trans configurations with a torsion angle ! close to ⇡, with a sparse population in cis conformation (! ⇠ 0). The degree of steric conflict of the two residues flanking the amide bond is typically larger in cis amides, resulting, for example, in only ⇠ 5 6% occurrence of cis peptide bonds in protein structures. 44,45 To estimate the relative probabilities for finding in different conformers in different conditions, we used WT-MetaD simulations.</p><p>As a fist step, we optimized the amide ! dihedral force field parameters, in order to obtain a trans-to-cis isomerization free energy profile consistent with the available experimental data (see extended details in the Methods section in the SI). 46 The trans-to-cis isomerization of ! angles consists of a local conformation change that is often compensated by local variations of the backbone angles and of the residues flanking the amide. 44 In order to asses how the free energy profile for the isomerization of ! is affected by the torsion of and , we selected these 3 dihedral angles as our CVs and ran WT-MetaD simulations activating/biasing the trans-to-cis transition of 2 (i.e., the torsion around the amide bond) in different conditions (Figure 3c-d). Preliminary WT-MetaD simulations showed that the free energy profile of the ! isomerization is not particularly influenced by the and torsions. Well-converged WT-MetaD runs allowed us to reconstruct the differences in free energies between the conformers ( G) and estimate the free energy barriers (to this end we used infrequent WT-MetaD simulations, as recrossing WT-MetaD simulations may underestimate the barrier heightssee extended Methods for details).</p><p>The results in Figure 3d compare four cases where: (i) 2 is free in solution (Figure 3d: dotted curve, cis conformer in pink), (ii) 2 is encapsulated in cage 1 (dashed curve, cis in dark pink), (iii) the isomerizing 2 is co-encapsulated in cage 1 with another trans 2 guest (encapsulated 2 dimer: dot-dashed curve, cis in light red), and (iv) 2 is co-encapsulated in cage 1 with co-guest 3 (dot-dashed curve, cis in light red). The results show that the stability of the conformers of 2 is significantly affected by confinement. The free energy differences between the cis and trans conformers (Figure 3d) indicate that, while trans is always the most stable configuration of the guest, the cis conformer is more and more stabilized as the crowding in the cage cavity increases. The transition barrier also decreases while increasing the crowding. This is captured by the G and the K conf values, as well as by the relative probability profiles P conf ! of Figure 3d. In particular the P conf ! plots the relative probability for different conformers (!) of 2 with respect to the trans conformer in all simulated complexes. We move from a cis:trans ratio of ⇠ 10 7 :1 for one 2 free in solution to ⇠ 10 5 :1 in the mono-encapsulated system (2 ⇢ 1), ⇠ 10 4 :1 when two 2 guests are co-encapsulated in the cage (2 2 ⇢ 1), to ⇠ 10 2 :1 in the 2 • 3 ⇢ 1 system. The 2 dimer encapsulation (2 2 ⇢ 1) system, where one of the two guests is kept fixed in a trans configuration in accordance with experiments, 10 falls in between the 2 ⇢ 1 and 2 • 3 ⇢ 1 cases. The strongly twisted at ! = ⇡/4 remains extremely unlikely in all systems, despite a similar thousand-fold stabilization via confinement. The most crowded case, 2 • 3 ⇢ 1, shows a ⇠ 10000⇥ increase in the probability for finding the more reactive cis conformer respect to the case where 2 is free in solution. This is remarkable, considered that experimentally this case is the one showing the strongest acceleration in the hydrolysis reaction. 10 Table 1: Thermodynamic and kinetic data for 2 isomerization in all simulated complexes. Free energy differences ( G trans!cis ) related to the trans-to-cis isomerization, equilibrium constants for the conformational change (K conf ), the height of the free-energy barriers ( G ‡ trans!cis ) from the trans state, characteristic timescales (t trans!cis and t cis!trans ) are reported.</p><p>a The second 2 trans guest was kept in trans conformation during the simulation.</p><p>These results provides a new perspective for interpreting the reactivity ranking obtained in Figure 3b. (i) The most probable conformer in all states, 2 trans , is also the least reactive.</p><p>(ii) The most reactive twisted conformers (! = ⇡/4 or, e.g., ⇡/2) are, at the same time, highly improbable, even at increased molecular crowding. (iii) The 2 cis conformer, moderately reactive (but sensibly more reactive that the 2 trans one), is unfavored in solution against</p><p>2 trans , but it becomes more and more relatively favored as the crowding increases upon confinement, emerging as the prominent reactive species in the cage.</p><p>The available experimental X-ray structures for these complexes show a 2 cis -2 trans dimer in the 2 2 ⇢ 1 case, and a 2 cis -twisted conformation in the 2 • 3 ⇢ 1 complex. This seems to indicate that in these complexes a 2 cis conformer is more favored than 2 trans . While this may seem to contradict the simulation results discussed above, it is worth noting that all the results collected up to this phase are valid only under the assumption that the encapsulated guests remain always within the cage cavity. Nonetheless, these are host-guest systems, in which the probability for finding the guests within the cage obeys a well-defined supramolecular equilibrium. Estimating the effective probability for finding the guests within the cage requires also studying the dynamics of guest encapsulation/exchange in-and-out the cavity.</p><p>As it will be demonstrated in the next section, accounting also for the intrinsic supramolecular dynamics of these host-guest systems provides results that are globally in very good agreement with all available experimental evidences.</p><!><p>In realistic conditions, the encapsulation/expulsion of guests as, e.g., 2 or 3 in/out the cavity of cage 1 may require crossing considerably high free energy barriers, 16,30 which makes them rare events in the timescales accessible via classical atomistic MD simulations. As recently done for other host-guest 16 and dynamic supramolecular systems, 31,47 we thus reconstructed the thermodynamics and kinetics for the processes of encapsulation/expulsion of amide 2 in/out the cavity of cage 1 by means of a well-suited WT-MetaD 48 simulation protocol (complete computational details are available in the Supplementary Information). 16,31,47,49,50 The extracted data are collected in Table 2 and Figure 4.</p><p>Table 2: Equilibrium and kinetics of the amide encapsulation/expulsion in/out cavity.</p><p>For each simulated host-guest complex, encapsulation free energies ( G), equilibrium constants K enc , expulsion free energy barriers ( G ‡ of f ), characteristic in-cavity residence times (t of f ), and the associated transition rates (k of f and k on ) estimated from the WT-MetaD simulations are reported. Comparing the encapsulation of the different isomers of 2 in the cavity of cage 1, either alone or when 1 also contains a co-guest (3 or 2 trans ), we could observe that in general the encapsulation of 2 cis is more favored than that of the 2 trans isomer in all studied cases (see Table 2 and Figure 4). The kinetic constants measured for the 2 trans encapsulation/expulsion in/out the cavity indicate that in all complexes the dynamics of the transitions are marginally affected by the presence of other guests in the cage (k of f and k on in the same orders of magnitude). On the other hand, the dynamics (and stability) of the 2 cis -complexes is more impacted by the presence of co-guests in the cage cavity, which are found to stabilize the 2 encapsulation in the cage cavity by ⇠ 2 4 orders of magnitude in the presence of 3 (lower k of f : more improbable/slower 2 expulsion out of cavity). Shown in Table 2, the estimated k of f for the expulsion of the 2 cis guest out from the cage cavity drops from ⇠ 7.1 ⇥ 10 1 s 1 , when only one 2 cis is present in the cavity of 1, to ⇠ 1 s 1 or ⇠ 5.9 ⇥ 10 2 s 1 , when 2 cis is co-encapsulated in the cavity of 1 with a 2 trans or with a 3 co-guest respectively. The k on , on the other hand, is found globally similar in all simulated cases (see Table 2).</p><p>Altogether, these data suggest that differences in the host-guest equilibrium in such systems in is mainly controlled by the interactions/affinity between the guest (2) and the effective host cavity, thought of as that accessible for the guest considering the cage and the presence of eventual encapsulated co-guests. From the calculated G, we could also estimate the host-guest affinity constants (K enc ) for all the host-guest systems (Table 2). It is worth noting that the K enc for the 2 cis conformers are in general orders of magnitude higher than those of the 2 trans complexes. In particular, this is evident for the 2 • 3 ⇢ 1 complexes.</p><p>In such a case there is a difference in the encapsulation Ks of ⇠ 5 orders of magnitude (Table 2). This means that the probability for having 2 cis co-encapsulated in the cavity together with 3 is ⇠ 100 0 000⇥ higher than that of finding a co-encapsulated 2 trans . Despite the fact that, in theory, the 2 trans conformer is found ⇠ 100⇥ more favored compared to the 2 cis one in the cage cavity (see Figure 3d: right secondary axis), such a statistical penalty for having a 2 trans conformer effectively encapsulated within the cage cavity -emerging from the host-guest equilibrium -explains why the experimentally obtained X-ray structures of these complexes always show a 2 cis encapsulated guest.</p><p>Nonetheless, the high K enc values indicate that in all the cases simulated herein the amide guest can be, in good approximation, assumed as always encapsulated in the cage cavity. In fact, from the thermodynamic data we can extrapolate the partition probability</p><p>The P in indicates the relative probability for having 2 in vs. out the cage cavity. In particular, P in tends to 1 when the K is high and the guest has a high probability of being encapsulated in the system, as it is the case in general for all host-guest complexes explored herein (P in ⇠ 1 in all cases, see also next section).</p><!><p>Based on all the parameters obtained from our simulations, we can define a reaction acceleration index, a, as the ratio between the observed reactivity with or without the presence of the cage in the system -i.e., when the reactant, guest 2, is encapsulated within the cage cavity (K(cage)) or when it is free in solution (K(sol)):</p><p>In the real system, amide hydrolysis can in principle take place both when 2 is en- capsulated in the cage cavity or when this is out of the cage (with the observed reaction coefficients determined by the probabilities for finding the different reactive conformersplanar or twisted -in the two environments). In general, the reaction acceleration a will thus depend on the likelihood that the hydrolysis of 2 occurs in vs. out of the cage. From our simulations we have seen that the conformational free energy landscape of the amide guest may change upon encapsulation (changing the relative free energy difference between cis and trans conformers). As a consequence, the probability for crossing the rotational barrier around the amide bond also changes. In particular, we could observe that the more reactive 2 cis conformer is more and more stabilized as the crowding increases in the cavity of cage 1 (Figure 3d). The simulations also show that the encapsulation of 2 cis within the cage cavity is considerably more stable than that of 2 trans , showing a higher affinity and retention time (Figure 4 and Table 2). Altogether, this indicates that it is more likely to observe 2 cis rather than 2 trans encapsulated within the cavity of the cage, which is consistent with the fact that the 2 cis conformer is present in the crystal structures obtained experimentally. 10 Such a dynamic complexity is represented in the scheme of Figure 5a. More in detail, the reactivity in the system depends on the propensity of the visited 2 conformers to react, their relative population in the different complexes, their probability of encapsulation (i.e., the relative population ratio between having 2 in the cage vs. in solution at the equilibrium), and the solvent molecules accessibility to the amide (i.e., the solvent is another key reactants) upon encapsulation. Noteworthy, all these parameters that can be extracted from our simulations.</p><p>In general, we can define a global reaction constant for the case when hydrolysis takes place within the cage cavity, K(cage), as the sum of the reaction constants (K ! (cage)) for all amide conformers (!) visited by the guest reactant 2 in the cage cavity:</p><p>where ! is the hydrolysis reaction constant associated to the possible amide conformers ! (see Figure 3b), P conf ! (cage) is the relative statistical weight for all different conformers ! in the cage cavity (Figure 3d), P in ! (cage) is the probability for effectively having each specific conformer ! in the reactive environment -in this case, inside the cage cavity (see Table 2)</p><p>-, and NW ! (cage) is the average number of contacts between solvent molecules (water or OH : key reactants for amide hydrolysis) and the amide's carbonyl (estimated as in Figure 2e).</p><p>Accordingly, the global reaction constant in the absence of the cage in the system (2 alone in solution), K(sol), can be defined as:</p><p>where in this case P conf ! (sol) and NW ! (sol) refer respectively to the relative probabilities for 2, when alone in the solvent, to assume the different conformers !, and the corresponding number of amide carbonyl-solvent molecules contacts. In this case, in the absence of the cage in the system, guest 2 is by definition always out of the cage, and P in ! (sol) = 1. Thus, Equation3 simplifies into:</p><p>Moreover, it is worth noting that, given the high G values in Figure 4c and Table 2, when the cage is present in the system, the guests can be also considered as always encapsulated within the cage cavity, so that in Equation 2 the P in (cage) term tends to ⇠1 (vide supra):</p><p>Given the high propensity to guest encapsulation (P in (cage) ⇠1), in this specific case the reaction acceleration in the system is found to be little dependent on the guest encap-sulation/expulsion equilibrium. On the other hand, the reactivity turns out to be rather controlled by the fact the guest is more favored to assume reactive conformations inside the cage cavity (compared to the case when this is free in solution). This is in full agreement with the available experimental evidences on these systems. 10 Finally, it is worth noting that while the summations in Equation 5and Equation 4 run in principle over all possible values of ! (have different reactivities -see Figure 3b), the data of Figure 3d clearly show that, due to the intrinsically high isomerization barrier, the relative probability for observing twisted (an extremely reactive) 2 conformers (e.g., ⇡/2, ⇡/4, etc.) is very low. These are distorted, very unstable conformers, with a survival lifetime which tend to 0, and for which the product ! • P conf ! ⇠ 0. The unique conformers with survival life and P conf ! 6 = 0 are 2 cis (! = 0) and 2 trans (! = ⇡). The latter, however, is substantially non-reactive (Figure 3b: ⇡ ⇠ 0), so that also in this case ⇡ • P conf ⇡ ⇠ 0. Based on these observations, in our case the reactivity of the system seems thus to be largely related to (i) how much over-stabilized is the reactive 2 cis conformer and (ii) how accessible is the amide to the co-reactant solvent molecules in such conformation in the cage cavity vs. in solution.</p><p>Combining these data, we estimate the reaction acceleration a for the various host-guest complexes reported in Figure 5b). We observe that, in this case, the reactivity increases with the crowding in the system. While a ⇠26-fold acceleration is computed for the monoencapsulated case (2 ⇢ 1), a double-encapsulation gives a ⇠64-fold increase for the 2 2 ⇢ 1 system. A dramatic a ⇠ 150 is obtained for the 2 • 3 ⇢ 1 complex. While such estimated a values may differ quantitatively from those obtained from the experiments (this can be expected, given the deviations of such ideal models from realistic systems/conditions), the trends can be still safely compared. Figure 5c shows a remarkable trend between our calculated acceleration data and the experimental ones. This validates our simulation approach.</p><p>It is worth noting that the mono-encapsulation case (2 ⇢ 1) does not have an experimental counterpart, due to the tendency of 2 to dimerize within the cage. Nonetheless, this extracase (where crowding is lower than in, e.g., 2 2 ⇢ 1 and 2 • 3 ⇢ 1) provides an additional case useful for comparison. In particular, the limited computed acceleration seen in this case supports the evidence that molecular crowding within the cage cavity is a key player for the reactivity in the host-guest system.</p><p>In order to obtain an insight into the key molecular determinants controlling the reaction acceleration in these host-guest systems, in Figures 5d-g we plot the computed a parameters against some of their key constitutive terms.</p><p>We have seen that the difference in affinity between cis and trans conformers among the different systems is the main factor determining the final reaction acceleration using Equation1, this being shown by the nearly perfect exponential correlation between a and the relative probability of finding the 2 cis conformer with respect to the 2 trans conformer in solution and in the different encapsulation complexes (Figure5d). The trend suggests that small incremental stabilizing effects on this conformation, e.g. by changing affinity and size of the co-guest, could result in potentially outstanding enhancements of reactivity for guest 2, keeping all other parameters constant.</p><p>Noteworthy, a quasi-exponential trend is observed between the computed reaction acceleration a and the encapsulation free energies ( G enc ) for 2 cis in all systems (Figure 5e).</p><p>In these systems, where the reactivity is observed to increase with the crowding inside the cage cavity, the a is also clearly related to the host-guest interaction (namely, to obtain a stable complexation, a strong host-guest affinity is necessary to compensate the crowding penalty associated to the binding). A similar trend can be observed also when looking at the weighted number of contacts between the host and the guest (Figure 5f, evaluated from the distributions of Figure 2c, i.e. a proxy for the host-guest interaction energy). If we consider the interaction between 2 and cage 1 to be consistent among all the investigated systems, we can trace the trend back to the interaction between guest and co-guest (or the absence thereof in the 2 ⇢ 1 case), with 3 showing a greater stabilizing effect for 2 cis within the cage cavity compared to another 2 co-guest. Noteworthy, as revealed by the obtained trends of Figures 5e-f, such favorable affinity can stabilize the reactive conformer of amide 2 to a higher extent, which results in a remarkable increase in the reaction acceleration a.</p><p>To obtain information on how much of the host-guest interaction is due to solvophobic effects, we calculated the reduction in the Solvent Accessible Surface Area (SASA) of the 2 cis conformer when this is encapsulated in the cavity of the various complexes vs. when this is alone in solution. 16,47,51 While a correlation with the computed reaction acceleration is observed (see Figure 5g), the trend becomes less neat. The trend is respected while moving from the amide in solution to mono-guest (2 ⇢ 1) and double-guest complexes (2 2 ⇢ 1 and</p><p>. However, the differences in acceleration between the various systems do not correlate in a neat manner with the SASA calculated for the various cases. This reveals that (i) non-specific hydrophobic effects alone are not sufficient to grasp the complexity of these reactive system, and suggesting that (ii), like in most of receptor-ligand complexes in Nature, specific molecular interactions are probably relevant in controlling the host-guest affinity.</p><!><p>Understanding reactivity in dynamic regimes and in systems in which reactants and products are in continuous exchange is a non-trivial task. Here we report a computational approach that allows us to reconstruct the reactivity in dynamic host-guest systems and to study at atomistic detail the key (molecular and dynamic) factors that control it. As a case study, we focus on the hydrolysis of amide guests encapsulated in the cavity of a coordination cage, for which experimental evidence for reaction acceleration has been recently reported. 10 By combining a multi scale modeling scheme with metadynamics simulations, we couple the study of the intrinsic dynamics of the host-guest system with that of the amide hydrolysis reaction. The approach allow us (i) to characterize the barriers to the hydrolysis reaction as a function of the conformation assumed by the amide, (ii) to study the conformations that the amide guest can assume and estimating their relative probabilities in vs. out the In the specific case study used here, we obtain clear evidence that the reaction acceleration is controlled by the crowding effects accompanying the guest encapsulation. We compared four cases, where the amide guest 2 is alone in solution, when it is encapsulated in the cage (2 ⇢ 1), or when it is co-encapsulated together with other co-guests in the cage cavity (2 2 ⇢ 1 and 2 • 3 ⇢ 1), we characterized all of them, and we estimated their hydrolysis acceleration factor. The results show unambiguously that the encapsulation of the amide in the cage cavity tends to stabilize the reactive conformers of the amide guest. This is key in such specific systems, considered the high encapsulation constants obtained for all studied cases (i.e., high probability to find the amide guest within the cage cavity).</p><p>We clearly observe how, when a co-guest is also co-encapsulated with the amide guest in the cage, the crowding in the cavity augments, and the reactive cis conformer of the amide guest is more and more stabilized. The acceleration scores estimated from our computations are found in remarkable trends with the reaction acceleration observed experimentally. Overall, our computational results are found in optimal agreement with the experimental results by Takezawa et al.. 10 , while it is worth noting that in our approach these emerge bottom-up, from a comprehensive study of the molecular and supramolecular dynamics of the host-guest system and of its key molecular equilibria. This provides a general character to this approach, as confinement-induced reaction acceleration (or deceleration) in such supramolecular (and intrinsically dynamic) host-guest system can only be explained by taking into account of all the dynamic processes that occur within them. The comprehensive picture of hydrolysis, and of how this may be modulated under confinement that we obtain here provides a general high-resolution framework for building structure-property relationships. This approach constitutes a useful general-purpose platform, useful to explore strategies towards the rational design of host-guest systems with a molecular-level control of chemical reactivity. Based on the results that we show herein, this approach offers a useful platform to explore, e.g., the effect of tuning the properties/features of the guest (e.g., hydrophobicity, symmetry, interactions, size, etc.) or of the cage cavity (e.g., structure, flexibility, hydrophobicity, etc.), as recently shown in other host-guest reactive systems. 16 Its versatility and generality may be also useful to explore ways to gain control over the system reactivity by tuning the host-guest dynamics by using, e.g., guests mixing and guest-guest encapsulation competitions, 10 which could allow tuning the residence time and fractions of encapsulated guests within the cage cavity and, consequently, the reaction in the system. In general, all these considerations underline how gaining control over the dynamics of these systems is key to control reactivity within them, and indicate how approaches such as that described herein can offer a relevant support towards the development of new types of reactive supramolecular systems.</p><!><p>Creation and parametrization of the molecular models</p><p>All atomistic model systems have been parametrized based on General Amber Force Field (GAFF) 1 , with the exception of the amide ! dihedral angle, which has been reparametrized to improve its accuracy as explained in the following section below. The donor-acceptor (Pd-N) bonds were parametrized using Seminario's method 2 following the Metal Center Parameter Builder (MCPB) protocol 3 as recently done in the atomistic models of other coordination cages. 4 The partial charges for the whole atomistic system (cage and the guests) were calculated using the RESP approach 5 as implemented in CP2K. 6 The quantum mechanical calculations for this purpose were performed using CP2K, with a BLYP-D2 functional 7,8 , paired with the Goedecker-Teter-Hutter pseudopotentials 9 and a triple-⇣ basis set with polarization functions (TZVP). 10 The atomistic parametrization was then carried out using the ANTECHAMBER software. 11 All the atomistic simulations in this work have been conducted in explicit solvent using the GROMACS-2020.2 software 12 patched with Plumed-2.7. 13 All systems were immersed in a simulation box filled with explicit TIP3P water molecules. 14 All simulated systems consist of a cubic box of different side length depending on the system: 37.2 Å for the amide in solution solvated by 1684 water TIP3P molecules, and ⇠51.0 Å with ⇠4290 water molecules for the other 3 host-guest systems. Moreover, for those models containing the cage, 12 molecules of NO 3 have been also added to neutralize the total charge present in the systems, consistent with the experimental conditions. 15 Reparametrization of ! dihedral potential terms of amide 2</p><p>Peptide bonds in protein structures are mainly found in trans conformation with a torsion angle ! close to ⇡. Due to the small population of the cis conformation 16,17 , the kinetics of isomerization of peptide bond has always been difficult to characterize experimentally and,</p><!><p>as a consequence, only limited data on the free energy barriers separating the two isomers are available. 18 Being generally derived from quantum mechanical calculations on model compounds, or based on experimental data from thermodynamic and kinetic studies, 19,20 the AMBER force field shows limitations in the estimation of the accurate free energy difference between the cis and trans conformations in peptide bond. Herein, this was evidenced and proven by preliminary metadynamics tests in this sense. As showed in section 3, the description of the cis-trans equilibrium for the amide 2 has a central role in the study of its reactivity, and thereby, our aim is to evaluate the accuracy of the potential terms involved in the process of isomerization of the ! dihedral angle. In the AMBER force field, the torsional potential energy term is expressed as the Pitzer potential 21 , a Fourier series term given by:</p><p>where V n , n, µ, and are, respectively, the dihedral force constants, periodicity, torsional angle, and phase angle.</p><p>To reproduce the torsional energy profile of the peptide bond, only the first two terms in this series are relevant: V 1 (n=1, = 0), and V 2 (n=2, = 180); the former describes the cis-trans equilibra, while the latter is responsible for the barriers to rotation about C-N bond.</p><p>In order to test the accuracy of the dihedral amide parameters, we compared the freeenergy surface (FES) for the torsion of an N-methylacetamide (NMA) amide -the simplest analog of the peptide bond within amides, that here we use as a reference to optimize the amide force field parameters -obtained performing WT-MetaD simulation with the experimental energy profile obtained with NMR studies. 18 In these WT-MetaD simulations, as the collective variable (CV) we chose the ! dihedral angle of the amide, with a bias factor of 30 deposited every 500 steps using Gaussians of initial height of 1. More precisely, these dihedral force field parameters have been modified until a good agreement was reached between our cis-trans isomerization FES and the experimental free energy profile for NMA (see Figure S1), modifying the main torsional parameters involved in ! bond rotation, while all the other non-bonded parameters were left unchanged (Table S1: in our procedure only the V 2 term for the general X-C-N-C torsion, and the V 1 and V 2 0 potential parameters specific of the H-N-C-O torsion have been modified). to keep a 300 K of temperature and 1 bar of pressure in the systems. The electrostatic interactions have been treated using the Particle Mash Ewald (PME). 25 The cutoff for the real part of the summation was 1.0 nm. The cutoff for the van der Waals interactions was set to 1.0 nm. All of the bonds involving hydrogen atoms were constrained using the LINCS algorithm. 26 The leap-frog integrator was used to integrate the equations of motions. All the analyses of Figure2 have been performed with Plumed-2.7. 13 . For the contact analysis a distance cutoff of 0.5 nm has been chosen between the involved groups, paired with a switching function with parameter D 0 =0.35 nm. The Solvent Accessible Surface Area (SASA) was computed with the default probe radius of 0.14 nm.</p><!><p>In these atomistic models, transitions such as, e.g., the isomerization of the amide, as well as amide encapsulation/expulsion in/out the cage are typically rare events, which cannot be effectively sampled via classical MD simulation. Recently, it has been demonstrated that the kinetics of rare events can be efficiently reconstructed by means of infrequent WT-MetaD simulations. [28][29][30] Provided that the the CV and setup of the WT-MetaD simulations are opportunely chosen, this approach relies on the fact that the real unbiased timescale of an event can be retrieved from the statistics obtained for the biased event, by calculating the transition time distributions.</p><p>We exploited this approach to calculate the characteristic timescales and the related kinetics of all the key processes studied herein -i.e., the amide hydrolysis rate limiting step, the cis-trans isomerization of the amide, and its encapsulation/expulsion in/out the cage (details on the WT-MetaD simulations setup for each of these studies are provided in the dedicated sub-sections below).</p><p>In particular, the unbiased transition time (t) can be calculated from the biased transition time (t W T ) extracted from each individual infrequent WT-MetaD run (in which the transition is biased/activated) as:</p><p>where V (s(R, t)) is the time dependent bias used during the simulation, and the exponential is averaged over the WT-MetaD run. The main idea behind this approach is to infrequently deposit the bias onto the free energy landscape to speed-up the transition and to effectively observe it at atomistic resolution during the run. At the same time, the V (s(R, t)) has to be deposited infrequently during the WT-MetaD, in such a way to prevent/minimize the deposition of bias on the transition barrier. The transition times (t) estimated from multiple infrequent WT-MetaD runs allow building a transition probability distribution, P n 1 , defining the probability to effectively observe at least one transition by time t:</p><p>where ⌧ is the characteristic timescale expected for unbiased transition. For a rare event, the statistics of transition times fit well with a Poisson distribution. 29,31 The kinetic rate constant (k) associated to such rare events can be then estimated from the characteristic transition timescales (⌧ ) as:</p><p>This allows to estimate the unbiased kinetics of a biased transition. The associated transition barrier ( G ‡ ), can be then estimated using the Eyring equation:</p><p>where  is the transmission coefficient (set to 1 in all cases studied herein, based on the fundamental no-recrossing assumption of transition state theory), k B is Boltzmann's S7 constant, and h is Planck's constant.</p><!><p>Ab initio metadynamics simulations have been performed using the CP2K package. 6,32 We employed the semi-empirical density-functional tight-binding (DF-TB) method, 33,34 in its self-consistent charge corrected variant SCC-DFTB. 35 Since our reactions include mostly organic molecules in water, we employed the well validated parametrization set miomod:nh. 36 Convergence of the SCF was set at 1.0 6 Ha. All simulated model systems used for the study of the hydrolysis reaction consist of a cubic box of side length 17.0 Å, containing amide 2 and 160 explicit water molecules, for a total of 510 atoms per simulation cell (Figure S3). Initial minimization and equilibration of this simulation box were performed for the neutral system.</p><p>Subsequently, the initial configuration for the hydroxide ion was obtained by deleting one proton from the water molecule closest to the amide bond. The Coordination Number (CN) of the oxygen of the OHwith respect to the hydrogens in the system was then constrained to be 1, in order to preserve the geometry of an hydroxide ion, following Crespo et al. 37 The CN was defined using a Fermi function of the following form: (1 [r/r 0 ] NN )/(1 [r/r 0 ] ND ),</p><p>where r 0 = 1.32 Å, NN = 16, and ND = 56 with respect to all of the hydrogens in the system. This CN ensures that the geometry of this species is constrained to be that of the hydroxide ion, which prevents complications arising from the identity of the hydroxide changing due to the Grotthuss mechanism, while still allowing the ion to react with amide.</p><p>Ab initio metadynamics simulations of the hydroxide ion attack of the amide have been conducted on different amide conformers, having different amide ! dihedral values which have been kept as contrained with an harmonic potential during the simulations. In particular, having initially set the constraints, every system (containing different ! amides) has been further thermalized for 3-4 ps. All simulations were run at 333 K in the NVT ensemble using the Canonical Sampling through Velocity Rescaling (CSVR) thermostat. 23 To estimate the characteristic rates of the OH attack/detachment from the carboxyl S8 In particular, we compared four cases, where the ! dihedral of amide 2 was constrained to 0, ⇡, ⇡/4 and ⇡/2. From the binding/unbinding transition timescales extracted from the 30 infrequent MetaD runs, we could reconstruct the characteristic unbiased kinetics for the events and estimate the characteristic unbinding/binding timescales, ⌧ of f and ⌧ on respectively. For all modeled cases, the kinetic constants for hydroxyde ion release from the amide has been calculated as k of f = 1/⌧ of f . The kinetic constant for hydroxyde ion attack (k on ) can be obtained the same way from ⌧ on -the obtained values have been then corrected considering that our simulation setup is consistent with a hypothetical OH concentration equal to the one of pure water (55.6 M), as the OH ion starts already (and remains) in close proximity of the amide. We could then estimate the equilibrium constant for the amide hydrolysis (K -see Table S2) as:</p><p>In particular, the case with !=⇡/2 demonstrated to be highly reactive and unstable, proving that this conformer of amide 2 is statistically irrelevant in this case.</p><p>In these ab initio infrequent WT-MetaD simulations we used the distance between O OH and the C=O carbon as the CV. We deposited hills (initial height = 2.0E-3 Ha) every 100 steps, using a bias-factor of 50. A potential wall was imposed to limit CV values larger than 3 Å. The CV was monitored to determine the transition events, with a cutoff of < 1.4 Å for ⌧ on and of > 2.8 Å for ⌧ of f . Table S2 reports the obtained data for the three dihedral angles.</p><p>Table S2: Reaction equilibrium and kinetic constants. To each system here studied we report the measured residence times (⌧ of f and ⌧ on ), kinetic constants (k of f and k on ), reaction constant (K reac ) and reactivity score ( ! ).</p><!><p>For the characterization of the barriers and kinetics of the trans-to-cis isomerization, we turned to infrequent WT-MetaD simulations. We ran 51 infrequent WT-MetaD simulations have been ran for each system where the isomerization of the amide has been activated.</p><p>In particular we, focused on the isomerization along ! and dihedral angles for each of our host-guest systems. This allowed us to compare how the encapsulation, and then the molecular crowding inside the cage cavity, affect the dynamics of isomerization of 2. In these infrequent WT-MetaD runs, the bias was deposited every 5000 steps (10 ps of simulation time) using Gaussians of initial height of 1.2 kcal/mol, of 0.23 rad, with bias factors between 6-16 depending on the system.</p><!><p>The isomers of amide 2 show different encapsulation/expulsion kinetics in/out the cage cavity, with different affinities ( G), retention times (⌧ ) and characteristic transition rates (k).</p><p>As expected, these quantities are influenced also by the presence of a co-guest (3, or another</p><p>2) inside the cavity of 1. For evaluation of the kinetics constant of the guests encapsulation/expulsion in/out the cage cavity, we conducted multiple infrequent WT-MetaD simulations in which the encapsulations/expulsion of the amide have been activated. In these WT-MetaD simulations, we used as the CVs the contacts between heavy atoms of guest and host-cage (CV1), the host-to-guest center-to-center distance (CV2), and the standard deviation of the contacts between heavy atoms of the guest and of the host-cage (CV3).</p><p>We conducted a first explorative WT-MetaD simulation, where the amide was biased to exchange in/out the cage cavity. Qualitatively, similar to what seen also in other hostguest 30 and dynamic supramolecular systems, 39 this simulation showed that the exchange of a guest from the cage cavity into the solvent is most likely a 2-steps process, where (i) the encapsulated guest is first expelled out of the cage cavity and it remains absorbed onto the cage surface (Figure S4a,b: in-to-out cavity transition), and then (ii), from such absorbed state, it jumps in solution. The same holds for the back (encapsulation) process, but in S11 opposite direction. As explained in the main text, for the purposes of this specific work, the exchange step (i) is the key one. In fact, this transition relates the state where 2 is encapsulated within the cage cavity vs. the closest state in free-energy where 2 is out of the cavity of 1 -i.e., the most likely transition (accompanied by the lowest exchange barrier) having consequences in terms of stabilization of the reactive 2 cis conformers seen in Figure 3d of the main paper). Indeed, as the crowding has a direct effect on the guest favored conformers and on their related reactivity, being out of the cage and adsorbed on the surface or in solution has negligible effects in terms of crowding for amide.</p><p>Reaching a robust convergence in a single WT-MetaD simulation for such a complex multi-step exchange process is prohibitive, and the FES shown in Figure S4a,b has thus just a qualitative/explorative value. As recently done also for other similar systems, 4,40 to characterize the key step (i), we thus opted to reconstruct the guest encapsulation/expulsion exchange profile, the associated G, transition barriers, and characteristic kinetics from multiple infrequent WT-MetaD simulations (see Figure S4c, and Figure 4 in the main paper).</p><p>In these infrequent WT-MetaD simulations, the bias was deposited every 5000 steps (10 ps of simulation time) using Gaussians of initial height of 0.12 kcal/mol, with bias factor ranging between 10 and 14 (depending on the system) for the expulsion out of the cage cavity, while we used 5 as the bias factor for the WT-MetaD simulations activating the guest encapsulation event. The deposed Gaussians had equal to 4.0, 0.04 nm, 2.0 along CV1, CV2 and CV3 respectively. For each system, the estimation of the kinetic constants (k) for the amide encapsulation/expulsion transitions in/out the cage cavity have been estimated from 50 WT-MetaD simulations. From these, we then could reconstruct all the associated thermodynamic quantities as described in the previous section above (see Equation S2-S5). As demonstrated by the data obtained for all cases, it is clear that in such host-guest systems the amide guest can be in general considered to be always inside the cage cavity (see Figure S4c, Figure 4 and Table 2 in the main paper) -i.e., in Equation 5 in the main paper P in ⇠ 1 in all simulated cases.</p>

ChemRxiv

Deciphering the regulatory logic of a stimulon

Although metabolic networks have been reconstructed on a genome-scale, the corresponding reconstruction and integration of governing transcriptional regulatory networks has not been fully achieved. Here we reconstruct such an integrated network for amino acid metabolism in Escherichia coli. Analysis of ChIP-chip and gene expression data for the transcription factors ArgR, Lrp, and TrpR showed that \xe2\x88\xbc82% of the genes they regulate are directly involved in amino acid metabolism. Further analysis shows that 19/20 amino acid biosynthetic pathways are either directly or indirectly controlled by these three transcription factors. Classifying the regulated genes into three functional categories of transport, biosynthesis, and metabolism leads to elucidation of regulatory motifs constituting the integrated network\'s basic building blocks. The regulatory logic of these motifs was determined based on the relationships between transcription factor binding and changes in transcript levels in response to exogenous amino acids. Remarkably, the resulting logic shows how amino acids are differentiated as signaling and nutrient molecules, and thus revealing the overarching regulatory principles of this stimulon.

deciphering_the_regulatory_logic_of_a_stimulon

<!>Genome-wide identification of TF-binding regions: Regulatory code analysis<!>Identification of regulons: topological analysis<!>Determination of causal relationships: functional analysis<!>The function of a stimulon: elucidation of overarching regulatory logic<!>Discussion<!>Bacterial strains and growth conditions<!>Chromatin immunoprecipitation and microarray analysis (ChIP-chip)<!>qPCR<!>ChIP-chip and expression data analysis<!>Motif searching<!>Raw experimental data

<p>Transcriptional regulatory networks (TRN) in bacteria govern metabolic flexibility and robustness in response to environmental signals1. Thus, causal relationships between transcript levels for metabolic genes and the direct association of transcription factors (TFs) at the genome-scale is fundamental to fully understand bacterial responses to their environment2,3. In particular, the molecular interaction between small molecules ranging from nutrients to trace elements and TFs governs the TRN and ultimately regulates the related metabolic pathways. From the causal relationships, a small set of recurring regulation patterns, or network motifs3,4 were identified and reconstructed to describe the design principles of complex biological systems. One primary discovery from this effort was the connected feedback circuit which coordinates influx (biosynthesis and transport) and efflux (metabolism) pathways that are jointly regulated by a TF sensing the relevant small molecule3. For example, a part of the global TRN is comprised of certain TFs (ArgR, Lrp, and TrpR) that sense the presence of exogenous amino acids (arginine, leucine, and tryptophan, respectively) and, in response, regulate the expression of a number of target genes5. Upon addition of these amino acids to the environment, the TFs exhibit enhanced, reversed, or unaffected regulatory modes3,6-8. These TF responses make these amino acids not just nutrients but also signaling molecules9.</p><p>Previously discovered network motifs3,4 represent a significant step forward in our understanding of complex biological behavior. However, they fail to appropriately elucidate the system wide response since they were either based upon incomplete information4, or were only specific to a single transcription factor and regulon3. This has resulted in an inability to appropriately understand complex regulatory phenomena existing across multiple transcription factors and regulatory signals. Hence, it is necessary to achieve a full elucidation of these interactions with systematic and integrated experimental analysis. Comprehensive elucidation of the causal relationships is achievable by integrated analysis of expression data obtained from microarray or sequencing (e.g., RNA-seq)10 with direct TF-binding information from chromatin immunoprecipitation coupled with microarrays or sequencing (ChIP-chip or ChIP-seq)3,11 under appropriate environmental conditions. Thus, we obtain and integrate genome-scale data from ChIP-chip for each TF and gene expression profiling to reconstruct regulons involved in amino acid metabolism at the genome-scale.</p><p>The elucidated regulatory logic falls into two categories that differentiate the role of amino acids as signaling and as nutrient molecules. Therefore, the reconstruction of the regulatory logic of the network motif allows us to establish the physiological role of each TF regulon and to determine how they govern the amino acid regulation in E. coli. Then, the integration of these multiple regulons into a unified network led to the first full bottom-up genome-scale reconstruction of a stimulon.</p><!><p>ArgR, Lrp, and TrpR are TFs involved in amino acid metabolism in E. coli6,7,12, responding to arginine, leucine, and tryptophan, respectively. The binding of the small effector molecule (here being the amino acids) to these TFs carries out the genome's regulatory code by enhancing or decreasing the TFs affinity for a specific genomic region and concurrently modulating the transcription of downstream genes. In the case of Lrp, the direct analysis of in vivo binding was fully described3 using chromatin immunoprecipitation coupled with microarrays (ChIP-chip) experiments. A total of 141 binding regions were analyzed, representing coverage of 74% of the previously identified regions3. However, similar genome-scale data for the other two major TFs in amino acid metabolism, ArgR and TrpR were unavailable. To determine their binding regions on a genome-wide level in an unbiased manner, we employed the ChIP-chip approach to E. coli cells harboring 8×myc-tagged ArgR or TrpR protein13. The resulting log2 ratios obtained from the ChIP-chip experiments identify the genomic regions enriched in the IP-DNA sample compared with the mock IP-DNA sample and thereby represent a genome-wide map of in vivo ArgR- and TrpR-binding regions (Fig. 1a).</p><p>Using a previously described binding region detection algorithm14, 61 and 8 unique and reproducible ArgR- and TrpR-binding regions were identified, respectively (Supplementary Table 1 and Supplementary Table 2). The 61 ArgR-binding sites detected included 13 sites previously characterized by DNA-binding experiments in vitro and mutational analyses in vivo15,16. For example, the ArgR-arginine complex transcriptionally represses gltBD, artPIQM operon, and artJ gene encoding arginine transport systems17,18. Our results confirmed that the ArgR-arginine complex binds to each of these promoter regions (Fig. 1b). In addition, the ArgR occupancy level at the promoter of the artJ gene is greater than that of artPIQM operon in the presence and absence of exogenous arginine (Supplementary Table 1). This result is in good agreement with the de-repression/repression ratio of 28 for PartJ and 3.2 for PartP previously reported for repressibility of the artJ and artP promoters18. Also, this result is consistent with recent microarray and qPCR experiments showing a significant arginine and ArgR-dependent down-regulation of both the artJ (about 50-fold) and artPIQM mRNA levels (about three to six-fold)17. In the case of TrpR, a total of five associations have been determined by DNA-binding experiments in vitro and mutational analyses in vivo7,19, all of which were also identified in our study (Fig. 1a and Supplementary Table 2). For instance, TrpR directly binds to the promoter regions of aroH and mtr involved in biosynthesis and transport of aromatic amino acids (Fig. 1b). Against the current genome annotation14, all of the ArgR- and TrpR-binding regions were observed within intergenic regions, i.e., promoter and promoter-like regions. The same preference was observed for Lrp-binding sites (Supplementary Table 1 and 2)3. DNA sequence motifs for each of the transcription factors were also re-derived based solely upon the ChIP binding regions and were in full agreement with previously described motifs (Supplementary Fig 2). Based on the fact that the increase in the intracellular arginine and tryptophan levels enhances ArgR and TrpR binding to its DNA targets20,21, the confirmation of previously discovered sequence motifs, and the full coverage of the known binding regions in our data we concluded that ArgR- and TrpR-binding regions identified here are bona fide binding sites.</p><p>Interestingly, as with gltBD, artPIQM, potFGHI, and mtr (Fig. 1b), we observed that Lrp directly binds to nine ArgR- and one TrpR-binding regions (Fig. 1c and Supplementary Fig. 1). For example, the direct binding of Lrp to the promoter region of the gltBD operon encoding glutamate synthase resulted in the activation of its transcription. In contrast, the role of ArgR-binding represents the negative regulation of the operon. Integrating binding regions and changes in transcript levels, the reciprocal mode3 in the transcriptional regulation of ArgR and Lrp was observed for cellular functions including putrescine transport (potFGHI), arginine transport (artPIQM), leucine response protein (lrp), arginine biosynthesis and utilization (argA and astCADBE), the formation of nucleoid (stpA), as well as glutamate biosynthesis and transport (gltBD and gltP). While Lrp activates the tryptophan transport (mtr), TrpR represses its transcription. In addition to confirming previously identified ArgR- and TrpR-binding regions, we found 47 and 3 novel ArgR- and TrpR-binding regions, which include the promoter region of potFGHI, encoding putrescine ABC transporter (Fig. 1b).</p><!><p>A regulon is defined as a group of genes whose transcription is controlled by a transcriptional regulator. The arginine regulon describing the genetic and regulatory organization of the genes involved in arginine biosynthesis in E. coli was used as an example in proposing the definition of the regulon in 196417,22. However, it has not been included in the definition of regulon whether each regulation is direct or indirect. So far, a total of 37, 56, and 10 genes have been characterized as members of regulons directly regulated by ArgR, Lrp, and TrpR, respectively15,16 Based upon regulatory codes described above, we significantly extended the size of these regulons and obtained 140, 283, and 15 target genes for each regulon. Since ArgR directly controls the transcription of lrp, the regulon size of each transcription factor can be described as ArgR (423) > Lrp (283) > TrpR (15). These regulons represent a hierarchical structure that can be used to identify the indirect effect of the TFs. For example, thrLABC operon involved in the threonine biosynthesis is directly activated by Lrp, either in the absence or presence of exogenous leucine. We observed that ArgR indirectly represses this operon in response to exogenous arginine; i.e., transcriptional repression without the direct binding of ArgR. It is therefore possible to partially elucidate the indirect regulation by ArgR based on the hierarchical regulatory network. ArgR represses Lrp leading to the indirect repression of the thrLABC operon. As shown in this example, integrated analysis of ChIP-chip and expression profiles allows us to fully understand the hierarchical TRN including the indirect regulatory effects.</p><p>Next, we classified the 438 target genes based on their functional annotation and found that most of these functions (∼82%) were assigned to amino acid metabolism and transport, as well as carbohydrate, nucleotide, and energy metabolism (Fig. 2). We are then able to show (Fig 3) that 19/20 amino acid biosynthetic pathways are directly or indirectly controlled by these three TF's. To do this we first mapped the directly regulated genes to known amino acid biosynthetic pathways and transport systems to determine their direct metabolic roles (Fig. 3a, b). ArgR directly regulates the transcription of all genes involved in the biosynthesis of arginine and histidine. It also regulates gltBD, aroB, aroK, and dapE involved in glutamate, aromatic amino acids, and lysine biosynthesis, respectively. The genes encoding the enzymes for the biosynthesis of branched chain amino acids are comprehensively regulated by Lrp, which also controls the transcription of gltBD and gdhA encoding glutamate synthase and glutamate dehydrogenase (glutamate biosynthesis), serC and serB encoding phosphoserine transaminase and phosphatase (serine biosynthesis), thrABC operon for aspartate kinase, homoserine kinase, and threonine synthase (threonine biosynthesis), argA for N-acetylglutamate synthase (arginine biosynthesis), and aroA for 3-phosphoshikimate-1-carboxyvinyltransferase (the chorismate formation for aromatic amino acid biosynthesis). TrpR regulates the transcription of genes involved in tryptophan biosynthetic pathway (trpLEDCBA operon), as well as aroH and aroL. In addition, it has been determined that TyrR directly regulates several genes in the aromatic amino acid biosynthesis (aroF, aroG, aroK, aroA, tyrA, and tyrB) in response to exogenous tyrosine15,16. Taken together, these four TFs control the biosynthesis of 12 amino acids. Furthermore, the biosynthesis of proline, glutamine, glycine, cysteine, and methionine is through branched biosynthetic pathways of glutamate, serine and aspartate (Fig. 3a). The remaining three amino acids (i.e., alanine, aspartate, and asparagine) are synthesized from glutamate as an amino donor (green dots in Fig. 3a). Therefore, biosynthetic pathways for all amino acids are directly or indirectly controlled by these four TFs.</p><p>Next, we classified the amino acids into ten groups based on the substrate specificity of each transport system, which are A (tyrosine, phenylalanine, tryptophan), B (arginine, histidine, lysine), C (glutamate, aspartate), D (leucine, isoleucine, valine), E (alanine, serine, glycine, threonine), F (proline), G (methionine), H (cysteine), I (asparagine), and J (glutamine) (Fig. 3b). As expected, the amino acids in the same group have a similar chemical structure, e.g. aromatic amino acids and branched chain amino acids in group A and group D, respectively. Transport systems for groups G-J are highly specific and were therefore classified into individual groups.</p><!><p>In general, genes for amino acid biosynthesis are repressed by each corresponding TF, whereas catabolic operons such as astCADBE, tdh-kbl, and gcvTHP are induced in response to the exogenous amino acids12,23. To determine the causal relationships between binding of a TF and the changes in RNA transcript levels of genes in the regulons, we integrated the binding regions of ArgR, TrpR, Lrp, and TyrR with the publicly available transcriptomic data (Fig. 4)3,17. We then determined activation or repression based upon the regulatory modes described previously3. Among genes in the ArgR regulon, about 18% genes were directly activated in response to the exogenous arginine, which include aroP and gltP genes encoding aromatic amino acids and glutamate/aspartate transporters. On the other hand, ArgR represses about 70% of its regulon members, including potFGHI, artJ, artPIQM, and hisJQMP encoding putrescine, arginine, lysine, ornithine, and histidine ABC transporters (Fig. 4). ArgR represses genes involved in the arginine and glutamate biosynthesis pathways, and unexpectedly, it directly down-regulates genes involved in histidine, aromatic amino acids, and lysine biosynthesis pathways. In case of amino acid utilization, ArgR induces astCADBE and puuEB operons encoding the metabolic pathways for arginine and putrescine, respectively. The remaining 12% of its regulon members had a direct association with ArgR without differential gene expression. Most of the remaining genes are currently annotated as genes of unknown function (Supplementary Table 1).</p><p>Gene expression profiles validated that Lrp directly regulates 283 genes. 45% and 55% of the Lrp-regulated genes were repressed and activated in response to the addition of the exogenous leucine3. As expected, Lrp controls the transport, biosynthetic and utilization pathways more globally than other transcription factors do. Lrp represses the transport systems for branched chain amino acids (brnQ, livKHMGF, and livJ), dipeptides (dppABCDF), and lipoproteins (lolCDE) but it activates a whole set of other transporters. Transporters that are activated by Lrp are aromatic amino acids (tyrP and mtr), arginine (artMQIP), glutamate (gltP), alanine, serine, glycine and threonine (cycA, tdcC, sdaC, and sstT), proline (proY), putrescine (potFGHI), dipeptide (dtpB), and oligopeptides (oppABCDF) (Fig. 4). In terms of amino acid biosynthetic pathways, Lrp represses all genes but the thrLABC operon for threonine biosynthesis. For amino acid utilization, Lrp activates all pathways for aromatic amino acids, arginine, aspartate, branched chain aromatic amino acids, alanine, glycine, serine, threonine, methionine, and putrescine. In case of the TrpR regulon, a total of 15 genes are directly regulated, of which 13 genes are repressed (Supplementary Table 2)16,24. TrpR also represses mtr encoding the tryptophan transporter as well as aroH, aroL, and trpABCDE involved in the tryptophan biosynthesis pathway. While TyrR activates the transport systems for aromatic amino acids (aroP, tyrP, and mtr), it represses tyrosine biosynthetic pathway comprising of aroG, aroL, aroF, tyrA, and tyrB (Fig. 4).</p><!><p>Based on the integrated analysis of TF-binding locations and gene expression profiles, we were able to connect transport, biosynthesis, and utilization of amino acids, and generate the connected bidirectional circuits (Fig. 5a). In the left feed-back circuit, TF-amino acid (TF-AA) complexes regulate the transcription of the transporters (T) and biosynthesis pathways (B), facilitating the influx of the amino acid molecules (AAin) from amino acids in the media (AAout) and precursors (AApre). In the right feed-forward circuit, TF-AA complexes control transcription of utilization genes (U) responsible for converting AAin into metabolites (M). Thus, the logical structures of the connected bidirectional circuit motifs can be described by a notation that uses three signs indicating repression (R) or activation (A) for each of T, B, and U (Fig. 5b). For example, the A-R-A circuit motif indicates that the transcription of transport, biosynthesis, and metabolic genes are activated, repressed, and activated, respectively, whereas the R-R-A circuit motif demonstrates that the transcription of both transport and biosynthesis are repressed and the metabolic genes are activated. The possible logical structures of the connected circuit motifs can be characterized depending on how the TF-AA complex activates or represses both influx (T and B) and efflux (U) in response to the exogenous amino acids. Based on the connected circuit motifs, we analyzed the behavior of logical structures of the transcription of transport, biosynthesis, and metabolic genes in responses to the exogenous arginine and leucine (Fig. 5b).</p><p>Surprisingly, there are only three influx-efflux combinations found between amino acid groups and TFs (Fig. 5c). For example, the connected circuit motif controlled by ArgR-arginine complex shows the R-R-A logical structure for group B amino acids (lysine, histidine, and arginine), whereas the logical structure of the motif is switched to A-R-R for glutamate and aspartate and A-R-A for other amino acids. On the other hand, the connected motif controlled by Lrp-leucine complex indicates the R-R-A logical structure for group D (valine, leucine, and isoleucine) and is again switched to A-R-R for glutamate and aspartate and A-R-A for other amino acids. For glutamate our primary observation was that the utilization was repressed given its role as a substrate for nine biosynthetic pathways (Fig. 3,4). However we acknowledge that the regulation is highly complex and not universally repressed. This logically follows from the critical and centralized role it plays throughout the metabolome25. Overall, we conclude that for two global transcription factors (ArgR and Lrp) in amino acid regulation, the connected circuit motif has an R-R-A logical structure for signaling molecules (i.e., arginine for ArgR and leucine for Lrp) and the A-R-A and A-R-R logical structures for other amino acids (Fig. 5c).</p><!><p>We reconstructed the regulons of ArgR, Lrp, and TrpR in E. coli individually and then integrated them to form the first genome-scale reconstruction of a stimulon. First, we set out to comprehensively establish the TF-binding regions on the E. coli genome experimentally and furthermore to elucidate any DNA sequence motif(s) correlated with the TF regulatory action. Second, we significantly extended the size of each regulon and obtained 140, 283, and 15 target genes for each regulon. Third, using changes in transcript levels on a genome-scale, we identified the regulatory modes for individual gene governed by each TF in responses to exogenous arginine, leucine, and tryptophan. The integrated analyses indicate that the functional assignment of the regulated genes is strongly enriched in the amino acid metabolism-related functions. As suggested previously, many of these genes are likely to be involved in the "feast or famine" adaptation for survival in nutrient-rich or depleted environments3,9. Fourth, we assigned the regulated target genes to three functional categories; transport, biosynthesis, and metabolism of amino acids. The classification allowed us to identify the connected circuit motif as a basic building block of the integrated network. Finally, we determined the regulatory logic of the connected circuit motif based on the causal relationships between the association of TFs and changes in transcript levels. These fall into two categories and thus allow for the differentiation between amino acids as signaling and nutrient molecules.</p><p>In general, transport systems along with biosynthetic and metabolic pathways convert external resources to basic building blocks to sustain life. The coordinated regulation of this primary process underlies expression of optimized metabolic states under different external conditions. Thus, we examined the logical structures of the metabolite-regulation connected circuit in response to the changes in the external amino acid availability in the reconstructed stimulon. We uncovered three unique logical structures that govern the amino acid biosynthesis and metabolism. The R-R-A logical structure was observed for signaling molecules whereas the A-R-A and A-R-R logical structures were determined for other amino acids severing as nutrient source (Fig. 5a, b). In principle, every metabolic pathway that includes transport, biosynthesis, and utilization functions could follow these logical structures. For example, the purine metabolism in E. coli contains a wide range of genes whose functions are transport (yieG), biosynthesis (cvpA-purF-ubiX, purHD, purMN, purT, purL, purEK, purC, hflD-purB, purA, and guaAB), utilization (apt), and a transcriptional regulator (purR). The metabolic functions of regulon members of PurR enriched into the purine metabolism and the connected circuit motif indicated the logical structures for signaling molecule in response to the exogenous purine26. It can be therefore envisioned that other potential metabolic pathways follow similar logical structures as determined for the amino acid metabolism in bacteria.</p><p>Bacterial cells import essential nutrients and inorganic ions such as galactose and iron due to the absence of the biosynthesis pathway. It is therefore of interest that the simple feedback circuit (SFL) motif, a connected circuit motif of transporter and utilization pathway by TF, is often observed in the regulatory circuits for these molecules27. If we assume the feedback circuit composed of influx and efflux combination, the logical structures of R-R-A, A-R-A, and A-R-R in the CFL motif can be reduced to R-A, A-A, and A-R, respectively. In E. coli, the galactose metabolic pathway is controlled by the galactose repressor (GalR) and galactose isorepressor (GalS), whereas iron homeostasis is controlled by the ferric uptake regulator (Fur)28,29 In the case of galactose metabolism, both GalR and GalS directly repress the transcription of galP encoding galactose permease. In a similar way, GalR partially represses the mglBAC operon encoding high-affinity, ABC-type transport system. When galactose is available in the medium, the DNA-binding by both GalR and GalS is inhibited, followed by the activation of those genes along with the genes for galactose utilization29. Therefore, the SFL motif exhibits the A-A logical structure, confirming the exogenous galactose as nutrient. In the iron homeostasis system in E. coli, intracellular iron binds to Fur, forming the active TF complex, which in turn activates the production of iron-using metabolic enzymes and also shuts down expression of iron transporters. Interestingly, the SFL motif for Fur regulon exhibits the R-A logical structure, similar to amino acids serving as signaling molecules described above. Therefore, we can conclude that iron acts as signaling molecule rather than nutrient.</p><p>In summary, we have described an integrative analysis of genome-scale data sets to comprehensively understand the basic principles governing a stimulon in the TRN of E. coli. The overarching regulatory principle elucidated enabled us to differentiate between metabolites as signaling and nutrient molecules. This important distinction between seemingly similar metabolites is non-intuitive and represents a triumph of genome-scale systems analysis. Similar analysis of other stimulons and large-scale regulatory networks may reveal that this regulatory principle is general. Thus, this approach to the analysis of regulation at the network level may reveal other fundamental non-obvious regulatory principles at work in genome-scale regulatory networks.</p><!><p>All strains used are E. coli K-12 MG1655 and its derivatives. The E. coli strains harboring ArgR-8myc, Lrp-8myc, and TrpR-8myc were generated as described previously13. Glycerol stock of ArgR-8myc strains were inoculated into W2 minimal medium containing 2 g/L glucose and 2g/L glutamine, and cultured overnight at 37 °C with constant agitation30. The cultures were inoculated into 50 mL of the fresh W2 minimal media in either the presence or absence of 1 g/L arginine and continued to culture at 37 °C with constant agitation to an appropriate cell density. E. coli strains harboring Lrp-8myc and TrpR-8myc were grown in glucose (2 g/L) minimal M9 medium supplemented with or without 20 mg/L tryptophan or 10 mM leucine, respectively3,31.</p><!><p>To identify ArgR-, Lrp-, and TrpR-binding regions in vivo, we isolated the DNA bound to ArgR protein from formaldehyde cross-linked E. coli cells harboring ArgR-8myc by chromatin immunoprecipitation with the specific antibodies that specifically recognizes myc tag (9E10, Santa Cruz Biotech)32. Cells were harvested from the exponential growth conditions in the presence or absence of exogenous arginine or tryptophan. The immunoprecipitated DNA (IP-DNA) and mock immunoprecipitated DNA (mock IP-DNA) were hybridized onto the high-resolution whole-genome tiling microarrays, which contained a total of 371,034 oligonucleotides with 50-bp tiles overlapping every 25-bp on both forward and reverse strands3,14. A ChIP-chip protocol previously described was used32,33 and microarray hybridization, wash, and scan were performed in accordance with manufacturer's instruction (Roche NimbleGen).</p><!><p>To monitor the enrichment of promoter regions, 1 μL immunoprecipitated DNA was used to carry out gene-specific qPCR3. The quantitative real-time PCR of each sample was performed in triplicate using iCycler™ (Bio-Rad Laboratories) and SYBR green mix (Qiagen). The real-time qPCR conditions were as follows: 25 μL SYBR mix (Qiagen), 1 μL of each primer (10 pM), 1 μL of immunoprecipitated or mock-immunoprecipitated DNA and 22 μL of ddH2O. All real-time qPCR reactions were done in triplicates. The samples were cycled to 94 °C for 15 s, 52 °C for 30 s and 72 °C for 30 s (total 40 cycles) on a LightCycler (Bio-Rad). The threshold cycle values were calculated automatically by the iCycler™ iQ optical system software (Bio-Rad Laboratories). Primer sequences used in this study are available on request.</p><!><p>To identify TF-binding regions, we used the peak finding algorithm built into the NimbleScan™ software. Processing of ChIP-chip data was performed in three steps: normalization, IP/mock-IP ratio computation (log base 2), and enriched region identification. The log2 ratios of each spot in the microarray were calculated from the raw signals obtained from both Cy5 and Cy3 channels, and then the values were scaled by Tukey bi-weight mean34. The log2 ratio of Cy5 (IP DNA) to Cy3 (mock-IP DNA) for each point was calculated from the scanned signals. Then, the bi-weight mean of this log2 ratio was subtracted from each point. Each log ratio dataset from duplicate samples was used to identify TF-binding region using the software (width of sliding window = 300 bp). Our approach to identify the TF-binding regions was to first determine binding locations from each data set and then combine the binding locations from at least five of six datasets to define a binding region using the recently developed MetaScope software14,35.</p><!><p>The ArgR-, Lrp-, and TrpR-binding motif analysis was completed using the MEME and FIMO tools from the MEME software suite36. We first determined the proper binding motif and then scanned the full genome for its presence. The elicitation of the motif was done using the MEME program on the set of sequences defined by the ArgR-, Lrp-, and TrpR-binding regions respectively37. Using default settings the previously determined ArgR38, Lrp3, and TrpR7 motif were recovered and then tailored to the correct size by setting the width parameter to 18-bp, 15-bp, and 8-bp respectively. We then used these motifs and the PSPM (position specific probability matrix) generated for each by MEME to rescan the entire genome with the FIMO program. The sequence logo generated from these sites.</p><!><p>All raw data files can be downloaded from http://systemsbiology.ucsd.edu/publications or Gene Expression Omnibus through accession numbers GSE26054.</p>

PubMed Author Manuscript

Challenges in Identifying the Dark Molecules of Life

Metabolomics is the study of the metabolome, the collection of small molecules in living organisms, cells, tissues, and biofluids. Technological advances in mass spectrometry, liquid- and gas-phase separations, nuclear magnetic resonance spectroscopy, and big data analytics have now made it possible to study metabolism at an omics or systems level. The significance of this burgeoning scientific field cannot be overstated: It impacts disciplines ranging from biomedicine to plant science. Despite these advances, the central bottleneck in metabolomics remains the identification of key metabolites that play a class-discriminant role. Because metabolites do not follow a molecular alphabet as proteins and nucleic acids do, their identification is much more time consuming, with a high failure rate. In this review, we critically discuss the state-of-the-art in metabolite identification with specific applications in metabolomics and how technologies such as mass spectrometry, ion mobility, chromatography, and nuclear magnetic resonance currently contribute to this challenging task.

challenges_in_identifying_the_dark_molecules_of_life

INTRODUCTION<!>CHROMATOGRAPHIC SEPARATIONS<!>MASS SPECTROMETRY<!>ION MOBILITY SPECTROMETRY<!>NUCLEAR MAGNETIC RESONANCE<!>ONE-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE<!>TWO-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE<!>NUCLEAR MAGNETIC RESONANCE DEREPLICATION<!>COMPOUND ISOLATION<!>DIFFERENTIAL ANALYSIS BY TWO-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY<!>THE SUMMIT APPROACH<!>CONCLUSIONS AND OUTLOOK

<p>Metabolomics is the newest omics field focused on the examination of metabolites in complex systems, with the goal of identifying pathway alterations that correlate with the onset and progression of specific processes, such as disease (1, 2). The metabolome is typically defined as the collection of small molecules in a given biological system that roughly falls under the ∼1,500-Da molecular weight window. Metabolites in the metabolome include endogenous molecules that are biosynthesized in primary metabolism, specialized secondary metabolite signaling molecules, lifestyle or environmental exposure molecules (the exposome), and molecules originating from the microbial community associated with the organism under study (the microbiome). Metabolomics can be either targeted to detect and quantify a set of known metabolites, or nontargeted, where the emphasis is to detect as many compounds as possible, even if associated with unknown chemical species. The metabolome, by definition, spans a vast diversity of chemistries, including lipids, sugars, amino acids, steroids, and a whole array of molecule types. These species exist in large concentration ranges that can be as high as millimolars and as low as femtomolars, suggesting that no single analytical method in existence today is able to detect, identify, and quantify all present species (3). Nuclear magnetic resonance (NMR) and liquid chromatography mass spectrometry (LC-MS) are the main platforms used for metabolomics, each with its own advantages and disadvantages in terms of sensitivity and peak capacity. These techniques are used individually in most studies but can also be combined to better yield metabolome coverage and enable more accurate metabolite identity annotation (4–6).</p><p>Despite rapid technological advances in both NMR and LC-MS, metabolite identification still remains the undisputed bottleneck in nontargeted metabolomics experiments. Only a small fraction, less than 2–10%, of the detected compounds in a nontargeted metabolomics study can be annotated reliably, with the chemical identity of most species detected remaining unknown (7). This vast chemical space has been described as the metabolome dark matter (8) and continues to be the focus of intense scientific research, in terms of new hardware and methods for improved separations and structural identification, but also new databases, libraries, and algorithms that can predict some of the analyte's molecular properties.</p><p>Much effort has been directed toward defining the confidence levels and minimum reporting standards associated with metabolite identification in any given metabolomics study. As early as 2007, Sumner et al. (9) proposed a four-level metabolite identification confidence scheme that divides metabolites into (a) identified compounds, (b) putatively annotated compounds, (c) putatively characterized compounds, and (d) unknown compounds. In level a, identified compounds are those that include at least two orthogonal molecular characterization methods that match a chemical standard [e.g., retention time (RT) and high-resolution mass spectrum, RT and NMR resonances, elemental formula and tandem MS (MS/MS) spectra, 1H and/or 13C NMR, and two-dimensional (2D) NMR spectra] (9). Generation of more than two pieces of such orthogonal metabolite characterization data is widely seen as providing good evidence in metabolite annotation, but it is not always possible due to a number of factors, including low signal-to-noise ratios, unavailability of chemical standards, and a lack of database coverage. Putatively annotated compounds are those that are identified by matching to literature or database information, but for which no chemical standard can be obtained for comparison purposes. Putatively characterized compounds are those for which only similarities to a given family of compounds can be established, but further information is not available [e.g., a glycerophospholipid characterized by the presence of an m/z 184 fragment in MS/MS experiments corresponding to the phosphocholine headgroup; or the neutral loss of 141 Da corresponding to the phosphatidylethanolamine headgroup (10, 11)].</p><p>Despite its usefulness, this four-level scheme lacks granularity and detail, so here we propose to further expand it through a score card (Table 1) that further refines the assignment of metabolite identification confidence in nontargeted metabolomics experiments using a points system. This system builds on advances in metabolomics instrumentation and algorithms described below, such as RT prediction, use of ion mobility (IM) collision cross sections (CCS), and prediction of MS/MS fragmentation and NMR spectra.</p><p>An example of how to apply the scoring approach described in Table 1 would be as follows: If an identity is proposed for an unknown metabolite by (a) matching the [M+H]+ and [M+Na]+ monoisotopic adduct ions (5 points) with an average 2.5-ppm error (10 points), followed by (b) an MS2 (10 points) and MS3 (5 points) match to the mzCloud database, (c) its experimentally measured CCS matched within tolerance to a database value (10 points), and (d) its 1H NMR spectrum matched to a database entry (15 points), a confidence score of (10 + 5) + (10 + 5) + (10) + (15) = 55 points could be assigned. Further improving identification confidence would require (a) increasing mass accuracy to 1–2 ppm for more than one adduct ion (5 points) while employing ultrahigh-resolution MS experiments to examine isotopic fine structure (20 points); (b) manually interpreting (5 points) the database-matched product ion spectrum to verify that observed fragment ions are compatible with known fragmentation pathway; (c) matching chromatographic RT to a chemical standard (10 points); (d) and performing 2D instead of one-dimensional (1D) NMR experiments (20 points). This would yield a more confident score of (5 + 20) + (10 + 10 + 5) + (10) + (10) + (20) = 90 points in total. By applying these more specific metabolite identification techniques to unknown species and combining the information they provide, a better confidence score is obtained. Strengths and limitations of each of these techniques, as applied to metabolomics, are discussed in the sections below.</p><!><p>Historically, chromatographic separations coupled to MS have offered one of the most versatile platforms for complex sample analysis and metabolite identification in nontargeted metabolomics studies (12). LC-MS is by far the most popular MS-based hyphenated technique in metabolomics due to its sensitivity, selectivity, reproducibility, and versatility for analyzing small molecules with a wide range of different physicochemical properties in biological samples (13, 14). Gas chromatography (GC)-MS, a predecessor to LC-MS, continues to be the obvious choice for fingerprinting volatile and low molecular weight compounds (15, 16). Analysis of less volatile compounds by GC-MS, however, requires additional preparation steps such as lyophilization, followed by chemical derivatization to increase thermal stability and volatility prior to analysis (17). To a lesser extent, capillary electrophoresis has also been utilized in metabolomics studies involving highly charged and polar ionogenic metabolites in small-volume biological samples (13, 18). Capillary electrophoresis is attractive in the sense that it separates compounds based on their intrinsic electrophoretic mobility, a parameter that reflects the charge and size of the analyte (18).</p><p>LC-MS offers the widest coverage and most efficient separation of complex metabolomes, with much enhanced peak capacity compared to MS alone (13). LC and MS are coupled by means of soft ionization techniques; electrospray ionization (ESI) (19–21) is the most common. ESI generally produces protonated or deprotonated gas-phase ions that can be used for accurate predictions of metabolite elemental formulae (22). In addition to in-source fragment ions, various adduct ions and multiply charged species that complicate data interpretation are also produced, leading to higher false-positive rates in metabolite identification (23). GC, on the other hand, is coupled to MS by means of electron ionization sources operated under vacuum and standardized at 70 eV, causing predictable fragmentation and rearrangement reactions that lead to highly reproducible mass spectra for comparison with libraries (16).</p><p>Reverse phase (RP) and hydrophilic interaction chromatography (HILIC) are by far the most commonly used chromatographic methods for LC-MS-based metabolomics (13, 24). In RPLC-MS, analytes are typically eluted by an aqueous-based mobile phase under a gradient of increasing organic solvent content from a hydrophobic stationary phase. In HILIC, analytes are eluted in order of increasing hydrophilicity from a hydrophilic stationary phase as the polarity of the mobile phase is increased by increasing the aqueous content.</p><p>Traditionally, chromatographic identification in metabolomics by either RP or HILIC approaches is best performed by RT matching with authentic chemical standards. However, not all metabolites are commercially available, and purchasing authentic standards for all possible metabolite candidates in a nontargeted study is incredibly costly and inefficient. Indeed, there are fewer reference standards available than the number of peaks that is detected in LC-MS experiments of biological samples (25–27). Although chemical synthesis can be attempted for specific cases of high-value unknowns, this degree of effort is seldom warranted.</p><p>GC-MS capillary columns are highly reproducible, which facilitates the compilation of standardized retention indices (28, 29) in libraries or public databases for compound identification (16, 30). In particular, Fiehn and collaborators (30) have greatly contributed to the metabolomics field by building FiehnLib libraries comprising mass spectra and retention indices from quadrupole and time-of-flight (ToF) GC-MS data. In contrast, it is difficult to catalog RT information from LC experiments in libraries due to the lack of procedural standardization and instrumentation involved, with varying LC pumps and injectors, columns, run temperatures, solvent gradients, mobile phase pH, and flow rates. In addition, column aging, temperature changes, MS detector drift, and other analytical factors may further compound RT variance in nontargeted LC-MS metabolomics experiments. Matrix effects caused by differences in biofluids matrix compositions can also lead to RT shifts. Spiking experiments with chemical standards can mitigate these effects (31), but in cases where chemical standards are not available, RT window prediction can complement metabolite identification efforts.</p><p>Different attempts have been reported in the literature (31–36) to integrate RT window prediction into the metabolite annotation process, most of which rely on quantitative structure–retention relationship (QSRR) modeling (37). The generalization of such predictive models depends on the application domain investigated, because using overly restricted domains for model building also leads to poor prediction in independent test sets (38). Molecular descriptors (39–42) also play a significant role in defining such application domains (38, 43, 44), as well as in defining the multiple algorithmic options and combinations of adjustable parameters involved in QSRR model building (42, 45). In general, RT factors (RF in the equation below, where T0 represents the chromatographic column void time) are calculated to allow comparison between different chromatographic systems (32, 36). Other studies utilize retention indices (28, 29), which are measures of relative RT based on reference compounds that elute immediately prior to and immediately following the analyte of interest (30, 45–48). RF=RT−T0T0. Cao et al. (31), for example, conducted QSRR modeling based on theoretical molecular descriptors and experimental RTs of 93 authentic compounds analyzed with HILIC LC-MS. A predictive QSRR model based on a random forest algorithm achieved high predictive accuracy, with mean and median absolute errors of 0.52 min and 0.34 min (5.1% and 3.2%), respectively. These authors applied this model to annotate features (RT, m/z) of perennial ryegrass (Lolium perenne) samples, significantly reducing the number of false-positive metabolite annotations that would be obtained if only accurate masses were considered. Predicted RTs were validated in this study using either authentic compounds or ion fragmentation patterns (31). Among the molecular descriptors utilized to build QSRR models, the partition coefficient (XLogP) was found to be the most relevant predictor in agreement with the HILIC model previously reported by Creek et al. (32). These authors converted the RT of 120 metabolite standards to retention factors that were input into a multiple linear regression model. The optimal QSRR model used six physicochemical variables and showed good predictive ability (cross-validated R2 = 0.82 and mean square error = 0.14) for RTs of metabolites with MW <400. Availability of predicted RTs translated into the removal of 40% of the false metabolite identifications based on accurate mass alone (32). The model was evaluated to putatively identify 690 metabolites in extracts of the protozoan parasite Trypanosoma brucei. Model limitations were associated with the applicability to only low-MW metabolites, since larger compounds were poorly predicted, most likely due to errors associated with predicted log D, the octanol-water partition coefficient calculated at a pH of 3.5. Based on their QSRR model, the authors produced a template file that allows users to calculate predicted RTs for a database of metabolites based on experimental RTs measured for standards.</p><p>QSRR modeling has also been applied to RPLC data. Bruderer et al. (34) evaluated RT prediction for a metabolomics database with 532 human metabolites. The authors built a model with only 16 compounds using logD2 (calculated based on log P and pKa) and the molecular volume as molecular descriptors. The developed model was evaluated for two different RP C18 columns and two pH conditions (pH = 3.0 and 8.0 for positive and negative ESI modes, respectively), achieving good prediction accuracy for a time window below 4 min (34). In addition, RT prediction combined with the data-independent acquisition (DIA) method known as sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) aided in annotating isobaric metabolites found in human urine, which increased identification confidence and reduced the number of false positives (34).</p><p>A different RT prediction model for RPLC was built by Wolfer et al. (44) based on 442 authentic standards, including fatty acids, nucleosides, sterols, sphingolipids, lipids, vitamins, cofactors, amino acids, aromatic biogenic acids, carbohydrates, catechols, neurotransmitters, and other metabolites to cover a large variety of polarity and chemical topology. The authors combined random forest and support vector regression models with 97 computationally determined descriptors derived from chemical structures. The model was further tested on an external validation set of 111 known compounds, predicting RTs with an average 13% error that reduced by 77% the number of incorrect candidates. The models were made available through a user-friendly interface to be integrated into existing workflows.</p><p>Identification of isobaric lipids is challenging even with high-resolution mass spectrometers. Aicheler et al. (33) developed an RT prediction model based on machine learning approaches that enabled the improved assignment of lipid structures and automated annotation of lipidomics data obtained by RPLC–high-resolution MS. A support vector regression model was built based on 201 lipids originating from mouse adipose tissue, including molecular structural features. The cross-validated model achieved good correlation (R = 0.989) between predicted and experimental RT in test samples (Figure 1) and allowed filtering out of more than half of the potential identifications, while retaining 95% of the correct candidates.</p><p>Most QSRR approaches have been successful at predicting RT with good accuracy. The main limitations of these strategies, however, are related to the lack of large and sufficiently diverse metabolite training sets for building predictive models that could cover the thousands of metabolites found in biological samples. In addition, some published models have lacked comprehensive external validation, thus risking overfitting (36). Further drawbacks are associated with the type of stationary phase material used (36, 43) that determines, together with the sample preparation protocol, the metabolome fraction that can be effectively resolved.</p><p>Alternative efforts have involved the development of tools that allow crowd sourcing of RT information across laboratories and chromatographic systems such as retention projection (49) and direct mapping (50) for GC-MS and LC-MS systems, respectively. The retention projection methodology for GC-MS data, for example, was shown to be threefold more accurate than retention indexing across five different laboratories under identical experimental conditions. This made it easier to account for unintentional differences between the various GC systems, such as temperature calibration errors, flow rate nonidealities, or variance in column dimensions (49). RT mapping for LC-MS data, developed under the name of PredRet, was done pairwise between LC systems using the same chromatographic method and provided higher accuracy than QSRR (50). However, QSRR models are able to a priori predict the RT of any given metabolite structure, whereas PredRet can only predict RT for compounds already in the database, i.e., those for which the RT has been previously determined in a comparable chromatographic system (50).</p><!><p>MS/MS experiments select a precursor ion for activation and fragmentation due to ion-neutral collisions with the gas filling the collision cell. The combination of chromatographic separations and MS/MS is one of the most powerful approaches to metabolite and natural product identification (51). In nontargeted metabolomics, these tandem mass spectra can be obtained in either a data-dependent acquisition (DDA) or DIA fashion (52). DIA approaches, such as MSE, benefit from larger precursor ion coverage (53), but they suffer from difficulties in matching precursor ions to product ions when chromatographic overlap is substantial. DDA, on the other hand, preferentially targets the most abundant precursor ions, resulting in poorer sampling of lower abundant precursor species. Repeated analysis can somewhat improve precursor ion coverage, but this improvement is typically only marginal due to redundant precursor ion sampling. Advanced precursor ion selection algorithms used in proteomics (54) could also benefit metabolomic studies. Broeckling et al. (55), for example, have proposed an alternative method for enabling the comprehensive MS/MS coverage of complex samples via data set–dependent acquisition. With this approach, real-time feedback between data processing and data acquisition was achieved using a combination of R, ProteoWizard, XCMS, and WRENS software, yielding a threefold improvement in the number of peaks mapped by MS/MS. Elaborate DDA approaches that combine with higher-energy collision dissociation have also been reported (56).</p><p>Targeted metabolomics approaches are different from nontargeted approaches in the sense that they typically rely on chemical standards with known structures, using triple quadrupole mass spectrometers in multiple reaction monitoring (MRM) mode, or hybrid quadrupole ToF/Orbitrap analyzers in SWATH or parallel reaction monitoring modes (57). Approaches that bridge the targeted and nontargeted methods, however, have been reported in the literature. Chen et al. (58) recently developed a hybrid method utilizing DIA for targeted quantitative metabolomics experiments. In this method, a sequentially stepped targeted MS/MS scan is used for improving coverage. In this type of scan, multiple product ion scans are acquired for all ions in the examined m/z ranges, selecting them as the chosen precursor ions. These scans are then followed by scheduled MRM scans for numerous ion pairs that are used for quantitation. Ferreira et al. (59) have also reported an approach that bridges classical targeted and nontargeted methods. This approach, named MRM profiling, makes use of numerous product-precursor ion transitions that are developed in a supervised fashion. By combining neutral loss and precursor ion scans, a list of more than 1,000 transitions is built from a pooled sample. Relative abundances of all product ions targeted in these MRM transitions are then measured for all samples and used to build univariate and multivariate diagnostic models for a specific condition, such as polycystic ovarian syndrome (60).</p><p>Precursor ion coselection is an underappreciated issue that commonly hinders metabolite identification in LC-MS/MS metabolomics, particularly with lipids. Most high-resolution mass spectrometers use a low-resolution quadrupole mass analyzer for mass selection prior to MS2 fragmentation. The selection window for the precursor ions is typically limited to 0.5–3 Da. When a high number of isobaric species chromatographically coelute, these are inevitably coselected and cofragmented, yielding a product ion spectrum that is a composite of all product ions yielded from the initial precursors in that window. One way to mitigate these interferences is to perform collision-induced dissociation (CID) experiments a posteriori from IM separations. Damen et al. (61) recently reported an ultraperformance LC (UPLC) method for the separation of closely related lipid molecular species using a stationary phase incorporating charged surface hybrid technology. The chromatographic method showed excellent RT reproducibility [intraassay relative standard deviation <0.385% and <0.451% for 20- and 10-min gradients, respectively (N = 5)]. The UPLC system was coupled to a hybrid quadrupole ToF mass spectrometer, equipped with a traveling wave ion mobility (TWIM) cell. Despite the use of a quadrupole mass analyzer for precursor ion selection, separations in the TWIM cell followed by transfer cell ion activation enabled the acquisition of cleaner low- and high-energy DIA MS/MS spectra that were more useful in terms of metabolite identification. Another approach to prevent precursor ion coselection is through the use of different variations of stored waveform inverse Fourier transform (SWIFT) ion excitation in Fourier transform ion cyclotron resonance (FT-ICR) (62). As early as 1995, O'Connor & McLafferty (63) reported on high resolution ion isolation using a capacitively coupled FT-ICR open cell. In these experiments, isotopic peaks of ubiquitin (8.6 kDa) and carbonic anhydrase (29 kDa) were isolated by SWIFT with an order of magnitude higher isolation power than previously reported in the literature. Another approach yielding high-resolution ion selection in FT-ICR MS is known as correlated harmonic excitation fields (64). With this approach, de Koning et al. (64) were able to achieve a resolution of ∼50,000 in the separation of deuterated toluene isotopes. Routine implementation of high-resolution precursor ion selection approaches such as those described above in a metabolomics context could significantly improve the odds of correct unknown identification via more selective CID experiments and higher confidence MS/MS database matching.</p><p>Although MS1 information coupled with local network enhancement analysis can be used for tentative metabolite annotation (65), high-quality tandem mass spectral libraries are becoming essential for more confident identification (66). A growing number of these libraries are currently available, including the Human Metabolome Database (HMDB) (67, 68), METLIN (69, 70), MassBank of North America (MoNA; http://mona.ftehnlab.ucdavis.edu/), LipidBlast (71), mzCloud (72), LIPID MAPS Structure Database (64, 73), Manchester Metabolomics Database (MMD) (23), and many others (74, 75). More general databases, such as PubChem (65, 76) and ChemSpider (65, 77), can also be incredibly useful for MS-based identification of unknowns. However, metabolite identification through mass spectral library searches is far from an automated task, and the analyst is typically forced to manually search each individual database and manually curate the obtained matches (if any) to ensure that differences in the type of mass spectrometer used and the collision energy employed are considered. Along these lines, Stein and coworkers (78) have proposed an approach for creating high-quality ESI tandem mass spectral libraries. The procedure involved the acquisition of tandem mass spectra for all major precursor ions in a direct infusion experiment. This was followed by assigning spectra to clusters and creating a consensus spectrum. Filtering through intensity-based constrains for cluster membership was then applied, together with peak testing, noise reduction, and examination by an experienced human evaluator, yielding a library of >9,000 compounds with ∼230,000 spectra.</p><p>When MS/MS database matches are not found, prediction of such spectra in silico can be advantageous to provide an added level of confidence to the proposed metabolite identity. To this purpose, Wolf and coworkers (79, 80) described the popular package named MetFrag, where a candidate list of metabolite identities is first obtained by searches of precursor ion masses, followed by ranking based on the agreement between in silico fragmentation spectra and experimental data. Initial evaluation of MetFrag showed that it was able to rank most of the correct compounds in the top three candidates produced by KEGG queries, producing better results than commercial software. In related work, Duhrkop et al. (81) described an approach named CFM-ID that combines computation and comparison of elemental formulae fragmentation trees with machine learning techniques. They reported a 2.5-fold increase in correct identifications compared with state-of-the-art methods when searching PubChem. Li's group (82) described a web interface (https://www.MyCompoundID.org) for metabolite identification based on both MS and MS/MS data for compounds in the HMDB and metabolites derived from these through in silico metabolic reactions. Fragmentation prediction for specific metabolite families, such as lipids, has certainly benefited subfields such as lipidomics (83–85), where chemical structures follow combinatorial rules. Prediction of electron impact spectra has also been achieved with a significant degree of success (86) based on an approach previously used for ESI data (87).</p><p>Combined prediction of various molecular properties, including retention indices, energy required to fragment 50% of a selected precursor ion, IM drift time, and CID spectrum, has been proposed by Grant and coworkers (88) through a package known as MolFind, but follow-up validation of such an approach through comparison with large experimental data sets has not been reported. Similar motivation led Hu et al. (89) to attempt the simultaneous prediction of both RTs and fragmentation patterns with the goal of identifying micropollutants.</p><!><p>In order to increase confidence in the identification of prioritized features after MS analysis, additional structural techniques, including infrared spectroscopy, NMR spectroscopy, and more recently, IM spectrometry (IMS), are often incorporated into nontargeted studies (90, 91) and are taken into account in our proposed identity confidence scoring scheme (Table 1). IMS is a gas-phase separation technique in which analytes are separated based on their rotationally averaged surface area or CCS. Briefly, IMS separations are conducted as analyte migration through an inert buffer gas under the influence of an applied electric field. Although specifics of gas composition, pressure, and applied field strength vary depending on the instrument configuration, interactions between these forces drive ion motion and separation in the IMS cell (92, 93). Because IMS distinguishes analytes based on their structural size in the gas phase, it is orthogonal to MS to a great extent, and it provides capabilities such as isomeric separations that are not possible with only the mass dimension (94, 95). Furthermore, IMS measurements occur on a millisecond timescale, which is readily nested into traditional LC and GC-MS workflows (96). Descriptions of various IMS platforms and configurations are available in several in-depth review articles (74, 97, 98) and are beyond the scope of this article.</p><p>Although growth in MS databases has steadily continued for several decades, CCS databases are still in their infancy. Since the commercialization of IMS-MS instrumentation in 2006 (73, 76), several studies have been devoted to collecting CCS values on a larger scale. These efforts have made CCS values available to the public, while also creating databases useful for nontargeted metabolomics experiments (77, 91, 99–101). Despite these advances, obtaining reproducible CCS values across various instrument platforms still remains challenging owing to several key factors that include variations in instrument design, experimental parameters, and calibration protocols. For example, only drift tube and differential mobility instruments can empirically measure CCS from first principles, meaning that no calibrants are needed for the values they produce. IMS platforms based on TWIM spectroscopy and trapped ion mobility spectroscopy (TIMS) instruments, however, must be calibrated with ions of known mobility before reliable CCS values can be generated (102–104). Also, the specific manner in which each instrument is calibrated can greatly influence the reproducibility of the reported CCS values. Several studies have previously described the challenges of choosing the correct calibrant ions for traveling wave devices with similar structural characteristics as the species being studied (105, 106). For example, Gelb et al. (107) observed that improper calibration of TWIMS with calibrant ions of a different chemical class and charge state could produce CCS measurements with an error in excess of 4% compared to using proper calibration protocols. Even after proper calibrant ions are chosen for a specific instrument and experiment, the optimal mathematical calibration procedure remains a topic of debate for several platforms (108, 109). As IMS-MS instrumentation continues to improve in terms of generating reliable and reproducible CCS values, slight variations in calibration procedures become increasingly critical. For example, a recent interlaboratory study from Paglia et al. (77) characterized TWIMS reproducibility to typically less than 3% relative standard deviation, suggesting that errors in CCS calibration could lead to errors in calculated CCS that are larger than the instrument's own reproducibility. In a similar fashion, a recent interlaboratory study by Stow et al. (100) demonstrated that new advancements in drift tube technology resulted in reproducibility of typically less than 1% relative standard deviation for CCS measurements between laboratories because no calibration was required.</p><p>Despite challenges in calibration procedures, once a reproducible CCS value is measured for a given analyte, matching a molecular feature to a library entry is a straightforward process when the analyte m/z and CCS are compared to entries based on analytical standards. If there is a structural match within a certain mass error and CCS tolerance, the molecule is considered a match. However, if a database search generates no matches, further work is needed. CCS databases are typically generated based solely on commercially available analytical standards. Unfortunately, the availability of such standards limits the size of databases, as many compounds either cannot be isolated or they are simply too expensive to obtain. For these molecules, CCS matching may only be feasible against predicted values generated by computational methods (110–112). Such computational approaches have shown promise in generating CCS values, usually with <2% agreement with experimental values (113). As more CCS values are published for the various IMS platforms, it is expected that predicted values will be able to fill the gap of unavailable standards for the identification of unknown metabolites.</p><p>IMS-MS also allows identification of metabolites through monitoring the placement of the analyte's observed CCS as a function of the measured m/z. For example, lipid molecules are typically characterized by their headgroup, length of the fatty acid tails, and the number of double bonds (114). These characteristics make the three-dimensional gas-phase structure of lipids quite rigid and, as a consequence, lipids have, on average, larger CCS values than peptides, carbohydrates, and nucleotides with similar masses, as shown in Figure 2 (99, 115). Several studies have noted these resulting mass/CCS ratios and have generated analytical trend lines describing the relationship between analyte mass and CCS values for a wide range of biomolecules (116, 117). In fact, if both the mass and CCS of an unknown analyte are accurately known, it may be possible to classify an unknown metabolite into a tentative biological class, after including other related factors such as mass defect and isotope ratio pattern. It is also worth noting that while there is significant overlap for several zones of the illustrated mass/CCS relationships in Figure 2, further advances in IMS resolving power and selectivity continue to increase the likelihood of producing better-resolved features in the CCS dimension.</p><!><p>NMR spectroscopy exploits the quantum mechanical interactions of atomic nuclei with an external magnetic field. These interactions arise because some nuclei have an intrinsic type of angular momentum called spin. In metabolomics applications, the most common nucleus to measure is 1H, with some applications using 13C (e.g., 118–121), 31P (e.g., 122), or other nuclei. All of these biological nuclei are spin 1/2, which means that when they are in an external magnetic field, they can adopt two energy levels separated by the resonance frequency (ω0) that is proportional to the magnetic field strength (B0), according to the equation ω0=−γB0, where γ is the gyromagnetic ratio, which is a physical constant for a specific nucleus. Thus, 1H resonates at 600 MHz in a 14.1-Tesla (T) magnet and 900 MHz in a 21.1-T magnet. Because of the quantum mechanical underpinnings of NMR spectroscopy, it provides atom-specific information, which makes it the method of choice for the structural characterization of unknown molecules. It can be quantitative, with the integrated value of each NMR proportional to the number of nuclei and thus the concentration of the molecule. NMR spectroscopy is also nondestructive and highly reproducible because the sample never comes into direct contact with the instrument. But all of these significant strengths come at a cost of overall sensitivity. Because it is a resonance phenomenon, NMR spectroscopy has a fundamental sensitivity that is limited by the Boltzmann equation NupNdown=e−ΔEkBT, where Nup and Ndown represent the number of nuclear spins in the upper and lower energy levels, ΔE is the energy gap between levels, kB is the Boltzmann constant, and T is the absolute temperature. For 1H at 600 MHz and room temperature, if the number of spins in the upper energy state is 1 million, there are only 1 million + 96 spins in the lower state, so only a small fraction of the sample contributes to the NMR signal. But the low energies associated with NMR spectroscopy also allow its noninvasive application in living systems through magnetic resonance imaging.</p><!><p>NMR metabolomics applications are typically done without chromatography or significant sample extraction steps. Therefore, the measured signals represent a complex mixture of metabolites in a sample. With modern spectrometers and probes, the practical lower limit of detection is about 10 µM in a 550-µL sample. The most common experiment in NMR metabolomics is a 1D 1H spectrum, which can have hundreds to sometimes thousands of overlapping peaks. These can be matched to databases with standard spectra of known metabolites. The most important public databases with NMR spectral libraries are the Biological Magnetic Resonance Data Bank (BMRB) (123) and HMDB (124). The primary difficulty in using these databases is that 1D NMR spectra can be heavily overlapped and, thus, there are almost always uncertainties in peak assignment using exclusively 1D methods.</p><!><p>Two-dimensional NMR offers significant advantages over 1D NMR. It not only reduces resonance overlap by spreading the signal into a second dimension, but it also can provide extra information about chemical bonding between nuclei. The drawback of 2D NMR is the length of time required for each experiment, so these are typically only used for pooled samples rather than every sample in a study. However, new approaches are improving the speed of 2D methods (125). One of the most useful 2D experiments in metabolomics is heteronuclear single quantum correlation (HSQC). The 2D HSQC experiment correlates 1H with 13C (or less common in metabolomics, 15N) that are covalently bonded. Each pair of bonded nuclei give a single peak in a 2D HSQC, and this provides a useful fingerprint of a mixture. Edison & Schroeder (126) wrote a more complete description of 2D NMR experiments and their interpretation.</p><!><p>Before the difficult step of unknown compound identification, it is important to first recognize and assign peaks that are known and in databases. This is called dereplication. There are several approaches to this, as both freely available and commercial packages. One of the most popular commercial software is Chenomx, which allows users to fit a library of reference standards to 1D 1H experimental metabolomics data. This is an excellent visualization tool but suffers from the problems mentioned above about 1D NMR and peak overlap. At least one study reports inconsistent results with Chenomx using the same data set and multiple analysts (127). Bruker Corporation offers a spectral database that includes a wide range of pH values for many common metabolites, and this can be used with both 1D and 2D data in its Assure software. The 2D data add confidence in annotation, but the cost of this solution may be beyond the budget of many labs.</p><p>Brüschweiler's laboratory has developed a suite of free web-based tools called COLMAR (complex mixture analysis by NMR; http://spin.ccic.ohio-state.edu/index.php/colmar). COLMAR has several functionalities that are useful for metabolomics. One of the simplest ways to use COLMAR is the 1H-13C-HSQC query, which takes a peak list from an HSQC spectrum and finds database matches using a combination of BMRB, HMDB, and internally curated data. HSQC matches are useful but can also be prone to misinterpretation, because they only report on a 1H–13C pair and do not include correlations between peaks. COLMAR adds to the HSQC query by allowing the addition of TOCSY (total correlation spectroscopy) or HSQC-TOCSY. The TOCSY experiments provide correlations between coupled 1H spins. Adding both HSQC and TOCSY or HSQC-TOCSY data significantly improves the confidence of dereplication of known metabolites in a mixture.</p><!><p>The most straightforward approach to NMR-based compound identification is to use a natural products-like strategy that involves purification of the unknown molecule. The purification steps are typically activity guided, essentially using the desired activity as a detector for the compound of interest. For example, the identification of the mating pheromone for Caenorhabditis elegans involved a series of low-resolution fractionations followed by assays of male-specific attraction (128). Once a fraction is sufficiently pure, both 2D NMR (and MS) data can be obtained and analyzed. There are several advantages of this strategy. First, the focus is on the compound of interest (i.e., the one with the desired biological activity or discriminating power). Second, the limits of detection are defined by the assay and not the NMR spectrometer. This is very important, because unknown molecules at concentrations lower than NMR detection limits can be concentrated and identified if sufficient material is available for the bioassay (129). Finally, the pure (or semipure) compound provides a straightforward way to relate NMR and MS data, which is important for a more reliable identification (see Table 1).</p><p>Although it is common to use some type of LC system for fractionation, it is not always best to start with analytical chromatography. It is often simpler to start with simpler solid-phase extraction (SPE) steps, which can be done on a larger scale than LC. Orthogonal SPE (e.g., C18 followed by ion exchange) can be quite effective at quickly simplifying mixtures, even with just a few fractions from each step. If necessary, the crude material from SPE fractionation can then be purified further using LC. This approach was used for the isolation of C. elegans (128) and Panagrellus redivivus (130) mating pheromones (and many other activity-guided fractionation studies).</p><p>Alternatively, the assay can be NMR or MS spectra in order to isolate an unknown peak of interest (131). In most metabolomics applications, important NMR or MS features are first determined from statistical analysis. If these features do not match databases, the same purification steps described for activity-guided fraction can be used, and fractions can be screened for the feature(s) of interest. Chemical fractionation is time consuming and not always possible without sample degradation (132).</p><!><p>Frank Schroeder's lab has developed a technique called differential analysis by 2D NMR spectroscopy (DANS) (133). A review of DANS and other approaches to NMR mixture analysis provides an additional overview (134). Briefly, DANS compares unfractionated high-resolution 2D NMR data sets (COSY) of two different genetic strains of organism, e.g., wild-type and a mutant of interest. In DANS, the data are manually overlaid, subtracted, and adjusted so that peaks common to both spectra will cancel, while peaks unique to one of the genotypes are retained. The DANS strategy does not include integration of MS data, but it does provide a very helpful overview of the major metabolic differences between the two genotypes and also then yields 2D NMR data that can be used to either partially or fully determine the structure of unknowns.</p><!><p>The Brüschweiler lab recently developed a powerful approach to link NMR and high-resolution MS data in metabolomics called structures of unknown metabolomic mixture components by MS/NMR (SUMMIT) (135). SUMMIT links MS and NMR data through computation (Figure 3). Chemical purification can be used but is not a requirement. The general idea is that both high-resolution MS and NMR data are collected on the same sample. The high-resolution MS can be obtained through either chromatography or direct infusion, e.g., with an FT-ICR instrument (136). Starting with a feature of interest from the MS data, it is possible to obtain a molecular formula directly from the intact precursor ion provided there is sufficiently high mass resolution.</p><p>Once a reliable molecular formula is known, it is possible to enumerate all structures that are consistent with that formula, e.g., by searching the ChemSpider database (http://www.chemspider.com). The difficulty with this step is that the number of possible molecules grows substantially with molecular weight. For example, C4H6O5 (e.g., maleic acid) yields 35 structures from ChemSpider. In contrast, ChemSpider yields 1,023 results for C21H30O3. Therefore, it is desirable for the number of possible structures to be reduced by other data, e.g., through association with a specific metabolic pathway through a metabolite-genome-wide association study.</p><p>The next step is to calculate the NMR chemical shifts of all possible structures from MS data. Calculations of NMR chemical shifts have become quite reliable with high-level ab initio or density functional quantum mechanical calculations (137). However, even semiempirical-based methods can provide reasonable results (135). The computed NMR chemical shifts are then compared against experimental NMR data for the closest match. This conceptually simple step can be complicated when the NMR data are from a complex and unfractionated metabolomics mixture, so a modification of this strategy would be to fractionate the NMR sample using similar chromatography to the LC-MS. By doing this step, the overall approach becomes similar to natural products fractionation described above with the additional step of using computational chemistry to determine the best structure rather than through traditional analysis.</p><!><p>Despite the limitations associated with each of the strategies described here, all of them have significantly contributed to addressing the most challenging problem in nontargeted metabolomics studies, which is to know the unknowns (27, 69, 138). Integration of the information produced by such advanced assays, however, is still largely lacking, thus preventing identification of metabolites in a high-throughput fashion. Expected advances in metabolomics informatics pipelines are expected to propel the field to a more mature stage, in a similar fashion to what has occurred in other omics fields such as genomics, transcriptomics and proteomics.</p>

PubMed Author Manuscript

mitochondrial pathway for biosynthesis of lipid mediators

The central role of mitochondria in metabolic pathways and in cell death mechanisms requires sophisticated signaling systems. Essential in this signaling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin, are oxidized by the intermembrane space hemoprotein, cytochrome c. We show that an assortment of oxygenated cardiolipin species undergoes phospholipase A2-catalyzed hydrolysis thus generating multiple oxygenated fatty acids, including well known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway including oxidation of polyunsaturated cardiolipins and accumulation of their hydrolysis products \xe2\x80\x93 oxygenated linoleic, arachidonic acids and monolyso-cardiolipins \xe2\x80\x93 is activated in vivo after acute tissue injury.

mitochondrial_pathway_for_biosynthesis_of_lipid_mediators

<!>Selective oxidation and hydrolysis of CL in mouse small intestine<!>Selective oxidation and hydrolysis of CL in rat brain<!>Generation of oxidized TLCL and its hydrolysis products in mitochondria<!>Identification of cytochrome c as the catalyst of CL oxidation and hydrolysis<!>Hydrolysis of oxidized TLCL by mitochondrial Ca2+-independent PLA2\xce\xb3<!>Hydrolysis of oxidized TLCL by PAF acetylhydrolase<!>Diversity of oxygenated FAs after hydrolysis of brain CLox generated by cyt c/H2O2<!>Discussion<!>Methods

<p>Mitochondria of eukaryotic cells contain machinery capable of oxidizing substrates in a coupled enzymatic and electrochemical process that effectively generates energy in the form of ATP. In addition to the powerhouse function, mitochondria are now viewed as the major regulatory platform involved in numerous intra- and extracellular metabolic and physiological pathways – from synthesis of major intracellular biomolecules to assembly of inflammasomes, immune responses, generation of reactive oxygen species, dynamic regulation of their own organization via fission/fusion and mitophagy as well as control and execution of apoptotic and necrotic cell death1. Although there is an intuitive necessity for the existence of specific signals for mitochondrial communications – the identification of these signals has not been fully achieved. An array of lipid mediators, with diversified and potent signaling effects on the normal homeostasis and responses to stress and disease, are generated through oxygenation of free polyunsaturated fatty acids (PUFA). Molecular machinery involved in the production of these bioactive oxygenated compounds has been mostly assigned to the cytosol2. Surprisingly, mitochondria have not been identified as a site of lipid mediators biosynthesis3.</p><p>The rate limiting step in the production of lipid mediators is the availability of oxidizable PUFA4. Normally esterified into cellular (phospho)lipids, free PUFA are released by Ca2+-dependent phospholipases A2 (PLA2) and act as substrates for oxygenation reactions by several enzymes, including, cyclooxygenases (COX), lipoxygenases (LOX), cytochrome P450 isoforms, and peroxidases5. A plethora of lipid regulators including prostaglandins, prostacyclins, thromboxanes, resolvins, protectins, maresins, leukotrienes, lipoxines, lipoxenes, levulo-glandins, among others, with multitude of physiologic effects are formed6, 7. Notably, the major precursor of lipid mediators, phosphatidylserine (PS)8, is lacking from mitochondria9. The majority of phospholipids constituting the inner and outer mitochondrial membranes (IMM and OMM) are manufactured outside of the organelle, with a notable exception of mitochondria-specific cardiolipin (CL). CL (1,3-bis(sn-3'-phosphatidyl)-sn-glycerol) is structurally unique as it contains two phosphatidyl groups linked to a glycerol backbone and four fatty acyl chains. With about 20 different, mostly PUFA residues available for esterification, the diversity of CLs and its oxygenated species may be very high thus making it an exceptionally good source of lipid mediators.</p><p>The final rate-limiting step in the production of CL takes place in the IMM to which the newly synthesized CL molecules are sequestered thus generating characteristic CL asymmetry10. Collapse of CL asymmetry and accumulation of its oxygenated products, have been identified as essential early steps of apoptosis culminating in the release of pro-apoptotic factors11. CL oxygenation is catalyzed by CL/cytochrome c (cyt c) complexes that act as a potent CL-specific peroxidase11 via an enzymatic mechanism similar to that of COX-2 catalyzed oxygenation of arachidonic acid (AA)12. Given that cyt c-driven reactions yield a highly diversified set of oxidized CL (CLox) products, including different stereo-isomers with hydroperoxy-, hydroxy-, epoxy- and oxo-functionalities13, 14, we hypothesized that mitochondrial CLs can be a source of bioactive lipid mediators. While lipid mediators, particularly eicosanoids, have been implicated in the regulation of intrinsic and extrinsic apoptotic pathways15, mitochondria have not been associated with their biosynthesis. Here we report that CL is oxidized to highly diversified polyunsaturated molecular species by cyt c thereby, representing a new biosynthetic pathway for lipid mediator production. A rich assortment of CLox species subsequently undergoes hydrolysis by Ca2+-independent iPLA2γ thus generating multiple oxygenated FAs (FAox) that include well known lipid mediators as well as oxygenated species of lyso-CLs with yet to be determined biological functions.</p><!><p>To ascertain whether mitochondrial CL can be a source of lipid mediators we performed lipidomics analysis of two different tissues - small intestine and brain - after whole body irradiation (WBI) and controlled cortical impact (CCI), respectively. In small intestine (a radiosensitive tissue) of C57BL6 mice exposed to 10 Gy of WBI, LC/MS analysis revealed: i) a decrease of oxidizable polyunsaturated CL molecular species (Fig. 1a), ii) generation of CLox (Fig.1b) and iii) accumulation of mono-lyso-cardiolipins (mCLs) (Fig. 1c) and FAox (Fig. 1d). CLox were mainly represented by molecular species containing one, two and three additional oxygen atoms and their structures were confirmed by MS/MS analysis (Fig. 1b). While mCLs mainly included non-oxidized molecular species, both mono- and di-oxygenated species of mCL (mCLox), particularly with mono- and di-oxygenated LA, were also detected (Supplementary Table S1, Supplementary Fig. S1) whereby the mono-oxygenated mCL derivatives were predominant (Fig. 1c). Quantitatively, the amount of detectable CLox (0.12 ± 0.05 pmol/nmol of total phospholipids) was ~2.5 times lower than the level of mCL (0.30 ± 0.18 pmol/nmol of total phospholipids). This indicates that the hydrolysis reactions – possibly catalyzed by Ca2+-independent iPLA2γ – were effective in converting CLox into mCLs. The major FAox species – mostly mono-oxygenated LA-derivatives - were 9-HODE and 13-HODE (Fig 1d). In addition, 9-KODE, 13-KODE, 9-HpODE, 13-HpODE, 12-HETE, 15-KETE, 15-HETE were also detectable albeit in significantly smaller amounts (Supplementary Fig. S2, Supplementary Table S2).</p><p>To ascertain whether iPLA2γ might be accountable for the hydrolysis of CLox leading to the generation of mCLs, we utilized 6E- (bromoethylene)tetrahydro-3R- (1-naphthalenyl)-2H-pyran-2-one, (R)-bromoenol lactone ((R)-BEL), an inhibitor of Ca2+-independent iPLA2γ in vivo16. We confirmed that (R)-BEL did not inhibit peroxidase activity of cyt c/CL complexes (Supplementary Table S3). (R)-BEL blocked the irradiation-induced accumulation of mCL by 94.9±0.7%. Notably, irradiation induced accumulation of two major lyso-phosphatidylcholines (LPC) (16:0-LPC and 18:0-LPC) was not significantly affected by (R)-BEL (data not shown). The amount of detectable CLox in the small intestine of mice pretreated with (R)-BEL and exposed to WBI was estimated as 0.29 ± 0.15 pmol/nmol of total phospholipids, a ~2.5-fold increase vs. its content in the absence of the inhibitor. Thus iPLA2γ has been mainly responsible for the hydrolysis of CLox and the production of mCL. Simultaneously, the total amount of released FAox was inhibited by 67.3±7.2%. In particular, 12-HETE and 15-HETE concentrations in irradiated intestinal samples from (R)-BEL treated animals were 45.7±16.0% and 76.3±28.0% of untreated irradiated intestinal samples. Expectedly, the decrease of oxidizable CL species was not significantly affected by (R)-BEL (data not shown). Overall, these data emphasize the major role of Ca2+-independent iPLA2γ in the generation of mCLs and also reflect the likely involvement of other pathways – independent of iPLA2γ – in the formation of FAox.</p><p>We next compared the effects of a cocktail of several reportedly effective inhibitors of COX-, LOX- and cytochrome P450-driven conventional biosynthetic mechanisms for lipid mediators species versus their generation via CL-dependent pathways. Specifically, we tested the effects of the inhibitors of: i) COX-1 and -2 - piroxicam (4-hydroxy-2-methyl-N-2-pyridinyl-2H-1, 2-benzothiazine-3-carboxamide-1,1-dioxide); ii) LOX - licofelone (6- (4-chlorophenyl)-2,3-dihydro-2,2-dimethyl-7-phenyl-1H-pyrrolizine-5-acetic acid); and cytochrome P450 epoxygenase/12-HETE activity (CYP2C) - MS-PPOH (N-(methylsulfonyl)-2- (2-propynyloxy)-benzene-hexanamide))17–20 on the formation of FAox in the small intestine of irradiated mice. Because all three enzymes – COX, P450 and 5-LOX – are iron-containing proteins of which the former two are hemoproteins, we assessed the effects of inhibitors on the catalytic peroxidase properties of cyt c/CL complexes in vitro. Within the range of concentrations corresponding to the doses applied in our in vivo experiments, these inhibitors did not suppress the peroxidase activity (Supplementary Table. S3). We found that the inhibitor cocktail partially decreased the levels of mono-oxygenated AA and oxo-LA compared with the group of mice that received no drugs before irradiation (Supplementary Table S4). The production of di-oxygenated LA did not appear to be affected by the mixture of inhibitors. This suggests that generation of lipid mediators from CL/CLox is accomplished through the enzymatic reactions distinct from the known Ca2+-dependent mechanisms realized via the release of free FAs and their subsequent oxygenation.</p><!><p>In rat brain, CLs were highly diversified and included mostly oxidizable polyunsaturated CLs (Fig. 2a). LC/MS analysis revealed that CCI caused depletion of oxidizable CLs containing LA, AA and docosahexaenoic (DHA) acids (Fig. 2a) and formation of CLox (Fig. 2b) and its hydrolysis products: mCL (Fig. 2c) and FAox (Fig. 2d). CLox were mainly represented by species containing one and two additional oxygen atoms (Fig.2b). MS/MS analysis identified mono-oxygenated species of CLox with either LAox or AAox (Fig. 2b). mCLs were represented by non-oxidized as well as oxidized mCLox species (Fig. 2c, Supplementary Table S1, and Supplementary Fig. S3). The CLox content was lower than the amount of mCL: 0.10 ± 0.02 and 0.26 ± 0.08 pmol/nmol of total phospholipids, respectively. In addition, the oxygenated species of non-esterified LA and AA were produced (Fig. 2d, Supplementary Fig. S4), whereby several lipid mediators were identified such as 9-KODE, 13-KODE, 9-HODE, 13-HODE, 9,12,13,-KEpOME, 9-HpODE, 13-HpODE, 12-HETE, 15-HETE (Supplementary Table S2). Markedly smaller amounts of oxygenated DHA were also detected (Fig. 2d). Quantitatively, the CCI-induced loss of CL species containing PUFA was in good correspondence with the accumulation of FAox (~5.1 and ~3.0 pmol/nmol of total phospholipids, respectively). This indicates that CL can be a source of CCI-induced production of FAox.</p><p>To ascertain whether the CL/CLox-dependent pathway may be cell type specific, we compared the responses of two types of brain cells – cortical neurons and one of the major components of glia, astrocytes, to a standard treatment with H2O2 in vitro. We found that neurons (Supplementary Fig. S5, Supplementary Table S5) contained more oxidizable polyunsaturated CL species than astrocytes (Supplementary Fig. S6, Supplementary Table S5). Accordingly, the amounts and speciation of CLox after H2O2 exposure was also markedly richer in the former than in the latter (Supplementary Figs. S5 and S6). Similarly, the levels and diversification of mCLs was also significantly greater in neurons than in astrocytes (Supplementary Figs. S5 and S6).</p><!><p>To further characterize pathways of CL peroxidation and hydrolysis we utilized C57BL6 mouse heart and liver mitochondria in which CL was accountable for ~15% of total phospholipids (Fig. 3). MS analysis demonstrated that oxidizable LA residues were present in all molecular species of CL (Supplemental Fig. S7). After exposure to a pro-oxidant, t-BuOOH, isolated mitochondria revealed the same pattern of characteristic CL changes as in vivo: i) decreased amounts of oxidizable CLs (Fig. 3a) accumulation of non-oxidized mCL (Fig. 3b) and LAox, (containing one and two oxygens) (Fig. 3c).</p><!><p>To directly assess the involvement of cyt c in CL peroxidation and hydrolysis products as a source of lipid mediators, we compared the production of mCL and FAox in cyt c+/+ and cyt c−/− mouse embryonic cells (MECs) during apoptosis triggered by non-oxidant (actinomycin D, ActD) or oxidant (t-BuOOH) stimuli (Fig. 4a). No significant differences were found in the total content and molecular speciation of CLs (approximately 3% of total phospholipids; 31.1±4.9 and 33.7±7.1 pmols CL/nmol total phospholipids, respectively) in cyt c+/+ and cyt c−/− cells (Fig. 4b and Supplementary Fig. S8). Treatments with ActD or t-BuOOH caused a significant decrease of oxidizable polyunsaturated species of CL and accumulation of CLox species with one and two oxygens in cyt c+/+ cells - but not in cyt c−/− - cells (Fig. 4b and Supplementary Fig. S8). Quantitatively, the amounts of CLox produced in cyt c+/+ cells challenged with either ActD or t-BuOOH were increased 2.6- and 3.0 -times compared to untreated controls. Analysis of hydrolysis products revealed the presence of both oxidized and non-oxidized mCL (Fig. 4c) as well as oxygenated LA (Fig. 4d). Mono- and di-oxygenated molecular species of mCLox were the predominant forms accumulating in cells upon the treatments (Fig.4c). Mono-oxygenated LA was the main product in ActD treated cyt c+/+ cells and was represented by a mixture of 9-HODE and 13-HODE (Fig. 4d). No significant increase in the levels of CLox and CL hydrolysis products was detected in ActD and t-BuOOH treated cyt c−/− cells (Fig. 4). Notably, ActD or t-BuOOH triggered apoptosis in cyt c+/+ cells as evidenced by a marked increase of PS externalization (Fig. 4a), whereas these pro-apoptotic effects were not found in cyt c−/− cells11.</p><p>MECs express two isoforms of cyt c – somatic (s-cyt c) and testicular (t-cyt c)21–23. Western blot analysis established that t-cyt c was accountable for ~45% of the total cyt c in MECs (Supplementary Fig. S9). In cyt c−/− cells, the amount of t-cyt c was ~1.5 times lower than in cyt c+/+ cells (0.83 ± 0.05 and 1.24 ± 0.08 ng/µg protein, respectively). As cyt c−/− cells rely more on glycolysis rather than mitochondrial respiration for ATP generation24 they maintain lower levels of mitochondria. Indeed, Western blots showed that the levels of several mitochondrial marker proteins (COX-IV, MnSOD, TIM23) were ~46%–83% lower in cyt c−/− cells versus cyt c+/+ cells (Supplementary Fig. S9). siRNA knock-down of the t-cyt c in cyt c−/− cells to ~50% of its content (Supplementary Fig. S9) did not affect the already low level of CL oxidation after ActD treatment. Similar to cyt c−/− cells the low level of mCL in cyt c−/− with knock-down t-cyt c remained unchanged after ActD exposure. These data suggest that t-cyt c is not a significant contributor to the generation of CL-driven lipid mediators.</p><!><p>Ca2+-independent iPLA2γ has been suggested as a likely candidate-catalyst capable to hydrolyze oxidized phospholipids in mitochondria25. To assess its role in the hydrolysis of CLox species, we bio-synthesized (using cyt c/H2O2 system) and purified (to the level of 99% using LC/MS) oxidized tetralinoleoyl-cardiolipin (TLCLox with 1–8 oxygens in four LA residues, see Supplementary Table S6 and Supplementary Fig. S10) and analyzed the products formed in the presence of a specific iPLA2γ inhibitor, (R)-BEL, in rat liver mitochondria (Fig. 3d). Mitochondria effectively hydrolyzed TLCLox as evidenced by i) decrease of TLCLox content (Fig. 3e) and ii) accumulation of oxygenated LA (with one and two oxygens) (Fig. 3f) as well as non-oxidized mCL and mCLox (Fig. 3g,h). The hydrolysis of TLCLox was effectively blocked by (R)-BEL (Fig. 3).</p><!><p>Mitochondria with externalized CL and/or CLox as well as cyt c can be released into circulation during injury/disease process26. Thus circulating CL and CLox can be a source of lipid mediators as well. Several phospholipases, including lipoprotein-associated PLA2 (LpPLA2) or platelet activating factor (PAF) acetylhydrolase (PAF-AH), have been shown to selectively hydrolyze oxidatively modified phospholipids to liberate FAox27. Recently, we have identified oxidized PS as a good substrate for Lp-PLA228. Because TLCL is the most predominant species of CL in circulation, we tested the ability of PAF-AH to release oxygenated FAs from TLCLox produced in cyt c driven reaction (Supplementary Fig. S10). LC/MS analysis revealed accumulation of mCL containing both LA and LAox (Figs. 5a, 5b, 5c), whereby LAox was represented by molecular species with one and two oxygens (Fig. 5d). Markedly less effective hydrolysis was detected when non-oxidized TLCL was treated with PAF-AH (data not shown). Thus, PAF-AH predominantly hydrolyzes oxygenated molecular species of CL to generate lipid signaling molecules formed in cyt c catalyzed reaction.</p><!><p>The high diversification and enrichment with PUFA of brain lipids makes neuro-CLs particularly interesting as a source of lipid mediators generated via CL oxidation by cyt c. Indeed, we identified 56 major molecular species of CL in lipid extracts from mouse brain (Supplementary Table S7) of which 55 were highly oxidizable polyunsaturated CL containing one to four PUFAs (Fig. 6a). To establish molecular identity and stereospecificity of peroxidizable PUFA in neuro-CLs we employed a mixture of PLA1 with PLA2 and established that the dominant PUFAs of CL were represented by AA and LA (Fig. 6a). DHA, EPA, eicosatrienoic and octadecatrienoic acids were detected as well but in relatively lower abundance. Cyt c/H2O2 caused significant decrease of PUFA-containing molecular species of CL (Fig. 6b and Fig. 6c). By employing PLA1 plus PLA2 as a tool to liberate FA from both sn-1 and sn-2 positions of CLox we identified by MS/MS analysis highly diversified molecular species of LAox and AAox (Fig. 6d) which included those detected in acutely injured tissues (brain after CCI and small intestine after WBI) (Supplementary Table S2) as well as in apoptotic cyt c+/+ cells. In addition, oxidatively truncated FA molecular species were detected (Supplementary Table S2). Overall, 31 molecular species of FAox have been identified as the products of brain CLox hydrolysis.</p><!><p>Oxygenated free PUFAs – generated by a wide array of enzymes, including cyclooxygenases (COX-1 and COX-2), lipoxygenases (5-LOX, 12-LOX and 15-LOX) and cytochrome P450 isoforms [reviewed in5, 7] - have numerous physiological roles. The potent biological effects of lipid regulators necessitate the maintenance of very low endogenous levels of free PUFA. As a result, their availability is the rate-limiting step in the generation of lipid mediators4. Release of PUFA precursors from their phospholipid storage sites is achieved via action of Ca2+-dependent phospholipases A2 (PLA2),30. We have reported a new Ca2+-independent pathway for selective generation of lipid mediators from a mitochondria-specific phospholipid, CL, whereby oxidation of FA residues is catalyzed by an intermembrane space protein, cyt c, directly in the esterified phospholipid. This is followed by hydrolysis of oxygenated CL species by two types of PLA2 specific towards oxidatively modified phospholipids. This novel pathway disobeys the major dogmas of lipid mediator biochemistry with regards to: i) oxygenation of a phospholipid rather than a free FA as the reaction substrate, ii) mitochondrial – rather than cytosolic - localization- iii) an electron carrier cyt c – rather than an oxidase - as the reaction catalyst, iv) the final – rather than the initial – hydrolytic stage using oxygenated species of CL by PLA2 to synthesize oxygenated PUFA, and v) Ca2+-independent - rather than Ca2+ dependent - nature of the pathway.</p><p>One can predict specific features of regulation of CL-dependent process that are markedly different from the "conventional" biosynthesis of oxygenated lipid mediators. The mitochondrial pathway is strictly dependent on the catalytic interactions between cyt c and CL. However, these two participants have limited access to each other. The former is confined to the intermembrane space while the latter is "cloaked" almost exclusively in the inner mitochondrial membrane31. Importantly, ~15% of cyt c is believed to be hydrophobically bound to the IMM likely via its binding to CL in the outer leaflet of the IMM and cannot be removed from mitochondria, as occurs to the majority of cyt c upon treatment with high ionic strength solutions32. The function of the tightly membrane-bound cyt c in normal mitochondria has not been identified. Given that CL-bound cyt c cannot act as electron acceptor from respiratory complex III33, we speculate that catalysis of CL-dependent formation of lipid mediators may represent an unrecognized function of cyt c.</p><p>For the peroxidase activity of cyt c/CL complexes, the presence of sufficient amounts of oxidizing equivalents such as H2O2 or lipid hydroperoxides ("peroxide tone"34, 35) is essential for triggering CL oxidation. Their supply may be driven by disrupted electron transport likely occurring as a consequence of cyt c binding with CL34. The complex has a redox potential ~400 mV more negative than free cyt c – the effect that precludes cyt c's function as an electron acceptor from mitochondrial complex III33. As a result accumulating reduced intermediates serve to donate electrons to molecular oxygen yielding superoxide radicals. Spontaneous or catalyzed dismutation of the latter generates H2O2 that feeds the catalytic peroxidase cycle of cyt c/CL complexes. This dependency on H2O2 as the source of oxidizing equivalents disappears with the accumulation of FA-hydroperoxides or CL-hydroperoxides that can act as more efficient substrates for the peroxidase half-cycle of the process35.</p><p>Cells can express, at different proportions, two isoforms of cyt c – s-cyt c and t-cyt c21–24. We found that t-cyt c does not significantly contribute to the production of lipid mediators. To investigate if there are potential differences in s-cyt c versus t-cyt c participation in lipid oxidation, we compared the interaction of two isoforms of cyt c with CL using sequence analysis, molecular structural modelling, as well as molecular docking and computational simulations. The sequences of s- and t-cyt c and 3D-structures displayed high similarity (Supplementary Fig. S12). Further, ligand docking analysis revealed close similarity between the two isoforms in binding a typical peroxidation substrate, TLCL, as judged by closely matching predicted binding energy values (Supplementary Table S8). Finally, coarse-grained molecular dynamics simulations of cyt c interactions with lipid bilayers revealed equally avid binding to TLCL-containing membranes at early stages in the simulation and lack thereof with lipid bilayers devoid of TLCL (Supplementary Fig. S12). This suggests that t-cyt c, like s-cyt c, should be competent towards peroxidase functions, including CL peroxidation.</p><p>The failure of t-cyt c to operate as a catalyst of CL oxidation in MECs may mostly due to its presence as apo-protein whereby its biosynthesis into the catalytically active holo-enzyme may be the limiting factor. According to Kim et al., three times more t-cyt c was detected by radioimmunoassay, than by spectral assessments of the catalytically competent hemoprotein36. Of note, the anti-t-cyt c antibody employed in our study does not distinguish between the apo- and holo-proteins. Differences in the localization of these two isoforms of cyt c in the intermembrane space and their differential interactions with CLs and other proteins may also be contributory to their dissimilar peroxidase activity towards CLs37, 38. The suggested higher pro-apoptotic activity of t-cyt c vs s-cyt c may be realized through their differential interactions with pro-caspase complexes (Apaf) and the formation of apoptosomes39.</p><p>A notable feature of cyt c-catalyzed oxygenation reactions described in this work is accumulation of not only of the expected hydroperoxy-CL and hydroxy-CL5 but also the presence of epoxy- and oxo-derivatives and truncated products of oxidative cleavage. Whether these derivatives are produced by enzymatic cyt c-catalyzed reaction or non-enzymatic process caused by degradation and release of cyt c's heme – is unknown. Similar products require additional enzymatic activities (i.e., epoxidase, epoxide hydrolases) in the traditional pathways triggered by COX and LOX5. Our results with pharmacological inhibitors of two different pathways COX/LOX/P450 and (R)-BEL support a role of CL oxidation as a source of the generated lipid mediators. In line with our data, disturbed CL re-acylation in mice with knock-down tafazzin - modeling Barth syndrome - was accompanied by the altered pattern of HETE and oxidized LA and DHA metabolites40.</p><p>The final step in biosynthesis of oxygenated free FAs requires hydrolysis of oxidatively modified CLs by PLA2. Several representatives from different groups of secretory PLA2 (sPLA2) of a large superfamily of PLA2 were shown to display hydrolyzing activity towards peroxidized phospholipids [reviewed in David et al.,29]. Among those, the best studied is LpPLA2 – a Ca2+-independent LpPLA2 or type VIIA PLA2 - which can be represented by intracellular and secreted forms30 and is active towards oxygenated long-chain and truncated forms of PC41. Oxidized PS has been identified as a representative of anionic peroxidized phospholipids readily hydrolyzable by Lp-PLA228.</p><p>CLs have been identified as signaling molecules in two major biological functions: mitophagy42 and apoptosis11. In both cases, CL gets externalized to the mitochondrial surface suggesting that any injury to plasma membrane may be associated with the release of these "CL-decorated" mitochondria into extracellular environments. Assuming that apoptosis commonly transitions to necrosis, mitochondria may – with their externalized CL and CLox – act as damage associated molecular patterns (DAMPs)26,43. Our studies reveal that not only CL and CLox but also CLox hydrolysis products – mCL and FFAox – may be released, along with mitochondria, from injured cells. Importantly, sufficient hydrophobicity of mCL retains its association with mitochondria. However, oxygenated mCLs may lose their association with the outer mitochondrial membrane and partition into the aqueous phase of extracellular compartments. Oxygenated FFAs also water-soluble, hence diffuse independently of mitochondrial surfaces. Release of CL44 and cyt c45 from injured host cells into the extracellular space, have been shown. Thus lipid mediators may also be generated extracellularly from CL with the contribution of cyt c and subsequent hydrolysis of CLox by Lp-PLA2. Overall, CL-dependent lipid mediators may be represented by a diversified variety of membrane-associated and freely diffusible and circulating signaling molecules whose identification and quantitative analysis will represent an intriguing opportunity for the future studies.</p><p>Brain has an unprecedented diversification of CLs13. By using brain CLs, we showed that a significant variety of lipid mediators could be generated by cyt c-catalyzed oxygenation process. In fact, we found that all eight well known LA-based lipid mediators were generated by cyt c/H2O2 (Supplementary Table S2). In the AA series, we were able to identify nine species of lipid mediators (Supplementary Table S2). A relatively smaller number of oxygenated derivatives of docosapentaenoic acid and DHA were detected in spite of the significant proportion of these FA residues in brain CLs. Also, terminally hydroxylated metabolites of AA, including 17-HETE, 18-HETE, 19-HETE, and 20-HETE were not found, implying that terminal hydroxylation reactions are not catalyzed by cyt c/H2O2 and are likely formed enzymatically after AA liberation.</p><p>The ranking order of FA resiues oxidation in the model biochemical system - where all the CL substrates were equally available - was DHA>AA>>LA. The amounts of DHA, AA and LA decreased almost 20-, 5- and 0.3-fold, respectively. The energies for H-abstraction in α-position to bis-allylic double-bonds for different FA with multiple double bonds decrease in the order LA<AA<DHA. Accordingly, the propagation rate constants for FA oxidation determined for LA (one bis-allylic group), AA (3 bis-allylic groups), EPA (4 bis-allylic groups) and DHA (5 bis-allylic groups) increase at ratios of 1, 3.2, 4.0, and 5.446. This is consistent with the assumption that the H-abstraction is essential for oxidation of different polyunsaturated species of CLs by cyt c's protein-immobilized Tyr radical: Tyr-O• +CL-H-> Tyr-OH +CL•. Thus, preferential oxidation of LA in brain CL's in vivo does not correlate with the chemical reactivity suggesting that CL oxidation in mitochondria is not a simple reaction of CLs with cyt c. Availability of CLs for oxidation by cyt c in the inter-membrane space likely defines the specificity of the reaction towards different CL species. Our findings with brain CLs may provide a long awaited explanation for a well-known large variety of CL species in the brain and some other tissues (eg, small intestine, lung) – by implying that they are used as precursors for biosynthesis of diversified lipid mediators47.</p><p>In conclusion, we describe a new pathway for generation of lipid mediators. This novel Ca2+-independent pathway localizes to mitochondria, involves cyt c-catalyzed oxygenation of CL esterified PUFA residues, is followed by hydrolysis of oxygenated CL species by two types of Ca2+-independent PLA2 specific towards oxidatively modified phospholipids.</p><!><p>This section describes key experiments only; an extended experimental section is provided in the Supplementary Methods.</p><p>Controlled cortical impact to the left parietal cortex in 17 day old rats was performed as described previously48. For all studies, a 6-mm metal pneumatically driven impactor tip was used. The velocity of the impact was 4.0 ± 0.2 m s−1, with a penetration depth of 2.5 mm. Whole body irradiation: C57BL/6NHsd female mice were irradiated with dose of 10 Gy using a J. L. Shepherd Mark 1 Model 68 cesium irradiator at a dose rate of 80 cGy/min as described previously49. 10 Gy of WBI is a LD100/30 for C56Bl6 mice, which means this dose causes death of all animals within 30 days after the exposure. Mice were euthanized 10 or 24 hrs later by CO2 inhalation. All procedures were approved by the IACUC of University of Pittsburgh and performed according to the protocols established.</p><p>Analysis of CL and mCL molecular species was performed using a Dionex Ultimate™ 3000 HPLC system coupled on-line to a linear ion trap mass spectrometer (LXQ, ThermoFisher Scientific, San Jose, CA) using Luna 3 µm Silica (2) 100 Å column (Phenomenex, Torrance, CA) as described previously28. To analyze oxygenated species, CL and mCL were isolated from total lipids by normal phase 2D-HPTLC using mobile phases as described by Rouser et al.,50. To prevent lipid oxidation during separation, chromatography was performed under N2 conditions on diethylenetriaminepentaacetic acid (DTPA) treated silica plates (5 × 5 cm, Whatman). CL and mCL were extracted from silica spots49 and used for reverse phase ESI-LC/MS analysis. LC/MS analysis was performed using a Dionex UltimateTM 3000 RSLCnano system coupled online Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA) using a C8 column (Luna 3 µm,100 Å, 150 × 2 mm, Phenomenex, Torrance, CA). For CLox an isocratic solvent system consisting of 2-propanol: water: triethylamine: acetic acid, 45:5:0.25:0.25, v/v was delivered at 150 µl/min for 20 minutes. For mCLox, a gradient of solvent A (acetonitrile: water: triethylamine: acetic acid, 45:5:0.25:0.25 v/v) and B (2-propanol: water: triethylamine: acetic acid, 45:5:0.25:0.25, v/v) was used at the flow rate of 150µl/min as follows: 0–10 min isocratic at 50% solvent B; 10–20 min linear gradient 50–100% solvent B; 20–32 min isocratic at 100% solvent B; 32–35 min linear gradient at 100–50% solvent B; 35–45 min isocratic at 50% solvent B. Spectra were acquired in negative ion mode using a spray voltage of 4.0 kV and a capillary temperature of 320 °C. Scans were acquired in data-dependent mode with an inclusion list for both CL/CLox, mCL/mCLox species, isolation width of 1.0 Da and a normalized collision energy of 24 in high energy collisional dissociation (HCD) mode. TMCL-(14:0)4 (Avanti Polar Lipids Inc., Alabaster, AL) and mCL-(14:0)3 were used as internal standards. The peaks with signal-to-noise (S/N) ratio of 3 and higher were taken into consideration.</p><p>Detection of oxidized FA. LAox was analyzed by LC/MS using a Dionex Ultimate™ 3000 HPLC system coupled on-line to LXQ linear ion trap mass spectrometer (ThermoFisher Scientific, San Jose, CA). A C18 column (Luna, 3 µm, 150 × 2 mm, Penomenex, Torrance, CA) and gradient solvents (A: tetrahydrofuran/methanol/water/CH3COOH, 25:30:50:0.1 (v/v/v/v) and B: methanol/water 90:10 (v/v)) containing 5 mM ammonium acetate were used. The column was eluted at a flow rate of 0.2 mL/min during first 3 min isocratically at 50% B, from 3 to 23 min with a linear gradient from 50% solvent B to 98% solvent B, then 23–40 min isocratically using 98% solvent B, 40–42 min with a linear gradient from 98% solvent B to 50% solvent B, 42–28 min isocratically using 50% solvent B for equilibration of the column. Spectra were acquired in negative ion mode using a spray voltage of 5.0 kV and a capillary temperature of 150 °C. Assay method for quantitative assessment of free AAox and DHAox was completed as previously described by Miller et al.51 Briefly, LC was performed using an acuity ultra-performance LC autosampler (Waters, Milford, MA). Separation of analytes was conducted on a UPLC BEH C18, 1.7µm (2.1 × 100 mm) column. A gradient mobile phase of 0.005% acetic acid, 5% acetonitrile in deionized water and 0.005% acetic acid in acetonitrile was used with a run time of 6.4 minutes. Analysis was performed using a TSQ Quantum Ultra (ThermoFisher Scientific, San Jose, CA) triple quadrupole mass spectrometer with heated electrospray ionization in negative selective reaction monitoring mode. Analytical data were acquired and analyzed using Xcalibur software.</p>

PubMed Author Manuscript

In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code†

The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-l-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels–Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins.

in_situ_epr_spectroscopy_of_a_bacterial_membrane_transporter_using_an_expanded_genetic_code†

<!>Conflicts of interest

<p>In situ investigation of proteins is key for comprehending the role of native environment on their structure and dynamics, but is a challenging task. To date, such studies especially on membrane proteins are underrepresented as they face many obstacles such as low expression yields and difficulty for specific labelling in the complex native membranes.1 To observe the protein of interest in the cellular environment spectroscopically, specific markers are required. Förster resonance energy transfer (FRET) can provide the average distance between fluorophores between rather bulky donor and acceptor fluorophores.2 As a complementary approach, site-directed spin labelling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a powerful biophysical tool,3–5 as the majority of cellular components are of diamagnetic nature, thus EPR-silent. In vitro, most common spin labelling approaches rely on nitroxide tags, as they are small, non-perturbing, and convenient to handle.6,7 In particular, their ability to report on rotational dynamics through line-shape analysis,8,9 and the possibility to perform distance determinations,10 provide unmatched spectroscopic characteristics. The best-known nitroxide spin label is the methanethiosulfonate spin label (MTSSL), which can be covalently attached to sulfhydryl groups of accessible cysteine residues in proteins.11 Most often genetic engineering of the protein of interest is required to eliminate undesired cysteines and, in turn, place new ones at designated sites.</p><p>In the past years, many in vivo EPR studies relied on the transfer of spin labelled proteins into cells, granting valuable insights into the behavior of proteins in their native environment. Beneficial reduction-stable paramagnetic centers include gadolinium,12 trityl13 or sterically shielded nitroxides.14 When aiming for in-cell approaches with membrane proteins, it is inevitable to perform spin labelling directly in the cellular environment. However, targeting cysteines or other native amino acids for spin labelling limits bioorthogonality, as these are ubiquitously present throughout cells. In turn, expansion of the genetic code by noncanonical amino acids (ncAA) is a promising alternative, which has proven its suitability for various biochemical and biophysical applications.15 For this purpose, orthogonal aminoacyl-tRNA-synthetase (aaRS)-tRNA pairs enable the selective charging of a nonsense suppressor tRNA (e.g. an amber codon (TAG)) with a ncAA.16 This technique introduces only minimal modifications into proteins and offers a wide range of highly selective reaction schemes.17 However, for EPR applications, the potential of ncAA-based bioorthogonal labelling is still in its infancy, especially in the context of living cells.18 A spin labelling scheme linking the ncAA p-Acetyl-l-phenylalanine to a nitroxide was pioneered in 2009,19 while the labelling of green fluorescent protein (GFP) by azide–alkyne cycloaddition in Escherichia coli (E. coli) represented significant advancements in this field.20,21 Further refinement of this technique has allowed even for distance measurements inside E. coli cells.22</p><p>Recently, we presented the first approach applying inverse electron-demand Diels–Alder click chemistry23–27 for SDSL of model proteins in vitro.28 The photoactivatable nitroxide for Diels–Alder (PaNDA) spin label (Fig. 1A) distinguishes itself by an o-nitrobenzyl-based photoremovable protecting group (PPG) for the TEMPO-based nitroxide.29–32 Upon UV irradiation at the desired timepoint, the PPG can efficiently release the nitroxide. The temporal control of the paramagnetic potential is expected to be especially useful to circumvent the rapid degradation of radicals in the reducing biological environment.33 PaNDA features a tetrazine moiety for rapid attachment to proteins without the need for potentially toxic catalysts. As a counterpart, the cyclooctene- or cyclooctyne-bearing ncAA derivatives TCO- and SCO-l-lysine are known for exceptionally high reaction rates and stability,34 and can be incorporated into the protein of interest by the tRNAPyl/PylRSAF synthetase.35</p><p>Here, we report on the application of PaNDA for spin labelling and Double Electron–Electron Resonance (DEER or PELDOR) spectroscopy of the cobalamin transporter BtuB in intact E. coli. BtuB is responsible for the transport of vitamin B12 (cobalamin) into the periplasm, and has been extensively studied in situ using MTSSL- and trityl-based EPR spectroscopy.36–42 BtuB lacks native cysteines, as the majority of outer membrane proteins does. Still, certain fractions of off-target labelling have consistently been detected when labelling whole cells using MTSSL. Consequently, BtuB is perfectly suited to capitalize on the use of ncAA in combination with the PaNDA spin label (Fig. 1A).</p><p>To enable ncAA incorporation, we modified a BtuB plasmid by installing an amber codon at position 404. This loop site is known from previous studies42 and is located on the extracellular side. Together with a second plasmid, which encodes the tRNAPyl/PylRSAF pair,35 BtuB-deficient RK5016 E. coli were transformed.43 The cotransformation using a constitutive and an inducible vector makes our expression system unique. While the constitutive expression of BtuB_F404 → ncAA in the minimal medium enables comparably slow protein expression and controlled membrane integration, the arabinose-induced expression of the PylRSAF synthetase ensures efficient ncAA incorporation. We expressed BtuB in the presence of 1 mM TCO- or SCO-l-lysine using 0.2% arabinose to induce the PylRSAF overnight. Sufficient expression yields of the full-length target protein (∼66 kDa) were detected only with SCO-l-lysine (SCO; Fig. 2A and Fig. S3, ESI†) for unknown reasons, as both ncAA yielded similar incorporation yields for other proteins.28 When the cotransformed RK5016 E. coli were grown in the absence of SCO-l-lysine during expression, no BtuB was observed for the amber mutant (Fig. S4, ESI†), which confirms the integrity of the expression system. Titrating the E. coli cells with TEMPO-modified cobalamin42 (TEMPO-CNCbl; Fig. 1B) allows for semi-quantitative assessment of expression levels due to its fast binding kinetics, and revealed ∼12 μM BtuB_F404 → SCO in the cell pellet (Fig. S7, ESI;† for BtuB_F404 (wt) ∼30 μM). The line shape of X-band EPR spectra also proves, that the incorporated SCO-l-lysine does not hinder TEMPO-CNCbl binding (Fig. S5–S7, ESI†).</p><p>To analyze the suitability of PaNDA for in situ spin labelling, after expression excess ncAA was removed by adding fresh minimal medium and by repeated washing steps. The labelling reaction was performed at a cell density corresponding to OD600 = 15 in the presence of 150 μM PaNDA for 45 minutes. Excess label was removed by pelleting and one washing step, before cells were transferred to a micropipette. As PaNDA is EPR-active only after irradiation, UV light of 365 nm was applied, which minimizes harm to bacteria44 (Fig. S13, ESI†) and is able to induce cleavage of the PPG29 within two minutes in vitro28 and, as found here, even in situ. Only after irradiation, we detected the characteristic nitroxide spectrum in the BtuB_F404 → PaNDA sample (Fig. 2B). The spin concentration is ∼6 μM, which corresponds to a spin labelling and deprotection yield of ∼50% in total. Compared to previous in vitro experiments with PaNDA28 and ncAA-mediated in vivo spin labelling studies,20,22 the reduced expression and labelling yield observed here is within the expected range. Moreover, we coexpressed BtuB_F404 (wt) and the PylRSAF in presence of SCO-l-lysine to check for potential off-target labelling of involved components. No attachment or labelling of PaNDA to the wildtype protein or to the E. coli cells was detected (Fig. 2C), which is clearly advantageous to previous experiments exploiting MTSSL labelling of BtuB.</p><p>Another beneficial feature of PaNDA is the PPG, which allows for advanced experimental schemes including temporally shifted activation of the nitroxide. To see whether this is feasible in situ, we expressed BtuB_F404 → SCO, performed labelling with PaNDA, and added TEMPO-CNCbl (Fig. 3). Notably, PaNDA labelling does not affect TEMPO-CNCbl binding. Complete depletion of the TEMPO-CNCbl-derived nitroxide signal was detected after ∼90 minutes. After irradiation of the cells to cleave the PPG, indeed the PaNDA-derived nitroxide signal was detected. We hypothesize, that the reason for the different decay rates is the different accessibility of the labels towards reducing agents. Altogether, this proves that the PPG is stable for at least three hours (including the labelling and sample preparation time) under in situ conditions.</p><p>Previously, distances in BtuB were determined either between two spin labelled cysteine residues, or in combination with TEMPO-CNCbl.36–42 As reference, we expressed BtuB_F404C and spin labelled with MTSSL in the isolated outer membranes (OM) and in situ resulting in the side chain BtuB_F404 → R1. After addition of TEMPO-CNCbl 4-pulse DEER experiments were performed (Fig. S14 and S15, ESI†). The resulting modulation depths are Δin situ = 7.4% and ΔOM = 2.7% and the maximum of the distance distributions overlays with the Multiscale Modeling of Macromolecules (MMM)45 simulation.</p><p>To perform EPR distance determination involving PaNDA, we produced BtuB_F404 → PaNDA both in situ and in the OM and added TEMPO-CNCbl. After irradiation, samples containing 20% d8-glycerol were frozen, and DEER was measured (Fig. 4 and Fig. S9–S11, ESI†). By doing this, we combined four challenging aspects in one experiment for the first time: (i) a membrane transporter as protein of interest (which suffer in general from low expression and challenging EPR spectroscopy), (ii) the use of ncAA, (iii) in situ spin labelling and (iv) in situ DEER measurement. Especially, low expression of the ncAA-containing BtuB reduces the protein concentration (Fig. 2A), resulting in low overall spin concentration as well as lower signal-to-noise ratio and short length of the obtained DEER trace. This leaves room for improvements in future experiments. However, especially with regard to modulation depths (Δin situ = 9.5% and ΔOM = 3.0%), the data we acquired confirm an adequate labelling degree and the general suitability of our approach for DEER measurements.</p><p>The DEER data for distance determination between BtuB_F404 → PaNDA and TEMPO-CNCbl looks similar in situ and in the OM (Fig. 4A). The corresponding distance distribution exhibits two maxima (d1 = 2.6 nm and d2 = 3.7 nm), and is relatively broad in the accessible range (Fig. 4B). The width of the distribution is expected due to the linker size resulting from the combination of the relatively long lysine-based ncAA and the PaNDA spin label. To further assess the extracted distance distribution, rotamers for the PaNDA-derived linkers were generated using the MtsslWizard46 software (Fig. 1C and Fig. S8, ESI†). They reveal that the first part of the experimental distance distribution overlays with the simulation, while the longer distance is not described (Fig. 4B). Previous experimental37 and computational47 findings however suggested high flexibility of the extracellular loops of BtuB. As we could not reproduce this by MTSSL-labelling (Fig. S14 and S15, ESI†), we suspect the PaNDA label to induce different loop conformations.</p><p>Moreover, we spin labelled BtuB_F404 → SCO with PaNDA, left out TEMPO-CNCbl, and measured DEER in situ. The resulting data indicates the homogenous distribution of BtuB on the E. coli surface (Fig. S12, ESI†).</p><p>In summary, SCO-l-lysine was incorporated into the membrane transporter BtuB in high yields, and the surface of living E. coli provides a suitable environment for PaNDA spin labelling as well as nitroxide activation via irradiation and spontaneous oxidation. The spin labelling and deprotection conditions developed for in situ EPR are completely biocompatible, and allowed for a DEER experiment involving the PaNDA label directly on the surface of E. coli.</p><p>This study provides the first spin labelling scheme for membrane proteins using an expanded genetic code, and the first application of Diels–Alder chemistry for spin labelling of proteins in the cellular environment.</p><p>This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant Agreement number: 772027 – SPICE – ERC-2017-COG). B. J. thanks DFG for the financial support through the Emmy Noether Program (JO 1428/1-1). We thank Frederike Krasucki for experimental contributions.</p><!><p>There are no conflicts to declare.</p>

PubMed Open Access

Palmitoylome profiling reveals S-palmitoylation\xe2\x80\x93dependent antiviral activity of IFITM3

Identification of immune effectors and the post-translational modifications that control their activity is essential for dissecting mechanisms of immunity. Here we demonstrate that the antiviral activity of interferon-induced transmembrane protein 3 (IFITM3) is post-translationally regulated by S-palmitoylation. Large-scale profiling of palmitoylated proteins in a dendritic cell line using a chemical reporter strategy revealed over 150 lipid-modified proteins with diverse cellular functions, including innate immunity. We discovered that S-palmitoylation of IFITM3 on membrane-proximal cysteines controls its clustering in membrane compartments and its antiviral activity against influenza virus. The sites of S-palmitoylation are highly conserved among the IFITM family of proteins in vertebrates, which suggests that S-palmitoylation of these immune effectors may be an ancient post-translational modification that is crucial for host resistance to viral infections. The S-palmitoylation and clustering of IFITM3 will be important for elucidating its mechanism of action and for the design of antiviral therapeutics.

palmitoylome_profiling_reveals_s-palmitoylation\xe2\x80\x93dependent_antiviral_activity_of_ifitm3

<!>Proteomic analysis of palmitoylated proteins in DC2.4 cells<!>IFITM3 S-palmitoylation on membrane-proximal residues<!>S-palmitoylation controls clustering of IFITM3<!>S-palmitoylation regulates IFITM3 antiviral activity<!>DISCUSSION<!>Cells, virus infections, transfections and flow cytometry<!>Metabolic labeling, immunoprecipitations and CuACC<!>LC-MS analysis<!>Microscopy<!>Cloning, quantitative PCR and topology diagram<!>Visualization and identification of palmitoylated proteins in DC2.4 cells<!>IFITM3 is S-palmitoylated on membrane-proximal cysteine residues<!>Palmitoylation-dependent clustering of IFITM3 in the ER<!>Antiviral activity of IFITM3 is regulated by palmitoylation

<p>Vertebrates have evolved sophisticated innate and adaptive mechanisms of immunity to combat microbial pathogens1. In response, viruses and pathogenic bacteria have acquired virulence factors that subvert or disarm host defenses1. For example, cellular membranes provide a simple barrier to infection, but viruses such as influenza virus have evolved specific proteins that fuse with membranes to allow viral replication inside host cells2. Alternatively, intracellular bacterial pathogens are taken up by phagocytic cells, but they then remodel cellular membranes to prevent their own degradation inside lysosomal compartments3. Cellular membranes are key interfaces for host resistance and prime targets for microbial virulence factors. We therefore performed large-scale profiling of palmitoylated proteins in phagocytic cells to identify membrane-associated proteins that contribute to immunity against microbial pathogens. We found that S-palmitoylation of interferon-induced transmembrane protein 3 (IFITM3) enhances its clustering in membranes and antiviral activity against influenza virus. Our results demonstrate that large-scale proteomic studies using chemical reporters of post-translational modifications can reveal unique regulatory mechanisms required for cellular resistance to infection.</p><p>Protein S-palmitoylation is a covalent fatty acid modification on cysteine residues that is essential for the proper targeting and function of many membrane-associated proteins4. Chemical methods with greater sensitivity than classical radiographic techniques have revealed new roles for palmitoylation5. Fatty acid analogs functionalized with azides or alkynes, termed chemical reporters, have enabled sensitive fluorescent detection and large-scale proteomic analysis of fatty-acylated proteins using bioorthogonal ligation methods, such as Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) (Fig. 1a)5. Fatty acid chemical reporters6,7 and acyl-biotin exchange methods8–11 have revealed a greater diversity of S-palmitoylated proteins in eukaryotic proteomes than was previously appreciated and suggest that many cellular pathways are regulated by protein S-palmitoylation. However, the palmitoylomes of phagocytic cells such as dendritic cells (DCs) have not been evaluated. DCs sense pathogen-associated molecular patterns, upregulate immunomodulatory genes and secrete cytokines to activate innate immune responses12. Furthermore, DCs are so-called professional antigen-presenting cells with unique abilities to phagocytose, process and present antigens to T cells13. The DC palmitoylome is therefore likely to include membrane-associated factors involved in innate or adaptive immunity to microbes.</p><!><p>To identify lipid-modified and membrane-associated proteins that may contribute to immune responses to microbial infections, we performed large-scale profiling of fatty-acylated proteins in the mouse DC line DC2.4 (ref. 14) using the palmitic acid chemical reporter alk-16 (ref. 7) and CuAAC (Fig. 1a). Alk-16 preferentially targets S-palmitoylated proteins6,7, whereas shorter alkynyl fatty acids selectively label N-myristoylated proteins7. In-gel fluorescence profiling of DC2.4 cell lysates reacted with an azide-functionalized fluorophore (az-rho, Supplementary Fig. 1a)7 demonstrated that a diverse repertoire of proteins are metabolically labeled by alk-16 (Fig. 1b). Cell lysates were then reacted with an azido-biotin cleavable affinity tag (az-diazo-biotin, Supplementary Fig. 1b)15 for enrichment of alk-16–labeled proteins with streptavidin beads, selective elution and gel-based proteomic identification by mass spectrometry (Supplementary Fig. 1c). Coomassie blue staining of proteins retrieved with streptavidin beads and sodium dithionite elution demonstrates the specificity of alk-16 and CuAAC labeling methods (Supplementary Fig. 1d). Proteins identified in three independent experimental runs (Supplementary Fig. 1d) were compiled and categorized into high- and medium-confidence lists on the basis of the number of assigned spectra, the fold increase above control samples and the number of experiments in which the protein was identified (Supplementary Tables 1 and 2). We selectively identified 157 proteins by alk-16 labeling as compared to control samples (Fig. 1c), with 60 and 97 proteins assigned to high- and medium-confidence lists, respectively (Supplementary Tables 1 and 2). Of these proteins, 52% of high- and 31% of medium-confidence hits have been reported using large-scale proteomic techniques with acyl-biotin exchange chemistry or chemical reporters in other cell types (Supplementary Tables 1 and 2)6,9,10. These proteins include calnexin, G protein subunits, transferrin receptor, N-ras, Lyn and CD9 (Fig. 1c and Supplementary Tables 1 and 2). Western blot analysis of alk-16–labeled and enriched DC2.4 cell lysates confirmed robust and specific recovery of calnexin (Supplementary Fig. 1e), the S-palmitoylated protein with the most alk-16 peptide spectral counts compared to control samples (Fig. 1c). Notably, IFITM3 (ref. 16) was among the candidate S-palmitoylated proteins associated with cellular responses to microbial infections (Fig. 1c).</p><!><p>Palmitoylation of IFITM3 was investigated further to confirm the high-confidence recovery of IFITM3 by alk-16 proteomics and to explore the potential role of palmitoylation in innate immunity. (Fig. 1c). IFITM3 is a predicted dual-pass transmembrane protein with four homologs, IFITM1, IFITM2, IFITM5 and IFITM6. These proteins range from 11 to 16 kDa, and homologs are present in diverse vertebrates ranging from humans to zebrafish. IFITM3 is commonly used as a marker for developing germ cells17, though its functional role is unclear18. Interaction of IFITM3 with the tetraspanins CD9 and CD81 was demonstrated in B cells, suggesting that this protein localizes to tetraspanin-enriched microdomains19. In addition to appearing in B cells, IFITM3 is also constitutively expressed in bone marrow, macrophages and some non-immune cells19. The IFITM3 promoter contains interferon-stimulated response elements that control its upregulation upon exposure to the antiviral cytokine interferon (IFN)-α20,21. Indeed, IFITM3 transcripts can be readily detected in unstimulated DC2.4 cells and are upregulated upon IFN-α activation (Fig. 2a). These results are consistent with our proteomic data suggesting that IFITM3 is expressed at basal levels in DC2.4 cells without additional stimulation (Fig. 1c). A report of IFITM1 antiviral activity against vesicular stomatitis virus suggested the IFITM protein family plays a role in cellular resistance to viral infections22. Notably, small interfering RNA–based screens of host factors involved in H1N1 influenza virus infection identified IFITM3 as a potent inhibitor of early influenza virus replication as well as of dengue and West Nile viruses16. The S-palmitoylation of IFITM3 and its potential roles in cellular resistance to virus infection have not been investigated.</p><p>IFITM3 amino acid sequence analysis indicates three transmembrane domain–proximal cysteine residues at positions 71, 72 and 105 as potential sites of S-palmitoylation (Fig. 2b). The membrane topology of IFITM3 depicted in Figure 2b is consistent with antibody staining of tagged N and C termini in nonpermeabilized cells16 as well as staining of the cytoplasmic domain in permeabilized cells19. Alk-16 labeling of HeLa cells transfected with hemagglutinin epitope (HA)–tagged IFITM3 followed by anti-HA immunoprecipitation, CuAAC with az-rho and in-gel fluorescence scanning indicated that IFITM3 is indeed fatty acylated (Fig. 2c). To hydrolyze alk-16–protein thioesters, half of the immunoprecipitated material was treated with neutral hydroxylamine (NH2OH). The resulting decrease in signal is consistent with S-palmitoylation of IFITM3 (Fig. 2c). The three cysteines present in IFITM3 were mutated singly or in combination to alanine and evaluated for alk-16 labeling. Mutation of cysteines 71 and 72 reduces alk-16 labeling, but dual mutation does not result in complete loss of palmitoylation (Fig. 2d). The Cys105 mutant also shows decreased alk-16 labeling, which is completely lost in the C71A C72A C105A triple mutant (palmΔ) (Fig. 2d). Bioinformatic analysis of available IFITM protein sequences revealed that the S-palmitoylated cysteine residues are present in most vertebrates including humans and zebrafish (Supplementary Fig. 2a). In fact, alk-16 labeling of human IFITM1, IFITM2 and IFITM3 suggests S-palmitoylation occurs on all three of these IFITM isoforms (Supplementary Fig. 2b). These results confirmed our alk-16 proteomic data from DC2.4 cells and demonstrated that IFITM3 is S-palmitoylated at three membrane-proximal cysteine residues.</p><!><p>We investigated the influence of S-palmitoylation on IFITM3 expression and distribution in HeLa cells. Immunofluorescence analysis revealed that IFITM3 is distributed into punctate clusters (Fig. 3a), whereas IFITM3-palmΔ showed more diffuse staining (Fig. 3a). Costaining of HA-IFITM3–transfected cells with other cellular markers suggests that IFITM3 localizes to the endoplasmic reticulum (ER) and is excluded from lysosomes, as shown by calreticulin and LAMP-1 staining, respectively (Fig. 3b). IFITM3 was also absent from early endosomes (EEA1 staining), Golgi (golgin-97 staining) and cholesterol-rich membranes (cholera toxin B staining) (Supplementary Fig. 3a). As IFITM3 has been shown to interact with the tetraspanin CD9 (ref. 19), we evaluated the distribution of HA-IFITM3 in cells expressing GFP-CD9 (Fig. 3b). IFITM3 partially colocalized with GFP-CD9 (Fig. 3b), another S-palmitoylated transmembrane protein (Supplementary Fig. 4a), in membrane compartments that are largely devoid of LAMP-1 staining (Supplementary Fig. 4b). Although IFITM3-palmΔ has a more diffuse staining pattern (Fig. 3a), in colocalization studies the lack of palmitoylation did not appear to grossly alter its cellular distribution in the ER or other cellular organelles (Supplementary Fig. 5). This suggests that palmitoylation controls clustering of IFITM3 in membranes rather than its trafficking to distinct cellular compartments. In summary, both IFITM3 and IFITM3-palmΔ primarily localize to the ER, where their clustering is controlled by S-palmitoylation.</p><!><p>We then determined whether S-palmitoylation was essential for IFITM3 antiviral activity toward influenza virus (H1N1, PR8 strain). Interestingly, infection experiments with pseudotyped viral particles previously demonstrated that the inhibitory activity of IFITM3 is specific to viral envelope glycoproteins, suggesting IFITM3 inhibits viral entry or internalization16. We therefore focused on early stages of influenza virus infection. HeLa cells transfected with HA-IFITM3 showed lower levels of influenza viral nucleoprotein (NP) mRNA expression 6 h after infection compared to vector-transfected cells, as measured by qRT-PCR (Fig. 4a). In contrast, viral NP production was mostly unaffected in cells transfected with HA-IFITM3-palmΔ (Fig. 4a), even though wild-type and palmitoylation-defective IFITM3 constructs were expressed at similar levels (Fig. 4a). To confirm these results, we evaluated influenza virus infection of cells by flow cytometry, measuring the protein levels of both HA-IFITM3 and influenza virus NP. Similar to the qRT-PCR results, cells expressing HA-IFITM3 had limited viral NP production 6 h after infection with influenza virus, whereas HA-IFITM3-palmΔ expression afforded comparable amounts of viral NP levels (upper right quadrant of flow cytometry plots) compared to control and non-transfected cells in the same cell culture (Fig. 4b and Supplementary Fig. 6). The observed antiviral activity of IFITM3 upon overexpression in mammalian cells against influenza virus is comparable to the previously reported decrease in viral HA, M2 and NP and is consistent with the cell-autonomous activity of IFITM proteins that was observed using small interfering RNA knockdown or deletion of IFITM3 (ref. 16). The antiviral activity of IFITM3 was also not a general effect of S-palmitoylated membrane protein overexpression, as transfection of cells with the tetraspanin CD9 had no significant influence on influenza virus replication compared to controls (Fig. 4b). The S-palmitoylation–dependent antiviral activity of IFITM3 was also observed at higher multiplicity of infection with influenza virus (Supplementary Fig. 7).</p><p>We also evaluated S-palmitoylation–dependent IFITM3 antiviral activity in HEK293T cells that are more susceptible to influenza virus infection. In accord with our data in HeLa cells (Fig. 4a,b), HEK293T cells expressing HA-IFITM3 (high) inhibited the influenza virus infection compared to non-transfected cells (low) as judged by flow cytometry analysis of influenza NP expression (Supplementary Fig. 8 and Fig. 4c). This antiviral activity was markedly diminished with the S-palmitoylation–deficient HA-IFITM3 construct and not observed for GFP-CD9 (Supplementary Fig. 8 and Fig. 4c). Similar results were also observed using antibody staining to the NS1 protein, another protein produced by influenza virus (Fig. 4c). Replicate experiments demonstrate that HA-IFITM3 expression in HeLa and HEK293T cells inhibits influenza virus infection approximately two-fold compared to non-transfected cells (Fig. 4c). These results are comparable to the two- to fivefold antiviral activity previously observed using A549 cells transduced with retroviral constructs expressing human IFITM isoforms16. The quantitative differences between our data and those published for A549 cells may be due to mouse and human IFITM isoform differences, location of epitope tags, analysis of different viral proteins, methods of overexpression and/or cell type analyzed. Nonetheless, our data demonstrate that mammalian cells expressing higher levels of IFITM3 are more resistant to influenza infection (Fig. 4). This antiviral activity of IFITM3 is dependent on S-palmitoylation, as mutation of modified cysteine residues to alanine abrogates more than 75% of this inhibitory activity (Fig. 4d). Overall, these results indicate that S-palmitoylation of IFITM3 is crucial for its full activity against influenza virus infection.</p><!><p>Palmitoylome profiling has revealed a post-translational mechanism important for host defense against viral infections. We discovered that S-palmitoylation of membrane-proximal cysteines on IFITM3 enhances its clustering in membranes and is crucial for its inhibitory activity toward influenza virus infection. Notably, S-palmitoylation does not appear to influence the protein levels of IFITM3, as judged by western blot, immunofluorescence and flow cytometry analyses (Figs. 2–4). In contrast to other S-palmitoylated transmembrane proteins such as LRP6 (ref. 23) or the anthrax toxin receptor24, for IFITM3 S-palmitoylation does not appear to grossly regulate its stability or trafficking but rather induces a clustering effect similar to that observed for tetraspanins25.</p><p>As IFITM3 shows specificity toward viral envelope proteins16 that are often themselves S-palmitoylated26–28 and clustered for membrane fusion with host cells2, S-palmitoylation may therefore be required for a multivalent display of these IFN effectors to block the activity of viral envelope proteins such as influenza virus hemagglutinin. Cysteine residues homologous to the S-palmitoylated residues found in mouse IFITM3 are present in IFITM protein isoforms in most vertebrates from humans to zebrafish, suggesting an evolutionarily conserved function for protein S-palmitoylation in innate immunity against microbial pathogens. In contrast to the phenomenon of viruses co-opting host palmitoylation machinery for infection26–28, S-palmitoylation of an IFN effector, IFITM3, restricts viral infection of host cells. Specific protein lipidation pathways may therefore be prime targets of microbial immune evasion strategies. The identification of S-palmitoylation–dependent IFITM3 clustering and antiviral activity should help elucidate the mechanism by which this family of IFN effectors inhibits virus replication and thus aid in the design of therapeutics targeted at pathogens such as influenza virus.</p><!><p>HeLa, HEK293T and DC2.4 cells were grown in DMEM with 10% (v/v) FBS. Cells were transfected using Lipofectamine 2000 (Invitrogen). Influenza A virus A/PR/8/34 (H1N1) was propagated in 9-d embryonated chicken eggs and titrated using MDCK cells. For flow cytometry, cells were fixed with PBS and 3.7% (w/v) paraformaldehyde, permeabilized with PBS and 0.2% (w/v) saponin and blocked with PBS and 2% (v/v) FBS. Cells were then incubated with anti-HA antibody (1/1,000, 16B12, Covance), washed three times and stained with goat anti-mouse antibody conjugated to Alexa-488 (1/1,000, Invitrogen). Viral protein was stained using mouse monoclonal antibodies against viral NP (clone HT103)29 directly conjugated to Alexa-568 or a mouse monoclonal antibody against viral NS1 (clone 1a7 (ref. 30), provided by J. Yewdell, US National Institutes of Health) conjugated to Alexa-647. Results were analyzed with FlowJo software.</p><!><p>Metabolic labeling of DC2.4 or HeLa cells with alk-16 or DMSO control was performed in DMEM and 2% (v/v) charcoal-filtered FBS. Chemical syntheses of alk-16 (ref. 7), az-rho7 and az-diazo-biotin15 have been reported previously. Alk-16–labeled cells were lysed with 0.1 mM triethanolamine (TEA) buffer containing 1% (w/v) Brij97 and Complete EDTA-free protease inhibitor cocktail at 5× concentration (Roche). We diluted 50 μg of protein from the alk-16–labeled and DMSO-treated samples to 34.5 μl with TEA and 1% (w/v) Brij 97 buffer. We added 10 μl 10% (w/v) SDS in H2O as well as CuAAC reactants including 1 μl of 5 mM az-rho (100 μM final concentration), 1 μl of freshly prepared 50 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (final concentration 1 mM), 2.5 μl 2 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (final concentration 100 μM) and 1 μl 50 mM CuSO4·5H2O (final concentration 1 mM), for a final volume of 50 μl. CuAAC reactions were allowed to proceed for 1 h at room temperature, and a chloroform-methanol precipitation was performed before SDS-PAGE. For immunoprecipitations, 400 μg of cell lysate was added to 15 μl of anti-HA antibody–conjugated agarose (Sigma) in a total volume of 150 μl and rocked at 4 °C for 1 h. Agarose was washed and resuspended in 44.5 μl 0.1 mM TEA and 4% (w/v) SDS, and CuAAC reactants were added as described above. In-gel fluorescence scanning was performed using a Typhoon 9400 imager (Amersham Biosciences). Western blots for HA-tagged proteins were performed using an anti–HA tag polyclonal rabbit antibody (1/1,000, 631207, Clontech).</p><p>For proteomic experiments, 5 mg of protein from DC2.4 cells were used. CuAAC reactants were added at the same concentrations as listed above except az-diazo-biotin was substituted for az-rho. Methanol-precipitated and washed protein pellets were resuspended in 1 ml of 0.1 mM TEA, 4% (w/v) SDS and 10 mM EDTA. Equal protein amounts were diluted 1/3 by volume with TEA and 1% (w/v) Brij97 buffer. We added 100 μl prewashed streptavidin agarose beads (Invitrogen) in TEA and 1% (w/v) Brij97 buffer to each sample. The protein and bead mixtures were incubated for 1 h at room temperature on a nutating mixer. The beads were then washed once with PBS and 0.2% (w/v) SDS, three times with PBS and twice with 250 mM ammonium bicarbonate (ABC). Beads were resuspended in 500 μl 8 M urea, and we added 25 μl 200 mM TCEP and 25 μl 400 mM iodoacetamide for capping of reactive cysteine residues. After 30 min, beads were washed twice with 250 mM ABC. Two elutions of proteins from beads were then performed using 250 μl of 25 mM sodium dithionite in 250 mM ABC containing 0.1% (w/v) SDS. Protein was concentrated using a YM-10 Centricon (Millipore). Samples were then subjected to SDS-PAGE and staining with Coomassie blue. DMSO and alk-16 lanes of the gel were then cut for trypsin digestion and peptide extraction. Extracted peptides were dried and resuspended in H2O and 0.1% (v/v) trifluoroacetic acid for mass spectrometry.</p><!><p>LC-MS analysis was performed with a Dionex 3000 nano-HPLC coupled to an LTQ-Orbitrap ion trap mass spectrometer (ThermoFisher). Peptides were pressure-loaded onto a custom-made 75-μm–diameter, 15-cm C18 reverse-phase column and separated with a gradient running from 95% buffer A (HPLC water with 0.1% (v/v) formic acid) and 5% buffer B (HPLC-grade CH3CN with 0.1% (v/v) formic acid) to 55% B over 30 min, next ramping to 95% B over 10 min and holding at 95% (v/v) B for 10 min. One full MS scan (300–2000 MW) was followed by three data-dependent scans of the nth most intense ions with dynamic exclusion enabled. Peptides from three independent experiments were identified using SEQUEST version 28 and were searched against the mouse International Protein Index (IPI) protein sequence database v3.45. Scaffold software (Proteome Software) was used to compile data from the three experimental runs.</p><!><p>For determination of IFITM3 localization, transfected HeLa cells were fixed with 3.7% (w/v) paraformaldehyde, permeabilized with 0.2% (w/v) saponin or 1% (v/v) Tween and blocked with 2% (v/v) FBS in PBS. Cells were incubated with individual antibodies for 30 min in sequence starting with mouse anti-HA (1/1,000, 16B12, Covance), then goat anti-mouse antibodies conjugated to rhodamine red (1/1,000, Invitrogen), then rabbit antibodies against specific cellular markers or cholera toxin B conjugated to Alexa-488 (Invitrogen) followed by secondary goat anti-rabbit antibodies conjugated to Alexa-488 (1/1,000, Invitrogen). All antibodies were diluted in PBS and 0.2% (w/v) saponin, except for cholera toxin B staining in which PBS and 1% (v/v) Tween 20 was used in all steps. Antibodies against calreticulin (1/500, ab2907, Abcam), LAMP1 (1/500, ab24170, Abcam), EEA1 (1/100, 2411, Cell Signaling) and golgin-97 (1/100, ab33701, Abcam) were used. Cells were incubated with TOPRO-3 (1/1,000, Invitrogen) as a final step.</p><!><p>IFITM3 and CD9 DNA sequences were amplified by PCR using cDNA prepared from DC2.4 RNA. PCR primers for IFITM3 and CD9 as well as mutagenesis primers for IFITM3 are listed in Supplementary Table 3. DC2.4 cells were treated with 500 ng ml−1 IFN-α2 (eBioscience) for 4 h to determine its effect on IFITM3 expression. Reverse transcription and quantitative PCR were performed using a previously described protocol31,32. Primers used for quantitative PCR can be found in Supplementary Table 4. The topology diagram of IFITM3 found in Figure 2b was drawn using TOPO2 transmembrane protein display software (http://www.sacs.ucsf.edu/TOPO2/).</p><!><p>(a) Metabolic labeling of cells with alk-16 palmitate reporter and subsequent CuAAC ligation with bioorthogonal detection tags for imaging or proteomics. (b,c) DC2.4 cells were incubated for 2 h with 50 mM alk-16 or DMSO as a control. In b, cell lysates were reacted with az-rho by CuACC, and proteins were separated by SDS-PAGE for visualization by fluorescence gel scanning. Coomassie blue staining demonstrates comparable loading. In c, cell lysates were reacted with az-diazo-biotin by CuAAC for enrichment of alk-16–labeled proteins with streptavidin beads and identification by mass spectrometry. For each identified protein, the ratio of peptide spectral counts from the alk-16 and DMSO samples was plotted. Several known palmitoylated proteins are shown in black. IFITM3, the highest-ranked candidate palmitoylated protein, is shown in red.</p><!><p>(a) Ifitm3 mRnA expression in DC2.4 cells after 4 h IFn-α treatment as measured by qRt-PCR. NT, non-treated. (b) predicted topology of IFITM3 showing the location of cysteine residues (gray shading) with respect to transmembrane domains. (c,d) HeLa cells were transfected with 2 μg vector, pCMV-HA-IFITM3 wild-type (WT) or cysteine mutants in six-well plates overnight and labeled with 50 μM alk-16 for 2 h. Cell lysates were subjected to anti-HA immunoprecipitation, reacted with az-rho by CuAAC, separated by SDS-PAGE and visualized by fluorescence gel scanning. Comparable protein loading was confirmed by anti-HA western blotting. In c, half of the indicated immunoprecipitations were treated with 2.5% (w/v) neutral NH2OH before boiling and gel loading to hydrolyze protein–alk-16 thioester linkages.</p><!><p>(a,b) HeLa cells grown on coverslips in 12-well plates were transfected with 1 μg pCMV-HA, pCMV-HA-IFITM3 or pCMV-HA-IFITM3-palmΔ and stained with anti-HA (red) and TOPRO-3 (blue). Insets are enlargements of the white-squared regions. Scale bars represent 10 μm. (b) Cells were also stained with antibodies against cellular markers, calreticulin and LAMP1 (green), or were co-transfected with GFP-CD9 (green).</p><!><p>(a) HeLa cells grown in six-well plates were transfected with 2 μg pCMV-HA, pCMV-HA-IFITM3 or pCMV-HA-IFITM3-palmΔ and infected with influenza virus (PR8 strain) with multiplicity of infection (MOI) of 1 for 6 h. ND, not detected; NI, non-infected. Influenza virus NP mRNA levels and Ifitm3 expression levels were examined by qRT-PCR. *P = 0.027 by Student's t-test; error represents s.d., n = 3. (b) HeLa cells grown in 12-well plates were transfected with 1 μg pCMV-HA, pCMV-HA-IFITM3, pCMV-HA-IFITM3-palmΔ or pEGFP-C1-CD9 and infected with influenza virus at an MOI of 1 for 6 h. Virus NP and IFITM3 protein levels were examined by flow cytometry using anti-NP and anti-HA staining, respectively. (c) HEK293T cells grown in 12-well plates were transfected with 1 μg pCMV-HA, pCMV-HA-IFITM3, pCMV-HA-IFITM3-palmΔ or pEGFP-C1-CD9 and infected with influenza virus at an MOI of 1 for 6 h. Non-transfected and transfected cells expressing the proteins of interest from the same culture were gated on (labeled "low" and "high," respectively) and analyzed for the percentage of these cells that were infected (Supplementary Fig. 8). Data from flow cytometric analysis of cells staining positive for influenza NP or NS1. (d) percentages in c were normalized such that the difference in infection rates for vector control and HA-IFITM3 high was set at 100% antiviral activity. Error in c and d represents s.d., n = 4. Note: the HA-tag epitope is derived from an H3 influenza virus strain and is not present in the PR8 strain of H1N1 influenza virus.</p>

PubMed Author Manuscript

Aggregation-free and high stability core–shell polymer nanoparticles with high fullerene loading capacity, variable fullerene type, and compatibility towards biological conditions

Fullerenes have unique structural and electronic properties that make them attractive candidates for diagnostic, therapeutic, and theranostic applications. However, their poor water solubility remains a limiting factor in realizing their full biomedical potential. Here, we present an approach based on a combination of supramolecular and covalent chemistry to access well-defined fullerene-containing polymer nanoparticles with a core-shell structure. In this approach, solvophobic forces and aromatic interactions first come into play to afford a micellar structure with a poly(ethylene glycol) shell and a corannulene-based fullerene-rich core. Covalent stabilization of the supramolecular assembly then affords core-crosslinked polymer nanoparticles. The shell makes these nanoparticles biocompatible and allows them to be dried to a solid and redispersed in water without inducing interparticle aggregation.The core allows a high content of different fullerene types to be encapsulated. Finally, covalent stabilization endows nanostructures with stability against changing environmental conditions.

aggregation-free_and_high_stability_core–shell_polymer_nanoparticles_with_high_fullerene_loading_cap

Introduction<!>Molecular design<!>Fullerene loading<!>Structural integrity of the nanoparticles<!>Radical scavenging activity<!>Protein adsorption<!>Hemolysis<!>Conclusions<!>Experimental details<!>Crosslinking of micelles<!>Conflicts of interest

<p>Fullerenes are a family of carbon cages. They are characterized by high electron affinities, reactive exteriors, and inert interiors. [1][2][3][4] The rst two characteristics enable them to quench reactive oxygen species, which are considered to be mediators of oxidative stress and cause for numerous chronic and acute diseases. 5 The interior space can entrap metal atoms such as gadolinium to assist in magnetic resonance imaging. 6 This approach of conning the active and toxic metal atoms to the carbon cage allows overcoming the stability and toxicity issues associated with the gadolinium chelates typically used for imaging purposes. 7 However, despite great potential, the applications of fullerenes in biomedical sciences remain rather limited due to their poor water solubility. The general approaches to enhance their water solubility include covalent modication of the exterior and supramolecular complexation with a variety of hosts (Fig. 1). [8][9][10][11][12][13][14][15][16][17] The former suffers from unknown toxicological proles of new fullerene derivatives and complex synthesis involving regio-and stereoisomers. Although Fig. 1 The most common strategies for preparing water-soluble fullerenes involve supramolecular complexation with a polymer chain or covalent modification of the carbon scaffold (top). The attributes of a fullerene-containing core-shell nanoparticle morphology as explored in this work (bottom).</p><p>great strides have been made recently with the help of 'click' chemistry, which allows for multi-fullerene molecules to be prepared by multi-step organic synthesis. 18 The latter approach suffers from low fullerene loading capacity ($1 wt%) and reduced in vivo stability of the noncovalent constructs. Interestingly, C 70 is shown to be a better candidate in terms of biological applications. 19 However, studies involving C 70 are scarce due to an even higher restriction on access to water-soluble larger fullerene structures. To address these issues, we designed a covalently stabilized block copolymer micellar system in which (i) host-guest interactions in the micellar core enable a high (8 wt%) fullerene loading capacity (Scheme 1), (ii) the exibility of the host units and their multiple numbers allow smaller (C 60 ) and larger (C 70 ) guest surfaces to be encapsulated irrespective of their sizes (Fig. 2), (iii) core-crosslinking renders the structures robust against changing environmental conditions, and (iv) a well-dened PEG-based shell provides solvation in water, hemocompatibility, and protection from unfavorable interparticle and protein-particle interactions. A preliminary property study is also carried out, which indicates that C 70 nanoparticles are more active than C 60 nanoparticles towards free radical scavenging in aqueous solutions. This observation further reinforces the idea that larger fullerenes offer superior biorelevant properties.</p><!><p>Corannulene (C 20 H 10 ) can be considered a fragment of fullerene C 60 . 20 However, its tour-de-force synthesis by Barth and Lawton predates the discovery of fullerenes by nearly two decades. 21 Since then, many properties of this fascinating molecule have been studied, including its capacity to host fullerene C 60 through complementarity of the curved p-surfaces. 23 To introduce this motif into a polymer chain, a post-polymerization modication strategy was developed (Scheme 2). For this, initially, an atom transfer radical polymerization of glycidyl methacrylate (GMA) monomer through a poly(ethylene glycol) (PEG)-based macroinitiator (PEG-Br) (M n(GPC) ¼ 8600, M w /M n ¼ 1.1) was carried out to access the reactive block copolymer (PEGb-PGMA) scaffold (M n(GPC) ¼ 12 700, M w /M n ¼ 1.2) (ESI Fig. S1 †). Area integration analysis in 1 H NMR spectroscopy indicates that the degree of polymerization for the GMA block was approximately 17 repeating units (ESI Fig. S2 †). The ring opening reaction between the epoxide group of PGMA and mercaptocorannulene then afforded host polymer 1 (M n(GPC) ¼ 17 800, M w /M n ¼ 1.2). 1 H NMR spectroscopy indicated that the postsynthesis modication reaction was quantitative; therefore, Scheme 1 Chemical structure of host polymer 1 encoded with corannulenes and its assembly with fullerenes through ball-and-socket interactions between concave and convex aromatic surfaces to furnish micellar nanoparticles.</p><p>Fig. 2 The hosting polymer block contains many corannulene units attached to the polymer backbone through a linker that can adjust upon receiving the guest molecule. These features allow accommodating different fullerene sizes in the micellar core.</p><p>approximately 17 corannulene units were incorporated into each diblock copolymer chain. This synthetic strategy was chosen because the ring opening reaction produces a reactive hydroxyl group that could be used for the core-crosslinking reaction once the amphiphilic block copolymer assembles into a micellar nanostructure. The choice of PEG as the shell component was due to its ability to avoid fouling with proteins and to impart water solubility and prolonged blood circulation kinetics to the nanoparticles. 24 Micelle formation and covalent stabilization 25 Above a certain concentration, block copolymers carrying hydrophilic and hydrophobic segments can assemble into a micellar structure in a solvent that selectively solubilizes only one of the polymer blocks. 26 The concentration is referred to as the critical micelle concentration (CMC). Micelles can be produced with different morphologies and functions. However, their supramolecular nature makes them susceptible to changing environmental conditions. For example, a solvent that can solubilize both polymer blocks can dissolve the supramolecular structure. In the context of biological applications, shear forces under ow conditions are also expected to disrupt such noncovalent assemblies. Crosslinking of the core or shell is required to stabilize the assembled structure against any changes in solvent, concentration, or temperature. This path leads to robust nanoparticles. 25,[27][28][29][30][31] To study micelle formation from polymer 1 under aqueous conditions, the uorescence emission of corannulene was monitored at 465 nm. As the polymer chains begin to assemble, the emission signal intensity decreases due to the aggregation-induced self-quenching process. 32 These data allows the determination of the CMC and indicate that at concentrations above 12 mg mL À1 , polymer 1 forms micellar structures (Fig. 3). Dynamic light scattering (DLS) studies were undertaken to investigate the micellar sizes. In dimethylformamide (DMF), a good solvent for both polymer blocks, a hydrodynamic diameter of <1 nm indicated individual polymer chains in the solution. In water, a solvent preferential for the poly(ethylene glycol) block, larger structures with an average hydrodynamic diameter of 37-38 nm could be observed, indicating micellar assembly. These micelles could be stabilized through crosslinking of the hydroxyl groups present in the core with adipic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) as the carboxyl activating agent and dimethylaminopyridine (DMAP) as the catalyst for the formation of the ester bonds. Upon crosslinking of the core, the micellar size decreased slightly (31-32 nm). This is most likely due to crosslinking-induced shrinkage of the core.</p><p>Since, such an intermolecular crosslinking process requires a very low fraction of the reactive sites, and the polymer chains contained the ester groups already (before formation of new ester bonds upon core-crosslinking), typical characterization tools such as NMR and IR spectroscopies were found to be of no help in characterizing the crosslinked structure. However, the success of the crosslinking process could be veried by dissolution of the micelles in DMFa solvent that is good for both polymer blocks and therefore capable of solubilizing the supramolecular micellar assembly into individual polymer chains (Fig. 3). Upon crosslinking, the micelles could be dispersed in DMF without destroying the nanostructure (Fig. 3). This indicated that the crosslinking process was successful in covalently stabilizing the micellar structure. However, larger particle sizes were observed (82-83 nm), presumably due to swelling of the crosslinked core in DMF.</p><!><p>Two different approaches were examined to incorporate fullerenes into the micellar structures (Fig. 4). In the rst approach, preformed micelles were exposed to C 60 /C 70 through sonication in water. Crosslinking was then performed to lock the C 60 /C 70 that entered into the core. Alternatively, polymer and C 60 /C 70 were mixed together in tetrahydrofuran, and then water was added to allow the formation of micelles. Finally, the supramolecular structure was crosslinked. In the second approach, the polymer chains can interact with the fullerenes before formation of the secondary structure. To study the loading capacity arising from these different preparation methods, UV-Vis spectroscopy was used (ESI Fig. S3 †). In water, the molar absorption coefficient (3) of C 60 is 49 000 at 340 nm, and 34 000 for C 70 at 384 nm. 15 This allows a comparison to be made and calculation of the concentration of fullerenes in the crosslinked nanoparticles to be performed. These data indicated that the rst approach resulted in the incorporation of approximately 1.22 wt% fullerenes into the particle core. The second approach, however, was superior and furnished 8.12 and 8.01 wt% C 60 and C 70 , respectively, into the nanoparticles.</p><p>To investigate the supramolecular complexation between fullerenes and corannulene through interaction of their curved surfaces, 13 C-enriched fullerenes were utilized, and the crosslinked nanoparticles were studied with the help of 13 C NMR spectroscopy (Fig. 5). In the case of C 60 nanoparticles, an upeld shi of 1.4 ppm (d ¼ 142.8 free C 60 ; d ¼ 141.4 complexed C 60 ), indicative of aromatic shielding effect and consistent with the previously reported supramolecular fullerene complexes, 33 was observed in the 13 C resonance of C 60 . To rule out any unspecic interactions as the cause for this shi, an external standard tube (of larger diameter than that of the original NMR tube) containing a pure solution of C 60 in deuterated tetrachloroethane was used. These samples showed signals from the uncomplexed fullerene (present as an external standard) and the signal from the C 60 nanoparticles also in deuterated tetrachloroethane. These data conrmed that the shi of 1.4 ppm was due to specic complexation of C 60 with corannulenes in the nanoparticle core. A broadening of the C 60 signal was also observed, possibly as a result of the restricted rotational freedom of complexed C 60 molecules in the nanoparticle core.</p><p>Unlike C 60 , which possesses only one type of carbon atom, C 70 possesses 5 different types of carbons. Furthermore, the commercially obtained 13 C-enriched C 70 sample was found to be contaminated with C 60 . Therefore, the 13 C NMR displayed a total of 6 signals from a mixture of fullerenes (ESI Fig. S4 †). The same external standard technique was employed to conrm the signal shi. This study indicated that in the nanoparticles, the signals shied by approximately 1, 1.2, 1.5, 1.1, and 1.1 ppm upon C 70 complexation. The signals were also relatively broad. These results again indicated that the fullerenes were localized in the particle core. Interestingly, when the two fullerenes are part of the same nanoparticle core, the shi in the C 60 signal is nearly half (0.7 ppm) that when there is no competition from C 70 . This indicates that C 70 is a preferred guest and binds more strongly to the multicorannulene host. In DLS, the Fig. 4 Different approaches for the encapsulation of fullerenes into the host polymer micelle. The top shows exposure of pre-formed micelles to fullerenes, which leads to low fullerene loading capacity. The bottom shows an alternative approach in which the micelles are allowed to form in the presence of fullerenes leading to a higher fullerene loading capacity. nanostructure sizes increase when the core nests the fullerenes (Fig. 6). In transmission electron microscopy (TEM), these cores could be visualized without the need for staining the samples due to their high electron densities (Fig. 7 and ESI Fig. S5 †).</p><p>Finally, corannulene was replaced with a simple phenyl group in polymer 1 (Scheme 3). This effort was directed at examining the antithesis that fullerene encapsulation in the micellar core required only a hydrophobic atmosphere and simple aromatic groups for stabilization. For this, the ring opening reaction was carried out using thiophenol to give polymer 2 (ESI Fig. S6 †). Polymers 1 and 2 were obtained from the same PEG-b-PGMA scaffold. Hence, they contained identical number of repeating units in the polymer chains (m z 113, n z 17) necessary for comparison. Polymer 2 successfully formed micelles in water (ESI Fig. S7 †). Importantly, however, it failed to show any fullerene uptake in its micellar core (ESI Fig. S8 †).</p><p>The 13 C NMR and UV-Vis spectroscopies, therefore, suggest that the specic convex-concave interactions between fullerenes and corannulene are key to encapsulating a large amount of fullerenes in the nanoparticle core.</p><!><p>Typically, aqueous dispersions of fullerene nanoparticles are prone to aggregation in the drying process, thereby losing their structural integrity. In the current molecular design, the particle shells are noncrosslinked and provide a molecularly dened layer of solvation (hydration) and protection to the crosslinked core against changes in concentration. The removal of the solvent and a redispersion therefore do not lead to any adverse effect on the integrity of the nanoparticles. This can be observed by drying the nanoparticulate matter and then redissolving it in water. The hydrodynamic volume before and aer this process remains unchanged (ESI Fig. S9-S11 †). This characteristic allows nanoparticles to be stored and transported in a powdered form. The aqueous solution can be prepared when required. Furthermore, once the solution is prepared, no changes are observed in the particle sizes even for months aer their dissolution (ESI Fig. S12 †).</p><p>Encouraged by these results, the thermal stability of the cargo was studied with the help of 13 C NMR spectroscopy (Fig. 8). The goal of these experiments was to heat the nanoparticles and determine whether uncomplexed fullerene could be detected in solution. If so, it would indicate that the guestloaded core is unstable at higher temperatures and releases the guest molecules into the solution. In these experiments, an external standard was not employed to avoid overlap between the free fullerene signals. Initially, the C 60 -containing nanoparticles were heated at 60 C for 24 hours, and no signal belonging to the free fullerene could be observed in the solution. Therefore, the temperature was increased to 100 C, and the sample was heated for 70 hours. In this case also, the complexed fullerene signal remained intact.</p><p>Finally, nanoparticle stability was evaluated against acidic and basic conditions. For this, nanoparticles were dissolved in PBS (pH ¼ 7.4) and citric acid/Na 2 HPO 4 (pH ¼ 2.5) buffers and monitored with the help of DLS. The nanoparticles did not display any change in their diameter under either conditions (ESI Fig. S13 †).</p><p>The ability to switch from bulk materials to aqueous solution and withstand demanding thermal/pH conditions relates to the core-shell morphology of the nanostructures and is unprecedented in the fullerene-based water-soluble nanoparticle arena.</p><!><p>Having access to fullerene nanoparticles, their free radical scavenging activity was assessed with the help of a 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. 34 In this assay, the interaction between an antioxidant and the pregenerated ABTS radical cation was examined with the help of UV-Vis spectroscopy (Fig. 9). Upon successful quenching of the radical cation, a decrease in the absorption intensity of the signals located at 645, 734, and 815 nm could be observed. The amount of antioxidant required to quench this intensity by 50% is dened as the inhibition concentration 50 (IC 50 ). This study produced interesting results. The micelles without C 60 show moderate antioxidant capacity (Fig. 9a), most likely due to (b). Polymer nanoparticles with fullerene C 70 (c). A comparison of the three systems to evaluate the amount needed to quench 50% of the free radicals in the system (d).</p><p>corannulenes in the micellar core (IC 50 $ 1000 mg mL À1 ). The presence of C 60 enhanced this capacity (IC 50 ¼ 529 mg mL À1 ) (Fig. 9b). In the case of C 70 , the visual color changes could already be appreciated beginning at 100 mg mL À1 (Fig. 9c). In this system, to reach IC 50 , a relatively lower amount of 384 mg mL À1 was required. C 70 -containing nanoparticles, therefore, are the most active system in this regard (Fig. 9d).</p><!><p>This aspect was studied with the help of bovine serum albumin (BSA) and the Bradford method to quantify the amount of the adsorbed protein onto the nanoparticles. 35 In this method, binding of the protein to a dye molecule changes its absorbance from red to blue region (ESI Fig. S14 †). If no protein binds, then the solution remains brown. Thus, the amount of complex present in solution is a measure of the protein concentration and can be estimated by use of an absorbance reading. To achieve this, the particles were incubated with BSA for 3 hours to achieve equilibration. Aer this time, the particles were removed from the solution by centrifugation, and the supernatant was examined by means of UV-Vis spectroscopy (595 nm) to quantify the amount of the 'free' (unadsorbed) protein. In this way, the amount of adsorbed protein was calculated to be 3.5 and 3.8% for nanoparticles containing C 60 and C 70 , respectively.</p><!><p>As a measure of biocompatibility, a hemolysis assay was performed to assess the lytic activity of nanoparticles using sheep red blood cells (RBCs). In this study, the released hemoglobin was detected by means of UV-Vis spectroscopy at a wavelength of 540 nm and compared to positive and negative controls. The negative control was PBS buffer, in which the RBCs do not disintegrate owing to the isotonic effect and a net zero movement of molecules across the membrane. 36 The positive control was deionized water, in which RBCs disintegrate fully due to a hypotonic effect and movement of pure water into the cells through osmosis, resulting in bursting of the cell walls. In the case of nanoparticles, however, no hemolysis was observed even at a concentration of 1000 mg mL À1 (ESI Fig. S15 †). This suggests that nanoparticles have no toxic effects on mammalian cell walls, which bodes well for their future applicability as diagnostic probes.</p><!><p>In summary, core-crosslinked polymer nanoparticles can be prepared by combining a supramolecular complexation and covalent stabilization strategy. The particles contain a high loading (8%) of smaller (C 60 ) and larger (C 70 ) fullerenes and provide an opportunity to compare their properties. Radical scavenging leads to the conclusion that C 70 particles possess higher antioxidant activity (IC 50 ¼ 384 mg mL À1 ). The particles can be stored in a powdered form in bulk and can be redispersed when required. They are stable even when heated to 100 C for 70 hours. They resist the accumulation of proteins (<4%) and are nonhemolytic even at high concentrations (1000 mg mL À1 ). It is anticipated that replacing normal fullerenes from the present design with endohedral fullerenes containing a contrast agent, such as Gd(III), will lead to nontoxic imaging probes with long circulation times due to their nanometer sizes and a poly(ethylene glycol) shell. Furthermore, the preparation of water-soluble nanoparticles with higher fullerenes 37 (e.g., C 76 and C 84 ) and the study of their biorelevant properties appear to be an enticing research direction.</p><!><p>Micelle formation First approach. Diblock copolymer 1 (3 mg) was rst dissolved in THF (150 mL). Aer adding this suspension dropwise into deionized water (3 mL) under stirring, the micellar solution was dialyzed (dialysis tube: cutoff 1 kDa) against deionized (DI) water for 2 days to remove the THF. The nal polymer concentration was about 1 mg mL À1 . Fullerene (1 eq. per corannulene unit) was added into the micellar solution (3 mg mL À1 ) and sonicated for 30 minutes. The solution was then ltered through a 0.8 mm syringe lter (cellulose acetate).</p><p>Second approach. Diblock copolymer 1 (3 mg) and fullerene (1 eq. per corannulene unit) were rst suspended in THF (150 mL) and stirred for 30 minutes. Aer adding this suspension dropwise into deionized (DI) water (3 mL) under stirring, the micelle solution was then ltered through a 0.8 mm syringe lter (cellulose acetate). The mixture was dialyzed (dialysis tube: cutoff 1 kDa) against deionized water for 2 days to remove the THF.</p><!><p>To a solution of adipic acid (0.15 mg, 1 mmol (0.5 eq. per hydroxyl unit)), EDCI (0.5 mg, 0.2 mmol) and DMAP (0.3 mg, 0.2 mmol) in DI water (50 mL) were added dropwise into the micellar solution (1 mg mL À1 ). The reaction mixture was stirred at room temperature overnight. Aer this time, the reaction mixture was dialyzed (dialysis tube: cutoff 1 kDa) against DI water for 1 day. The crosslinked micelles were lyophilized to dryness.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Discovery of chemical markers for improving the quality and safety control of Sinomenium acutum stem by the simultaneous determination of multiple alkaloids using UHPLC-QQQ-MS/MS

Sinomenium acutum stem is a popular traditional chinese medicine used to treat bone and joint diseases. Sinomenine is considered the only chemical marker for the quality control of S. acutum stem in mainstream pharmacopeias. However, higenamine in S. acutum stem is a novel stimulant that was banned by the World Anti-Doping Agency in 2017. Therefore, enhancing the quality and safety control of S. acutum stem to avoid potential safety risks is of utmost importance. In this study, a fast, sensitive, precise, and accurate method for the simultaneous determination of 11 alkaloids in S. acutum stem by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHpLc-QQQ-MS/MS) was established. this method successfully analyzed thirty-five batches of S. acutum stem samples. The average contents of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine were 24.9 mg/g, 6.35 mg/g, 435 μg/g, 435 μg/g, 288 μg/g, 44.4 μg/g, 22.5 μg/g, 21.1 μg/g, 15.8 μg/g, 9.30 μg/g, and 8.75 μg/g, respectively. Multivariate analysis, including principal component analysis (PCA), orthogonal partial least square method-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA), were performed to characterize the importance and differences among these alkaloids in S. acutum stem samples. As a result, sinomenine, magnoflorine, coclaurine, acutumine, and higenamine are proposed as chemical markers for quality control. Higenamine and coclaurine are also recommended as chemical markers for safety control. This report provides five alkaloids that can be used as chemical markers for improving the quality and safety control of S. acutum stem. it also alerts athletes to avoid the risks associated with consuming S. acutum stem. Abbreviations EIC Extracted ion chromatogram ESIElectrospray ionization

discovery_of_chemical_markers_for_improving_the_quality_and_safety_control_of_sinomenium_acutum_stem

<!>Discussion<!>Materials and methods<!>Standard solution preparation.

<p>The precision was determined by continuously testing one concentration of the mixed standard solution six times. Sample Q29 was separated into six portions, and each portion was extracted separately to evaluate the repeatability of this method. The test solution of sample Q29 was employed to evaluate the stability over 0, 2, 4, 6, 8, and 12 h in one day at room temperature. The results for all the compounds, which are summarized in Table 2, indicate that the instrument has good precision, the method is repeatable, and the compounds in the sample solution are sufficiently stable for accurate and precise analysis within 12 h at room temperature (Supplementary information).</p><p>Six portions (approximately 0.25 g each) of sample Q29 were accurately weighed and extracted separately for the recovery test. The results (Table 2) show that the recovery rates of all 11 alkaloids were within the range of 97.7-103.5% with RSDs not more than 5%, indicating that this method is accurate enough to measure the contents of these 11 compounds in S. acutum stem. Multivariate analysis. Principal component analysis (PCA) analysis showed that the cumulative contribution of the first three principal components was 64.3%. The 3D score scatter plot (Fig. 4A) showed that the 11 alkaloids could be divided into three groups, i.e., sinomenine in Group 1, magnoflorine in Group 2, and all the others in Group 3. The orthogonal partial least square method-discriminant analysis (OPLS-DA) model www.nature.com/scientificreports/ resulted in a (1 + 3) component with the variables of R2 (X) of 0.614, R2 (Y) of 0.792, and Q2 of 0.633. As shown in Fig. 5A, the samples were differentiated into two groups by the OPLS-DA model. The low content group is on the left side of the score plot, while the high content group is on the right side. In the loading plot (Fig. 5C), sinomenine, magnoflorine, higenamine, coclaurine, and acutumine were further from the origin. The plot in Fig. 5D displays the variable importance for the projection values (VIPs) of all variables. The VIPs of OPLS-DA demonstrated that sinomenine and magnoflorine had the greatest influence. Hierarchical cluster analysis (HCA) showed that the 11 alkaloids could be divided into three groups, as shown in Fig. 6. Sinomenine and magnoflorine were present at high levels. Higenamine, acutumine, and coclaurine were present at moderate levels. Magnocurarine, sinoacutine, columbamine, jatrorrhizine, palmatine, and 8-oxypalmatine were present at low levels.</p><!><p>In this study, thirty-five batches of S. acutum stem samples were acquired from local hospitals or pharmacies in China and were determined by the optimized UHPLC-QQQ-MS/MS method. Hua Zhou authenticated all the samples, and the voucher specimens are stored at the State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology). The sample number, sample origin, and collection location for each sample are shown in Table 3, and the samples are highly representative of this herb.</p><p>The average total content of these 11 alkaloids was 32.6 ± 7.17 mg/g. These alkaloids can be empirically divided into three groups (Fig. 7) based on their abundance. Sinomenine and magnoflorine, which are present at the milligram level and account for 76.6 and 19.5%, respectively, of total content, can be classified as high-abundance compounds. Coclaurine, acutumine, and higenamine, which are present at the microgram level and account for 1.34, 1.34, and 0.885%, respectively, of the total content, can be classified as moderate-abundance compounds. Moreover, the remaining six alkaloids, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine, although at the microgram level, account for less than 0.374% of the total content and therefore can be classified as low-abundance compounds. Magnoflorine is a homolog of sinomenine, and they have similar phenanthrene structures and similar biosynthetic pathways 22 , which could be the reason that they are in the same group. A similar explanation can be applied in the case of higenamine and coclaurine 11 . www.nature.com/scientificreports/ PCA can simplify complex information by replacing the original variable index with a small number of comprehensive indicators utilizing dimension reduction. In the present study, PCA was used to analyze the distribution pattern of the 11 alkaloids in the S. acutum stem. The result of the cumulative contributions meant that the original information of the dataset was basically retained. The 2D loading scatter plot (Fig. 4B), which provided useful information to identify the essential features in the PC1 and PC2 dimensions, showed that sinomenine, magnoflorine, coclaurine, acutumine, and higenamine were located at the edges of the axes, demonstrating the greater correlations with PC1 and PC2 and implying that these five compounds were essential.</p><p>OPLS-DA is a supervised discrimination method. Figure 5B shows the modeling used to predict the specific components with the most significant influence on the samples. It demonstrated the considerable quality of the model. Samples with more substantial VIP (> 1.0) are generally more related to sample classification. We found that the VIP values of sinomenine and magnoflorine were more than 1.0, and those of higenamine and coclaurine were very close to 1, and these compounds were in the top four. Therefore, as indicated by the plots in Fig. 5C,D, sinomenine, magnoflorine, higenamine, and coclaurine played an essential role in the S. acutum stem samples.</p><p>The HCA results were also consistent with the results of content determination, PCA, and OPLS-DA above. Therefore, multivariate analysis verified that sinomenine, magnoflorine, higenamine, coclaurine, and acutumine were the main chemical components of the S. acutum stem.</p><p>According to the Chinese Pharmacopeia, the following three requirements should be used for the selection of potential chemical markers. The content of the marker in the medicinal material should be higher than 0.02%. The corresponding specific or active ingredients selected as markers for content determination should be involved in the function or bioactivity of the Chinese medicines. A multicomponent detection method should be used when a single component cannot reflect the medicinal materials' overall activity 23 . The pharmacological activities of sinomenine 2,24-26 , magnoflorine 14,27 , coclaurine 28 , acutumine 29 , and higenamine 30,31 well represent the main indications or bioactivities of S. acutum stem, which are anti-inflammation, analgesia, anti-hypertension, anti-arrhythmia, anticancer, and immunomodulation 32 . Therefore, based on the multivariate analysis results and the bioactivities of these components, it was reasonable and representative to identify these specific alkaloids, i.e., sinomenine, magnoflorine, higenamine, acutumine, and coclaurine, as chemical markers of S. acutum stem. These alkaloids are recommended for improving the quality control of S. acutum stem in pharmacopeias and for use as relevant standards in the future. The use of sinomenine and magnoflorine as chemical markers was also consistent with the published literature conclusions 2,33 . The other three alkaloids proposed together with sinomenine and magnoflorine better reflect the overall quality of this herb. www.nature.com/scientificreports/ For the safety control of S. acutum stem, higenamine and coclaurine are proposed as chemical markers. Before discussing the safety, we determined that the sinomenine content in each of the samples tested in this study met the requirements of the pharmacopoeias 3,4 . These samples were qualified under the existing standards.</p><p>In this study, higenamine and coclaurine were found in S. acutum stem at high levels. Therefore, athletes should consider this herbal medicine and its products with caution. The WADA stipulates that all selective and nonselective beta-2 agonists, including all optical isomers, are prohibited. Higenamine is a β-androgenic receptor agonist. It possesses lipolytic activity and can improve cardiac left ventricular function. Therefore, it can promote the growth of skeletal muscle. This is the main reason that higenamine is explicitly banned as a novel stimulant ingredient 8 .</p><p>The average contents of higenamine and coclaurine in the S. acutum stem were 288 μg/g and 435 μg/g, respectively, in the present study. The recommended oral dosage of S. acutum stem is 6-12 g per day according to the Chinese Pharmacopoeia. A previous study reported that the maximum urinary concentration of higenamine in humans was 0.2-0.4 ng/mL within 10-12 h after oral administration of the herbal product containing 19.8 μg higenamine, and the maximum urinary concentration of coclaurine was 0.3-1.0 ng/mL (corresponding to 4.5 μg of coclaurine) 21 . Therefore, we initially speculated that the maximum urinary concentration ranges of higenamine and coclaurine that could be detected after oral S. acutum stem treatment for 10-12 h were 17.5-69.9 ng/mL and 39.5-264 ng/mL, respectively, if the ingredients were completely extracted. The anti-doping organization required that the concentration of higenamine in urine should not be more than 10 ng/mL 34 . Apparently, when athletes take regular doses of S. acutum stem, the urinary concentration of higenamine exceeds the stimulant detection threshold. Even worse, because of the enzymatic conversion between coclaurine and higenamine, the total concentration of stimulants may also increase in the human body. Therefore, according to the existing www.nature.com/scientificreports/ standards, these qualified herbs still pose a notable safety risk. With the widespread use of S. acutum stem and its products, there is a high risk that unintentional higenamine doping could be detected. This situation also reminds industry and regulatory bodies that higenamine and coclaurine can be used as chemical markers for the safety control for S. acutum stems. It is better to warn athletes to use caution on the packaging of related products. This study also alerts athletes that they should be very cautious when they are seeking assistance from herbal medicines to relieve inflammation and pain caused by sports injuries.</p><!><p>Reagents and chemicals. Sinomenine, magnoflorine, sinoacutine, columbamine, and coclaurine as chemical reference standards were purchased from Chengdu Chroma Biotechnology Co., Ltd. (Chengdu, P.R. China). Higenamine, acutumine, magnocurarine, jatrorrhizine, palmatine, and 8-oxypalmatine as reference standards were purchased from Shanghai Chenyi Biotechnology Co., Ltd. (Shanghai, P.R. China). All compounds were at a purity of ≥ 98.0%, and the chemical structures are given in Fig. 8. Formic acid from Sigma-Aldrich Corporation (St. Louis, MO, USA), acetonitrile from Anaoua Chemicals Supply (Houston, TX, USA), and methanol from Anaoua Chemicals Supply (Cleveland, OH, USA) were of HPLC grade. Ethyl alcohol from Anaoua Chemicals Supply (Cleveland, OH, USA) and other chemicals and reagents were of analytical grade.</p><p>instrument and analytical conditions. An ultrasonic cleaner (i-Quip, Shanghai, China) was used to extract the alkaloids from the samples. A Milli-Q ultrapurification system (Millipore, Bedford, MA, USA) was used to produce ultrapure water. A UHPLC system (1290 series, Agilent Technologies, Santa Clara, CA, USA) coupled with a QQQ-MS/MS (6460 series, Agilent Technologies, Santa Clara, CA, USA) was used to quantitatively detect the 11 alkaloids. Chromatographic separation was performed on a Waters ACQUITY UPLC HSS C18 SB column (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, MA, USA) at 30 °C with a mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B) in the following gradient: 0-4 min, 16-16% B; 4.01-7 min, 30-30% B; 7.01-10 min, 70-70% B. The injection volume was 2 μL, and the flow rate was 0.35 mL/min. Mass Hunter software (Agilent Technologies, Santa Clara, CA, USA) was used for optimization and quantification. The 11 alkaloids were detected using multiple reaction monitoring (MRM) and an electrospray ionization (ESI) source in positive ion mode. The transitions of the 11 compounds are shown in Table 2. The other parameters were as follows: drying gas (N 2 ) flow rate, 11.0 L/min; drying gas temperature, 300 °C; nebulizer, 15 psig; and capillary voltage, 4,000 V.</p><!><p>Appropriate amounts of the reference standards of the 11 alkaloids were dissolved in 70% ethanol to prepare stock solutions. The mixed standard solution was obtained by accurately mixing the 11 stock solutions and diluting them with 70% ethanol. The final concentrations of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine and jatrorrhizine in the mixed solution were 156, 63.4, 4.90, 7.62, 4.02, 0.656, 0.546, 0.723, 0.437, 0.342 and 0.247 μg/mL, respectively. Two microliters of the mixed standard solution was injected into the UHPLC- The sample powder (0.5 g passed through a 50 mesh sieve) was accurately weighed in a conical flask with a stopper. The flask was supplemented with 20.0 mL of 70% ethanol, well stoppered, and accurately weighed. Next, the flask was ultrasonicated for 20 min (power 150 W, frequency 20 kHz), cooled to room temperature, weighed again, and the weight was restored to its initial value with 70% ethanol. The sample solution was shaken thoroughly and filtered through 0.22-μm microporous filtration membranes. Two hundred microliters of the obtained filtrate was diluted to 1.0 mL with 70% ethanol to afford the test solution.</p>

Scientific Reports - Nature

A homoleptic alkynyl-protected [Ag₉Cu₆(^<i>t</i>BuCC)₁₂]⁺ superatom with free electrons: synthesis, structure analysis, and different properties compared with the Au₇Ag₈ cluster in the M₁₅⁺ series

We report the first homoleptic alkynyl-protected AgCu superatomic nanocluster [Ag 9 Cu 6 ( t BuC^C) 12 ] + (NC 1, also Ag 9 Cu 6 in short), which has a body-centered-cubic structure with a Ag 1 @Ag 8 @Cu 6 metal core. Such a configuration is reminiscent of the reported AuAg bimetallic nanocluster [Au 1 @Ag 8 @Au 6 ( t BuC^C) 12 ] + (NC 2, also Au 7 Ag 8 in short), which is also synthesized by an anti-galvanic reaction (AGR) approach with a very high yield for the first time in this study. Despite a similar Ag 8 cube for both NCs, structural anatomy reveals that there are some subtle differences between NCs 1 and 2. Such differences, plus the different M 1 kernel and M 6 octahedron, lead to significantly different optical absorbance features for NCs 1 and 2. Density functional theory calculations revealed the LUMO and HOMO energy levels of NCs 1 and 2, where the characteristic absorbance peaks can be correlated with the discrete molecular orbital transitions. Finally, the stability of NCs 1 and 2 at different temperatures, in the presence of an oxidant or Lewis base, was investigated. This study not only enriches the M 15 + series, but also sets an example for correlating the structure-property relationship in alkynyl-protected bimetallic superatomic clusters.

a_homoleptic_alkynyl-protected_[ag₉cu₆(^<i>t</i>bucc)₁₂]

Introduction<!>Results and discussion<!>Conclusions

<p>Superatomic coinage bimetallic nanoclusters (NCs) with atomic precision are currently being extensively investigated due to their tunable structure, 1,2 enhanced stability, [3][4][5] and signicantly modied physicochemical properties, [6][7][8] compared with homonuclear parent clusters. 9-13 Among these monolayerprotected clusters, the most studied combination is Au-Ag, as they are fully miscible in bulk. 14,15 It is well known that a plethora of stable Au NCs have been synthesized and characterized in the past few decades, 16,17 yet stable Ag NCs comprise a rather recent entry in the coinage metal NC eld. 18,19 In contrast to Au or Ag, they are far fewer examples of Cucontaining coinage alloy NCs, particularly AgCu molecules. As for the capping agent for protection, mixed ligands can yield a more complicated structure and hence obscure the structureproperty relationship establishment; therefore, molecules of single structure type particularly thiolate compounds have been widely employed to prepare AgCu NCs. For instance, in 2016, Zheng's group reported the rst thiolated chiral threeconcentric-shell cluster containing free valence electrons, [Ag 28 Cu 12 (SR) 24 ] 4À (SR ¼ 2,4-dichlorobenzenethiolate). 20 In 2018, Zhu's group showcased the controllable synthesis of the AgCu bimetallic NC [Ag 40.13 Cu 13.87 S 19 ( t BuS) 20 ( t BuSO 3 ) 12 ], which consisted of a Cu 10 Ag 2 S 7 core, a M 42 ( t BuS) 20 ( t BuSO 3 ) 12 shell, and another 12 bare S atoms. 21 Recently, Bao et al. prepared the [Ag 13 Cu 10 (SAdm) 12 ] 3+ (Adm ¼ -SC 10 H 15 ) NC, which has a Ag 13 core and a Cu 10 (SAdm) 12 shell. 22 Recently, alkynyl molecules have been emerging as a new type of ligand for preparing coinage metal NCs, 23,24 mainly because alkynyl molecules can generate more diverse surface binding moieties and some undiscovered molecular clusters with magic numbers, [25][26][27][28][29][30] eventually leading to drastically different functionalities. [31][32][33] In terms of AuAg NCs, Wang and Zheng groups documented the fabrication of the Au 34 Ag 28 (-PhC^C) 34 NC and its use as a model catalyst to explore the signicance of surface ligands in promoting catalysis. 34 In another report, Wang et al. discovered that when incorporating alkali metal ions or copper atoms into the alkynyl-protected body-centered cubic (BCC) [Au 7 Ag 8 ( t BuC^C) 12 ] + NC (which is also NC 2 in this study), site preference can be observed. 35 Recently, Yuan et al. found that the alkynyl-protected monomeric (AuAg) 34 can be assembled into 1D polymers with Ag-Au-Ag bonds between neighboring clusters through a solventmediated approach. 36 In the AgCu regime, the alkynyl ligand has also been utilized. For instance, Williams and co-workers reported two halide-ion-templated heterometallic Ag 8 Cu 6 rhombic dodecahedron clusters and investigated the spectroscopic properties and reactivity of these clusters along with those of the parent Ag 14 NCs. 37 Mak's group employed the designed tetranuclear precursors [(R-C^C-C^C)Ag] 4 (R ¼ i Pr, t Bu, and Ch) to construct a series of heteropolynuclear silver(I)copper(I) diynyl complexes that bear a common trigonal-planar CuAg 3 NC core, and such complexes exhibited long-lived emission upon photoexcitation in various media at room temperature and 77 K. 38 In a recent study, Zang's group discovered that the o-carboranealkynyl-protected [Cu 6 Ag 8 (C 4 -B 10 H 11 ) 12 Cl]NO 3 NC can serve as a perfect hypergolic material, as its ignition delay time can be shortened to 15 ms. 39 However, in the above examples of AgCu NCs, all the Ag and Cu atoms are present as the +1 charge state, and no free electrons exist in these clusters. According to the superatom theory, 40 these molecules are regarded as M(I) clusters or complexes rather than superatoms with free electrons. Due to the presence of free electrons, the superatoms have quite different structures and physicochemical properties, and hence they can nd different applications in catalysis, 41,42 optoelectronic devices, 36 biomedical regimes 31 and so on. To the best of our knowledge, no case of a homoleptic alkynyl-protected superatomic AgCu NC with free electrons has been reported so far.</p><p>Herein, we report the rst case of a homoleptic alkynylprotected AgCu superatomic NC, namely [Ag 9 Cu 6 ( t BuC^C) 12 ] + (NC 1, also Ag 9 Cu 6 in short), which has a BCC-based structure with a three-layered Ag 1 @Ag 8 @Cu 6 metal core conguration. Such a structure is reminiscent of the reported [Au 7 Ag 8 (t BuC^C) 12 ] + (NC 2, also Au 7 Ag 8 in short) molecule with a Au 1 @Ag 8 @Ag 6 metal architecture, and it is rst-time synthesized by an anti-galvanic reaction (AGR) approach with a ultrahigh yield in this study. The structural differences between NC 1 and NC 2 result in signicantly different optical absorption properties. Comprehensive DFT calculations disclosed the discrete LUMO and HOMO energy levels of NCs 1 and 2, where the characteristic absorbance peaks of NCs 1 and 2 can be correlated to the specic molecular orbital transitions. Finally, the stability of NCs 1 and 2 at room temperature and 60 C, in the presence of H 2 O 2 or CH 3 ONa, was investigated and compared.</p><!><p>NC 1 was rst prepared by following a modied "Two-in-One" method (see experimental details in the ESI †). 43 Briey, in the presence of NaSbF 6 , t BuC^CAg(I) is reduced by (PPh 3 ) 2 CuBH 4 in the mixed solvent of dichloromethane and acetonitrile. The reaction was aged for 12 h during which the solution gradually changed from colorless to yellow, and nally to dark blue. A blue block crystal was obtained by diffusing methanol into the dichloromethane solution containing the crude product. It is worth noting that by introducing one metal in the precursor and another metal in the reducing agent, such a "Two-in-One" method might be universal for synthesizing bimetallic NCs. NC 2 was synthesized by the AGR approach between organometallic t BuC^CAg(I) and Au 22 ( t BuC^C) 18 NC under mild conditions. The synthetic protocols regarding the t BuC^CAg(I) precursor and Au 22 ( t BuC^C) 18 can be found in the ESI. †</p><p>The chemical compositions of NCs 1 and 2 were veried by electrospray ionization mass spectrometry (ESI-MS) in positive mode. As shown in Fig. 1 ] + (cal. MW: 3214.8515 Da, deviation: 0.0005 Da), respectively. Also, the isotopic distributions of the two NCs match perfectly with the simulated results (inset in Fig. 1A and B). One may notice that there are some fragments in the ESI-MS spectra of NC 1, and the peak analysis in Fig. S1 ] + , respectively. For NC 2, there is a much less pronounced peak, and the peak assignment analysis in Fig. S2 † shows that it can be assigned to [Au 7 Ag 8 ( t BuC^C) 10 ] + , which is probably generated by losing two ligands (-t BuC^C) from parent NC 2.</p><p>Moreover, the ngerprint absorbance peaks of NC 1 are located at 544, 579, and 620 nm, and its characteristic absorbance feature along with the simulated pattern will be discussed next. Nevertheless, we monitored the absorbance change during the formation of NC 1. As shown in Fig. S3A, † upon reduction, an absorbance peak at 477 nm gradually appeared with the maximal value reached at 1 h. Aer that, such an absorbance peak gradually diminished and the characteristic peak at 579 nm from NC 1 gradually emerged. The 477 nm absorbance peak indicates that some intermediate may exist; however, several attempts to isolate it were not successful. As depicted in Fig. S3B, † the sharp color transition from yellow to dark yellow, slight pink and eventually blue can be clearly visualized. To better understand the AGR process from Au 22 (t BuC^C) 18 to NC 2, the reaction process was also monitored using time-resolved UV-visible absorption spectra. As shown in Fig. S4A, † the absorbance features of Au 22 NC disappeared immediately upon the addition of t BuC^CAg(I), while a new absorption band at $537 nm emerged. There are two obvious color changes at the time point of t BuC^CAg(I) addition and in the period from 4 to 8 h (Fig. S4B †). Aer 2 h, the characteristic peak at 487 nm of NC 2 gradually emerged, meanwhile the absorbance peak at 537 nm gradually diminished. It has been postulated but not ascertained that there might be some critical intermediate during the AGR process, which is still under investigation.</p><p>The electronic structures of NCs 1 and 2 were subsequently probed by X-ray photoelectron spectroscopy (XPS), and the results are presented in Fig. S5, S6 and Table S1. † As depicted in Fig. S5A, † the XPS survey scan prole conrmed the coexistence of Ag and Cu elements. The Ag/Cu atomic ratio is estimated as 10.33/6.90, in good agreement with the theoretical value (9/6). It can be noted that the binding energy of the Ag 3d 5/ 2 electrons is located at 368.41 eV, higher than that of bulk Ag (367.9 eV) and lower than that of Ag(I) (368.87 eV) (Fig. S5B †). 44 It suggests that the valence state of Ag in NC 1 is between 0 and +1. Meanwhile, the binding energy of Cu 2p 3/2 (933.12 eV) agrees well with that of Cu(I) (932.45-933.48 eV), implying that six Cu atoms are present as Cu(I) (Fig. S5C †). 45,46 For NC 2, from the XPS survey scan prole (Fig. S6A †), the Ag/Au atomic ratio can be estimated as 8.27/7.28, in good agreement with the theoretical value (8/7). As shown in Fig. S6B, † the binding energy of the Au 4f 7/2 electrons is located at 84.50 eV, in between those of bulk Au (84.0 eV) and Au(I) (86.0 eV), 47 suggesting that the M 1 core in NC 2 is Au(0). In addition, the binding energy of the Ag 3d 5/2 electrons is located at 368.83 eV, indicating that the valence state of Ag in NC 2 is +1 (Fig. S6C †). 45 Subsequently, the atomic packing structure of NC 1 was examined using a single-crystal X-ray diffractometer. As illustrated in Fig. S7, † NC 1 crystallizes in the space group R 3, and each unit cell has a SbF 6 À counterion, indicating that NC 1 possessed a +1 charge state. More detailed structural parameters are summarized in Table S2. † The structural anatomy of NC 1 is shown in Fig. 2A, which contains nine silver atoms, six copper atoms and twelve tert-butylacetylene ligands, and hence the molecule can be formulated as [Ag 9 Cu 6 ( t BuC^C) 12 ]SbF 6 .</p><p>The six copper atoms and twelve alkynyl ligands form six t BuC^C-Cu-C^C t Bu motifs on the surface of this quasispherical structure of NC 1 (Fig. 2B). Interestingly, all tertbutylacetylene ligands bind with Cu atoms via s bonding, and with Ag atoms in the p manner (Fig. 2C). Note that such linear motifs were observed for the rst time in the alkynyl-protected AgCu bimetallic NCs, even though similar linear motifs have been previously documented in several alkynyl-protected AuAg NCs, including Au 7 Ag 8 , 35 Au 24 Ag 20 , 48 Au 34 Ag 28 , 34 (AuAg) 34 , 36 Au 57 Ag 53 , 49 and Au 80 Ag 30 . 50 As illustrated in the space-lling structure (Fig. 2D), six Cu sites and eight Ag sites are exposed, which might serve as open active sites for catalysis. Despite the orientation of the rigid ligand on the surface of NC 1 being similar to that of NC 2, the exposure extent of Ag atoms in NC 2 is somehow more than that in NC 1 (Fig. S8A and B †). However, for NC 2, even with the same ligand, it is more expansive, probably owing to the much larger radius of the Au atom (Fig. S8C and D †) than that of the Cu atom.</p><p>Next, the anatomical structure of NC 1 is compared with that of NC 2 (the detailed structural parameters of NC 2 are summarized in Table S3 †). As shown in Fig. 2E, NC 1 adopts a core-shell-shell conguration of Ag 1 @Ag 8 @Cu 6 , similar to that of NC 2, as both of them can be classied as the BCC-based M 15 + series with an M 1 kernel@Ag 8 cube@M 6 octahedron architecture. However, the AuAg core in NC 2 is slightly constricted compared to the AgCu core in NC 1, as the average adjacent Ag-Ag bond length is 3.333 Å and 3.271 Å in the Ag 8 cube for NC 1 and 2, respectively (Fig. S9 †). In addition, the average bond length from the central Ag to the Ag atoms in the Ag 8 cube is 2.887 Å, comparable with that of bulk Ag (2.889 Å).</p><p>Note that, compared with the reported Ag-Ag bond lengths in alkynyl-protected Ag NCs listed in Table S4, † there is argentophilic Ag-Ag interaction in the Ag@Ag 8 cube for NC 1. Moreover, as illustrated in Fig. S10A, † the capped Cu atom isn't located exactly above the centre of the Ag 4 plane, which is different from the surface of NC 2 (Fig. S10B †). That means, besides the atom differences in the M 1 kernel and M 6 octahedron, NC 1 and NC 2 have some subtle structural differences, and such subtle differences may affect their physicochemical properties as well. In addition, the structure of the thiolateprotected M 15 NC Au 15 (SR) 13 also has been theoretically proposed, but it is quite different from that of NC 1 and NC 2, as it consists of a tetrahedral Au 4 core, a [Au 7 (SR) 7 ] ring, and two [Au 2 (SR) 3 ] "staple" motifs. 51 Next, we rst compared the optical absorbance properties of NC 1 and NC 2. For NC 1, as shown in Fig. 3A and S11, † there are four prominent peaks at 333 nm (3.72 eV), 544 nm (2.28 eV), 579 nm (2.14 eV), and 620 nm (2.00 eV), a broad absorption peak at 422 nm (2.94 eV), and a weak shoulder at 357 nm (3.47 eV). The energy bandgap derived from the absorbance spectrum is $1.80 eV. For NC 2, as presented in Fig. 3B and S11, † there are also four prominent peaks at 3.13 nm (3.96 eV), 422 nm (2.80 eV), 477 nm (2.60 eV), and 506 nm (2.45 eV), and a weak shoulder at 339 nm (3.65 eV), while the energy bandgap is $2.22 eV. It is worth noting that, despite some similar absorbance patterns, the absorbance features are drastically different for NC 1 and NC 2 in terms of the peak position and optical bandgap. Such huge discrepancies can be probably attributed to the structural differences. As both NCs adopt an M 1 @Ag 8 @M 6 metal conguration, the M 1 kernel (Ag vs. Au) can make a dramatically different contribution to the absorbance, and the outer M 6 octahedron (Ag 6 vs. Au 6 ) not only modulates the geometrical conguration, but also inuences the electronic structure (discussed next). Given the standard absorbance curve (Fig. S12A and B †) of the two NCs, according to Lambert-Beer's law, the molecular absorptivity (3) of NC 1 (3 ¼ 0.35 Â 10 4 M À1 cm À1 ) and NC 2 (3 ¼ 0.78 Â 10 4 M À1 cm À1 ) can be determined, as summarized in Table S5. † Therefore, through calculation, the yield of NC 1 was 41.05% (based on Ag, and the yield is 41.74% based on Cu), and the yield of NC 2 was up to 86.72% (based on Au). The details of the calculation process can be found in the ESI † (Fig. S13 and Tables S6, S7 †). It is worth noting that the yield of NC 2 here is much higher than that of the previously reported method. This is mainly due to the fact that the reported method is a "bottom-up" direct reduction approach, in which other polydisperse clusters are also produced, while the AGR method here can yield more homogeneous products, and NC 2 is the main oxidation product (from 4e of Au 22 NC to 2e of Au 7 Ag 8 NC). In addition, we also studied the photo-luminescence properties of the two M 15 NCs. As shown in Fig. S14, † NC 2 strongly emits in the near-IR region (l max ¼ 818 nm), in good agreement with the previous report. 35 However, there is no obvious emission peak for NC 1.</p><p>To elucidate the relationship between the electronic structure and optical properties of NCs 1 and 2, we carried out time dependent-density functional theory (TD-DFT) calculations. The optimized structure based on the crystal structure is used as a model for TD-DFT calculation. As shown in Fig. 3E and F, there is no change in structure aer optimization, except for a slightly distorted orientation of the ligands. The distribution of the electronic cloud map of the highest occupied molecular orbital (HOMO) of NC 1 is quite similar to that of NC 2, whereas the cloud density position in the lowest unoccupied molecular orbital (LUMO) of NC 1 is quite different from that of NC 2. From the cloud density distribution, it can be clearly noted that charge transfer occurs from ligands to the metal core for NC 2, resulting in free electron localization in the metal core. While in stark contrast, charge transfer happens from the metal core to ligands for NC 1, which leads to a non-radiative loss of excited state electron energy. Such different charge transfer behaviors are probably the main cause of the different luminescence properties. From Fig. 3E, the molecular orbitals of NC 1 revealed a jelliumatic shell closing at the HOMO state of 1S with two electrons and the LUMO state of 1P, in which there was conversion from S to P. It is also in good accord with the electron counting results, as the metal core offers een delocalized electrons; while twelve electrons are delocalized at the metalcore bonds, one electron has to be deducted to form a cation, and hence NC 1 can be literally considered as a two-electron jelliumatic molecule. The absorption feature of NCs 1 and 2 was also theoretically simulated. As shown in Fig. 3C and D, four prominent peaks namely a, b, g, and d can be clearly recognized for both NCs, and the detailed transitions corresponding to the signicant peaks are listed in Tables S8 and S9 † for NCs 1 and 2, respectively. For NC 1, an optical bandgap of ca. 1.80 eV is extrapolated according to the value of the absorption edge, which is close to the absorption peak at 1.72 eV (a) in the simulated spectrum. Note that the band a could not be merely considered as a HOMO to LUMO transition, but might be resulted from three transition modes (HOMO to LUMO, HOMO to LUMO + 1, and HOMO to LUMO + 2) with nearly equal contribution values (96.2%, 96.1%, and 95.6%) (Fig. S15A †). Similar transition modes are observed in NC 2 (Fig. 3D and S16A †), and the excitation energy (DE ¼ 2.04 eV) is also close to the optical bandgap (exp. 2.22 eV). The b peak at 2.22 eV of NC 1 can also be attributed to three transition modes (HOMO to LUMO + 3, HOMO to LUMO + 4, and HOMO to LUMO + 5), whereas the b peak at 2.89 eV in NC 2 can be assigned to two transition modes (HOMO À 8 to LUMO and HOMO À 8 to LUMO + 1) with the contribution of 42.3% and 12.5%, respectively (Fig. S15B and S16B †). In addition, the g peak of NC 1 comprises een transition modes (e.g., HOMO À 6 (67.7%), HOMO À 7 (55.9%), HOMO À 1 (43.5%) to LUMO + 2 and so on) (Fig. S15C †). In contrast, the g peak of NC 2 can be attributed to six transition modes, including HOMO to LUMO + 4 (69.0%), LUMO + 8 (59.4%), and LUMO + 9 (56.7%) (Fig. S16C †). The d peak of NC 1 is mainly contributed by the HOMO À 28 to LUMO + 2 (48.2%) transitions (Fig. S15D †), while the d peak of NC 2 is predominantly contributed by two transition modes (i.e., HOMO À 6 to LUMO + 4, 21.4%, and HOMO À 19 to LUMO + 2, 24.1%) (see Fig. S16D †). One can conclude that even if NCs 1 and 2bear a similar M 15 + geometrical conguration, the optical absorption properties are quite different, mainly owing to the differences in the M 1 kernel (Ag vs. Au) and M 6 octahedron (Cu vs. Au), and probably the subtle difference in the Ag 8 cube as well. Furthermore, we also calculated the electronic structures of NCs 1 and 2 to unravel the relationship between the electronic structure and the optical absorption properties of M 15 + NCs.</p><p>According to the Kohn-Sham (KS) molecular orbital energy level diagram (Fig. 4A and B), there is an obvious energy gap between the HOMO and the remaining occupied orbitals for the two NCs, which may be because the two electrons at the HOMO orbital with higher energy tend to relax to the HOMO -1 orbital.</p><p>It is worth noting that for the frontier unoccupied molecular orbitals (including the LUMO), the Ag(sp) atomic orbital makes the most signicant contribution in both NCs. That means, for both NCs, Ag 8 cubic atoms are the major contributor to the unoccupied molecular orbitals. It indicates that the unoccupied molecular orbitals of the M 15 + clusters might be localized at some specic position (i.e., M 8 cube), while the M 1 kernel and M 6 octahedron make less contribution, reminiscent of the case of M 21 (SCH 3 ) 15 . 52 This explains that there are triplet peaks located at high wavelength for both NCs. However, for the remaining occupied molecular orbitals (not including the HOMO) particularly the deep occupied orbitals, the Cu(d) atomic orbital makes the most important contribution in NC 1, while Au(d) and Ag(d) both contributed signicantly in NC 2.</p><p>For the HOMO, drastic differences can be observed, Ag(sp) and Au(sp) atomic orbitals make the most signicant contribution to the HOMO orbital in NCs 1 and 2, respectively. The absorption band a in NC 1 is primarily attributed to intraband Ag(sp) to Ag(sp) transitions, while the interband Au(d) to Ag(sp) transitions lead to the a band in NC 2. This nding reveals that the central M(0) atom of M 15 NCs plays a signicant role in the HOMO energy. Moreover, the HOMO energy of NC 1 is higher than that of NC 2, probably due to the fact that, compared with Ag, the central Au atom has higher cohesive force thus causing NC 2 to have lower HOMO energy than NC 1. Despite the similar structural scaffold of the two NCs, the subtle differences in metal composition and structural coordination mode might result in a vast difference of their stability. The stability of NCs 1 and 2 was investigated by monitoring the time-resolved UV-vis absorbance spectra at different temperatures, in the presence of an oxidant (e.g., H 2 O 2 ) or Lewis base (e.g., CH 3 ONa). Fig. 5 shows the intensity of the peaks at 579 nm for NC 1 and 487 nm for NC 2 versus time. At room temperature, the absorbance of NCs 1 and 2 remained almost unchanged for 24 h (Fig. S17A and B †), indicating that the two NCs can be stable under ambient conditions. As shown in Fig. 5A, the relative absorbance intensity of NC 2 preserved 81% of its initial value in 24 h, however, NC 1 retained 94%, indicating much less decomposition. It was because both NCs are situated at the lowest energy state, as conrmed by DFT calculations of the vibration frequency of NCs 1 and 2 (Table S10 †). When incubated at 60 C (Fig. 5B), the relative intensity of NC 1 remained 81% at 24 h, slightly higher than that of NC 2 (78%), indicating excellent thermal stability for both NCs. However, the absorption intensity of NC 1 dropped much faster than that of NC 2 in the rst 5 h (Fig. S17C and D †), indicating that it is more susceptible to heat. However, in the following 5-24 h, the relative intensity of NC 1 remained almost unchanged. Previous studies have documented that the thermal stability of bimetallic NCs not only depends on the extent of free electron centralization, 4,53 but also the metal-ligand interaction needs to be considered, 54 specically, the interaction between the surface metal atoms (Ag, Au or Cu) and the alkynyl ligand must be taken into account for these two title NCs. As shown in Fig. S9, † the s and p bonds between the metal and carbon atoms of the surface binding motif in NC 1 (Cu1-C1: 1.856 Å; Cu1-C2: 1.884 Å; Ag1-C1: 2.395 Å; Ag2-C2: 2.355 Å) are slightly stronger than those in NC 2 (Au1-C1: 1.983 Å; Au1-C2: 1.980 Å; Ag1-C1: 2.495 Å; Ag2-C2: 2.505 Å). Therefore, the more compact structure of NC 1 can prevent decomposition at higher temperature. Furthermore, NC 1 is more stable than NC 2 in the presence of the Lewis base (1 wt% CH 3 ONa in EtOH), but rapidly decomposed upon the addition of the oxidant (30 wt% H 2 O 2 ). As shown in Fig. 5C, NC 1 decomposed slightly faster than NC 2 in the rst 5 h, and both reached the same intensity at 8 h, aer that, NC 2 decomposed slightly faster. The higher relative intensity (84% vs. 80%) indicates that NC 1 possessed slightly superior stability in the presence of the Lewis base. During this process, the color of NC 1 showed no change, while the NC 2 solution gradually turned from orange to light orange, as visualized in Fig. S18A and B. † Note that the cohesive force between Ag and Au in NC 2 is higher than that between Cu and Ag in NC 1, making NC 2 more vulnerable to the Lewis base, as CH 3 ONa is a nucleophilic agent, and hence it can attack the surface of NC 2 more favorably. However, NC 2 is more robust than NC 1 upon adding H 2 O 2 aqueous solution (Fig. S18C and D †). As shown in Fig. 5D, NC 1 decomposed completely in 1 h, suggesting that NC 1 is sensitive to H 2 O 2 , probably because the Cu(I) atoms on the surface of NC 1 can be easily oxidized into Cu(II). In contrast, the corresponding Au(I) atoms in NC 2 possessed strong antioxidation capacity. However, it still lost 35% of the initial value, and such a decomposition can be presumably attributed to the fact that the Ag atoms in the Ag 8 cube are easily attacked by the lone pair electrons of the peroxy radical (O 2 2À ). 55 Such a phenomenon occurs even more seriously for NC 1, leading to an accelerated and complete decomposition in 1 h.</p><!><p>In conclusion, a novel homoleptic alkynyl-protected AgCu superatom [Ag 9 Cu 6 ( t BuC^C) 12 ] + was synthesized for the rst time via a one-pot reaction with high yield (>40%). X-ray crystallographic analysis revealed that it possesses a BCC-based Ag 1 @Ag 8 @Cu 6 conguration. Also, the BCC-based [Au 7 Ag 8 (t BuC^C) 12 ] + cluster with a Au 1 @Ag 8 @Au 6 metal core was also prepared by an AGR approach with a ultrahigh yield (>86%). DFT calculations revealed that the different absorption features of the two NCs can be attributed to the differences in the M 1 kernel (Ag vs. Au), M 6 octahedron (Cu vs. Au), and the subtle differences in the Ag 8 cube. The characteristic absorbance peaks of NCs 1 and 2 are successfully correlated with the specic molecular orbital transitions. NC 1 possessed superior stability to NC 2 at both room temperature and elevated temperature, and NC 1 also showed better tolerance to the Lewis base but is much more sensitive to the oxidant. We envision that this study can stimulate more research efforts on Cucontaining bimetallic superatomic NCs in terms of their synthesis, structural analysis, property exploration and beyond.</p>

Royal Society of Chemistry (RSC)

Biochemical characterization of Metnase\'s endonuclease activity and its role in NHEJ repair

Metnase (SETMAR) is a SET-transposase fusion protein that promotes non-homologous end joining (NHEJ) repair in humans. Although both SET and the transposase domains were necessary for its function in DSB repair, it is not clear what specific role Metnase plays in the NHEJ. In this study, we show that Metnase possesses a unique endonuclease activity that preferentially acts on ssDNA and ssDNA-overhang of a partial duplex DNA. Cell extracts lacking Metnase poorly supported DNA end joining, and addition of wt-Metnase to cell extracts lacking Metnase markedly stimulated DNA end joining, while a mutant (D483A) lacking endonuclease activity did not. Given that Metnase overexpression enhanced DNA end processing in vitro, our finding suggests a role for Metnase\'s endonuclease activity in promoting the joining of non-compatible ends.

biochemical_characterization_of_metnase\'s_endonuclease_activity_and_its_role_in_nhej_repair

<!>Cells, enzymes, oligonucleotides, and antibodies<!>Chemicals and DNA substrates<!>Generation of Metnase over- and under-expressing cell lines<!>SDS-PAGE and Western blot analysis<!>Preparation of cell extracts<!>DNA cleavage assay<!>Glycerol gradient centrifugation<!>DNA end joining assay coupled to E. coli colony formation<!>Metnase prefers ssDNA to dsDNA for its DNA cleavage activity<!>Metnase preferentially cleaves ssDNA overhang of a partial duplex DNA<!>A Metnase mutant defective in DNA cleavage failed to support DNA end joining in vitro<!>Metnase enhances processing of non-compatible ends in NHEJ repair in vitro<!>Discussion

<p>DNA double-strand breaks (DSB) are the primary cytotoxic lesions repaired by non-homologous end joining (NHEJ) in mammals (1, 2). NHEJ repair involves processing and ligation of two free DNA ends, requiring Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, and XRCC4/DNA Ligase IV (3-13). Upon DSB damage, the Ku70/80 complex first binds to the DNA ends, and recruits DNA-PKcs (3, 4, 6, 7, 11, 12, 14, 15). Autophosphorylation of DNA-PKcs is required for NHEJ, and Artemis, WRN, and XRCC4 are also phosphorylated in a DNA-PKcs-dependent manner (16). Artemis possesses nuclease activity and interacts with DNA-PKcs, and may be required for DNA end processing prior to DNA end joining (16). The recruitment of the XRCC4-DNA Ligase IV (Lig4) complex is essential for the final ligation step (17, 18). XLF, also known as Cernunnos, is a newly identified NHEJ factor known to stimulate Lig4 through its interaction with XRCC4 (12, 19, 20).</p><p>In vitro NHEJ repair analysis (21-25) has been a useful tool for identification of additional repair factors, but was limited to joining of linearized plasmid DNA with compatible ends (21). A PCR-based intermolecular end joining assay (22, 26, 27), on the other hand, allows to measure joining of both compatible and non-compatible ends (27). Recent studies with in vitro end joining approach suggested that, although NHEJ repair is dependent on the Ku complex, DNA-PKcs, and XRCC4/Ligase4, it also requires additional factors for end joining (22, 28, 29). Metnase, also known as SETMAR, is a novel SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] and transposase fusion protein (30, 31). The chimeric fusion of the Hsmar1 transposase with a SET domain is a unique event that occurred in the evolution of anthropoid primates approximately 50 million years ago and is not found in prosimian monkeys or other mammals (30). Metnase possesses many but not all of the activities of a transposase, including sequence-specific DNA binding and DNA looping, the assembly of a paired end complex (PEC), the cleavage of a 5′-end of the TIR element, and the promotion of integration at a TA dinucleotide target site (30, 32-36). A transposase domain containing the DDE acidic motif conserved among retroviral integrase and transposase families (37, 38). It also possesses histone lysine methyltransferase (HLMT) activity at histone 3 lysine 4 and lysine 36 (39, 40) associated with chromatin opening (41-43) at DNA damage sites (40). Metnase's involvement in NHEJ repair came from an in vivo study showing that overexpression of Metnase increased NHEJ repair, while it did not produce any significant changes in homologous recombination repair (HRR) (39). Similarly, cells treated with Metnase-siRNA showed a significant reduction for in vivo NHEJ repair activity (39). Metnase over-expression resulted in a 3-fold survival advantage after ionizing radiation compared to vector controls (39), further evidence of a linkage between Metnase and NHEJ. Metnase is also involved in genomic integration of foreign DNA (39, 44) that depends on some of the NHEJ factors (45, 46). A deletion of either SET or the transposase domain abrogated Metnase's function in DNA repair, indicating that both domains are required for this function (39). However, whether Metnase plays a direct role in NHEJ, or enhanced the activity of other NHEJ components, is not well defined.</p><p>In this study, we show that Metnase possesses a unique endonuclease activity that preferentially acts on ssDNA and ssDNA overhang of a partial duplex DNA. Cell extracts lacking Metnase exhibited significantly lowered end joining activity, which was comparable to those seen in extracts lacking DNA-PKcs or Ku80. Addition of wt-Metnase but not the mutant (D483A) restored DNA end joining activity with cell extracts lacking Metnase. These data imply that Metnase plays a direct role in the joining of both compatible and non-compatible ends.</p><!><p>HEK-293 cells, mouse Ku80-/-, DNA-PK-/-and ATM-/- cells were previously described (39, 47). Restriction enzymes (BamH I, Hind III, Kpn I, EcoR V, and Pst I) were obtained from Promega (Madison, WI). The oligonucleotides were obtained from the Integrated DNA Technologies (Coralville, IA). An anti-Metnase antiserum (polyclonal) was generated from rabbits using two peptides representing amino acids 483-495 and 659-671 (39). An anti-FLAG antibody was obtained from Sigma (St. Louis, MO). The oligonucleotides and the 5′-fluoresent labeled DNA were obtained from the Integrated DNA Technologies (Coralville, IA).</p><!><p>The following suppliers provided the listed items: [γ-32P]-ATP (3000 Ci/mmol) from Perkin-Elmer and Analytical Science (Boston, MA), DE81 filters from Whatman Bio System (Maidstone, England), heparin-Sepharose from Amersham Biosciences (Piscataway, NJ), and Bradford reagents and protein molecular weight markers were purchased from Bio-Rad (Hercules, CA). Closed-circular pBluescript (pBS) II SK+ duplex phagemid DNA (3.0 kbps) was linearized with indicated restriction enzyme(s), and purified on agarose gel twice using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Concentrations of recovered DNA were determined by spectrophotometer.</p><!><p>Human embryonic kidney (HEK-293) cells expressing either wt-Metnase or the mutant (D483A) were generated by a stable transfection of HEK-293 cells with either pcDNA3.1-V5/His (Invitrogen) for the control or pcDNA3.1-Metnase-V5/His for the over-expression of Metnase (39). Transfectants were selected in 800ug/ml G418 for 14-21 days, and individual colonies were expanded and analyzed for Metnase expression by Western blot using a polyclonal antibody specific for Metnase (39). Metnase underexpressors were generated by stable transfection of HEK-293 cells with either pRNA-U6.1/Hyg (Genescript) for the control or pRNA-U6.1/Hyg-siRNA-Metnase to reduce the expression of endogenous Metnase. Transfectants were selected in 150 μg/ml hygromycin for 10-14 days. Reverse transcription-PCR was used as the initial screen for clones that had a reduced expression of Metnase compared to the control U6 clone. The primers for Metnase and 18S and the sequence for the Metnase siRNA were described previously (39).</p><!><p>Protein fractions were analyzed by 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE). For western blot, proteins were transferred to polyvinylidene difluoride (PVDF) membrane, probed with an anti-FLAG (monoclonal mouse IgG, Sigma) or an anti-Metnase antibody (polyclonal rabbit IgG) followed by horseradish peroxidase-conjugated secondary antibody. Protein was visualized by using the ECL system (Amersham Biosciences).</p><!><p>Whole cell extracts were prepared from HEK-293 cells as described previously (21). Briefly, HEK-293 cells expressing different levels of Metnase were harvested at 80-90% confluency, and washed three times in ice-cold PBS and once in hypotonic lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 5 mM DTT). Cells were resuspended in 500 μl of hypotonic buffer, incubated on ice for 20 min and homogenized after addition of protease inhibitors (0.17 μg/ml phenylmethyl-sulfonyl fluoride, 1 μg/ml aprotinin, 0.01 units/ml trypsin inhibitor, 1 μg/ml pepstatin, 1 μg/ml chymostatin, 1 μg/ml leupeptin). Following 20 min incubation on ice, 0.5 volume of high-salt buffer (50 mM Tris-HCl, pH 7.5, 1 M KCl, 2 mM EDTA, 2 mM DTT) was added to the cell lysates prior to centrifugation for 3 hr at 42,000 rpm in a Beckman SW50.1 rotor. The supernatant was dialyzed against E buffer (20 mM Tris-HCl pH 8.0, 0.1 M KOAc, 20% glycerol (v/v), 0.5 mM EDTA, 1 mM DTT) for 3 hours and was fast-frozen and stored at −80°C.</p><!><p>DNA cleavage assay was carried out using the previously described procedure with modification (35). Briefly, reaction mixtures (20 μl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 5% glycerol, BSA (10 μg), and 2 mM MgCl2 were incubated with indicated amounts of wt-Metnase or the mutant in the presence of 200 fmol of 5′-32P-DNA. After incubation at 37°C for indicated amount of time, reaction mixtures were analyzed by 12 % polyacrylamide gel electrophoresis containing 8M urea for DNA cleavage.</p><!><p>Immunopurified wt-Metnase was sedimented through a linear 10-35 % (vol/vol) glycerol gradient at 45,500 rpm for 26 hrs at 4°C. Fractions (175 ul/each) were collected from the bottom, and the aliquots were examined for DNA cleavage activity and also run on 10% SDS-PAGE for western-blot analysis.</p><!><p>Reactions mixtures (100 μl) contained 50 mM Tris-HCl (pH 7.5), 0.5 mM Mg-acetate, 60 mM potassium acetate, 2 mM ATP, 1 mM DTT, and 100 μg/ml BSA. Where indicated, 2.5 mM dNTPs were included in the reaction mixtures. Cell-free extracts were pre-incubated for 5 min at 37°C before addition of 1.0 μg of DNA substrate. Following incubation for 1 hr at 37°C, DNA products were deproteinized, purified by QIAprep Kit (Qiagen, Valencia, CA), and transformed into E. coli for colony formation. For the transformation, we used the high efficiency DH5α competent cells (> 1.0 × 108 cfu/μg). Where indicated, PCR amplification of end joining products was performed using Taq DNA polymerase (Promega, Madison, WI) and the two primers (M13 Reverse and T7 primers).</p><!><p>Metnase, a SET-transposase fusion protein, is a DSB repair factor that possesses most of the transposase's activities, including 5′-terminal inverted repeat (TIR)-specific DNA binding, DNA looping via the assembly of a paired end complex (PEC), the cleavage of a 5′-end of the TIR element, and the promotion of integration at a TA dinucleotide target site (30, 32-36). We previously showed that Metnase, unlike mariner transposase (48), possesses TIR-independent endonuclease activity that converts circular duplex DNA to nicked circular and/or linearized duplex DNA and also cleaves duplex DNA (35). To understand the role of Metnase's TIR-independent endonuclease, we examined its non-TIR DNA cleavage with ss-and dsDNA. When Metnase was incubated with 50mer of either ssDNA or dsDNA (see Fig. 1A for details), Metnase exhibited its endonucleolytic activity that targets several key cleavage sites as shown previously [35]. Metnase showed a significantly higher DNA cleavage activity with ssDNA, as compared to dsDNA (Fig. 1B). This was a surprising result because Metnase has a conserved DDE motif in the transposase domain that targets TIR-specific dsDNA (33, 49). We then compared wt-Metnase with a mutant (D483A) defective in DNA cleavage (35) for ssDNA cleavage to see whether ssDNA cleavage activity observed in Fig. 1B belongs to Metnase. Although we saw no difference in protein purity between flag-wtMetnase and the mutant (flag-D483A) on SDS-PAGE (Fig. 1C), the mutant showed little or no cleavage activity under the conditions where wt-Metnase exhibited >90% cleavage of ssDNA substrate (Fig. 1D). To further confirm Metnase-mediated ssDNA cleavage activity, immunopurified wt-Metnase was subjected to a glycerol gradient centrifugation (see Materials & Methods for the details), and the fractions were examined for wt-Metnase and ssDNA cleavage activity. The peak fractions (#12-14) of wt-Metnase (Fig. 1E, bottom panel) correlated well with ssDNA cleavage activity (Fig. 1E, top panel), suggesting that ssDNA cleavage activity observed in Fig. 1B was mediated by Metnase.</p><!><p>To find out physiologic relevance of Metnase's ssDNA cleavage activity, we next examined Metnase for cleavage of partial duplex DNA (30mer of oligonucleotides annealed to either 3′- or 5′-end of 5′-32P-labeled 50mer DNA) that mimics DSB damage (Fig. 2A). If Metnase possesses a preferential ssDNA overhang cleavage activity, we would expect to see >30 nts cleavage products with a partial duplex DNA with 3′-overhang (Fig. 2B, lanes 1-3), while <20 nts cleavage products would be generated in the presence of a partial duplex DNA with 5′-overhang (Fig. 2B, lanes 4-6). Indeed, Metnase showed a preferential cleavage of ssDNA region of a partial duplex DNA with 5′-overhang, while it acted on both ss- and dsDNA regions of a 3′-overhang DNA (Fig. 2B). A kinetic analysis also showed that Metnase preferentially cleaves on ssDNA overhang of a partial duplex DNA (Fig. 2C). To see functional implication of Metnase's DNA cleavage activity in DSB repair, we also examined Metnase for cleavage of various DNA substrates (listed in Table 1) that can be generated after DSB damage and/or during DNA repair (7, 11, 13, 14, 16, 28). Although all DNA substrates used here contain ssDNA region in their structures, Metnase showed a preferred ssDNA overhang cleavage activity with flap and pseudo Y DNA substrates (Fig. 3A, lanes 2 & 8). When Metnase was incubated with hairpin DNA, loop DNA, and duplex DNA with ssDNA gap (see Table 1 for DNA structures in details), it did not show any DNA cleavage at the ssDNA region (Fig. 3A, lanes 6, 12, & 14). This result, along with the observation shown in Fig. 2, suggests that Metnase possesses a unique endonuclease activity that preferentially acts on ssDNA overhang of a partial duplex DNA. The finding that Metnase has no hairpin or DNA loop opening activity is an important functional difference between Metnase and Artemis since the latter has a preferred hairpin opening activity (50, 51), and suggests that Metnase does not have a role in opening hairpin intermediates in V(D)J recombination. We also examined whether Metnase has a directional preference (5′-vs. 3′-) in its ssDNA overhang cleavage activity with flap and/or pseudo Y DNA. Metnase showed ssDNA overhang cleavage with both 5′- and 3′-flap/pseudo Y DNA (Fig. 3B, lanes 4 & 8), although intensity of 3′-overhang cleavage products (>30 nts) was somewhat diminished probably due to an additional DNA cleavage towards 5′-end, producing 2 nts cleavage product. Metnase-mediated DNA cleavage with various DNA substrates summarized in Fig. 3C suggests that Metnase has a unique endonuclease activity that preferentially acts on ssDNA overhang of a partial duplex DNA.</p><!><p>Since Metnase stimulates NHEJ repair (32, 39, 44), we next examined whether Metnase's DNA cleavage activity is involved in DNA end joining. For this, cell extracts containing different level of Metnase were examined for end joining activity using an intramolecular end joining coupled to E. coli colony formation, where circular duplex DNA generated from end joining of linear plasmid DNA in the presence of cell extracts was quantitatively measured by ampicillin-resistant E. coli colonies following DNA isolation and transformation (Fig. 4A) (52). As shown previously (21, 22), colony formation requires the presence of ATP and Mg+2, but was completely inhibited in the presence of wortmannin, a specific inhibitor of PI-3 kinase family (Fig. 4B). DNA end joining activity was supported by cell-free extracts prepared from wild-type mouse fibroblast and ATM-/- but not those from DNA-PKcs-/- and Ku80-/- cells (Fig. 4C), suggesting that the intramolecular end joining measured by a coupled to E.coli colony formation occurs via NHEJ repair pathway. In keeping with the previous observation (22), joining of non-complementary ends was less effective than the compatible end joining but was stimulated in the presence of dNTPs (Fig. 4D), suggesting that DNA polymerase action facilitates intramolecular joining of non-compatible ends in vitro (22, 53, 54). We then examined whether Metnase expression level influences joining of non-compatible ends in a cell-free system. Cell-free extracts prepared from HEK-293 cells overexpressing wt-Metnase stimulated joining of non-compatible ends by 25-50%, while extracts prepared from cells transfected with Metnase-specific siRNA showed 8-20 fold lower DNA end joining activity than the control extracts (Fig. 5B). More importantly, addition of purified wt-Metnase to cell extracts restored DNA end joining activity, while addition of the mutant protein (D483A) lacking DNA cleavage activity (Fig. 1D) (35) poorly restored end joining activity (Fig. 5B), suggesting that Metnase's DNA cleavage activity is involved in DNA end joining in vitro.</p><p>To see in vivo relevance of Metnase's DNA cleavage activity, we next examined cells stably expressing the mutant (D483A) for the joining of linearized plasmid DNA in vivo. DNA end joining activity was measured by rejoining of a plasmid DNA that was linearized within the β-galactosidase gene (39). Overexpression of wt-Metnase increased precise and total end joining by 2.3 and 2.6 fold, respectively, whereas cells expressing the mutant (D483A) showed a very little influence on DNA end joining (Fig. 6B). Cells expressing wt-Metnase or the mutant (D483A) were also examined for genomic integration of exogenous DNA by measuring the assimilation and passage to progeny of a selective marker. A stable expression of wt-Metnase in HEK-293 cells did not affect plating efficiency (data not shown), however, it increased genomic integration of a plasmid DNA by 4-5 fold, while over-expression of the mutant (D483A) had little or no effect on genomic integration (Fig. 6C). Together, our results suggest that DNA cleavage activity of Metnase has a positive role in DNA end joining in vivo.</p><!><p>To further understand the role for Metnase's DNA cleavage activity in NHEJ, we examined whether Metnase directly influences DNA end processing during NHEJ. For this, linearized plasmid DNA containing different non-compatible ends [Kpn I-Pst I (3′ & 3′), Bam HI-Hind III (5′ & 5′), and Bam HI-Pst I (3′ & 5′)] was incubated with cell extracts containing different levels of Metnase. Following in vitro end joining reactions coupled to E. coli colony formation, 18 colonies were randomly selected and analyzed for DNA end processing by PCR amplification and DNA sequencing analysis. The PCR product of a control plasmid DNA in the absence of cell extracts was 210 nts in size, whereas most, if not all, of the end joining products showed smaller than 210 nts of PCR products (Supplemental data to Table 2). Minimal base-pair loss (0-15 nts) was observed in end joining products with cell extracts underexpressing Metnase (siRNA-Met), whereas end joining products with cell extracts overexpressing Metnase showed base-pair loss of up to 85 nts (Table 2). This result suggests a role for Metnase in DNA end processing, which likely promotes joining of non-compatible ends in a cell-free system.</p><!><p>NHEJ repair involves a direct rejoining of two separated DNA ends and comprises the major DSB repair pathway in mammals. Earlier studies identified Ku70/80 heterodimer, DNA-PKcs, and XRCC4/Ligase4 are required for NHEJ repair in vitro (16, 22, 27, 28, 54-58). However, given that DNA end joining often requires processing of non-compatible ends additional factors are necessary for completion of NHEJ in a cell-free system (22, 28, 29). We previously reported that Metnase forms DSB foci with other repair factors and stimulates DSB repair and genomic integration of foreign DNA in vivo (32, 39, 44, 59). In this study, we showed that Metnase possesses a unique endonuclease activity that plays a positive role in the joining of non-compatible ends in cell-free system.</p><p>Upon DNA damage, Metnase is introduced to the DSB damage sites via physical interaction with a binding partner, human Pso4, and colocalizes with other DSB repair factors (32). Although it is not clear what role Metnase plays in DNA end joining, Metnase-mediated stimulation of NHEJ repair in vivo requires both the SET and the transposase domains (39). Metnase directly mediates dimethylation of H3K36 at DSB sites, and enhanced the association of early DNA repair factors such as NBS1 and Ku70 (40), suggesting that Metnase's HLMT activity is involved in NHEJ. Given that thousands of potential Metnase binding (TIR and TIR-like) sites present in human chromosomes (30, 33, 60), a possibility exists that over- or underexpression of Metnase affects DNA end joining simply by influencing the expression of other repair-related genes in vivo. The in vitro end joining experiments presented in this study, however, showed extracts prepared from cells treated with Metnase-siRNA failed to support DNA end joining in vitro, while addition of wt-Metnase fully restored DNA end joining activity (Fig. 5), suggesting that Metnase has a critical role in NHEJ repair.</p><p>Cell extracts overexpressing wt-Metnase not only stimulated DNA end joining (Fig. 6) (32, 39, 61, 62), but also enhanced DNA end processing based on DNA sequencing analysis of end joining products (Table 2). In contrast, cell extracts lacking Metnase showed opposite results, indicating that Metnase stimulates DNA end joining via processing of non-compatible ends. DNA end processing facilitates end joining by increasing the chance for partial annealing between two non-compatible ends. Several DSB repair factors such as the Mre11/Rad50/Nbs1 (MRN) complex, Artemis, and the Werner syndrome protein (WRN) have been suggested to be involved in the processing of non-compatible ends (50, 51, 63, 64). The Mre11/Rad50/Nbs1 (MRN) complex and Artemis possess a 3′-5′ exonuclease (and endonuclease) activity and ssDNA-specific 5′-3′ exonuclease, respectively. The Werner syndrome protein (WRN) is a RecQ-like DNA helicase that also possesses 3′-5′ exonuclease activity (65, 66). Furthermore, DNA-PKcs phosphorylates WRN (67), and the Ku complex stimulates WRN's exonuclease activity (68), suggesting that WRN may also participate in DNA end processing. MRN's exonuclease activity is for mismatched DNA ends and pauses at sites of microhomology (69), while its endonuclease is to open fully paired hairpin DNA (64). Artemis possesses an endonuclease activity specific for hairpins and 5′ or 3′ overhangs following phosphorylation by DNA-PKcs (50, 51), suggesting that it plays a role in V(D)J recombination repair and perhaps in removing the 5′ and 3′ overhangs of non-compatible ends during NHEJ repair. Given that Metnase possesses no hairpin or loop opening activity (Fig. 3), it does not play a role in V(D)J recombination.</p><p>While Metnase contributes to DNA end joining through an enhanced processing of non-compatible ends, its DNA cleavage activity cannot explain Metnase's stimulatory role in the joining of compatible ends. Similar to DNA-PK- and Ku80-defective cells (Fig. 4C), cell extracts lacking Metnase failed to support joining of compatible ends (data not shown) (39), suggesting that Metnase also has a role in the joining of compatible ends, perhaps by promoting recruitment of the XRCC4-Lig4 complex (59), an essential player in the ligation step through a physical interaction upon DNA damage. The DNA binding property of Metnase may assist in the localization of DNA Ligase IV at the free DNA ends. In this case, Metnase is epistatically above end-processing and subsequent joining, but perhaps below free end recognition and protection, in the NHEJ cascade.</p>

PubMed Author Manuscript

Application of olefin metathesis in the synthesis of functionalized polyhedral oligomeric silsesquioxanes (POSS) and POSS-containing polymeric materials

This mini-review summarizes the applications of olefin metathesis in synthesis and functionalization of polyhedral oligomeric silsesquioxanes (POSS) and POSS-containing polymeric materials. Three types of processes, i.e., cross metathesis (CM) of vinyl-substituted POSS with terminal olefins, acyclic diene metathesis (ADMET) copolymerization of divinyl-substituted POSS with α,ω-dienes and ring-opening metathesis polymerization (ROMP) of POSS-substituted norbornene (or other ROMP susceptible cycloolefins) are discussed. Emphasis was put on the synthetic and catalytic aspects rather than on the properties and applications of synthesized materials.

application_of_olefin_metathesis_in_the_synthesis_of_functionalized_polyhedral_oligomeric_silsesquio

<!>Introduction<!><!>Introduction<!><!>Introduction<!><!>Introduction<!><!>Introduction<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!><!>Cross metathesis of vinyl-substituted silsesquioxanes<!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Ring-opening metathesis polymerization (ROMP) of POSS-functionalized monomers<!><!>Conclusion

<p>This article is part of the thematic issue "Progress in metathesis chemistry III".</p><!><p>Silsesquioxanes are nanostructures described by the empirical formula RSiO3/2, where R represents hydrogen, alkyl, alkenyl, aryl, arylene or their functionalized derivatives. A number of silsesquioxane structures have been reported including random, ladder, cage and partial cage structures. Silsesquioxanes with specific cage structures are commonly referred as polyhedral oligomeric silsesquioxanes (POSS). From among POSS structures the most thoroughly studied is a cubic silsesquioxane unit, denoted also as T8. It contains an inorganic cubic core composed of eight Si atoms at the vertices, connected through O atoms along the edges, chemically bonded with eight different or similar organic substituents so that it represents a truly hybrid architecture. The cubic silsesquioxane unit is characterized by a three-dimensional nanoscopic size structure with approximate Si–Si distance equal to 0.5 nm and an approximate R–R distance of 1.5 nm (Figure 1). The synthesis, structure and properties of POSS have been extensively reviewed [1–3].</p><!><p>Cubic octasilsesquioxane.</p><!><p>Proper selection of organic substituents R allows the modification of solubility of POSS in reaction media, its compatibility with polymers, biological systems, or surfaces. The introduction of one or more reactive groups into the POSS structure permits their further chemical modification. Because of the ease of the synthesis as well as the commercial availability of polyhedral oligomeric silsesquioxanes containing vinyl groups (which is a common functional group used in organosilicon chemistry), POSS are often functionalized through the chemical processes of C=C bond transformation, e.g., hydrosilylation, Heck coupling, silylative coupling and olefin metathesis.</p><p>Olefin metathesis, i.e., catalytic exchange of double bonds between carbon atoms, is a powerful tool in organic synthesis. The use of metathesis in organic and polymer synthesis is comprehensively described in excellent monographs [4–6]. However, the literature does not offer a more detailed review on the application of metathesis in the synthesis of functionalized polyhedral oligomeric silsesquioxanes (POSS). The lack of a pertinent overview in this field has prompted us to summarize the reported applications of olefin metathesis in the synthesis and functionalization of oligomeric silsesquioxanes and POSS-containing polymeric materials. This review is focused on the synthetic and catalytic aspects rather than on the properties and applications of the resulting materials.</p><p>Vinylsilanes show a specific reactivity towards alkylidene ruthenium complexes because of a strong effect of the silyl group on the properties of the double bond. In general, the substituents at the silicon atom determine the regioselectivity of the vinylsilane cycloaddition to the Ru=C bond. The knowledge of this untypical reactivity is pivotal for the application of metathesis for the modification of vinylsilanes, vinyl-substituted siloxanes, spherosilicates and silsesquioxanes. The appropriate choice of substituents permits the control of the process to a certain degree. The reactivity of vinylsilanes with different substituents at silicon towards alkylidene ruthenium complexes is illustrated in Scheme 1 [7].</p><!><p>Reactivity of vinylsilanes in the presence of ruthenium alkylidene complexes; a) cross metathesis, b) homometathesis, and c) decomposition of β-silylruthenacyclobutane.</p><!><p>According to Scheme 1a, as a result of the reaction of trialkoxy-, tris(trimethylsiloxy)-, trichloro- or dichloromethyl-substituted vinylsilanes with Grubbs catalyst of first or second generation (A), the active methylene complex B and the corresponding (E)-1-phenyl-2-(silyl)ethene are formed. The methylene complex B in the presence of styrene undergoes metathetic conversion to benzylidene complex A and ethene. When dichloro-substituted vinylsilanes are used, the pathway shown in Scheme 1b is also possible. Metathesis of dichloro-substituted vinylsilanes with Grubbs catalyst A leads to styrene and (silyl)methylidene complex C. Formation of (silyl)methylidene complex C has not been confirmed by spectroscopic methods. The reaction of the postulated complex C with vinylsilane gives the corresponding (E)-1,2-bis(silyl)ethenes and the methylene complex B. The methylene complex B may react with vinylsilane to form ethene and regenerate complex C. In the presence of vinylsilanes containing alkyl substituents the Grubbs catalyst undergoes fast decomposition as a result of β-transfer of the silyl group in the appropriate β-(silyl)rutenacyclobutane complex to ruthenium followed by reductive elimination of the corresponding propene derivative (Scheme 1c). The transformation resulted in complexes that do not contain a carbene ligand and do not show catalytic activity in metathesis.</p><p>The most important consequences of the above-described reactivity in metathesis of vinyl-substituted siloxanes, spherosilicates and silsesquioxanes are presented in Figure 2. It should be indicated that one of the consequences of the described reactivity is the inactivity of vinylsilsesquioxane in homometathesis.</p><!><p>The scope and limitations of metathesis in transformations of vinyl-substituted siloxanes and silsesquioxanes.</p><!><p>The limitations apply to silanes containing a double bond located directly at the silyl group and do not apply to allylsilanes and other alkenylsilanes, which behave like terminal olefins and readily undergo metathesis.</p><p>Application of metathesis in chemistry of unsaturated derivatives of POSS is limited to three types of processes, i.e., cross metathesis (CM) of vinyl-substituted POSS with terminal olefins, acyclic diene metathesis (ADMET) copolymerization of divinyl-substituted POSS with α,ω-dienes and ring-opening metathesis polymerization (ROMP) of POSS-substituted norbornene (or other ROMP susceptible cycloolefins, Scheme 2).</p><!><p>Application of olefin metathesis in the synthesis and modification of POSS-based materials: a) functionalization of vinyl-substituted POSS via cross metathesis; b) synthesis of POSS-containing polymers via acyclic diene metathesis; c) synthesis of POSS-containing copolymers via ROMP.</p><!><p>Nearly all metathetic transformations described in this review have been performed in the presence of commonly used ruthenium-based catalysts (Figure 3). In contrast, there are only a few examples of application of molybdenum-based complexes in modification of silsesquioxanes (Figure 3), which can be explained as related to the sensitivity of these complexes toward atmospheric oxygen, moisture and functional groups of reagents.</p><!><p>Olefin metathesis catalysts used in transformations of silsesquioxanes.</p><!><p>The first metathetic transformations of vinyl-substituted silsesquioxanes and spherosilicates (Figure 4) were reported by Feher in 1997 [8]. In the presence of molybdenum alkylidene complex Mo-1 octavinylsilsesquioxane (OVS) underwent cross metathesis of terminal and internal olefins, functionalized olefins (such as allyltrimethoxysilane, ethyl undec-10-enylate, oct-7-enyltrimethoxysilane, 5-bromopentene, pent-4-en-1-ol) and styrene.</p><!><p>Octavinyl-substituted cubic silsesquioxane (OVS) and spherosilicate.</p><!><p>Moreover, the catalytic activity of the first generation Grubbs' catalyst (Ru-1) was demonstrated in CM of OVS with pent-4-en-1-ol and 5-bromopentene. It has been found that terminal alkenes undergo cross metathesis much more readily and are clearly better than internal alkenes from the cost perspective. However, internal alkenes are less volatile and cannot produce any ethene, which makes them interesting starting materials. A slight vacuum had to be applied to reactions with terminal alkenes in order to remove ethene, because ethene would strongly slow down the desired cross metathesis and inactivate Schrock-type metathesis catalysts. CM of OVS with styrenes proceeded stereoselectively. A mixture of cis- and trans-isomers was obtained in the transformations of other olefins tested. Spherosilicate was shown to undergo CM with pent-1-ene and styrene in the presence of Mo-1. No data on the activity of Ru-1 in metathesis transformation of spherosilicates was provided.</p><p>In 2004 Marciniec reported the first efficient cross metathesis of octavinylsilsesquioxane (OVS) occurring in the presence of first generation Grubbs' catalyst (Ru-1, Scheme 3) [9].</p><!><p>Cross metathesis of OVS with terminal olefins (stereoselectivity as discussed in the text).</p><!><p>Octavinylsilsesquioxane (OVS) has been effectively transformed via cross metathesis with styrene, 1-hexene and allyltrimethylsilane. The reactions were carried out in the presence of first-generation Grubbs catalyst at room temperature using a 12- or 24-fold molar excess of olefin relative to silsesquioxane. The reaction with styrene led to the formation of the expected product with an exclusive E-stereochemistry around the newly formed C=C double bond, while aliphatic α-alkenes (1-hexene, allyltrimethylsilane) gave a mixture of stereoisomers (E/Z = 94:6). Additionally, when 1-hexene was used as reacting partner, the product of the cross metathesis was accompanied by considerable amounts of those of olefin homometathesis. Under optimized conditions, CM of OVS with styrene proceeds quantitatively despite the low loading of the catalyst (0.5 mol % relative to the vinylsilyl group, Scheme 3) [9]. Effective cross metathesis was observed when OVS was treated with vinyl sulfide in the presence of second generation Grubbs' catalyst (Ru-2). The product was obtained in 91% isolated yield, however, the process required a temperature elevation to 60 °C and the use of a catalyst amount of 4 mol % [9].</p><p>Laine has described the cross metathesis of OVS with a series of substituted styrenes (Scheme 4) [10].</p><!><p>Cross metathesis of OVS with substituted styrenes.</p><!><p>Cross metathesis was carried out using a 1.5-fold excess of commercially available functionalized styrenes and 0.5 mol % of Ru-1. The reaction mixtures were stirred for 72 h to ensure complete conversion of the silsesquioxane. The quantitative conversion of the substrate can be achieved by blowing a gentle stream of nitrogen above the reaction mixture to remove the ethylene formed. The resulting 4-bromostyryl derivatives were subsequently modified via Heck coupling with a set of 4-substituted styrenes to give the next generation of functionalized derivatives. The authors also demonstrated the possibility of further functionalization of an amino-substituted derivative via the reaction with 3,5-dibromo or dinitrobenzoyl chloride. The proposed synthetic method based on the gradual development of the organic part can be used for the synthesis of new star polymers, dendrimers or hyperbranched molecules. Further examples of the use of cross metathesis of OVS with styrenes in order to form functionalizable dendrimer cores have been reported by Cole-Hamilton [11]. Procedures allowing the syntheses of POSS derivatives with synthetically useful functional groups in multigram quantities have been proposed (Scheme 5).</p><!><p>Modification of OVS via CM with styrenes.</p><!><p>A similar procedure permits the synthesis of a series of vinylbiphenyl chromophore-decorated cubic oligosilsesquioxanes [12–13]. In the process conditions applied (methylene chloride at 55 °C, Ru-1) cross metathesis has been accompanied by competitive olefin homometathesis. The authors have developed a method for the isolation and purification of the expected materials and obtained the desired derivatives (Figure 5) with isolated yields exceeding 60%.</p><!><p>Vinylbiphenyl chromophore-decorated cubic silsesquioxanes.</p><!><p>Chromophore-functionalized silsesquioxane-core dendrimers were obtained to investigate their photophysical properties [12,14]. In the synthesized compounds chromophore properties were only slightly influenced by the core. The possibility of fine-tuning of the photophysical properties of the POSS-based dendritic molecule not only by changing the chromophore but also by providing tailored steric interactions between bridges and/or chromophores was proved [14]. Interestingly, the 4'-vinylbiphenyl-3,5-dicarbaldehyde group modified macromolecule (Figure 5d) displayed the ability to become luminescent when exposed to reducing agents such as NaBH4, LiAlH4 or BH3 [13].</p><p>Procedures for high yield and selective modification of octavinylsilsesquioxane (OVS) via CM with a variety of substituted styrenes, including the ones bearing highly π-conjugated substituents such as phenyl, 1-naphthyl, 9-anthracenyl and 2-thienyl have been reported by Marciniec [15]. For all styrene derivatives tested, the procedures described permitted highly regioselective metathesis leading to exclusive formation of the E-isomer. Cross-metathesis experiments were performed under mild reaction conditions (CH2Cl2, 40 °C, 24 h), in the presence of the first generation Grubbs catalyst (Ru-1). Under such conditions, a fully selective course of the reaction was observed.</p><p>Núñez has described the synthesis of fluorescent POSS derivatives with carboranylstyrene fragments attached to each corner. The procedure involves CM of OVS with carboranylstyrene compounds with different substituents (Ph, Me, or H, Scheme 6) [16].</p><!><p>Cross metathesis of OVS with carboranylstyrene.</p><!><p>The reactions catalyzed by Ru-1, occurred with quantitative conversion and excellent regio- and stereoselectivity leading to exclusive formation of E-isomers. However, CM was accompanied by a minor amount of homometathesis. Fortunately, the product of homocoupling could be easily separated from the desired CM products. The presence of the carborane clusters was shown to enhance the thermal stability of the materials. Absorption and emission data of carborane–POSS hybrids indicate a large red-shift with respect to the precursors. Dautel and Moreau have synthesized octakis[2-(p-carboxyphenyl)ethyl] (Scheme 7) and octakis[2-(4-carboxy-1,1'-biphenyl)ethyl]silsesquioxane via cross-metathesis methodology [17]. In the presence of palladium and dihydrogen the synthesized derivatives undergo, under mild conditions, hydrogenolysis of the benzyl ester group to the carboxylic acid and hydrogenation of the C=C double bonds at the silicon atoms (Scheme 7). The ability of the obtained derivatives, in particular the carboxylic acids, to generate nanostructured materials through self-organization processes was tested. The X-ray crystal structures of the octaester showed an interpenetrated compact packing of the molecular building blocks without any specific supramolecular interaction. The structure of the octaacid was found to contain hydrogen-bonded ribbons, thanks to the two-dimensional character of the acid and the directionality of the hydrogen bond pattern of the acid dimer.</p><!><p>Synthesis of octakis[2-(p-carboxyphenyl)ethyl]silsesquioxane via CM and subsequent hydrogenation.</p><!><p>Cross metathesis of monovinyl-substituted POSS with olefins has been reported for the first time by Marciniec [18]. It was demonstrated that monovinylheptaisobutyl-substituted octasilsesquioxane (monovinyl-POSS) underwent highly efficient CM with styrenes as well as vinyl and allyl organic derivatives in the presence of Ru-1 (Scheme 8).</p><!><p>Cross metathesis of monovinyl-POSS with olefins.</p><!><p>The reactions were performed in refluxing methylene chloride in the presence of usually 1 mol % of first generation Grubbs catalyst (Ru-1) and led to the formation of the expected products with isolated yields ranging from 85% to 97%. In all cases the exclusive formation of E-isomers was detected and the formation of competitive olefin homometathesis was not observed. The reactions were carried out using a small excess of olefin (1.5–3 equiv) to ensure complete conversion of the reactants. In the reaction of monovinyl-POSS with allylbenzene, CM was accompanied by double bond migration, which results in reduction of the isolated yield of the CM product (85%) and the formation of minor amounts (15%) of 1-propenylbenzene. No similar isomerization was observed in the reaction of POSS with allyltrimethylsilane. Further research enabled Marciniec to extend the scope of the reaction by reporting efficient CM of monovinyl-POSS with a series of substituted styrenes. The reported procedures permit efficient and selective functionalization of mono- and octavinylsilsesquioxanes with π-conjugated substituents via cross metathesis (Scheme 9) [15].</p><!><p>Cross metathesis of monovinyl-POSS with highly π-conjugated substituted styrenes.</p><!><p>In 2016 Marciniec reported the synthesis of a series of new cubic POSS in which one vertex silicon atom was replaced by a germanium atom bearing a vinyl group [19]. Monovinylgermasilsesquioxanes were successfully converted into the corresponding styryl derivatives via CM with styrenes (Scheme 10). Under optimized reaction conditions complete conversion of reacting partners and selective formation of CM products with exclusive E-arrangement around the C=C double bonds was observed.</p><!><p>Cross metathesis of monovinylgermasilsesquioxane with styrenes.</p><!><p>The most suitable catalyst for CM was found to be Ru-1, in whose presence no undesirable competitive reaction of olefin homometathesis occurred. Full conversion of monovinylgermasilsesquioxane required the use of 1 mol % of the catalyst. The reactions described are the first examples of metathesis activity of vinylgermanium compounds.</p><p>More than a decade ago Yoshida developed a new class of silsesquioxyl compounds containing rigid Si–O–Si bonds, called double-decker silsesquioxanes [20–21]. This class of compounds has recently been reviewed [22]. Marciniec found that divinyl-substituted double-decker silsesquioxanes (DDSQ-2SiVi) can be functionalized via cross metathesis and provided a series of examples of effective CM of DDSQ-2SiVi with styrenes and selected allyl derivatives (Scheme 11) [23].</p><!><p>Cross metathesis of DDSQ-2SiVi with olefins.</p><!><p>Under optimized reaction conditions (Scheme 11), CM led to the exclusive formation of E-isomers and was not accompanied by competitive homometathesis. This selectivity was obtained thanks to the use of the Ru-1 catalyst, moderately active in homometathesis of the olefins studied. Effective transformation was observed for substituted styrenes. Expected products were isolated with yields in the range of 88–95%. When allyl derivatives (allyltrimethylsilane, allylbenzene and allyl alcohol) were tested as olefinic partners, incomplete conversions of reactants (55–60%) were observed, despite the increased catalyst loading (2 mol %). Effective metathesis transformation was observed also in the presence of Ru-2 but then considerable amounts of olefin homometathesis product were formed. The presence of a methyl group at the vinylsilyl moiety was responsible for the lack of activity, which was consistent with earlier studies (Scheme 1). The scope of the reaction was further extended to the palette of olefins containing conjugated systems of π-bonds (Scheme 12) [24].</p><!><p>Cross metathesis of DDSQ-2SiVi with substituted styrenes.</p><!><p>Irrespective of the type of olefin used, under optimized conditions all reactions proceeded with high yields and stereoselectivity, leading to exclusive formation of the E,E-isomer. Marciniec reported the synthesis of divinylgermasilsesquioxane (DDSQ-2GeVi) and proved effective functionalization of such compounds by cross metathesis with a series of 4-substituted styrenes and allylbenzene, in the presence of Ru-1 (Scheme 13) [19].</p><!><p>Cross metathesis of (DDSQ-2GeVi) with olefins.</p><!><p>Under optimized conditions reactions led to fully chemo- and stereoselective formation of disubstituted germasilsesquioxanes. The ability of alkyldisiloxyvinylgermane to be converted in metathesis is worth noting as the analogous vinylsilane does not undergo metathesis.</p><p>In 2010 Laine reported a procedure enabling the synthesis of polyhedral vinylphenyl-substituted deca- and dodecasilsesquioxanes (denoted T10 and T12, respectively) [25]. Divinyl octa- or decaphenylsubstituted T10 and T12 derivatives (mixture of isomers) were demonstrated to effectively undergo cross metathesis with 4-bromostyrene in the presence of Ru-1 (Scheme 14).</p><!><p>CM of divinyl-substituted T10 and T12 with 4-bromostyrene (selected isomers are shown).</p><!><p>Attempts of homometathesis of vinylsilsesquioxanes have failed, which is understandable in view of the above presented scheme of reactivities of vinylsilanes (Scheme 1). The possibility to modify vinyl and styryl derivatives of silsesquioxanes via Heck reaction has been proved. The Heck coupling of 4-bromostyrene and vinyl-POSS derivatives leads to the formation of oligomeric products containing a silsesquioxane core in the polymer backbone. The deca- and dodecavinyl derivatives of T10 and T12, respectively, undergo cross metathesis with 4-bromostyrene in the presence of Ru-1 to form 4-bromostyryl derivatives, which in turn can be modified by Heck coupling with styrene to produce stilbenevinyl derivatives (Scheme 15) [26]. Laine has proposed a procedure for the separation of T10 and T12 derivatives, which enabled detailed photophysical studies of pure T10 and T12 core-based materials [26].</p><!><p>Synthesis of vinylstilbene derivatives of T10 and T12 via a sequence of CM and Heck coupling.</p><!><p>Detailed photophysical studies of chromophore-functionalized T10 and T12 silsesquioxanes have shown that the cage size and/or the symmetry can strongly affect photophysical properties [26]. In the subsequent paper the authors describe the use of OVS or mixtures of T10 and T12 units in the synthesis of hydroxyphenyl-terminated silsesquioxanes. Such derivatives were obtained via cross metathesis with 4-acetoxystyrene or via a sequence of cross metathesis with 4-bromostyrene and Heck coupling with 4-acetoxystyrene. The resulting acetoxy compounds were then hydrolyzed to produce hydroxy-functionalized derivatives. These compounds, after purification, were reacted with adipic acid chloride to form POSS-moiety containing highly crosslinked polyesters with some porosity [27].</p><p>Czaban-Jóźwiak and Grela have studied the metathetic transformation of allyl-substituted cubic silsesquioxane [28]. In search for the optimum catalyst a variety of ruthenium complexes were tested in the CM of allylsilsesquioxane with tert-butyl acrylate and (Z)-1,4-diacetoxybut-2-ene as model olefins (Scheme 16).</p><!><p>Cross metathesis of allyl-POSS with tert-butyl acrylate and (Z)-1,4-diacetoxy-but-2-ene.</p><!><p>For the majority of the ruthenium catalysts tested, despite the mild reaction conditions, high yields were observed. No reaction or lower yields of the test reaction products were observed for first generation catalysts and indenylidene complexes. For further research, active in preliminary tests and commercially available second generation Grubbs–Hoveyda catalyst Ru-3 and its nitro derivative Ru-4 were selected. The same authors were able to successfully functionalize allylsilsesquioxane with more challenging, three different steroid derivatives. The reactions were performed in toluene at 100 °C in the presence of 2 mol % of Ru-3 or Ru-4 (Scheme 17, substrate a or in CH2Cl2 at 45 °C in the presence of 2 mol % of Ru-4 (Scheme 17, substrates b and c). The products were obtained with yields of 62–72% as a mixture of Z/E isomers in the ratio of 20:80. Efficient homometathesis of allylsilsesquioxane occurring in toluene at 100 °C in the presence of 0.5 mol % of Ru-4 was noted. The observed activity of allylsilsesquioxane in homometathesis is understandable because allylsilanes (unlike vinylsilanes) behave in metathesis like terminal olefins.</p><!><p>Cross metathesis of allyl-POSS with olefins.</p><!><p>There are scarce reports on the application of ADMET in the synthesis of oligomers or polymers containing a POSS unit in the main chain. Marciniec disclosed ADMET copolymerization of DDSQ-2SiVi with dienes in the stereoselective synthesis of a new class of vinylene–arylene copolymers containing double-decker silsesquioxanes in the main chain (Scheme 18) [24].</p><!><p>Acyclic diene metathesis copolymerization of DDSQ-2SiVi with diolefins.</p><!><p>The products were polymers characterized by Mn in the range from 9100 to 18300 Da and Mw in the range from 13600 to 46100 Da. Thermogravimetric analyses indicate a high level of thermal resistance of the obtained systems, reaching the temperature values over 550 °C. Analogous ADMET copolymerization of divinylgermasilsesquioxanes with 4,4'-divinylbiphenyl or 4,4''-divinylterphenyl can be used in the synthesis of stereoregular trans-germasilsesquioxyl–vinylene–phenylene oligomers (Scheme 19) [19].</p><!><p>Acyclic diene metathesis copolymerization of DDSQ-2GeVi with diolefins.</p><!><p>This method permitted obtaining a polymer with Mw in the range from 9057 to 11033 Da and polydispersity index (PDI) = 1.5.</p><!><p>The chemistry of inorganic–organic hybrid materials has emerged as a fascinating new field of modern nanotechnology. The inclusion of POSS cages into the polymeric material can significantly improve such properties of the polymer as thermal and oxidative resistance, surface properties, improvement of mechanical properties as well as reduced flammability, heat release and viscosity during processing [29]. Synthesis, properties and applications of POSS-containing materials are the subject of numerous reviews [30–37]. From among the methods for preparation of organic–inorganic hybrid materials, polymerization or copolymerization is particularly convenient to incorporate POSS units into polyolefins.</p><p>Ring-opening metathesis polymerization (ROMP) is the type of olefin metathesis chain-growth polymerization that uses metathesis catalysts to generate polymers from cyclic olefins [38–41]. To obtain polymers functionalized with POSS in the side chain, a susceptible to the ROMP monomer connected via a suitable linker to the silsesquioxane cage should be used. Due to the ease of polymerization and functionalization, norbornene derivatives are the most often used monomers.</p><p>The aim of this section is to indicate the applications of ROMP in the synthesis of hybrid materials containing the POSS moiety covalently bonded to organic polymeric chains rather than the discussion of the properties of the obtained materials.</p><p>The synthesis of polymers by ROMP is carried out almost exclusively in the presence of ruthenium-based catalysts Ru-1–Ru-6 because of their tolerance to moisture, atmospheric oxygen and most functional groups as well as commercial availability. The choice of solvents is determined by the solubility of monomers, with methylene chloride, chloroform, and toluene being the most commonly used. Polymerization is terminated by addition of ethyl vinyl ether to the reaction mixture. Ruthenium residues from the obtained copolymer are removed on a short alumina plug.</p><p>In 1999 Lichtenhan reported ring-opening metathesis copolymerization of POSS-functionalized norbornene with norbornene in the presence of the Mo-based catalyst Mo-2 (Figure 3, Scheme 20) [42]. The polymerization was carried out in CHCl3 under nitrogen atmosphere. The reactions were terminated by the addition of benzaldehyde. A series of random copolymers with different weight percentage of POSS containing comonomer were synthesized.</p><!><p>Ring-opening metathesis copolymerization of norbornenylethyl-POSS with norbornene.</p><!><p>Ruthenium alkylidene catalyst Ru-1 was successfully used by Caughlin who reported ring-opening metathesis polymerization of heptacyclopentylnorbornenylethyloctasilsesquioxane and its copolymerization with cyclooctene [43]. The obtained copolymer was subsequently hydrogenated to afford polyethylene–POSS random copolymer (Scheme 21). Thermogravimetric analysis of the polyethylene–POSS copolymers under air showed a significant improvement of the thermal stability relative to that of polyethylene.</p><!><p>Synthesis of a polyethylene–POSS copolymer via ring-opening metathesis copolymerization of norbornenylethyl-POSS with cyclooctene and subsequent hydrogenation.</p><!><p>In subsequent studies Caughlin used ring-opening metathesis copolymerization of POSS-functionalized norbornene with 1,5-cyclooctadiene in the presence of Ru-1 for the synthesis of a series of random copolymers in which POSS loading varied in the range from 0 to 53 wt % (Scheme 22) [44]. Polymers with a weight-average molecular mass in the range from 67000 to 88000 Da were obtained.</p><!><p>ROMP of norbornenylethyl-POSS with 1,5-cyclooctadiene.</p><!><p>In the random copolymers obtained, the associative interactions between the particles were shown to result in the formation of ordered nanostructures. TEM micrographs indicate that the copolymers assemble into small, randomly oriented lamellae with lateral dimensions of approximately 50 nm and a thickness of ca 3–5 nm that corresponds to twice the diameter of a POSS nanoparticle. With increasing POSS concentration, the nanostructures extend to longer continuous lamellae having lateral lengths in the order of microns. Ruthenium alkylidene catalyst Ru-5 was successfully used in copolymerization of cubic silsesquioxane bearing four β-styryl and four (3-phenyloxiran-2-yl) substituents with dicyclopentadiene (DCPD) [45]. Moreover, octanorbornenyl cubic silsesquioxane was found to undergo ring-opening metathesis copolymerization with DCPD. Due to limited solubility only 0.1 mol % of POSS was used in the copolymerization. Such a small content of the POSS-containing comonomer caused, however, an increase in Tg up to 15 °C in relation to that of polyDCPD. Similar examinations were reported by Coughlin who used first generation Grubbs catalyst (Ru-1) for copolymerization of POSS-functionalized norbornene with DCPD (Scheme 23) [46]. During polymerization, PPh3 had to be added to reduce the activity of Ru-1.</p><!><p>Copolymerization of POSS-functionalized norbornene with DCPD.</p><!><p>Dicyclopentadiene and norbornenylethyl-POSS or tris(norbornenylethyl)-POSS (Scheme 24) have been copolymerized over a range of POSS loadings. In the copolymers obtained using mononorbornylethyl-POSS, the aggregates containing three to four POSS molecules were observed for high POSS loadings. When tris(norbornenylethyl)-POSS was used as comonomer, the POSS remained uniformly dispersed over all loadings. No improvements in thermal properties were observed in the copolymers obtained.</p><!><p>Copolymerization of tris(norbornenylethyl)-POSS with DCPD.</p><!><p>Another POSS-containing monomer – N-(propyl-POSS)-7-oxanorbornene-5,6-dicarboximide was tested in ring-opening metathesis copolymerization with 3-(trifluoromethyl)phenyl-7-oxanorbornene-5,6-dicarboximide in the presence of second generation Grubbs catalyst (Ru-2, Scheme 25) [47].</p><!><p>Copolymerization of N-(propyl-POSS)-7-oxanorbornene-5,6-dicarboximide with 3-(trifluoromethyl)phenyl-7-oxanorbornene-5,6-dicarboximide DCPD.</p><!><p>The use of specified proportions of the two comonomers allowed obtaining a series of copolymers with different POSS contents characterized by average molecular weights in the range of 42,000–200,000 Da and PDI values in the range of 1.3–1.9. The surface morphology and thermal properties of hybrids were found to be affected by the POSS macromer. TEM analysis of copolymer films revealed the presence of POSS agglomerates. An analogous macromer bearing POSS-bound via phenylene linker was used in the synthesis of a series of polymers and copolymers with 3-(trifluoromethyl)phenyl-7-oxanorbornene-5,6-dicarboximide (Scheme 25) [48]. It was found that the increase in the content of POSS units in the copolymer results in a decrease in thermal stability and Tg values. TEM and AFM microimages show spherical POSS aggregates uniformly dispersed within the copolymer. POSS-substituted polynorbornenes, in which POSS groups are linked to the polynorbornene backbone through the flexible spacer with different lengths, were subjected to homopolymerization by ROMP and copolymerization with norbornene substituted with a butyl ester group, to determine the effect of the spacer length on POSS crystallization ability and the composition dependence of physical properties of the copolymers [49]. A series of homopolymers and random copolymers were synthesized in the presence of third generation Grubbs catalyst Ru-6 in CH2Cl2, at room temperature (Figure 6) [49].</p><!><p>Homopolymers and copolymers having POSS groups attached to the main chain via flexible spacers of different lengths.</p><!><p>It has been demonstrated that the length of the spacer affects the crystallizability of POSS groups so that the use of a reasonably long spacer to link the POSS groups to the main chain can make POSS groups crystallizable.</p><p>Kim and Kwon have shown that ring-opening metathesis copolymerization of norbornenylethyl-POSS with methyltetracyclododecene in the presence of first generation Grubbs catalyst (Ru-1) is a practical route to the synthesis of block copolymers containing POSS nanoparticles (Scheme 26) [50]. ROMP of norbornenylethyl-POSS produced the corresponding homopolymer in relatively controlled molecular weights (Mn = 17,900–26,300 Da) and narrow molecular weight distributions (in the range Mn/Mw = 1.19–1.29). Copolymerization was employed by a sequential monomer addition. At first, the POSS-NBE was introduced into the reaction system containing the catalyst and after its complete conversion methyltetracyclododecene was added. The reaction was terminated with ethyl vinyl ether as soon as the second monomer was fully converted. A series of copolymers with different POSS-NBE content were obtained. The PDI values were in the range of 1.32–1.53 with average molecular weights of ca. 48000–63000 Da.</p><!><p>Ring-opening metathesis copolymerization of POSS-NBE with methyltetracyclododecene.</p><!><p>The synthesized POSS containing nanocomposites displayed significant improvements in their thermal stability relative to that of the polynorbornenes formed in the absence of POSS cages. Xu has reported an example of the synthesis of POSS-containing block copolymers via "living" ROMP [51]. Copolymerization of norbornenylethyloctasilsesquioxane with 2-endo-3-exo-5-norbornene-2,3-dicaboxylic acid trimethylsilyl ester was performed in the presence of Ru-1. The block copolymer was obtained via sequential monomer addition (Scheme 27). After hydrolysis of the ester function, the polymer was isolated by precipitation.</p><!><p>Synthesis of block copolymer via ROMP by sequential monomer addition.</p><!><p>As a result two block copolymers were obtained. The one containing 5% of POSS units was characterized by Mn = 26200 Da and PDI = 1.16 and the other one bearing 10% of POSS-substituted monomeric units, has a number average molecular weight Mn = 33200 Da and a polydispersity index PDI = 1.23.</p><p>The possibility of employing ROMP as a key step in the synthesis of a polynorbornene-based mesogen-jacketed liquid crystalline polymer (MJLCP) containing polyhedral oligomeric silsesquioxane (POSS) in the side chain was demonstrated by Shen and Fan (Scheme 28) [52]. The reaction was performed in the presence of third generation Grubbs catalyst Ru-6 under inert atmosphere. The synthesized polymer showed various phase structures including POSS crystal and a hexagonal columnar phase, which, depending on temperature, can coexist with each other. The POSS crystal was shown to have a tremendous effect on the liquid crystalline behavior of the polymer.</p><!><p>Synthesis of a liquid crystalline polymer with POSS core in the side chain.</p><!><p>Wang has reported living ROMP of a series of monomers bearing a polymerizable norbornene dicarboxyimide group attached via an appropriate linker to 1–4 POSS units [53]. Copolymerization of POSS-bearing monomers with norbornene containing pendant poly(ethylene oxide) group permitted the synthesis of a number of block copolymers, containing blocks of hydrophobic nature (POSS containing block) and those of hydrophilic nature (polyether containing block, Scheme 29). The block copolymer was synthesized via sequential monomer addition starting from the POSS-containing macromer. The synthesis of the copolymers was carried out under mild reaction conditions in the presence of Ru-6. It was shown that the polymers obtained can self-assemble in THF solution into aggregates, when water was added.</p><!><p>Sequential synthesis of copolymers of polynorbornene containing POSS and PEO pendant groups.</p><!><p>Lee has performed a series of sequential ring-opening metathesis copolymerization of norbornene-exo-2,3-dicarboximido)dodecanoylamino)propylheptaisobutyl-POSS and exo-5-norbornene-2-carbonyl-end poly(benzyl methacrylate, Scheme 30) [54] and obtained rodlike POSS−bottlebrush block copolymers containing crystalline POSS pendants in one block and amorphous polymeric grafts in another block. Hierarchical self-assembly of rodlike copolymer was studied from the point of view of its utility in producing highly ordered 1D photonic crystals.</p><!><p>Synthesis of rodlike POSS−bottlebrush block copolymers [54].</p><!><p>Surface-initiated ROMP was used to grow an organic corona phase on the surface of CdSe/ZnS quantum dots [39]. Functionalization of the surface with the octenyldimethylsilyl group allowed the attachment of a ruthenium alkylidene complex as a catalyst. Subsequent ROMP of norbornenylethylisobutyl cubic silsesquioxane or norbornenedicarbonyl chloride produced different molecular weights and narrow polydispersity homo- or copolymer layers directly onto the quantum dots (Scheme 31) [55].</p><!><p>Surface-initiated ROMP producing copolymer layers on the surface of CdSe/ZnS quantum dots.</p><!><p>Olefin metathesis, a universal tool in organic and polymer synthesis, offers numerous advantages for the synthesis of POSS-based materials. Ruthenium-based olefin metathesis catalysts tolerate the presence of water, air and nearly all functional groups. Commercially available vinylsilsesquioxanes can be easily modified and/or functionalized by cross metathesis. According to the CM product-selectivity model [56], vinylsilsesquioxane is an olefin type III (it does not undergo homodimerization). The correct choice of olefin and catalyst permits selective CM. Another metathetic transformation – acyclic diene metathesis copolymerization – permits introduction of a POSS group to the copolymer main chain. This methodology has not been thoroughly studied so far. In turn, ring-opening metathesis (co)polymerization is a convenient tool for introducing a number of functional groups, including POSS, in the side chain of polymers. This method is limited by the small number of monomers susceptible to ROMP. In view of the dynamic development of the studies on synthesis and properties of inorganic–organic hybrid materials, it is reasonable to expect that olefin metathesis thanks to its advantages and charm will find numerous further applications in the synthesis of POSS-based materials.</p>

PubMed Open Access

Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G1 Arrest/S Phase Delay and Inhibition of Apoptotic Pathway During Chemotherapy in Bone Marrow

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the number and duration of G1 phase in PCa cells. Importantly, GAS6 further increased the number of G1 arrested cells during docetaxel chemotherapy. GAS6 altered the signals of key cell cycle regulators: Cyclin B1 (G2/M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry) were all downregulated, while p27, p21, Cyclin D1, and CDK4 (G0/G1 phase) were upregulated. Importantly, these signaling events were further accentuated during docetaxel treatment in the presence of GAS6. Moreover, the apoptotic response of PCa cells to GAS6 was examined during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis. However, this apoptotic response was abrogated in PCa cell cultures in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. In addition, docetaxel induced significant levels of Caspase-3 and PARP cleavages in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which may have important implications for targeting metastatic disease.

growth_arrest-specific_6_(gas6)_promotes_prostate_cancer_survival_by_g1_arrest/s_phase_delay_and_inh

INTRODUCTION<!>CELL CULTURE<!>PROLIFERATION ASSAY<!>FUCCI-PC3 CELLS<!>CELL DEATH ASSAYS<!>QUANTITATIVE RT-PCR<!>ELISA<!>IMMUNOSTAINING<!>WESTERN BLOT<!>STATISTICAL ANALYSES<!>GAS6 EXPRESSED BY OSTEOBLASTS IN BONE MARROW, WHICH INHIBITS PCA CELL PROLIFERATION AND EXPRESSION OF CELL CYCLE MARKERS<!>GAS6 INDUCES G1 CELL CYCLE ARREST IN PCA CELLS IN RESPONSE TO DOCETAXEL CHEMOTHERAPY<!>GAS6 UPREGULATES G1 CELL CYCLE ARREST SIGNALS AND DOWNREGULATES S PHASE ENTRY SIGNALS DURING DOCETAXEL CHEMOTHERAPY IN PCA CELLS<!>GAS6 EXPRESSED BY OSTEOBLASTS CONTRIBUTES TO THE PROTECTION OF PCA CELLS FROM DOCETAXEL-INDUCED APOPTOSIS<!>DISCUSSION

<p>Prostate cancer (PCa) is the second leading cause of cancer deaths in American males [Pienta and Esper, 1993]. Death of most PCa patients is associated with bone metastasis [Koutsilieris, 1993]. PCa cells are known to develop resistance to chemotherapy, particularly within the marrow [Petrioli et al., 2003; Taichman et al., 2007; Sweeney et al., 2015]. Therefore, understanding the mechanisms of tumor cell survival and drug-resistance is a key component in targeting cancer cells more effectively.</p><p>The homing and lodging of disseminated tumor cells (DTCs) in the bone marrow and the survival of these cells in this microenvironment are essential steps to establish PCa bone metastases [Jung et al., 2015]. Recent studies suggest that many hematopoietic and mesenchymal cells participate in the cellular and molecular events required for the establishment of metastases and maintenance of tumor progression in marrow [Taichman et al., 2002; Sun et al., 2005; Kucia et al., 2005 ; Shiozawa et al., 2011; Jung et al., 2013]. Once in the marrow, DTC fate including G0-G1 growth arrest or dormancy and reactivation may be governed by the signals from the metastatic microenvironment. As conventional cancer therapeutics largely target proliferating cells, dormant DTCs may be innately protected from chemotherapeutic insults setting the stage for subsequent relapse [Quesnel, 2008; Yumoto et al., 2014]. Previously, we demonstrated that growth arrest-specific 6 (GAS6) secreted by osteoblasts inhibits PCa proliferation [Shiozawa et al., 2010]. These data suggest that once the DTCs enter an environment where GAS6 levels are high, interactions between GAS6 and its receptors may regulate PCa dormancy [Shiozawa et al., 2010]. Consistent with these observations, we reported that GAS6 levels are significantly higher in the femur and tibia vs the humeri of SCID mice corresponding to the prevalence at which metastatic PCa lesions occur following intravenous inoculation [Jung et al., 2012]. We also demonstrated that the binding of PCa cells to osteoblasts in bone marrow induces TANK binding kinase 1 (TBK1) expression, which induces the cell cycle arrest and enhances chemotherapeutic resistance of PCa cells (Kim et al., 2013]. These findings suggest that identifying novel dormancy-associated pathways are crucial to prevent PCa recurrence and provide a more effective therapeutic strategy for PCa.</p><p>Chemotherapy using docetaxel is a standard treatment option for patients with metastatic castration-resistant prostate cancer. More recently, docetaxel has also shown an impressive survival benefit when given soon after diagnosis of metastatic hormone-sensitive prostate cancer [Sweeney et al., 2015]. However, all patients eventually develop chemotherapy resistance, which reduces survival in patients with advanced prostate cancer [Hong, 2002; Sweeney et al., 2015]. Docetaxel functions in part by disrupting the microtubule network in cells, which is essential for cell division during mitosis [Yoo et al., 2002; Li et al., 2004]. In addition, docetaxel alters protein targets involved in cell survival, normal physiological functions, and oncogenesis (Li et al., 2004]. Docetaxel also increases cytokine production in PCa cell cultures and circulating cytokines in the castration-resistant PCa patients [Mahon et al., 2015]. CXCL12/CXCR4 signaling is known to prevent docetaxel-induced microtubule stabilization via p21-activated kinase 4 (PAK4)-dependent activation of LIM domain kinase 1 in PCa cells [Bhardwaj et al., 2014]. Further, the inflammatory cytokine CCL2 enhances the development of resistance to docetaxel-induced cytotoxicity in PCa cells [Qian et al., 2010]. Moreover, protein inhibitors of activated signal transducer and activator of transcription (STAT) factors 1 (PIAS1), a crucial survival factor, significantly increased in docetaxel resistant PCa cells and in tissue of patients after docetaxel chemotherapy [Puhr et al., 2014]. Docetaxel also promotes the upregulation of the cell cycle inhibitor (p19) and downregulation of cyclins (cyclin A and cyclin B1) in head and neck cancer cells [Yoo et al., 2002]. Similar results were observed in PCa cells with the upregulation of cyclin-dependent protein kinase (CDK) inhibitors (p21 and p27) and downregulation of cyclins (cyclin A2, cyclin E2, and cyclin F), CDK4, and cell division cycles (CDC2, CDC7, CDC20, and CDC25B) [Li et al., 2004]. Thus, understanding the mechanisms underlying the extrinsic or intrinsic cellular signaling process responsible for docetaxel resistance is urgently needed.</p><p>In the present study, we explored that GAS6, expressed by osteoblasts, regulates the cell cycle and apoptosis in PCa cells during chemotherapy in the bone marrow. We demonstrate that GAS6 significantly increases the number of G1 arrested cells by altering signaling networks associated with G1 arrest and S phase delay. Furthermore, we demonstrate that GAS6 contributes to the protection of PCa cells from docetaxel-induced apoptosis in cell culture and similarly the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. In addition, we show that GAS6 can protect PCa cells from apoptotic signaling via Caspase-3 and PARP cleavage. Our results suggest that GAS6 contributes to the regulation of PCa cell survival during chemotherapy in the bone marrow microenvironment.</p><!><p>Human PCa cell lines (PC3, DU145) were obtained from the American Type Culture Collection (Rockville, MD). GFP expressing PCa cell lines (PC3GFP and DU145GFP) were established by lenti viral transduction. Murine osteoblast cells were established as previously reported [Jung et al., 2011]. All prostate cancer cell lines were routinely grown in RPMI 1640 (Life Technologies, Carlsbad, CA), and murine osteoblast cells were grown in α-MEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS, GEMINI Bio-Products, Sacramento, CA), 1% penicillin-streptomycin (P/S, Life Technologies) and maintained at 37°C, 5% CO2, and 100% humidity.</p><!><p>PCa cells (PC3 or DU145) (3 x 103) were seeded onto 96-well culture plates with RPMI 1640, 1% FBS, and 1% P/S and then the cells were treated with human recombinant GAS6 (0–3μg/ml (cat. 885-GSB-050, R&D Systems, Minneapolis, MN) for 3 days. Proliferation was measured by the XTT Assay kit (cat. TOX2, Sigma, St. Louis, MO).</p><!><p>To develop a method to monitor the cell cycle in prostate cancer cells, we transduced a human prostate cancer cell line, PC3 with lentiviruses containing the fluorescent ubiquitination-based cell cycle indicator (Fucci) vectors (Clontech, Mountain View, CA). Cells contain both a chromatin licensing and DNA replication factor 1 (CDT1)-Cherry reporter and a Geminin-Cyan reporter. Early S phase cells are double-positive for the reporters, fluorescing yellow. M phase is colorless due to the destruction of both Geminin-Cyan and CDT1-Cherry [Sakaue-Sawano et al., 2008]. pRetroX-G1-Red vector (cat. 631463, Clontech) and pRetroX-SG2M-Cyan vector (cat. 631462, Clontech) were packaged into lentivirus at the University of Michigan Vector Core Facility. Lentiviral pRetroX-G1-Red vector and lentiviral pRetroX-SG2M-Cyan vector were coinfected into PC3 cells. Infected cells were selected for 7 days in media containing 1μg/ml Puromycin and analyzed by FACS analysis. Cell cycle monitoring was performed in Fucci-PCa cell culture with direct GAS6 (1–2μg/ml) treatment or with the co-culture of osteoblasts (wild-type OB (GAS6+/+ OB) or GAS6 deficient OB (GAS6−/− OB)) following treatment with anticancer drug, docetaxel (Taxotere, 1μg/ml, cat. NDC0409-0201-10, Hospira, Lake Forest, IL). Additionally, Fucci-PC3 cell imaging was captured by video. Fucci-PC3 cells were cultured for 24 hours in RPMI with 10% FBS, 1% P/S and then, treated with Vehicle or GAS6 (2μg/ml) for 24 hours. Five spots of cells in each group were set for tracking. Video images were taken for 24 hours at 15 min intervals using a Deltavision Elite Microscope (GE Healthcare Life Science, Pittsburgh, PA). The duration of G1 phase in single cells was measured (n=20/group).</p><!><p>Cell death assays were performed in PCa cell cultures treated with GAS6 or in coculture of PCa cells with osteoblasts. First, PCa cells (1 x 105 cells/well) were seeded onto 12-well culture plates for 24 hours. Cells were cultured for 24–48 hours following GAS6 (2μg/ml) treatment, and then treated with the docetaxcel (1μg/ml) for 24 hours. For the cocultures, GAS6+/+ OB or GAS6−/− OB (1 x 105 cells/well) was seeded onto 12-well culture plates for 24 hours. Thereafter, GFP expressing PCa cells (1 x 105 cells/well) were added to the wells. Cells were cultured together for 48 hours, and then treated with the anticancer drug, docetaxcel (1μg/ml), for 24 hours in the coculture system. Apoptosis was measured by flow cytometry (FACSAria IIu three laser flow-cytometer, Becton Dickinson, Mountainview, CA) using by selecting PCa cells with the GFP reporter and with the PE Annexin V Apoptosis Detection Kit I (cat. 559763, BD Biosciences, San Jose, CA). Assays were performed in triplicate and the results are representative of three independent experiments. Assays were performed in triplicate and the results are representative of three independent experiments. In tumor sections from PCa cells were injected into vertebral bodies (vossicles) from wild-type (GAS6+/+) or GAS6 knockout (GAS6−/−) mice [Jung et al., 2011], apoptosis of PCa cells was measured by Cell Meter TUNEL Apoptosis Assay Kit (cat. 22844, AAT Bioquest, Sunnyvale, CA).</p><!><p>Total RNA was extracted from cells using the RNeasy mini or micro kit (Qiagen, Valencia, CA) and converted into cDNA using a First-Strand Synthesis Kit (Invitrogen). Quantitative PCR was performed on an ABI 7700 sequence detector (Applied Biosystems) using TaqMan Universal PCR Master Mix Kit (Applied Biosystems) according to the directions of manufacturer. TaqMan MGB probes, PLK1 (Hs00153444_m1) and STK15 (Hs01582072_m1) (Applied Biosystems) were used. β-actin (Hs01060665-g1) was used as an internal control for normalization of target gene expression.</p><!><p>An antibody sandwich ELISA was used to evaluate GAS6 expression in bone marrow from GAS6+/+ or GAS6−/− mice as a negative control by following the directions of the manufacturer (cat. DY986; R&D Systems). Bone marrow extracellular fluids were obtained by flushing femora, and tibiae with 500μl of ice-cold PBS, and the supernatant was harvested by centrifugation at 400g for 5 minutes. GAS6 levels were normalized to total protein.</p><!><p>Cells, tumor sections and long bone sections were used for immunostaining. Cells were fixed and permeabilized with Perm/Wash Buffer (cat. 554723, BD Biosciences). Tumor sections were blocked with Image-iT FX signal enhancer for 30 min and incubated for 2 hours at room temperature with primary antibodies combined with reagents of Zenon Alexa Fluor 488 (green) or 555 (red) labeling kit (Invitrogen). GAS6 (cat. AF885, R&D System) antibody was used as primary antibody. After washing with PBS, the slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Images were taken with Olympus FV-500 confocal microscope.</p><!><p>PCa cells were cultured in RPMI 1640 with 10% FBS and 1% P/S. Whole cell lysates were prepared from cells, separated on 4–20% Tris-Glycine gels and transferred to PVDF membranes. The membranes were incubated with 5% milk for 1 hour and incubated with primary antibodies overnight at 4°C. Primary antibodies used were as follows: p-CHK2 (1:1,000 dilution, cat. 2197, Cell Signaling), CHK2 (1:1,000 dilution, cat. 2662, Cell Signaling), Cyclin B1 (1:1,000 dilution, cat. 4138, Cell Signaling), Cyclin D1 (1:1,000 dilution, cat. sc-753, Santa Cruz), CDK4 (1:1,000 dilution, cat.12790, Cell Signaling), p27 (1:1,000 dilution, cat. 3686, Cell Signaling), p21 (1:1,000 dilution, cat. 2947, Cell Signalng), p-CDC25A (1:1,000 dilution, cat. sc-101655, Santa Cruz, Santa Cruz, CA), CDC25A (1:1,000 dilution, cat. 3652, Cell Signaling), Cyclin E1 (1:1,000 dilution, cat. sc-481, Santa Cruz), and CDK2 (1:1,000 dilution, 1:1,000 dilution, cat. sc-163, Santa Cruz,), Caspase-3 (1:1,000 dilution, cat. 9662, Cell Signaling), and PARP (1:1,000 dilution, cat. 9542, Cell Signaling). Blots were incubated with peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, 1:2,000 dilution, Cell Signaling) for 1 hour, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). Membranes were reprobed with monoclonal anti-β-actin antibody (1:1,000 dilution; cat. 4970, Cell Signaling) to control for equal loading.</p><!><p>Results are presented as mean ± standard deviation (s.d.). Significance of the difference between two measurements was determined by unpaired Student's t-test, and multiple comparisons were evaluated by the Newman-Keuls multiple comparison test. Values of p < 0.05 were considered significant.</p><!><p>We examined the extent to which GAS6 expressed by osteoblasts in the bone marrow of the long bones of wild-type (GAS6+/+) mice and GAS6 deficient (GAS6−/−) mice. GAS6 was expressed by osteoblasts in the marrow of GAS6+/+ mice, but not in the marrow of GAS6−/− mice (Fig. 1A). GAS6 protein levels in the marrow were also examined. It was determined that the GAS6 protein levels were significantly greater in the marrow of the GAS6+/+ vs GAS6−/− mice (Fig. 1B). Next, to test the effects of Gas6 on PCa cell proliferation, the proliferation assays were performed along with gene expression profiling assays for cell cycle markers. GAS6 inhibited PCa cell proliferation in a dose dependent manner in vitro (Fig. 1C). Levels of mRNA for PLK1 and STK15 indicative of cells in G2/M phase were dramatically decreased in the PCa cells in response to GAS6 (Fig. 1D, E). These findings suggest that GAS6 expressed by osteoblasts in bone marrow inhibits PCa proliferation.</p><!><p>Previously we demonstrated that GAS6 secreted from osteoblasts plays a critical role in establishing PCa cell dormancy [Shiozawa et al., 2010]. Here we further explored how GAS6 regulates the cell cycle in PCa cells. For these investigations, the cell-cycle specific Fucci-vectors were employed in PC3 cells and Fucci expression was used to isolate cells at different stages of the cell cycle [Sakaue-Sawano et al., 2008] by examining FACS profiles of cells in G1, S, and G2/M (Fig. 2A). In standard cultures, we found that 2μg/ml of GAS6 increased the percentage of cells in G1 phase significantly at 48 and 72 hours (Fig. 2B). We also examined that the duration of cell arrested time in G1 phase in live-cell imaging of GAS6 treated Fucci-PC3 cells for 24 hours.</p><p>We found that GAS6 treated Fucci-PC3 remained in the G1 phase for 20.8 hours 15 minutes vs 9.1 hours for vehicle treated cells (Fig. 2C, D). The cell cycle monitoring was next performed for docetaxel treated cells with the presence or absence of GAS6. More G1 arrested cells were identified after docetaxel and GAS6 treatments at 24 hours and 48 hours (Fig. 2E). In addition, the cell cycle monitoring was performed in cocultured Fucci-PC3 cells with GAS6 expressing wild-type osteoblasts (GAS6+/+ OB) or GAS6 deficient osteoblasts (GAS−/− OB) following treatment with docetaxel. Importantly, more G1 arrested cells were found in coculture with wild-type osteoblasts (GAS6+/+ OB) compared to GAS6 deficient osteoblasts (GAS−/− OB) in both vehicle and docetaxel treated conditions (Fig. 2F). Collectively, the data suggest GAS6 expressed by osteoblasts may regulate the growth arrest during docetaxel chemotherapy in bone marrow microenvironment.</p><!><p>Docetaxel is known to downregulate genes associated with the cell proliferation, while upregulating genes associated with cell cycle arrest and induction of apoptosis [Li et al., 2004]. Therefore we examined the protein expression of several core cell cycle regulators including cyclins, CDKs, CDK inhibitors, and CDCs by Western blot in PCa cell cultures treated with docetaxel and/or GAS6. We found that Cyclin B1 (G2/M marker) and CDC25A, Cyclin E1, CDK2 (S phase entry regulator) were downregulated, while p27, p21, Cyclin D1, and CDK4 (associated with expression at G0/G1) were upregulated in GAS6 treated PCa cells (Fig. 3A, B). These signaling events were further accentuated in PCa cells treated with docetaxel and GAS6. These data suggest that GAS6 promotes G1 cell cycle arrest and a delayed entry into S phase.</p><!><p>PCa cells are known to develop resistance to chemotherapies, particularly in the marrow. To explore whether GAS6 plays a role in this process, we first examined the percentage of apoptotic cells following treatment with docetaxel and/or GAS6 by Annexin V staining. As expected, more PCa cells underwent apoptosis following docetaxel treatment compared to vehicle treatment (Fig. 4A, B). However, fewer PCa cells underwent apoptosis in the presence of GAS6 for both vehicle and docetaxel treatment (Fig. 4A, B). Additionally, we performed an apoptosis assay on cocultures of PC3 cells with GAS6+/+ OB or GAS−/− OB following docetaxel treatment. Fewer apoptotic cells were identified when PCa cells were cocultured with GAS6+/+ OB vs GAS−/− OBs in both vehicle and docetaxel treatment (Fig. 4C, D). To validate these in vitro studies, next we performed TUNEL staining on tumors grown in a GAS6+/+ or GAS6−/− bone environment (e.g. vossicles) as described in previous studies [Jung et al., 2012]. Fewer apoptotic tumor cells were found in the tumors established by PC3 cells within GAS6+/+ vossicles compared with GAS6−/− vossicles (Fig. 4E, F). Finally we examined the extent to which GAS6 regulates docetaxel-induced apoptosis signaling in PCa cells. For these investigations, Caspase-3 and PARP levels in PCa cells following treatment with docetaxel and/or GAS6 were examined by Western blots. We found that docetaxel induces significant levels of Caspase-3 and PARP cleavages in PCa cells. Importantly, GAS6 protected caspase-3 and PARP from cleavage in the PCa cells (Fig. 4G, H). Collectively, these data suggest that GAS6 expressed by osteoblasts in bone marrow plays a significant role in the regulation of PCa cell survival during docetaxel chemotherapy.</p><!><p>Recent studies suggest that signals from the bone marrow microenvironment protect PCa cells from chemotherapy. Here we demonstrate that GAS6 expressed by osteoblasts regulates cell cycle and apoptosis of PCa cells during chemotherapy. We found that GAS6 significantly increased G1 arrested PCa cells by signals associating with G1 cell arrest and S phase delay. Furthermore, we found that GAS6 contributes to the protection of PCa cells from docetaxel-induced apoptosis in vitro cultures and a GAS6-expressing bone environment protects PCa cell apoptosis within the primary tumors in vivo studies. Finally, we found that GAS6 prevents the activation of docetaxel-induced apoptotic signaling, Caspase-3 and PARP. Our results suggest that GAS6 expressed by osteoblasts in the bone marrow plays a significant role in regulation of PCa cell survival during chemotherapy.</p><p>PCa frequently takes more than 5 years to progress to lethal metastatic disease or biochemical recurrence after curative surgery or radiation therapy [Amling et al., 2000; Morgan et al., 2009; Roberts and Han, 2009] indicating that DTCs may enter into a state of cellular dormancy for long periods within metastatic sites [Quesnel, 2008; Yumoto et al., 2014]. Yet how DTC cells become dormant, acquire resistance to anticancer drugs, and ultimately cause tumor recurrence/metastatic relapse remain critical questions. Previously, we demonstrated that GAS6 secreted from osteoblasts plays a critical role in establishing PCa cellular dormancy [Shiozawa et al., 2010]. Here, we further demonstrated the mechanism by which GAS6 regulates cell cycle of PCa cells in response to chemotherapy. Indeed, we found that GAS6 increases the number and duration of G1 arrested cells by downregulation of Cyclin B1 (G2/M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry) and upregulation of p27, p21, Cyclin D1, and CDK4 (G0/G1 phase) in PCa cells during docetaxel chemotherapy.</p><p>Intensive studies have identified mechanisms of target molecules or signals associated with the anti-proliferative effects and anti-tumorigenic activities for the PCa treatment. Docetaxel-chemotherapy targets proliferating cancer cells, which are associated by core cell cycle regulators including cyclins, CDKs, CDK inhibitors, and CDCs [Kawamata et al., 1995; ELDeiry et al., 1995; Nilsson and Hoffmann, 2000; Poggioli et al., 2001; Erlanson and Landberg, 2001; Yoo et al., 2002; Li et al., 2004; Roy et al., 2008; Chiu et al., 2009]. Double knockdown of the cyclin-dependent kinase inhibitor 1A (p21) and the cyclin-dependent kinase inhibitor 1B (p27) in DU145 cells show higher tumor growth rate than the comparable growth of either p21 or p27 knockdown [Roy et al., 2008]. Another important cell cycle regulator, CDC25A, is highly expressed during S phase entry of the cell cycle in many cancers [Busino et al., 2004; Lavecchia et al., 2009]. CDC25A has oncogenic properties, is highly expressed in human PCa, and its expression level correlates with high Gleason scores and metastatic PCa [Chiu et al., 2009]. The expression of CDC25A in PCa cells suppresses PSA expression and functions as an AR corepressor, suggesting that it may also be a possible anticancer target [Chiu et al., 2009]. Further reports show that docetaxel promotes the upregulation of a CDK inhibitor (p19) and downregulation of the cyclins (cyclin A and cyclin B1) in the head and neck cancer cells [Yoo et al., 2002] and the upregulation of CDK inhibitors (p21 and p27) and downregulation of cyclins (cyclin A2, cyclin E2, and cyclin F), CDK4, and cell division cycles (CDC2, CDC7, CDC20, and CDC25B) in PCa cells [Li et al., 2004]. Despite this evidence, docetaxel continues to be an effective agent of growth inhibition and induction of apoptosis in part by the upregulation of growth arrest signals during the chemotherapy. Yet these actions also lead to the generation of cell cycle arrest signals, which induce cellular dormancy and ultimately increase drug-resistance. Here we found that in docetaxel-chemotherapy treated PCa cells, GAS6 further downregulates active cell cycling phase regulators including Cyclin B1 (G2/M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry). In complement, we saw upregulation of arresting cell cycle phase regulators including p27, p21 Cyclin D1, and CDK4, (G0/G1 phase). Taken together, GAS6 promotes induction of drug-resistant signals in docetaxel-treated PCa cells. Future cancer therapeutic studies will be required to identify selective agents capable of inducing cancer cell apoptosis without inducing dormancy within the context of these complex microenvironmental cues.</p><p>The ability of docetaxel to promote cancer cell apoptosis is dependent on its function in cell death signaling [Jin and EL-Deiry, 2005]. During the G2/M phase arrest of cancer cells, docetaxel binds to microtubules with high affinity, activating JNK signaling causing B-cell lymphoma 2 (Bcl-2) phosphorylation, thereby promoting a cascade of events that ultimately leads to apoptotic cell death [Haldar and Basu, 1997]. Increased JNK signaling also increases dephosphorylation of Bad, which associates with Bcl-2, releasing Bcl-2 from Bax. Unbound Bax translocates to the inner mitochondrial membrane forming Bax/Bax pores, allowing release of cytochrome c and activation of caspases leading to apoptosis [Wolter et al., 1997]. Reduction of multidrug resistance-associated protein-1 (MRP-1) by pre-treatment of 1,25-dihydroxy vitamin D3 (1,25-VD) enhanced the docetaxel antitumor activity through Bcl-2 signaling pathway in PC3 cells [Tinga et al., 2007]. Docetaxel is also able to induce apoptosis through different caspase families in PCa cells; Caspase-3 and Caspase-7 in LNCaP and TSU-Pr1 cells vs. Caspase-8 in PC3 cells [Marcelli et al., 1999; Muenchen et al., 2001]. Furthermore, Docetaxel-induced apoptosis has also been associated with the increases of Survivin, GADD45A, Fas/Apo-1, and FOXO3A in PCa cells [Li et al., 2004]. Apoptosis is guided by a complex series of cellular events and initiation of these events is tightly regulated by the balance of cell death and survival signals. The balance of these signaling mediators is not yet fully understood, but is essential in understanding mechanisms that govern cell fate such as apoptosis and cancer cell resistance.</p><p>Just as the docetaxel-inducing apoptotic signaling pathways have been elucidated, there is increasing evidence that docetaxel can confer resistance to cancer cells. For example, PIAS1 is a crucial survival factor, which was significantly increased in docetaxel resistant cells and in tissue of patients after docetaxel chemotherapy. Downregulation of PIAS1 results in increased apoptosis, suggesting the importance of PIAS1 in maintaining cell survival [Puhr et al., 2014]. Moreover, GAS6 induces activation of AKT/PKB leading to the phosphorylation of Bad as well as activation of ERK, JNK/SAPK, and p38 MAPK in serum starved NIH3T3 cells [Goruppi et al., 1999]. In addition, GAS6 secreted by osteoblasts rapidly induces the phosphorylation of ERK in PC3 cells in standard culture conditions, which provides evidence that GAS6 can promote PCa cell survival [Shiozawa et al., 2010]. However, the balance between cell survival signals and apoptotic signals in cell fate remains poorly understood. In this investigation, we further demonstrated that docetaxel significantly increases the apoptotic signals, cleaved Caspase-3 and PARP in PCa cells, while GAS6 protected PCa cells from Caspase-3 and PARP cleavages. These data suggest that GAS6 may be a critical molecule in suppressing the activation of key apoptotic signaling mediators, thus promoting cell survival. Therefore, targeting GAS6 or GAS6 signaling during cancer therapy for metastatic diseases may be helpful in elimination of quiescent PCa DTCs, as well.</p><p>In summary, this work provides evidence that supports a crucial role for GAS6 in PCa cell survival during chemotherapy within the bone marrow microenvironment (Fig. 5). Importantly, this work contributes to the understanding of dormancy signals that facilitate chemotherapeutic resistance in PCa cells, which may have important implications for optimizing therapeutic strategies against metastatic disease and tumor recurrence.</p>

PubMed Author Manuscript

Amine Analysis Using AlexaFluor 488 Succinimidyl Ester and Capillary Electrophoresis with Laser-Induced Fluorescence

Fluorescent probes enable detection of otherwise nonfluorescent species via highly sensitive laser-induced fluorescence. Organic amines are predominantly nonfluorescent and are of analytical interest in agricultural and food science, biomedical applications, and biowarfare detection. Alexa Fluor 488 N-hydroxysuccinimidyl ester (AF488 NHS-ester) is an amine-specific fluorescent probe. Here, we demonstrate low limit of detection of long-chain (C9 to C18) primary amines and optimize AF488 derivatization of long-chain primary amines. The reaction was found to be equally efficient in all solvents studied (dimethylsulfoxide, ethanol, and N,N-dimethylformamide). While an organic base (N,N-diisopropylethylamine) is required to achieve efficient reaction between AF488 NHS-ester and organic amines with longer hydrophobic chains, high concentrations (>5 mM) result in increased levels of ethylamine and propylamine in the blank. Optimal incubation times were found to be >12 hrs at room temperature. We present an initial capillary electrophoresis separation for analysis using a simple micellar electrokinetic chromatography (MEKC) buffer consisting of 12 mM sodium dodecylsulfate (SDS) and 5 mM carbonate, pH 10. Limits of detection using the optimized labeling conditions and these separation conditions were 5–17 nM. The method presented here represents a novel addition to the arsenal of fluorescent probes available for highly sensitive analysis of small organic molecules.

amine_analysis_using_alexafluor_488_succinimidyl_ester_and_capillary_electrophoresis_with_laser-indu

1. Introduction<!>2.1. Materials<!>2.2. Labeling Reactions<!>2.3. Capillary Electrophoresis<!>2.4. Data Analysis and Figure Generation<!>3. Results and Discussion<!>4. Conclusions

<p>Quantitative compositional analysis of specific primary amines is applied in food science and agriculture to characterize samples for quality control. Primary amines in soil samples provide information about the available sources of organic and bioorganic N in an ecosystem [1], a boon for management of agriculture, and a central aspect to researching systems that involve N or the nitrogen fixation cycle. Primary amines and amino acids are also used to indicate reactions in food processing and to directly indicate nutritional value and quality of products [2–5]. A large majority of known bioactive molecules and neurotransmitters are primary amines, amino acids, or low molecular weight metabolites of these species [6], so primary amine analysis is of continually increasing interest for metabolomics, pharmaceuticals, and detection of hazardous agents in biowarfare. In situ analysis of primary amines is additionally of great interest for investigating planetary chemistry [7, 8] as well as the synthesis and origin of prebiotic amino acids [9].</p><p>For compositional amine analysis, a separation method must typically be applied to resolve specific amines within a sample. Many applications require field-deployable amine compositional analyses, such as clinical devices, detection of harmful biological agents, and in situ astrobiology and planetary science experiments. Capillary electrophoresis (CE), when used in conjunction with laser-induced fluorescence (LIF), provides a fast and easily miniaturizable technique. CE-LIF also provides the opportunity to incorporate the separation and detection steps in line with extraction instruments and microfluidics devices. This is an attractive feature for lab-on-a-chip approaches and a necessary step for developing field-deployable detection instruments for the clinic, biowarfare, and in situ astrobiology experiments. In fact, CE is already a targeted implementation of lab-on-a-chip analysis for bioterrorism defense [10], μCE-LIF has been fully automated and miniaturized towards future in situ Martian and other planetary missions [7, 11], and CE-LIF's direct compatibility with dialysates and biological fluids has been applied to clinical samples and in vivo biomedical research [2, 12, 13]. However, the latter two applications often require detection of amines with varied solubility in aqueous media. Micellar electrokinetic chromatography (MEKC) enables separation and therefore analysis of hydrophobic longer-chain amines, while preserving analytic capability of shorter and more hydrophilic amines. MEKC-LIF, only differing from CE-LIF by addition of a surfactant, presents the opportunity to extend implementation of these established approaches to field-deployable instrument development to detect a broader spectrum of targets in complex samples.</p><p>Fluorescence detection of primary amines provides a quick and potentially highly sensitive, quantitative analysis. Particularly, fluorescence detection of amines by CE-LIF with excitation at 488 nm has demonstrated limits of detection (LOD) from μM to nM [3] depending on the target amine, fluorescence probe, and optimization conditions. The speed of fluorescence detection ensures that the rate-limiting step for analysis is upstream in sample collection, preparation, or separation. However, to use this method, amines without autofluorescent properties must be chemically derivatized with a fluorescent probe. The commercially available dye, AlexaFluor 488 (AF488), is optimally excited with a 488 nm laser line (extinction coefficient of 73,000 cm−1 M−1 at 494 nm) and has an emission maximum at 525 nm, making it easy to apply with standard light sources, instrumental settings, and filters. Low AF488 self-quenching enables highly sensitive analysis, and the N-hydroxysuccinimidyl ester (NHS-ester) functionality makes it highly amine-specific. In aqueous and biological samples, the spontaneous derivatization reaction of AF488 NHS-ester circumvents the need for additional reagents while the high amine specificity provides advantages over other amine-reactive dyes such as isothiocyanates, which also react with sulfhydryl groups, and dyes which react directly with amines and change their chemical structure and fluorescent properties upon derivatization (e.g., 4-chloro-7-nitro-1,2,3-benzoxadiazole). The derivatization through amine esterification, promoted by the NHS leaving group, allows for modular selection of other fluorophores to optimize fluorescent properties for varied equipment. AF488 is a water soluble probe that is pH insensitive over a range that extends from below 4 to above 10, making it suitable for a range of applications. The dye is negatively charged overall, facilitating separation of tagged analytes by electromigration.</p><p>Here, we describe a novel method for amine analysis using MEKC-LIF in conjunction with a labeling protocol employing AF488 NHS-ester. We prepared and analyzed samples of nonylamine, hexadecylamine, and octadecyl amine to test the applicability of this method to aliphatic amines with reduced solubility in aqueous media and no detectable autofluorescence. Samples were separated and detected using a commercial Beckman Coulter P/ACE MDQ system with 488 nm LIF detection. Labeling conditions were optimized, including organic solvent, the concentration of the base diisopropylethylamine (DIEA), and the incubation time. Suitable separation characteristics were found using MEKC with sodium dodecyl sulfate as the surfactant, and the resulting analytical technique was characterized.</p><!><p>All chemicals were of analytical reagent grade and were used as received. AlexaFluor 488 succinimidyl ester (AF488 NHS-ester) was purchased from Invitrogen Corporation (Carlsbad, CA), diluted to 20 mM in N,N-dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO), and stored at −20°C. Sodium carbonate (NaCO3, Sigma-Aldrich) was used to prepare 50 mM aqueous solutions with 18 MΩ·cm water. The pH was adjusted using 1 M NaOH (Sigma-Aldrich) and measured using a glass electrode and a digital pH meter (Orion 290A, Thermo; Waltham, MA). Sodium dodecyl sulfate (SDS) was acquired from Sigma Aldrich and used to prepare a 100 mM stock in 18 MΩ·cm water. Amines for standard solutions, including nonylamine (C9-NH2), dodecylamine (C12-NH2), hexadecylamine (C16-NH2), and octadecylamine (C18-NH2), were purchased in pure form from Sigma Aldrich and used to prepare 10 mM solutions in ethanol (Sigma-Aldrich). N,N-diisopropylethylamine (DIEA, Sigma-Aldrich) was diluted to 10 mM in ethanol, DMF, and dimethylsulfoxide (DMSO, Sigma-Aldrich). The stock solutions were combined as needed to result in the solutions used.</p><!><p>Labeling reactions were conducted by combining the appropriate volumes of 20 mM AF488, 10 mM DIEA, amine, and solvent. Reactions were incubated in the dark overnight (16–24 hrs) unless otherwise indicated. After incubation, reactions were diluted into the separation buffer at a 5 : 100 ratio unless otherwise indicated.</p><!><p>Capillary electrophoresis (CE) separations were conducted on a Beckman Coulter P/ACE MDQ capillary electrophoresis system equipped with 488 nm laser-induced fluorescence (LIF) detection. The capillary was rinsed using pressure with the separation buffer for two (2) minutes, and then the sample was injected (pressure) for 5 seconds. Separations were conducted at 15 kV for 15 minutes. After separation, the capillary was rinsed using pressure with pure water for five minutes. Capillary conditioning using 1 M NaOH was conducted with a 5-minute rinse as needed. Separation buffers tested included 10 mM carbonate (pH 10) and 10 mM carbonate with 12 mM SDS (pH 10).</p><!><p>Resulting electropherograms were generated and exported in comma-separated values format using 32 Karat software (Beckman Coulter Inc.). These files were imported into PeakFit (Systat) for smoothing (0.1% Loess) and baseline correction prior to peak fitting. The resulting smoothed and baseline corrected electropherograms or data from peak fitting was imported into Origin (OriginLabs) to generate figures. Chemical equations were drawn in ChemBioDraw Ultra. All raw figures were imported into Adobe Illustrator for image cleanup.</p><!><p>Figure 1 shows the labeling reaction between AF488 NHS-ester and a primary amine. This reaction is base-catalyzed and was found to proceed for C9-NH2 and shorter amines in 10 mM aqueous carbonate, pH 10. Longer-chain amines (C12-NH2 and longer) were found to be insoluble in aqueous solutions without surfactant and therefore did not label to any detectable extent. For this reason, we examined organic solvents for labeling reactions with DIEA included to provide a basic environment. The fluorescence intensities of amines labeled in 10 mM DIEA in ethanol, DMF, and DMSO and then separated in 10 mM carbonate, 12 mM SDS, pH 10, are shown to be normalized to the DMSO fluorescence intensity in Figure 2. Labeling proceeded to nearly the same extent, within error, in all three organic solvents. DMSO may provide slightly better labeling than ethanol. DMSO is often favored in extraction and sample preparation for its solvating ability and stability, so it is encouraging to see that labeling proceeded optimally in DMSO. However, these results also indicate that choice of solvent can be dictated by concerns such as downstream analysis method, safety, and ease of evaporation with marginal reduction in labeling.</p><p>To explore the impact of DIEA concentration on labeling efficiency, its concentration in ethanol was varied from 0 to 48.75 μM in a solution that contained 1 μM amine and 25 μM AF488 NHS-ester. Figure 3 shows the results of CE separation after an overnight incubation of the solutions. While very low levels of amine were labeled without any DIEA, there is no change, within error, of the amount labeled in solutions containing between 12.5 and 48.75 μM DIEA. However, some contamination in the shorter chain amine region (C2-NH2, C3-NH2) was observed to increase with increasing DIEA concentration. These results indicate that DIEA concentration can and, when possible, should be kept to a minimum.</p><p>While overnight incubations are logistically simple for an operator and for potential automated implementations, sometimes it is preferable to obtain results from a sample within a shorter window of overall time. Therefore, we examined the impact of incubation time on the labeling reaction extent. Figure 4 shows the relative fluorescence intensity of the amine peaks diluted into separation buffer and immediately separated via CE at incubation times from 1 hr to 30 hr. Over 90% final intensity was achieved within 6 hours for the amines studied. While the samples were prepared in such a way to potentially yield pseudo-first-order kinetics, the data do not fit a rate law first-order in amine concentration and in fact most closely fit a rate law second-order in amine concentration. This does not seem like a physical likelihood given stoichiometry of the reaction and the standard mechanism proceeding via a tetrahedral intermediate. Therefore, we cannot make any statements about true kinetic parameters of the reaction based on our data and recommend a true kinetics study as part of future work with the AF488 NHS-ester fluorescent probe.</p><p>Based on the above experiments, the optimum conditions for our work using AF488 NHS-ester as a fluorescent probe for amine analysis are 12.5 μM DIEA in DMF with at least 6 hr and preferably overnight incubation. Electropherograms of amines separated in 10 mM carbonate, 12 mM SDS, pH 10, after labeling with the optimized conditions, are shown in Figure 5. While the separation conditions were not optimized nor studied in great detail in this work, separation characteristics are given in Table 1. Limits of detection (S/N = 3) were determined using the optimized labeling conditions and these separation conditions: 17 nM C16-NH2 and 5.7 nM C18-NH2. A contamination issue with our C9-NH2 stock prevented determination of its LOD during the timeframe of this work. This work provides a foundation for further work more fully optimizing an AF488-based amine assay for a desired application.</p><!><p>Amine detection using LIF with AF488 as the reactive probe enables potentially novel analytical techniques as it provides an alternative to other probes' fluorescent at 488 nm. The optimized reaction conditions are mild and fast (90% efficiency within 6 hrs) while enabling excellent limits of detection (nM or ppb). While AF488 is not fluorogenic and therefore cannot be used as a fluorescent probe without some form of postreaction separation, we have demonstrated that this separation can be achieved using a simple, standard MEKC solution and a commercial CE instrument. The limits of detection achievable using the commercial system and initial CE separation conditions were in the low nM range, or single parts-per-billion, making this technique more than sufficiently sensitive for multiple applications. Further work remains to fully characterize the kinetics of the labeling reaction. Additionally, the current separation method is insufficient for the simultaneous analysis of C16-NH2 and C18-NH2; thus, further work is required to develop application-specific optimized separation methods.</p>

PubMed Open Access

Exploring Ion Permeation Energetics in Gramicidin A Using Polarizable Charge Equilibration Force Fields

All-atom molecular dynamics simulations have been applied in the recent past to explore the free energetics underlying ion transport processes in biological ion channels. Roux and co-workers, Kuyucak and coworkers, Busath and coworkers, and others have performed rather elegant and extended timescale molecular dynamics simulations using current state-of-the-art fixed-charge (non-polarizable) force fields in order to calculate the potential of mean force defining the equilibrium flux of ions through prototypical channels such as Gramicidin A. An inescapable conclusion of such studies has been the gross overestimation of the equilibrium free energy barrier, generally predicted to be from 10 \xe2\x80\x93 20 kcal/mole depending on the force field and simulation protocol used in the calculation; this translates to an underestimation of experimentally measurable single channel conductances by several orders of magnitude. Next-generation polarizable force fields have been suggested as possible alternatives for more quantitative predictions of the underlying free energy surface in such systems1. Presently, we consider ion permeation energetics in the gramicidin A channel using a novel polarizable force field. Our results predict a peak barrier height of 6 kcal/mole relative to the channel entrance; this is significantly lower than the uncorrected value of 12 kcal/mol for non-polarizable force fields such as GROMOS and CHARMM27 which do not account for electronic polarization. These results provide promising initial indications substantiating the long-conjectured importance of polarization effects in describing ion-protein interactions in narrow biological channels.

exploring_ion_permeation_energetics_in_gramicidin_a_using_polarizable_charge_equilibration_force_fie

<p>All-atom molecular dynamics simulations have been applied in the recent past to explore the free energetics underlying ion transport processes in biological ion channels. Roux and co-workers1–4, Kuyucak and coworkers5, Busath and coworkers6, and others have performed rather elegant and extended timescale molecular dynamics simulations using current state-of-the-art fixed-charge (non-polarizable) force fields in order to calculate the potential of mean force defining the equilibrium flux of ions through prototypical channels such as Gramicidin A. Such studies overestimate the permeation free energy barrier, generally predicting maximum heights from 10 – 20 kcal/mole depending on the force field and simulation protocol used. This translates to an underestimation of experimentally measurable single channel conductances by several orders of magnitude. Next-generation polarizable force fields7–9 have been suggested as possible alternatives for more quantitative predictions of the underlying free energy surface in such systems1. Presently, we consider ion permeation energetics in the gramicidin A channel using a polarizable force field.</p><p>We apply a charge equilibration polarizable force field for lipids, water, and protein; the small, hard potassium cation is treated as a non-polarizable entity. This is a sufficiently justified approximation as the polarizability of potassium10 is 0.83 Å3, which is smaller than the polarizability of solvent, lipid, and protein constituents. Moreover, without performing full hydration free energy calculations to assess the ion-water and ion-protein interactions, we computed gas-phase interaction energies for K+ with TIP4P-FQ (solvent model employed currently) and the N-Methylacetamide molecule (an often-used proxy for interactions with a peptide backbone). The current force field combination yields a TIP4P-FQ to ion interaction energy of −15.7 kcal/mole and an NMA to potassium ion interaction energy of −28.5 kcal/mole. The respective ab initio values (calculated for this work at the MP2/cc-pVTZ level) are −15.65 and −30.02 kcal/mole and experimental binding affinity (enthalpy) values are −16.2 and −29.83 kcal/mole, respectively. Thus the current force field captures the relative driving force for partitioning between bulk solvent and peptide channel (at least at the level of matching gas-phase binding affinity from experiment and ab initio calculations)11.</p><p>Further details of the simulation system and MD protocol are given in Supplementary information. Structural integrity of the gA channel is monitored via the root-mean-squared deviation from the initial structure, obtained by equilibrating the crystal structure (PDB entry 1JNO)12. From Figure 1 it is evident that over the course of extended time-scale MD simulations, the structure of the channel is robust for the calculations at hand. Data is shown for simulations with a free gA channel in a DMPC bilayer; similar behavior is observed for the channel structure with restrained ion. Figure 2 shows the one-dimensional potential of mean force computed using umbrella sampling with post-simulation data processing using the WHAM equations13, 14. The current polarizable force field shows a dramatic decrease in the central barrier to ion permeation. Furthermore, we stress that no corrections to the PMF (for ionic strength and system periodicity) have been included (though the effects of such corrections will no doubt further reduce inherent barriers; such a study is outside the scope of the present Communication, and is reserved for a future study). Assuming a constant channel K+ diffusion coefficient (reduced by one-tenth from the bulk value), we estimate a maximum conductance of 57pS, in semiquantitative agreement with the experimental value of 24 pS (picoSiemens)1,3 (Supplementary Information). The global minimum occurs at 9.5Å relative center of mass separation, in excellent agreement with the solid state N15 NMR chemical shift anisotropy experiments of Tian et al15. Moreover, the site at 7.5 Å is seen to be of low free energy (almost commensurate in stability to the global binding site) but separated by a significant free energy barrier of 5 kcal/mole. This further coincides with the NMR measurements15 suggesting an internal binding site of significantly reduced signal relative to the external binding sites (conjectured to be of equal free energy). We note that non-polarizable force fields in general predict similar locations of local minima, but the relative energetics are force field-dependent. For the CHARMM nonpolarizable force field, the global minimum is 12.5 Å, while the current polarizable force field shows a global minimum at 9.5 Å.</p><p>Since the subtle, local interactions between the ion and coordinating ligands and channel water primarily impact ion translocation within this channel, we consider in Figure 3 dipole moment distributions of ion-coordinating water molecules and backbone carbonyl groups; Figure 3 (top panel) shows the distributions for water molecules only in the channel for the cases where the water is interacting only with backbone carbonyl groups and when the water is also coordinating the ion. Likewise, for the carbonyl groups (Figure 3, bottom panel), we investigate the dipole moment for groups coordinating one water molecule and those coordinating the ion. We also consider specific residues in order to highlight the residue dependence (local environmental dependence) of the water and carbonyl electrostatics that is possible to capture with polarization effects. The bottom panel of Figure 3 shows the shift in carbonyl dipole moment between coordination with water (solid curve) and ion (dashed curve). This shift is on the order of 0.2 Debye, and this in conjunction with the enhanced water dipole moments agrees with the deep free energy minima corresponding to the binding sites along the channel axis. The induced dipole thus provides a compensatory stabilization for the ion being desolvated. This stabilization is lacking in non-polarizable force fields. Furthermore, the binding site stability is evidenced by the larger barriers presenting as the ion moves out of the binding sites; for the inner binding sites, the polarizable force field PMF shows barriers of 4–5 kcal/mole (i.e. moving towards the inner channel from sites at 9.5, 7.5, and 5 Å).</p><p>SUPPORTING INFORMATION AVAILABLE Supporting information containing simulation protocol and information on force fields is given on the web.</p><p>Backbone rmsd for gA embedded in a DMPC bilayer. RMSD relative to PDBID:1JNO structure12.</p><p>1-D Potential of Mean Force. The x-axis corresponds to the z-component of the center of mass separation between the gA dimer and K+ ion.</p><p>Water dipole moment distributions (top panel) for water coordinating with ion (solid line) and channel waters coordinating with gA backbone carbonyl groups. The numbers 15, 69, and 652 label channel waters starting at the channel opening and moving to the channel center. Bottom panel shows distributions for backbone carbonyl groups (residue TRP13) coordinating with channel waters (solid line) and restrained channel ion (dashed line).</p>

PubMed Author Manuscript

Synthesis and spectroscopic properties of new bis-tetrazoles

Syntheses of N,N′-phenyltetrazole podands link with aliphatic chains containing oxygen, nitrogen and sulphur atoms, are described. The complexing properties of these compounds towards metal cations (Fe2+, Cu2+, Zn2+, Co2+, Ni2+) were investigated by absorption and infrared spectroscopy. The UV–Vis titrations were performed to estimate the stability constant values of the respective complexes with Cu2+ ion. Changes in UV–Vis absorption spectra and IR spectra of compound 6 under various concentrations of Cu2+ ion in methanol suggest formation of very unstable complex. The structure of ligand 2 has been deduced by X-ray crystallography.

synthesis_and_spectroscopic_properties_of_new_bis-tetrazoles

Introduction<!><!>Synthesis<!><!>X-ray structural analysis of the 1,5-bis[2-(1H-tetrazol-1-yl)phenoxy]-3-oxapentane (2)<!><!>Spectrophotometric studies of the metal cation complexation<!><!>Conclusion<!>Experimental section<!>Syntheses<!>General procedure for synthesis the bis-tetrazoles<!>1,2-Bis[2-(1H-tetrazol-1-yl)phenoxy]ethane (1)<!>1,5-Bis[2-(1H-tetrazol-1-yl)phenoxy]-3-oxapentane (2)<!>1,8-Bis[2-(1H-tetrazol-1-yl)phenoxy]-3,6-dioxaoctane (3)<!>1,5-Bis[2-(1H-tetrazol-1-yl)phenoxy]-3-phenylazapentane (4)<!>1,5-Bis[2-(1H-tetrazol-1-yl)phenoxy]-3-(4-toluenesulfonyl)azapentane (5)<!>1,5-Bis[2-(1H-tetrazol-1-yl)phenyl(thio)]-3-phenylazapentane (6)<!>1,5-Bis[2-(1H-tetrazol-1-yl)phenyl(thio)]-3-oxapentane (7)<!>1,5-Di(4-toluenesulfonyloxy)-3-phenylazapentane<!>1,5-Bis(2-nitrophenoxy)-3-phenylazapentane<!>1,5-Bis(2-aminophenoxy)-3-phenylazapentane<!>1,5-Di(4-toluenesulfonyloxy)-3-(4-toluenesulfonyl)azapentane<!>1,5-Bis(2-nitrophenoxy)-3-(4-toluenesulfonyl)azapentane<!>1,5-Bis(2-aminophenoxy)-3-(4-toluenesulfonyl)azapentane<!>1,5-Bis[2-aminophenyl(thio)]-3-phenylazapentane hydrochlochloride<!>1,5-Bis[2-aminophenyl(thio)]-3-oxapentane hydrochloride<!>Spectrophotometric studies of metal cation complexation<!>Supplementary material

<p>Numerous proposals have been made over a considerable period for the production of the synthetic tetrazoles from nitriles, amines and amides. The tetrazole function is metabolically stable and this feature and a close resemblance between the acidic character of the tetrazole group and the carboxylic group has inspired syntheses of potential therapeutic agents. Due to the possibility of deprotonation, protonation and alkylation amongst other reactions, the properties of tetrazoles can be considerably varied and result in numerous possible derivatives. The tetrazole ring is resistant to the action of acids, bases and reducing agents. Tetrazolate anions have dual reactivity, form stable complexes with metals and halogens. They have wide applications as corrosion inhibitors [1], analytical reagents, high-energy materials [2, 3] and component of ionic liquid and gas generating compositions [4]. Tetrazoles play important role in coordination chemistry as ligands [5], in medicinal chemistry as metabolically stable substitutes for carboxylic acids. However, tetrazoles themselves exhibit no pharmacological activity; many of their derivatives possess interesting biological (anti-hypertensive, anti-allergic and antibiotic) activities [6]. In recent years, particular attention has been directed to mono- or bidentate tetrazole ligands as molecular hosts in generating supramolecular arrays and functionalized poly-tetrazoles such as sensors.</p><p>Methods of synthesis monosubstituted tetrazoles, which include 1-, 2- and 5-monosubstituted tetrazoles, were the main subject of various studies. The choice of synthetic methods of 1-substituted tetrazoles is limited. One of the most known methods of synthesis 1-substituted tetrazoles is cycloaddition of isocyanides to hydrazoic acid. This method was improved replacing hydrazoic acid by a more effective reagent, trimethylsilyl azide (Me3SiN3) [7]. Another method involves the reaction of amines with ethyl orthoformate and sodium azide in glacial acetic acid. Aromatic, aliphatic and heteroaromatic amines can be used for preparation of tetrazoles and bis-tetrazole derivatives. 2-Substituted tetrazoles are usually synthesized by alkylation or acylation of tetrazole and that reactions are characterized by low selectivity and poor yield. 5-Substituted tetrazoles can be prepared in several ways; the commonest method of synthesis involves the cycloaddition of nitriles to azides. The reaction is general and is widely used in the synthesis of tetrazoles derivatives in the design of new drugs containing a tetrazole ring. Nitriles possessing various functional groups and dinitriles can be used as initial compounds. Interesting is synthesis of substituted tetrazoles in multistep reaction of aldehydes with iodine in aqueous ammonia, followed by addition of sodium azide in the presence of Lewis acids (e.g., ZnCl2 or ZnBr2) [8] in the mixture of water, ammonium chloride and N,N-dimethylformamide. In classical synthesis of tetrazoles, hydrazoic acid or sodium azide reacts with imidoyl chloride intermediate which is formed in reaction of amide with phosphorus(V) chloride [9]. Noteworthy is one-step conversion of secondary monoamides to tetrazoles under Mitsunobu reaction conditions with azides, especially with tributyl- or trimethylsilyl azide which are safe and soluble in organic solvents [10, 11]. An alternate method employs trifluoromethanesulfonic anhydride and sodium azide for imidoyl derivatives preparation, and thus is converted to tetrazoles [12]. It is commonly known that application of this methodology to the bisamides is impractical because of poor yield and low solubility of this type of compounds in the typical Mitsunobu reaction medium (e.g., anhydrous THF or dichloromethane).</p><!><p>General formula of the macrocycles</p><p>Synthesis of bis-[2-(1H-tetrazo-1-yl)phenoxy]-ethane derivatives</p><p>Synthesis of bis-[2-(1H-tetrazo-1-yl)phenoxy)-3-azapentane derivatives</p><p>a Synthesis of bis-[2-(1H-tetrazo-1-yl)phenyl(thio)]-3-phenylazapentane, b synthesis of bis-[2-(1H-tetrazo-1-yl)phenyl(thio)]-3-oxapentane</p><!><p>In all cases tetrazole residues were obtained in reaction of amines with ethyl orthoformate and sodium azide in glacial acetic acid. The reaction occurs under mild conditions and the yield of products is very high. The best yield over 97 % was obtained in case of compounds 1–3. Final products (1–5) were purified by crystallization. The compounds are white (1–3) or brown (4) solids. The oil compounds 6–7 were purified by column chromatography.</p><!><p>a 1H NMR spectra of 6 in CDCl3, b 1H NMR spectra of 1,5-bis[2-aminophenyl(thio)]-3-phenylazapentane hydrochlochloride in d-MeOH</p><!><p>Experimental data were collected on the KM4 κ-geometry diffractometer equipped with Sapphire 2 CCD detector (Oxford Diffraction). Enhanced Mo Kα1 X-ray radiation source with a graphite monochromator was used. Measurements were carried out in four ω-scan runs—scan width 0.75º, exposure time 300 s per frame was rather long due to weak diffracting power of all the specimen. Determination of the unit cell and data collection was carried out at room temperature, i.e., 293(2) K. All preliminary calculations were performed using CrysAlis RED and CrysAlis CCD software package (Oxford Diffraction, 2010).</p><p>The structure was solved by direct methods. Refinement was made against all reflections by the full-matrix least squares procedure based on F 2. All of the non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were refined as riding with isotropic U values fixed as 1.2 times Ueq of the respective pivot atom. Due to low value of absorption no correction was applied. The calculations were carried out using the SHELX-97 (G. Sheldrick, 1997) program package, run under WinGX 1.70.00 (L. Farrugia, 1999) Windows shell program.</p><!><p>Molecular structure of 2, displacement ellipsoids drawn at 50 % probability level</p><p>Stacking interactions in crystals off compound 2 in vicinity of inversion centre</p><p>Crystal data and structure refinement for podand 2</p><!><p>In recent years the number of publications and patents describing the structure and physicochemical properties of ligands including in their structure tetrazole heterocyclic fragment has grown intensely. This is due to the wide range of practical applications of these compounds. The high physiological activity and low toxicity of tetrazoles makes it possible to regard their metal complexes as substances of versatile biochemical and pharmaceutical destination. Complex stability depends on ligands structure, essentially position and type of electron donating atoms. Compounds 1–3 consist of two 1-N-phenyltetrazole residues linked by polyether chain. Ligand 1 in comparison to 2 and 3 forms the most rigged system. More flexible its analogues 1–2 have more degrees of freedom during complex formation. In further studies other electron donating atoms such as nitrogen (compounds 4 and 5) and sulfur (compounds 6 and 7) were introduced into molecule structure. That modification should improve selective recognition of transition and heavy metal cations.</p><!><p>The absorption maxima (λmax) and corresponding extinction coefficients of new bis-tetrazole in MeOH at 298 K</p><p>The changes in UV–Vis spectra in the presence of metal chloride (tenfold excess) for compounds a 1 (c = 2 × 10−5 mol dm−3); b 2 (c = 2.5 × 10−5 mol dm−3); c 3 (c = 2 × 10−5 mol dm−3) d UV–Vis titration of 2 (c = 2.5 × 10−5 mol dm−3) with copper(II) chloride (c = 10−4 mol dm−3, titration step 0.01 ml) in methanol at 298 K</p><p>UV–Vis titration of 1,5-bis[2-(1H-tetrazol-1-yl)phenyl(thio)]-3-phenylazapentane (c = 2.5 × 10−5 mol dm−3) with copper(II) chloride (c = 10−4 mol dm−3, titration step 0.01 ml)</p><p>IR spectra (film) of pure ligand 6 (a) compared with its complex with copper(II) (b)</p><p>MIR spectra (film) of compound 6 (upper spectrum) and its complex</p><p>a Mid range FT-IR spectrum (film) of the ligand 2 b ligand with FeCl2 c ligand with FeCl3</p><!><p>A novel group of bis-tetrazole containing aromatic units and aliphatic chains with sulphur, nitrogen and oxygen atoms was synthesized. The bis-tetrazole was synthesized by using amines and ethyl orthoformate, sodium azide in glacial acetic acid. It was found that the yield of the products is polyether's chains occurrence dependent. The X-ray structure of compound 2 was reported. Complexing properties of compounds 1–7 have been studied by spectrophotometric titration and IR.</p><!><p>All materials and solvents were of analytical reagent grade. Thin layer chromatographies (TLC) were performed on aluminium plates covered with Silica gel 60 F 254 (Merck). 1H NMR spectra were taken on Varian instrument at 200 and 500 MHz. IR spectra were recorded on Genesis II FTIR (Mattson) instrument. UV–Vis measurements were carried with the use of UNICAM UV 300 Series spectrophotometer. Elemental analyses were obtained on EAGER 200 apparatus. The melting points were uncorrected. All measurements were performed out at room temperature.</p><!><p>The starting diamines: 1,2-bis(2-aminophenoxy)ethane, 1,5-bis(2-aminophenoxy)-3-oxapentane, 1,5-bis[2-aminophenyl(thio)]-3-oxapentane hydrochloride and 1,8-bis(2-aminophenoxy)-3,6-dioxaoctane were synthesized as described earlier in literature [13–16].</p><!><p>A mixture of respective diamine (5 mmol), sodium azide (0.78 g, 12 mmol) and triethyl orthoformate (2.49 ml, 15 mmol) was dissolved in 10 ml glacial acetic acid and was stirred and heated at 90 °C for 9–10 h. It was subsequently cooled to room temperature and the resulting solution was poured into 50 ml of water and the precipitate was filtered off. The crude product (1–5) was purified by recrystallization using distilled water. Compounds 6 and 7 were purified by column chromatography on silica gel. As an eluent, methylene chloride (at the beginning) and then a mixture of methylene chloride–methanol (50:1) was used.</p><!><p>Yield 99 % (1.73 g), white solid, mp 215–216 °C. 1H NMR (500 MHz, d-DMSO): δ = 4.35 (s, 4H, CH2), 7.04 (d, J = 8.31 Hz, 2H, Ar), 7.10 (m, 2H, Ar), 7.41 (m, 2H, Ar), 7.62 (d, J = 7.33 Hz, 2H, Ar), 8.95 (s, 2H, tetrazole) ppm. IR (KBr): 3137, 3090, 2962, 2900, 1510, 1489, 1473, 1397, 1288, 1252, 1171, 1133, 1085, 1036, 746 cm−1. Anal. calcd. for C16H14N8O2: C 54.85, H 4.03, N 31.98. Found: C 54.91, H 4.08, N 31.87.</p><!><p>Yield 98.5 % (1.94 g), white solid, mp 85–88 °C. 1H NMR (500 MHz, CDCl3): δ = 3.81 (t, J = 4.4 Hz, 4H, CH2), 4.27 (t, J = 4.39 Hz, 4H, CH2), 7.15 (m, 4H, Ar), 7.46 (m, 2H, Ar), 7.82 (d, J = 6.35 Hz, 2H, Ar), 9.34 (s, 2H, tetrazole) ppm. IR (KBr): 3127, 3069, 2952, 2891, 1512, 1473, 1453, 1399, 1297, 1253, 1175, 1142, 1132, 1084, 745 cm−1. Anal. calcd. for C18H18N8O3: C 54.82, H 4.60, N 28.41. Found: C 54.78, H 4.54, N 28.37.</p><!><p>Yield 97 % (2.12 g), white solid, mp 110–112 °C. 1H NMR (500 MHz, d-DMSO): δ = 3.71 (m, 6H, CH2), 4.25 (m, 6H, CH2), 7.16 (m, 2H, Ar), 7.32 (d, J = 7.69 Hz, 2H, Ar), 7.50 (m, 2H, Ar), 7.67 (d, J = 6.31 Hz, 2H, Ar), 9.71 (s, 2H, tetrazole) ppm. IR (KBr): 3150, 3073, 2948, 2888, 1507, 1470, 1448, 1394, 1294, 1256, 1166, 1131, 1100, 750 cm−1. Anal. calcd. for C20H22N8O4: C 54.79, H 5.06, N 25.56. Found: C 54.73, H 5.12, N 25.48.</p><!><p>Yield 90.6 % (0.25 g), brown solid, mp 146–148 °C. 1H NMR (500 MHz, d-DMSO): δ = 3.58 (m, 4H, CH2), 4.15 (m, 4H, CH2), 6.61 (m, 4H, Ar), 7.12 (m, 2H, Ar), 7.29 (d, J = 8.06 Hz, 2H, Ar), 7.55 (t, J = 7.69 Hz, 3H, Ar), 7.66 (d, J = 7.69 Hz, 2H, Ar), 9.77 (s, 2H, tetrazole) ppm. IR (KBr): 3147, 3081, 2935, 2883, 1599, 1510, 1473, 1453, 1289, 1251, 1165, 1128, 1088, 751 cm−1. Anal. calcd. for C24H23N9O2: C 61.39, H 4.94, N 26.85. Found: C 61.47, H 4.99, N 26.93.</p><!><p>Yield 46 % (0.075 g), brown solid, mp 103–108 °C. 1H NMR (500 MHz, d-DMSO): δ = 2.37 (s, 3H, CH3), 3.65 (m, 4H, CH2), 4.14 (m, 4H, CH2), 6.88 (m, 4H, Ar), 7.39 (d, J = 8.06 Hz, 2H, Ar), 7.76 (d, J = 8.06 Hz, 2H, Ar), 8.06 (m, 4H, Ar), 8.88 (s, 2H, tetrazole) ppm. IR (KBr): 3135, 3078, 2975, 2881, 1539, 1463, 1453, 1330, 1289, 1251, 1185, 1158, 1088, 1043, 970, 894, 740, 694, 650, 548 cm−1. Anal. calcd. for C25H25N9O4S: C 54.83, H 4.60, N 23.02, S 5.86. Found: C 54.79, H 4.68, N 23.13, S 5.91.</p><!><p>Yield 45 % (0.45 g) of yellow oil. TLC (methylene chloride–methanol, 30:1). 1H NMR (500 MHz, CDCl3): δ = 2.86 (t, J = 6.84 Hz, 4H, CH2), 3.29 (t, J = 7.32 Hz, 4H, CH2), 7.17 (t, J = 7.32 Hz, 3H, Ar), 7.45 (m, 5H, Ar), 7.53 (m, 3H, Ar), 7.57 (d, J = 7.32 Hz, 2H, Ar), 8.90 (s, 2H, tetrazole) ppm. IR (film): 3129, 3096, 3065, 3010, 2927, 1693, 1596, 1496, 1462, 1356, 1279, 1200, 1052, 1045, 997, 874, 754 cm−1. Anal. calcd. for C24H23N9S2: C 57.46, H 4.62, N 25.13. Found: C 57.39, H 4.57, N 25.28.</p><!><p>Yield 65 % (0.26 g) of yellow oil. 1H NMR (500 MHz, CDCl3): δ = 2.89 (t, J = 5.86 Hz, 4H, CH2), 3.41 (t, J = 6.35 Hz, 4H, CH2), 7.46 (m, 4H, Ar), 7.54 (t, J = 6.84 Hz, 2H, Ar), 7.65 (d, J = 7.82 Hz, 2H, Ar), 9.05 (s, 2H, tetrazole) ppm. IR (film): 3137, 3080, 2945, 2555, 1584, 1526, 1474, 1146, 1163, 1297, 1143, 1087, 1007, 761, 467 cm−1. Anal. calcd. for C18H18N8OS2: C 50.69, H 4.25, N 26.27, S 15.03. Found: C 50.73, H 4.32, N 26.19, S 15.14.</p><!><p>N-Phenyldiethanolamine (7.25 g, 40 mmol) was suspended in 100 ml pyridine and cooled with ice at 0 °C. To stirring mixture, three portions of 4-toluenesulfonyl chloride (30.64 g, 160 mmol) were added over a period of 30 min. The mixture was allowed to stay at 5 °C for 24 h. After ice addition, crude product was precipitated. Pure product was obtained by crystallized from petroleum ether. Yield 18.05 g (92 %) of white solid, mp 91–93 °C. 1H NMR (500 MHz, CDCl3): δ = 2.43 (s, 6H, CH3), 3.57 (t, J = 6.19 Hz, 4H, CH2), 4.11 (t, J = 6.02 Hz, 4H, CH2), 6.51 (d, J = 8.06 Hz, 2H, Ar), 6.74 (t, J = 7.32 Hz, 1H, Ar), 7.15 (t, J = 8.3 Hz, 1H, Ar), 7.26 (d, J = 7.61 Hz, 5H, Ar), 7.71 (d, J = 8.3 Hz, 4H, Ar) ppm. IR (KBr): 2958, 1925, 1598, 1505, 1579, 1230, 1150, 1096, 965, 889, 851, 819, 778, 747, 665, 554, 502 cm−1.</p><!><p>A mixture of 2-nitrophenol (1.11 g, 8 mmol), 1,5-di(4-toluenesulfonyloxy)-3-phenylazapentane (1.95 g, 4 mmol) and anhydrous potassium carbonate (1.1 g) in N,N-dimethylformamide (5 ml) was heated at 100 °C for 8 h. After 8 h TLC (eluent, petroleum ether–diethyl ether, 1:2 v/v) confirmed that the starting material had completely been used. The mixture was then diluted with water; the precipitate was separated, washed with water, dried and next recrystallized from propan-2-ol. Yield 1.67 g (99 %) of pale yellow solid, mp 111–113 °C. 1H NMR (200 MHz, CDCl3): δ = 4.02 (t, J = 5.08 Hz, 4H, CH2), 4.31 (t, J = 5.06 Hz, 4H, CH2), 6.76 (m, 3H, Ar), 7.00 (m, 3H, Ar), 7.26 (t, J = 8.01 Hz, 3H, Ar), 7.48 (m, 2H, Ar), 7.82 (m, 2H, Ar) ppm. IR (KBr): 2930, 2882, 1608, 1510, 1347, 1272, 1252, 1195, 1168, 1038, 902, 849, 740, 694, 696, 611 cm−1.</p><!><p>1,5-Bis(2-nitrophenoxy)-3-phenylazapentane (0.42 g, 1 mmol) was suspended in 3.2 ml of ethanol/concentrated hydrochloric acid. To reaction mixture, SnCl2.2H2O (2.03 g, 9 mmol) in 2.5 ml concentrated hydrochloric acid was added dropwise over a period of 30 min. The mixture was refluxed for 8 h with reaction progress control by TLC (diethyl ether). After cooling the reaction mixture was diluted with water and extracted with methylene chloride. The extract was dried with MgSO4 and evaporated under reduced pressure. Pure product, beige–yellow oil, 0.26 g (71 %) was recrystallized from propan-2-ol. 1H NMR (200 MHz, CDCl3): δ = 3.91 (t, J = 5.86 Hz, 4H, CH2), 4.21 (t, J = 5.86 Hz, 4H, CH2), 6.73 (m, 6H, Ar), 6.80 (t, J = 7.32 Hz, 3H, Ar), 6.86 (d, J = 7.81 Hz, 2H, Ar), 7.26 (t, J = 7.31 Hz, 2H, Ar) ppm. IR (film): 3450, 2930, 2880, 1598, 1507, 1453, 1347, 1312, 1272, 1252, 1190, 1163, 1035, 900, 849, 740, 694, 696 cm−1.</p><!><p>Diethanolamine (3.83 ml, 40 mmol) was suspended in 60 ml pyridine and cooled with ice at 0 °C. To stirring mixture, three portions of 4-toluenesulfonyl chloride (45.86 g, 240 mmol) were added over a period of 30 min. The mixture was allowed to stay at 5 °C for 24 h. After ice addition, crude product was precipitated. Pure product was obtained by crystallized from benzene. Yield 9.0 g (39.6 %) of green solid, mp 95–98 °C. 1H NMR (200 MHz, d-DMSO): δ = 2.37 (s, 3H, CH3), 2.42 (s, 6H, CH3), 3.28 (t, J = 5.85 Hz, 4H, CH2), 3.99 (t, J = 5.86 Hz, 4H, CH2), 7.35 (d, J = 7.81 Hz, 2H), 7.48 (d, J = 7.81 Hz, 4H), 7.56 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 8.3 Hz, 4H) ppm. IR (KBr): 2953, 1936, 1597, 1494, 1453, 1357, 1307, 1197, 1097, 1089, 859, 815, 738, 654, 517 cm−1.</p><!><p>A mixture of 2-nitrophenol (2.22 g, 16 mmol), 1,5-di(4-toluenesulfonyloxy)-3-(4-toluenesulfonyl)azapentane (4.5 g, 8 mmol) and anhydrous potassium carbonate (2.21 g) in dimethylformamide (10 ml) was heated at 100 °C for 8 h. The mixture was then diluted with water; the precipitate was separated, washed with water, dried and next recrystallized from propan-2-ol. Yield 3.96 g (99 %) of pale beige solid, mp 117–119 °C. 1H NMR (200 MHz, CDCl3): δ = 2.31 (s, 3H, CH3), 3.87 (t, J = 5.37 Hz, 4H, CH2), 4.31 (t, J = 5.37 Hz, 4H), 7.02 (m, 4H), 7.15 (d, J = 7.81 Hz, 2H), 7.52 (t, J = 8.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 6.35 Hz, 2 H) ppm. IR (KBr): 2966, 2882, 1608, 1523, 1486, 1356, 1335, 1280, 1257, 1156, 1088, 1048, 1014, 967, 895, 748, 716, 696, 641, 547 cm−1.</p><!><p>1,5-Bis(2-nitrophenoxy)-3-(4-toluenosulfonylo)azapentan (0.25 g, 0.5 mmol) was suspended in 1.6 ml of ethanol/concentrated hydrochloric acid. To reaction mixture, SnCl2·2H2O (1 g, 4.5 mmol) in 1.25 ml concentrated hydrochloric acid was added dropwise over a period of 30 min. The mixture was refluxed for 6 h with reaction progress control by TLC (methylene chloride–methanol, 10:1). After cooling the reaction mixture was diluted with water and extracted with methylene chloride. The extract was dried with MgSO4 and evaporated under reduced pressure. Pure product, beige–yellow oil, 0.176 g (71 %) was recrystallized from propan-2-ol. 1H NMR (200 MHz, CDCl3): δ = 2.40 (s, 3H, CH3), 3.70 (t, J = 5.37 Hz, 4H, CH2), 4.14 (t, J = 5.37 Hz, 4H, CH2), 6.64 (m, 2H, Ar), 6.86 (m, 6H, Ar), 7.35 (d, J = 7.32 Hz, 2H, Ar), 7.26 (d, J = 7.81 Hz, 2H, Ar) ppm. IR (film): 3434, 2976, 2880, 1618, 1527, 1453, 1335, 1322, 1287, 1255, 1187, 1159, 1085, 1044, 970, 894, 740, 694, 656, 550 cm−1.</p><!><p>2-Aminothiophenol (1.07 ml, 10 mmol), 1,5-di(4-toluenesulfonyl)oxy-3-phenylazapentane (2.44 g, 5 mmol) and anhydrous potassium carbonate (1.38 g) in dimethylformamide (10 ml) were heated for 24 h at 100 °C. The solvent was removed under reduced pressure and the crude product was diluted with water and extracted with methylene chloride. The extract was dried with MgSO4 and evaporated under reduced pressure. Bisamine was then dissolved in methanol and concentrated hydrochloric acid (0.32 ml) to provide red solid of 1,5-bis[2-aminophenyl(thio)]-3-phenylazapentane hydrochloride (2.32 g, 98 %), mp 168–171 °C. 1H NMR (500 MHz, d-MeOH): δ = 3.11 (m, 4H, CH2), 3.68 (m, 4H, CH2), 7.02 (m, 3H, Ar), 7.31 (t, J = 7.63 Hz, 3H, Ar), 7.43 (m, 5H, Ar), 7.66 (d, J = 7.63 Hz, 2H, Ar) ppm. IR (KBr): 3420, 2827, 2580, 2547, 1972, 1599, 1555, 1503, 1494, 1444, 1280, 1214, 1182, 1141, 759, 691, 452 cm−1.</p><!><p>A mixture of 2-aminothiophenol (2.14 ml, 20 mmol), 1,5-dichloro-3-oxapentane (1.17 ml, 10 mmol) and anhydrous potassium carbonate (2.76 g) in 10 ml of dimethylformamide was heated for 24 h at 80 °C. The reaction progress was monitored by TLC using mixture of petroleum ether–ethyl acetate (4:1 v/v) as a mobile phase. The reaction mixture was cooled to room temperature and extracted with methylene chloride (2 × 20 ml) and water (20 ml). The organic phase was dried over MgSO4 and concentrated under reduced pressure. Pure product was obtained with the use of column chromatography with the same mixture of solvents as TLC. The product was subsequently dissolved in methanol and concentrated hydrochloric acid to give white solid of 1,5-bis(2-aminophenylsulfide)-3-oxapentane hydrochloride, mp 197–199 °C. Yield 2.75 g (70 %). 1H NMR (200 MHz, CDCl3): δ = 2.9 (t, J = 6.0 Hz, 4H, CH2), 3.41 (t, J = 6.05 Hz, 4H, CH2), 6.18–7.12 (m, 8H, Ar). IR (KRr): 3437, 2900, 2560, 1992, 1584, 1556, 1526, 1474, 1446, 1297, 1097, 1007, 757, 467 cm−1. The synthesis was performed analogously to the synthesis compounds described in literature.</p><!><p>UV–Vis titration was carried out by addition of metal chloride to the bis-tetrazole solution. Titrations were carried out in 1 cm path length quartz cuvette keeping constant volume of the ligand solution (2 ml). Titration step 0.01 ml.</p><!><p>Complete crystallographic data of structure have been deposited with the Cambridge Crystallographic Data Centre, CCDC Nos. 859174. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-3336-033, e-mail:deposit@ccdc.cam.ac.uk).</p>

PubMed Open Access

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death*

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.

glyceraldehyde-3-phosphate_dehydrogenase_(gapdh)_aggregation_causes_mitochondrial_dysfunction_during

Introduction<!>Relation between NO-induced GAPDH Aggregation and Mitochondrial Dysfunction in SH-SY5Y Cells<!><!>Formation of GAPDH Aggregates Occurs at Mitochondria<!><!>NO-induced GAPDH Aggregation Directly Causes Mitochondrial Dysfunction in Vitro<!><!>NO-induced GAPDH Aggregation Directly Causes Mitochondrial Dysfunction in Vitro<!>Aggregates of GAPDH Mediate Mitochondrial Dysfunction in SH-SY5Y Cells<!><!>GAPDH Aggregate-mediated Mitochondrial Dysfunction Participates in cyt c Release and Nuclear Translocation of AIF<!><!>GAPDH Aggregates Induce Necrotic Cell Death via PTP Opening in SH-SY5Y Cells<!><!>GAPDH Aggregation Does Not Affect Oxidative Stress-induced Proteasome Activity, ER Stress-related Protein Expression, or Autophagy<!><!>GAPDH Aggregation Does Not Affect Oxidative Stress-induced Proteasome Activity, ER Stress-related Protein Expression, or Autophagy<!><!>GAPDH Aggregation Does Not Affect Oxidative Stress-induced Proteasome Activity, ER Stress-related Protein Expression, or Autophagy<!><!>Discussion<!><!>Chemicals, Antibodies, and Plasmids<!>Cell Culture and Cell Viability<!>Subcellular Fractionations<!>Semiquantification of Mitochondrial Depolarization<!>Assessment of GAPDH Aggregation<!>Cell Immunofluorescence<!>Expression and Purification of Recombinant GAPDH<!>Preparation of Aggregates of GAPDH<!>Preparation of Isolated Mitochondria from Mice<!>Transmission Electron Microscopy<!>Measurement of GAPDH Enzyme Activity<!>In Vitro Mitochondrial Swelling Assay and Mitochondrial Depolarization Assay<!>Examination of Cytosolic Cyt c and Nuclear AIF<!>Apoptosis/Necrosis Assay<!>Proteasome Activity<!>Detecting Levels of Major ER Stress Proteins<!>Examination of NO-induced Autophagy<!>Statistical Analysis<!>Author Contributions<!>

<p>Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that is responsible for the sixth step of glycolysis (1). In addition to this metabolic function, GAPDH is now recognized as a multifunctional protein that exhibits other functions, including transcriptional (2) and posttranscriptional gene regulation (3), intracellular membrane trafficking (4), and cell death (5, 6). In the GAPDH-mediated cell death pathway, the involvement of GAPDH in nuclear translocation and its aggregation under oxidative stress have been proposed (7–9). The active-site cysteine (Cys-152) seems to play a crucial role in both pathways. For example, GAPDH binds to Siah (seven in absentia homolog) through oxidation/S-nitrosylation of Cys-152 and translocates into the nucleus in response to oxidative stress, such as that from nitric oxide (NO) (10). Nuclear GAPDH activates p300/CREB (cAMP-response element-binding protein)-binding protein (CBP) (11) and poly(ADP-ribose) polymerase-1 (12). Additionally, oxidative stressors initiate amyloid-like GAPDH aggregation via intermolecular disulfide bonds at Cys-152 (13–15).</p><p>The accumulation of unfolded proteins can cause protein aggregation in the aged brain, and these aggregates facilitate the formation of pathological amyloid deposits, which is a key cause of several neurodegenerative/neuropsychiatric disorders (16, 17). Aggregated GAPDH in the brain is also amyloidogenic, and GAPDH amyloidal aggregates colocalize with Lewy bodies in Parkinson's disease (18, 19) and with senile plaques and neurofibrillary tangles in Alzheimer's disease (20–23). Based on these findings, we suggested previously a critical role for GAPDH aggregation in oxidative stress-induced neuronal cell death both in vitro and in vivo (14, 15, 24, 25). Further, GAPDH aggregation is likely related to the pathogeneses of amyotrophic lateral sclerosis and Huntington's disease (26, 27). However, the detailed mechanisms for cell death induced by GAPDH aggregation in the context of these pathogeneses remain unclear.</p><p>It has been posited that abnormal protein aggregation leads to mitochondrial dysfunction, proteasome inhibition, endoplasmic reticulum (ER)3 stress, and autophagy, which ultimately cause cell death (28–32). Notably, ∼5–20% of the total GAPDH under physiological conditions is generally bound to the mitochondria in most species (33, 34). Further, treatment of isolated mitochondria with GAPDH directly causes their dysfunction (35) through the activation of voltage-dependent anion channels, which are known components of the mitochondrial permeability transition pore (PTP) (36). PTP opening leads to mitochondrial depolarization and the release of cell death mediators from the intermembrane space, such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF) (37). From these observations, we focused on mitochondria to elucidate the cell death pathway that is mediated by GAPDH aggregation.</p><p>We demonstrated previously that NO-induced cell death is attenuated by a GAPDH mutant with a substitution of Cys-152 to alanine (C152A-GAPDH) in a dominant-negative manner (24). Therefore, this study was designed to investigate the underlying mechanism by which this occurs. Our experiments using C152A-GAPDH revealed the involvement of mitochondrial dysfunction, as well as the subsequent release of cyt c and nuclear translocation of AIF via PTP opening, in NO-induced necrotic cell death mediated by GAPDH aggregation.</p><!><p>As an oxidant, we selected NOC18, an NO generator (14). The IC50 for NOC18-induced decrease of cell viability in SH-SY5Y cells was ∼200 μm (Fig. 1A). Treatment with NOC18 caused GAPDH oligomer formation in cells and in insoluble fractions in a concentration-dependent manner (Fig. 1B). To validate the effect of GAPDH aggregation on mitochondria, we assessed mitochondrial membrane potential in cells using the anion dye rhodamine 123 (Rho 123). Compared with vehicle treatment, 200 μm NOC18 significantly increased Rho 123 fluorescence, indicating a disruption of the mitochondrial membrane, which was enhanced in a concentration-dependent manner with NOC18 (Fig. 1C). We next examined the correlation between the formation of GAPDH oligomers and the disruption of mitochondrial membrane potential; the result clearly indicated a strong correlation (R2 = 0.8036, Fig. 1D). These results indicate a relation between NO-induced GAPDH aggregation and mitochondrial dysfunction. Thus, in the following experiments, sufficient aggregates of GAPDH and mitochondrial dysfunction were observed after 48 h of treatment with 200 μm NOC18.</p><!><p>Relation between NO-induced GAPDH aggregation and mitochondrial dysfunction in SH-SY5Y cells. A, effect of treatment with the indicated concentrations of NOC18 for 48 h on cell viability in SH-SY5Y cells. Data are presented as mean ± S.D. (n = 3–5). B, concentration-dependent effect of NOC18 treatment in SH-SY5Y cells on the formation of insoluble GAPDH oligomers. Cells were treated with the indicated concentrations of NOC18 for 48 h followed by insoluble fractionation and subsequent non-reduced Western blotting (WB). C, concentration-dependent effect of NOC18 treatment on mitochondrial depolarization measured by Rho 123 fluorescence. Fluorescence images of cells treated with vehicle or 200 μm NOC18 are shown in the top panels. The graph represents Rho 123 fluorescence of cells treated with the indicated concentrations of NOC18. Values were normalized by the number of cells stained with Hoechst 33342. Data are presented as mean ± S.D. (n = 6). *, p < 0.05; **, p < 0.01, relative to vehicle treatment, Dunnett's test. Scale bar, 10 μm. D, correlation between the amount of GAPDH oligomers and Rho 123 fluorescence (R2 = 0.8036).</p><!><p>To investigate the origin of the aggregates of GAPDH that induce mitochondrial dysfunction, we used Western blotting to study whether these aggregates exist within mitochondrial fractions in NOC18-treated SH-SY5Y cells (Fig. 2A). Successful mitochondrial fractionation was confirmed by the presence of cytochrome c oxidase (complex IV (CIV)) and the absence of histone H2B (a marker for nuclear fraction) and triosephosphate isomerase (a marker for cytosolic fraction). A large amount of GAPDH was present in the mitochondrial fraction, as reported previously (Fig. 2A, left panel) (35). Further, we verified the presence of GAPDH aggregates in the mitochondrial fraction from SH-SY5Y cells. Although vehicle-treated control cells had only monomeric GAPDH (37 kDa), GAPDH oligomers and a lesser number of GAPDH monomers were present in the mitochondrial fraction of NOC18-treated cells (Fig. 2A, right panel). Similarly, we used immunofluorescence to assess whether GAPDH was localized near the mitochondria using the cell-permeable mitochondrial-selective dye MitoTracker Red (Fig. 2B). Whereas the juxtaposition of the GAPDH signal was around the mitochondria in vehicle-treated cells, NOC18-treated cells showed punctate GAPDH-positive signals that were consistent with aggregates of GAPDH, which partially merged with MitoTracker Red, indicating mitochondrial localization of GAPDH aggregates (Fig. 2B, right panels). To identify where the aggregates of GAPDH occurred in cells, we examined the amounts of oligomeric GAPDH in mitochondrial fractions with or without DTT, which reduces GAPDH oligomers and causes dissociation to monomers (15). In the absence of DTT (under a non-reduced condition), treatment with NOC18 significantly increased the amount of GAPDH oligomers and decreased the amount of GAPDH monomers in mitochondrial fractions, whereas in the presence of DTT (under a reduced condition), these changes were abolished, as assessed by both Western blotting and Coomassie Brilliant Blue staining (Fig. 2C). These results suggest that NO-induced aggregates are derived from mitochondrial GAPDH.</p><!><p>Formation of GAPDH aggregates occurs at mitochondria. A, confirmation of mitochondrial fractionation using CIV as a mitochondrial marker, H2B as a nuclear marker, and TPI as a cytosolic marker. GAPDH was present in the mitochondrial fractions (left panel) from SH-SY5Y cells, which were subjected to non-reduced Western blotting (WB) for GAPDH (right panel). B, images of cell immunofluorescence of GAPDH (green) and MitoTracker Red (red) are shown. GAPDH-positive punctate signals in cells treated with NOC18 partially colocalize with MitoTracker Red. Scale bar, 5 μm. C, Western blotting for GAPDH and Coomassie Brilliant Blue (CBB) staining of mitochondrial fractions in the absence (non-reduced) or presence (reduced) of 100 mm DTT. The graphs represent quantitative results of Western blotting analysis regarding GAPDH oligomers (Oligo.) and monomers (Mono.). Data are mean ± S.D. (n = 3). *, p < 0.05, relative to NOC18(−), Student's t test.</p><!><p>We next evaluated whether GAPDH aggregation leads directly to mitochondrial dysfunction. It has been reported that the detectable amount of GAPDH bound to mitochondria differs depending on the method of isolation (34). Therefore, we attempted to obtain GAPDH-free mitochondria to accurately assess the direct action of GAPDH aggregates on mitochondria. According to the protocol reported previously (38), successful isolation of mitochondrial fractions was achieved and confirmed by transmission electron microscopy (Fig. 3A, left panel). The absence of GAPDH in the isolated mitochondria was ascertained by Western blotting (Fig. 3A, right panel) and by measuring GAPDH enzyme activity (Fig. 3B). We showed recently that the addition of the GAPDH mutant C152A-GAPDH inhibits NO-induced amyloidogenic aggregation of wild type GAPDH (WT-GAPDH) in vitro (24). Therefore, we treated the solutions of isolated mitochondria with aggregates of WT-GAPDH or a mixture containing aggregates of WT- and C152A-GAPDH. Mitochondrial dysfunction was monitored by the degree of mitochondrial swelling and mitochondrial membrane depolarization. The treatment of isolated mitochondria with aggregates of WT-GAPDH significantly decreased the turbidity of the solutions, indicating mitochondrial swelling (Fig. 3C, red line and squares) relative to vehicle treatment (Fig. 3C, black line and circles). The mixture containing aggregates of WT- and C152A-GAPDH significantly reduced the degree of swelling compared with that of WT-GAPDH alone (Fig. 3C, blue line and triangles). We also found that treatment with aggregates of WT-GAPDH caused robust mitochondrial membrane depolarization, indicated by an increase in Rho 123 fluorescence (Fig. 3D, red line and squares), and the mixture containing aggregates of WT- and C152A-GAPDH significantly attenuated this mitochondrial membrane depolarization (Fig. 3D, blue line and triangles). Further, we morphologically examined the effects of aggregates of GAPDH on isolated mitochondria using transmission electron microscopy (Fig. 3E, left panels), and mitochondrial swelling was assessed quantitatively (Fig. 3E, right graph). Compared with vehicle treatment, marked mitochondrial swelling was observed after treatment with aggregates of WT-GAPDH, whereas treatment with a mixture containing aggregates of WT- and C152A-GAPDH attenuated but did not abolish the formation of this abnormal morphology (Fig. 3E). Similar to treatment with aggregates of WT-GAPDH, the swelling of isolated mitochondria was observed after treatment with 1 mm Ca2+, which was used as a positive control. These results indicate that NO-induced GAPDH aggregation directly participates in mitochondrial dysfunction.</p><!><p>NO-induced aggregates of GAPDH cause mitochondrial dysfunction in vitro. A, transmission electron microscopy of isolated mitochondria (left panel). Scale bar, 400 nm. Western blotting (WB) shows the absence of GAPDH in isolated mitochondria (right panel). B, measurement of GAPDH enzyme activity confirms the absence of GAPDH in isolated mitochondria. C and D, effects of GAPDH aggregation on mitochondrial swelling (C) and depolarization (D). Isolated mitochondria were treated with vehicle (black line and circles), aggregates of WT-GAPDH (0.3 mg/ml, red line and squares), or an aggregate mixture of WT- (0.3 mg/ml) and C152A-GAPDH (0.75 mg/ml, blue line and triangles) for the indicated time periods. Mitochondrial swelling was measured by absorbance at 540 nm. Mitochondrial depolarization was measured by Rho 123 fluorescence. Data are mean ± S.D. (n = 4). **, p < 0.01, relative to vehicle treatment; ##, p < 0.01, relative to treatment with aggregates of WT-GAPDH, Dunnett's test. E, transmission electron microscopy of isolated mitochondria treated with vehicle, aggregates of WT-GAPDH, aggregates derived from a mixture of WT- and C152A-GAPDH, and 1 mm Ca2+ (left panels). Scale bar, 400 nm. A mitochondrial cross-sectional area was determined (right). Data are mean ± S.D. (n = 4). *, p < 0.05, relative to treatment with vehicle; #, p < 0.05, relative to treatment with aggregates of GAPDH, Student's t test. F and G, effect of CsA on GAPDH aggregate-induced mitochondrial swelling and depolarization. Isolated mitochondria were treated with vehicle or aggregates of GAPDH (0.3 mg/ml) or CsA (1 μm) for 2 min prior to treatment with aggregates of GAPDH and incubated for 30 min. Mitochondrial swelling and depolarization were measured as described for C and D above. Data are mean ± S.D. (n = 4). *, p < 0.05, and **, p < 0.01, relative to the treatment with vehicle, Dunnett's test; #, p < 0.05, relative to the treatment with aggregates of GAPDH, Student's t test.</p><!><p>One of the most convincing proposed mechanisms underlying mitochondrial swelling and depolarization is the PTP-induced mitochondrial swelling model (39). Based on this model, using cyclosporin A (CsA), which binds to cyclophilin D and inhibits the opening of PTP (39), we examined whether aggregates of GAPDH induce mitochondrial dysfunction via PTP opening. The treatment of isolated mitochondria with aggregates of WT-GAPDH for 30 min elicited mitochondrial swelling and depolarization, whereas these alterations were largely prevented by the addition of CsA (Fig. 3, F and G). Together, these results suggest that aggregates of GAPDH directly evoke mitochondrial dysfunction, resulting in the swelling and depolarization of mitochondria via the opening of PTP.</p><!><p>To investigate the involvement of aggregates of GAPDH in mitochondrial dysfunction at the cellular level, we used doxycycline (DOX)-inducible WT- or C152A-GAPDH-overexpressing SH-SY5Y cells. Western blotting performed with an anti-GAPDH antibody showed that NOC18-induced GAPDH aggregation in mitochondrial fractions was augmented by overexpression of WT-GAPDH (by 2-fold) but was significantly attenuated by C152A-GAPDH overexpression (by 0.8-fold) (Fig. 4A). In addition, NOC18-induced mitochondrial membrane depolarization was measured in these cells by Rho 123 fluorescence (Fig. 4B). Similar to the results shown in Fig. 3D, NOC18-induced mitochondrial membrane depolarization was significantly increased (by 2-fold) in cells overexpressing WT-GAPDH but inhibited by C152A-GAPDH overexpression (by 0.6-fold) (Fig. 4B). These results indicate that NO-induced aggregation of overexpressed GAPDH induces mitochondrial dysfunction in cells.</p><!><p>GAPDH aggregation mediates mitochondrial dysfunction in SH-SY5Y cells. A, effects of overexpression of WT- and C152A-GAPDH on mitochondrial GAPDH aggregation in SH-SY5Y cells. DOX-inducible WT- or C152A-GAPDH-overexpressing cells were treated with NOC18 and then subjected to mitochondrial fractionation followed by Western blotting (WB). The amount of GAPDH oligomers was quantified and presented in the graph on the right. Data are mean ± S.D. (n = 4). *, p < 0.05, relative to DOX(−), Student's t test. B, mitochondrial depolarization measured by Rho 123 fluorescence in NOC18-treated WT- or C152A-GAPDH-overexpressing cells. Data are mean ± S.D. (n = 4). **, p < 0.01, relative to DOX (−), Student's t test. Scale bar, 100 μm.</p><!><p>Initiation of cell death induced by oxidative stress from NO involves mitochondrial membrane depolarization and causes the subsequent release of two cell death mediators from the mitochondria: cyt c, which goes into the cytosol, and AIF, which translocates into the nucleus (39). Therefore, we investigated whether cyt c release into the cytosol and/or nuclear translocation of AIF were caused by GAPDH aggregation. Western blotting showed that when SH-SY5Y cells were treated with NOC18, the levels of cytosolic cyt c were significantly increased (Fig. 5A). Further, NOC18-induced release of cyt c was significantly augmented by the overexpression of WT-GAPDH, whereas the release was decreased by overexpression of C152A-GAPDH (Fig. 5B). This result was also observed by immunofluorescence. In the absence of DOX, cyt c partially merged with MitoTracker Red. This mitochondrial cyt c decreased, likely by diffusion into the cytosol, by overexpression of WT-GAPDH in DOX-treated cells. However, the overlap of MitoTracker Red and cyt c was retained by overexpression of C152A-GAPDH (Fig. 5C). Similarly, we confirmed that NOC18 induced nuclear translocation of AIF in SH-SY5Y cells (Fig. 5D). The levels of AIF in the nuclear fraction from cells treated with NOC18 were significantly increased by overexpression of WT-GAPDH and decreased by overexpression of C152A-GAPDH (Fig. 5E). Immunofluorescence showed that overexpression of WT-GAPDH reduced the merging of AIF signals with those of MitoTracker Red and increased nuclear localization of AIF following NOC18 treatment (Fig. 5F). Moreover, the merging of AIF and MitoTracker Red signals was retained by overexpression of C152A-GAPDH, which did not induce nuclear translocation of AIF. Additionally, CsA treatment significantly inhibited NOC18-induced cytosolic cyt c release (Fig. 5G) and AIF nuclear translocation (Fig. 5H). These results imply that GAPDH aggregation participates in the cytosolic release of cyt c and the nuclear translocation of AIF through PTP-dependent mechanisms under oxidative stress.</p><!><p>GAPDH aggregate-mediated mitochondrial dysfunction participates in release of cyt c and nuclear translocation of AIF. A, release of cyt c into the cytosol of SH-SY5Y cells was induced by treatment with NOC18 for 48 h, as evaluated by cytosolic fractionation and Western blotting (WB, left panel). The graph presents values calculated as the ratio of cyt c band intensity relative to TPI band intensity. Data are mean ± S.D. (n = 3). **, p < 0.01, relative to control, Student's t test. B, effects of the DOX-induced overexpression of WT- and C152A-GAPDH on cyt c release after NOC18 treatment. Data are mean ± S.D. (n = 3). *, p < 0.05, relative to DOX(−), Student's t test. C, immunofluorescence of cyt c (green) and MitoTracker Red (red) in DOX-inducible WT- or C152A-GAPDH-overexpressing SH-SY5Y cells treated with NOC18 for 48 h. Scale bar, 10 μm. D, nuclear translocation of AIF was assessed by Western blotting of nuclear fractions. Values were calculated as the ratio of AIF band intensity to H2B band intensity. Data are mean ± S.D. (n = 3). **, p < 0.01, relative to vehicle treatment, Student's t test. E, effects of overexpression of WT- and C152A-GAPDH on nuclear translocation of AIF induced by NOC18 treatment. Data are mean ± S.D. (n = 3). *, p < 0.05, **, p < 0.01, relative to DOX(−), Student's t test. F, immunofluorescence for AIF (green) and MitoTracker Red (red) in DOX-inducible WT- or C152A-GAPDH-overexpressing SH-SY5Y cells. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. G and H, effect of CsA on NOC18-induced release of cyt c and nuclear translocation of AIF, respectively. Data are mean ± S.D. (n = 3). *, p < 0.05, **, p < 0.01, relative to CsA(−), Student's t test.</p><!><p>We next evaluated the type of cell death induced by GAPDH aggregates using propidium iodide (PI)/annexin V staining. A large percentage of cell death induced by NOC18 treatment was necrotic (about 36%, PI+/annexin V− cells), whereas early apoptosis (PI−/annexin V+ cells) and late apoptosis/necrosis (PI+/annexin V+ cells) were hardly seen (Fig. 6A). To determine the type of cell death induced by GAPDH aggregates, we employed either WT- or C152A-GAPDH-overexpressing cells. Necrosis was significantly increased in cells overexpressing WT-GAPDH but was decreased by C152A-GAPDH overexpression (Fig. 6, B and C). Notably, there were no significant differences in other types of cell death (Fig. 6, B and C). Greater necrosis following WT-GAPDH overexpression was completely abolished by CsA treatment (Fig. 6D), indicating that NOC18-induced GAPDH aggregates cause necrotic cell death via PTP opening.</p><!><p>GAPDH aggregates induce necrosis via the opening of PTP. A, NOC18-induced ell death type was examined by PI/annexin V-FITC staining (left graph). Representative images of necrosis, late apoptosis/necrosis, and early apoptosis are shown in the right panels. B and C, effect of overexpression of WT- or C152A-GAPDH on type of cell death (early apoptosis, late apoptosis/necrosis, or necrosis). Data are mean ± S.D. (n = 3). *, p < 0.05, relative to DOX(−), Student's t test. D, effect of CsA on NOC18-induced necrotic cell death in DOX-inducible WT- GAPDH-overexpressing SH-SY5Y cells. Data are mean ± S.D. (n = 3). *, p < 0.05, relative to DOX(−); n.s., not significant by Student's t test.</p><!><p>In addition to mitochondrial dysfunction, several lines of evidence suggest that the proteasome is also involved in the toxicity of amyloidogenically aggregated proteins, such as amyloid-β and α-synuclein, and these aggregated proteins attenuate the activity of proteasomes in neurons and contribute to cell death (38, 40). Therefore, we studied the effects of GAPDH aggregation on trypsin- and chymotrypsin-like proteasome activities (Fig. 7). We first confirmed the decrease of proteasome activities in NOC18-treated SH-SY5Y cells (Fig. 7A). However, the decline of these proteasome activities induced by NOC18 treatment was not altered by overexpression of either WT- or C152A-GAPDH (Fig. 7B).</p><!><p>Proteasome activity is not affected by GAPDH aggregation. A, reduction of trypsin-like and chymotrypsin-like protease activities caused by the treatment of SH-SY5Y cells with NOC18 for 48 h. Data are mean ± S.D. (n = 4). *, p < 0.05, **, p < 0.01, relative to vehicle treatment, Student's t test. B, effects of the overexpression of WT- and C152A-GAPDH on trypsin-like and chymotrypsin-like protease activities with NOC18 treatment. Data are mean ± S.D. (n = 4). n.s., not significant by Student's t test. A.U. indicates arbitrary units.</p><!><p>We also evaluated the influence of GAPDH aggregation on ER stress, which is considered critical for aggregated protein-induced cell death, by assessing the expressions of the ER chaperone glucose-regulated protein 78 (GRP78; also called KDEL) and the ER-related proapoptotic protein C/EBP homologous protein (CHOP) (41, 42). An up-regulation of the GRP78 and CHOP protein levels was observed in NOC18-treated SH-SY5Y cells (Fig. 8A), which was not altered in cells overexpressing WT- or C152A-GAPDH (Fig. 8B).</p><!><p>Endoplasmic reticulum stress-related protein expression is not affected by GAPDH aggregation. A, up-regulation of GRP78 and CHOP in SH-SY5Y cells treated with NOC18 for 48 h, as evaluated by Western blotting. The graphs present the ratios of GRP78 or CHOP band intensity relative to β-actin band intensity. Data are mean ± S.D. (n = 3). *, p < 0.05, relative to vehicle treatment, Student's t test. B, effects of overexpression of WT- and C152A-GAPDH on the expression of GRP78 and CHOP with NOC18 treatment. Data are mean ± S.D. (n = 3); n.s., not significant by Student's t test. A.U. indicates arbitrary units.</p><!><p>Lastly, we examined the effects of GAPDH aggregation on autophagy. In SH-SY5Y cells, the induction of autophagy by treatment with NOC18 was confirmed by modification of microtubule-associated protein 1 light chain 3B (LC3), observed as an increase in the amount of the faster migrating form, LC3-II (Fig. 9A). The conversion from LCS-I to LCS-II is considered a marker of autophagy (43). Concomitantly, the induction of autophagy was morphologically ascertained by the presence of punctate structures stained with monodansylcadaverine (MDC) (Fig. 9B), which accumulates in autophagic vacuoles (43). By contrast, neither the increase in LC3-II nor the presence of MDC-positive vacuoles was altered by overexpression of WT- and C152A-GAPDH (Fig. 9, C and D). Additionally, the viability of WT- and C152A-GAPDH-overexpressing cells was tested in the presence or absence of 3-methyladfenine (3-MA), an autophagy inhibitor. Similar to results from our previous report (24), we observed exacerbation and amelioration of cell viability under NOC18 treatment following overexpression of WT- and C152A-GAPDH, respectively; these effects were not observed upon treatment with 3-MA (Fig. 9E). Together, these observations indicate that proteasome activity, ER stress-related protein expression, and autophagy do not contribute to mechanisms that mediate GAPDH aggregate-induced cell death with NOC18 treatment.</p><!><p>Induction of autophagy is not affected by GAPDH aggregation. A, NOC18-induced autophagy was measured by the conversion of LC3-I (18 kDa) to LC3-II (16 kDa) as assessed by Western blotting (WB). The graph presents values calculated as the ratio of LC3-II band intensity to β-actin band intensity. Data are mean ± S.D. (n = 3). **, p < 0.01, relative to vehicle treatment, Student's t test. B, fluorescence of MDC staining of NOC18-treated SH-SY5Y cells. Arrows show punctate signals, indicating the induction of autophagy. Scale bar, 10 μm. C, effects of overexpression of WT- and C152A-GAPDH on the induction of autophagy with NOC18 treatment as evaluated by Western blotting. Data are mean ± S.D. (n = 3). D, fluorescence of MDC staining in DOX-inducible WT- and C152A-GAPDH-overexpressing SH-SY5Y cells with NOC18 treatment. Arrows show MDC-positive vesicles, indicating the induction of autophagy. Scale bar, 10 μm. E, effects of autophagy inhibitor 3-MA (1 mm) on the cell viability in WT (left graph)- or C152A (right graph)-GAPDH-overexpressing SH-SY5Y cells treated with NOC18 for 48 h. Data are mean ± S.D. (n = 4). **, p < 0.01, relative to DOX(−), Student's t test. A.U. indicates arbitrary units.</p><!><p>In this study, we have demonstrated that NO induced the formation of GAPDH aggregates at mitochondria, leading to mitochondrial dysfunction (Figs. 1 and 2). Moreover, experiments using GAPDH-free isolated mitochondria showed that GAPDH aggregates directly induced mitochondrial swelling and depolarization via PTP opening (Fig. 3). Additionally, reducing GAPDH aggregation by expressing a dominant-negative mutant, C152A-GAPDH, ameliorated NO-induced mitochondrial depolarization (Fig. 4), subsequent cytosolic release of cyt c and nuclear translocation of AIF (Fig. 5), and necrotic cell death in a PTP-dependent manner (Fig. 6). By contrast, NO-induced alterations of proteasome activity, ER stress-related protein expression, and autophagy were not affected by GAPDH aggregation (Figs. 7–9). These results suggest a crucial role for GAPDH aggregation in mitochondrial dysfunction in cells under oxidative stress.</p><p>NO-induced GAPDH aggregation appears to be composed of mitochondrial GAPDH. Our results show that under treatment with the NO generator NOC18 at a concentration leading to 50% reduction in cell viability, aggregates of GAPDH emerged along with mitochondrial dysfunction (Fig. 1). Furthermore, NO-induced aggregates of GAPDH localized to the mitochondria; these aggregates were derived from mitochondrial GAPDH rather than recruited from other organelles (Fig. 2). In contrast, it was reported that GAPDH translocates to the mitochondria of cerebellar granule cells and several tumor cell lines during apoptotic stimulation, such as with serum deprivation or treatments with staurosporine, etoposide, and lonidamine (35). This difference may be due to the type of stimulus. Indeed, aggregates of GAPDH are generated by various stimuli, such as hydrogen peroxide, dopamine, peroxynitrite, and S-nitrosoglutathione (13), indicating that a variety of mechanisms underlie mitochondrial dysfunction.</p><p>Aggregates of GAPDH directly induce mitochondrial dysfunction. In our experiments using isolated mitochondria free from endogenous GAPDH, treatment with aggregates of GAPDH led to mitochondrial swelling and depolarization. These effects were attenuated by the addition of C152A-GAPDH, which reduces the amount of aggregation (Fig. 3). Alternatively, treatment with soluble GAPDH did not evoke mitochondrial dysfunction (data not shown), which contradicts the findings of Tarze et al. (35). However, their findings (35) were obtained from commercially available GAPDH. It is known that GAPDH is prone to aggregate unless prepared with a reducing reagent such as DTT (14, 15). It is possible that the GAPDH (25 μg/ml) used in their study was already aggregated. Indeed, we found that with a high concentration (100 μg/ml) of soluble GAPDH prepared using our established procedure for purification of fully reduced GAPDH, which can trigger GAPDH aggregation even in the absence of NOC18, induced mitochondrial dysfunction (data not shown). These observations suggest that aggregated GAPDH has more deleterious effects on mitochondria than does soluble GAPDH.</p><p>How do aggregates of GAPDH cause mitochondrial dysfunction? Our data from isolated CsA-treated mitochondria indicate that GAPDH aggregate-induced mitochondrial swelling and depolarization are due to PTP opening (Fig. 3, F and G). Several studies have reported on the mechanisms of mitochondrial dysfunction induced by other aggregated proteins such as amyloid-β, α-synuclein, and mutant huntingtin via PTP (44–46). However, exactly how aggregated protein could open the PTP is not clear. S-Nitrosylated GAPDH reportedly acts as a mitochondrial trans-S-nitrosylase in the heart (47). Hence, to explore the influence of the trans-S-nitrosylase activity of GAPDH aggregates, we first determined the S-nitrosothiols (SNO) of GAPDH aggregates using Saville's method described in Ref. 48. The result indicated that GAPDH aggregates have almost no S-nitrosothiols (0.10 ± 0.02 SNOs/tetramer), similar to soluble GAPDH (0.10 ± 0.01 SNOs/tetramer (supplemental Fig. S1A)). To further investigate the ability of GAPDH aggregate to act as a mitochondrial trans-S-nitrosylase, SNO levels of mitochondrial proteins were examined using isolated mitochondria and the biotin switch method. Compared with mitochondria incubated with vehicle, those treated with GAPDH aggregates did not show different SNO-protein band patterns (supplemental Fig. S1B). Although it is possible that the trans-S-nitrosylase activity of GAPDH may influence intracellular mitochondrial function, taking into consideration that GAPDH aggregates directly cause dysfunction in isolated mitochondria (Fig. 3), the association of a mechanism other than trans-S-nitrosylation could be considered.</p><p>Mitochondrial GAPDH aggregation may be a key regulator of NO-induced necrotic cell death concomitant with the release of cyt c and AIF from the mitochondria. Our data from DOX-inducible WT- or C152A-GAPDH-overexpressing SH-SY5Y cells demonstrate that the amount of mitochondrial GAPDH aggregation correlates with mitochondrial dysfunction resulting in cyt c release into the cytosol and nuclear translocation of AIF (Figs. 4 and 5). Cyt c release is thought to occur via permeability transition-associated mitochondrial swelling and the subsequent rupture of the outer mitochondrial membrane (49). Similarly, nuclear translocation of AIF from the mitochondrial intermembrane occurs concurrently with the disruption of mitochondrial membrane potential (50). These findings support our results showing that PTP inhibition by CsA treatment suppressed cytosolic cyt c release and nuclear translocation of AIF (Fig. 5, G and H). Interestingly, a large population of cell death induced by GAPDH aggregates was necrotic (Fig. 6), even though both cyt c and AIF are generally regarded as apoptotic molecules. Severe ATP depletion and plasma membrane integrity loss are primary drivers of necrosis, but activation of downstream apoptotic signaling is also thought to be a contributor (51). There is growing evidence that AIF is the key molecule in programmed necrosis (termed "necroptosis") (52). Hence, there is a need for further investigation into the contributions of cyt c and/or AIF to GAPDH aggregate-induced necrosis.</p><p>It is well known that aggregates of amyloidogenic protein are involved in proteasome activity (38) and ER stress (41, 42). Aggregates of GAPDH may also contribute to the proteasome inhibition and ER stress responsible for NO-induced cell death (53, 54), as the aggregates in our study were not strictly localized to the mitochondria (Fig. 2B). However, our results indicate that neither NO-induced proteasome inhibition nor ER stress is affected by altering the amount of GAPDH aggregation (via overexpression of WT- and C152A-GAPDH (Figs. 7 and 8)). In addition, autophagy has an important role in NO-induced cell responses (55) and is regulated by GAPDH expression via an increase of Atg12 expression (56). Nonetheless, although NO stress induced autophagy, it was not mediated by the amount of GAPDH aggregation (Fig. 9).</p><p>Nuclear translocation of GAPDH is not likely to participate in the death of SH-SY5Y cells treated with NOC18. We assessed whether WT- or C152A-GAPDH overexpression influences the nuclear translocation of GAPDH induced by NOC18-treatment (supplemental Fig. S2). Treatment of naive SH-SY5Y with NOC18 induced distinct nuclear translocation of GAPDH (supplemental Fig. S2, A and B). The amount of nuclear GAPDH was increased by overexpressing WT-GAPDH but not by overexpressing C152A-GAPDH (supplemental Fig. S2, C and D). Further, we assessed the viability of WT- and C152A-GAPDH-overexpressing cells in the presence or absence of deprenyl, an inhibitor of GAPDH nuclear translocation, and showed that nuclear translocation of GAPDH does not regulate cell viability (supplemental Fig. S2E). Consequently, these results support those from our previous study showing that high levels of oxidative stress are unrelated to the nuclear translocation of GAPDH (14).</p><p>In summary, the present study demonstrates that NO-induced aggregates of GAPDH mediate mitochondrial dysfunction and subsequent necrotic cell death concomitant with cyt c release and AIF nuclear translocation (Fig. 10). It is well recognized that NO has critical roles in the pathogeneses of several diseases, such as Alzheimer's disease, Parkinson's disease, and stroke (57). Interestingly, there is some evidence suggesting the involvement of GAPDH aggregation in Alzheimer's and Parkinson's diseases (18, 21, 23). We have also found that aggregates of GAPDH form abundantly after middle cerebral artery occlusion in a mouse stroke model in which oxidative stress is responsible for neuronal cell death (in preparation). Thus, the findings in the present study could help in understanding the molecular mechanism underlying oxidative stress-related diseases and provide new therapeutic approaches for brain damage.</p><!><p>Schema of GAPDH aggregate-induced mitochondrial dysfunction. Aggregates of GAPDH are formed at mitochondria, which directly induce a decrease in mitochondrial membrane potential (Δψ) and mitochondrial swelling via the opening of a PTP. Mitochondrial dysfunction results in both cytosolic release of cyt c and nuclear translocation of AIF, leading to necrosis. Meanwhile, aggregates of GAPDH do not influence proteasome activity, the ER stress cascade, or the induction of autophagy.</p><!><p>Unless otherwise noted, the chemicals used were of analytical grade. Antibodies were as follows: mouse monoclonal anti-GAPDH (MAB374) and rabbit polyclonal anti-histone H2B (catalog No. 07-371) from Millipore; rabbit polyclonal anti-AIF (AF1475) from R&D Systems; mouse monoclonal anti-cyt c (catalog No. 556433) from BD Biosciences; rabbit polyclonal anti-Myc (A-14, sc-789), rabbit polyclonal anti-CHOP (F-168, sc-575), and rabbit polyclonal anti-His (Omni-probe M-21, sc-499) from Santa Cruz Biotechnology; mouse monoclonal anti-CIV (1D6E1A8) from Invitrogen; monoclonal anti-KDEL (GRP78) (10C3) from Stressgen; monoclonal anti-β-actin (AC15) from Sigma; mouse monoclonal anti-LC3 (2G6) from NanoTools; and polyclonal anti-triosephosphate isomerase (TPI) prepared in house (58).</p><p>The cloning of human WT GAPDH cDNA was performed as reported previously (4). For bacterial expression, cDNA was cloned into pBAD-HisA (Invitrogen) using the SacI and KpnI sites. For mammalian cell line expression, the cDNA was cloned into pcDNA4-TO-Myc/HisA (Invitrogen) using the EcoRI and EcoRV sites. Using WT-GAPDH as a template, the alanine-substituted mutant C152A-GAPDH was generated with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol, as reported previously (15). 3-MA was purchased from Sigma. Deprenyl was kindly provided by Fujimoto Pharmaceutical (Osaka, Japan).</p><!><p>Human neuroblastoma SH-SY5Y cells (ATCC) were grown in DMEM/F12 supplemented with 10% FBS, 2 mm glutamine, and antibiotics-antimycotics (Invitrogen) at 37 °C in a 5% CO2 humidified incubator. Generation of stable cell lines for inducible expression of GAPDH was established as reported previously (15). SH-SY5Y cells were cotransfected with pcDNA6/TR (Invitrogen) and pcDNA4-TO/Myc-HisA vectors carrying WT- or C152A-GAPDH using Lipofectamine 2000 (Invitrogen) or HilyMax (Dojindo). Stable cells resistant to both blasticidin (20 μg/ml) and zeocin (100 μg/ml) were cultivated. Inducible expression of Myc-tagged GAPDH was performed by treatment with DOX (1 μg/ml) for 3–6 days. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay kit (Promega) according to the manufacturer's protocol (15).</p><!><p>Subcellular fractionations were carried out according to the following procedures. After a 48-h treatment with either control vehicle (0.1 n NaOH) or the NO generator NOC18 (Dojindo), cells were washed twice with PBS and then incubated for 5 min in ice-cold PBS containing 40 mm iodoacetamide to protect unmodified thiols from oxidation during fractionation. All subsequent steps were performed at 4 °C. For isolation of the insoluble fraction, cells were scraped in 500 μl of buffer A (10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 3 mm MgCl2, 0.05% Nonidet P-40, 0.5% Triton X-100, 40 mm iodoacetamide, 1 mm PMSF, and protease inhibitor mixture (Roche Diagnostics)). After 30 min, the suspensions of cells were homogenized vigorously for 15 s with rocking. The total lysates were centrifuged at 20,400 × g for 10 min, and both the total cell lysates and pellets were collected to prepare insoluble fractions. To detect insoluble GAPDH oligomers in cells, the pellets obtained from total cell lysates were resuspended in 200 μl of buffer B (10 mm HEPES-KOH, pH 7.4, 25 mm NaCl, 3 mm MgCl2, 300 mm sucrose, 40 mm iodoacetamide, 1 mm PMSF, and protease inhibitor mixture (Roche Diagnostics)), washed three times by centrifugation (3000 × g for 10 min), and again resuspended in the buffer. After the addition of 100 μl of buffer B, the pellets were sonicated for 30 s and finally obtained as insoluble fractions. These fractions were stored at −80 °C for further use. Protein concentrations of the samples were determined using the Bradford assay (Bio-Rad).</p><p>Mitochondria were isolated from cells as reported previously (37) with some modifications. Briefly, after treatments with or without 200 μm NOC18 for 48 h, cells were washed twice with PBS and then homogenized in isolation buffer (5 mm HEPES-KOH, pH 7.5, 210 mm mannitol, 70 mm sucrose, 1 mm EDTA, and 110 μg/ml digitonin). Homogenates were centrifuged at 5000 × g for 20 min at 4 °C, and then the cell pellet was resuspended in isolation buffer. The suspensions were homogenized and centrifuged (2000 × g for 5 min at 4 °C). Supernatants were further centrifuged at 11,000 × g for 10 min at 4 °C, and the resultant pellets were used as mitochondrial fractions.</p><!><p>This procedure was performed as described previously (59) with minor modifications. Cells were treated with 5 μm Rho 123 for 30 min at 37 °C with 5% CO2. Cells were washed with PBS and stained with Hoechst 33342. The fluorescence of Rho 123 was detected by a confocal scanning microscope (C1si-TE2000-E, Nikon, Japan). For semiquantification of fluorescence of Rho 123, four to seven microscopic fields were selected randomly, and the fluorescence values (excitation wavelength, 485 nm; emission wavelength, 535 nm) were measured using an Ez-C1 free viewer (Nikon). Concomitantly, the total number of cells stained with Hoechst 33342 (2 μg/ml) in the same field was counted, and substantial values of fluorescence of Rho 123 were normalized.</p><!><p>To detect aggregates of GAPDH in insoluble and mitochondrial fractions, each fraction was mixed with low SDS sample buffer (final concentration: 62.5 mm Tris-HCl, pH 6.8, 0.5% SDS, 10% glycerol, and 0.002% bromphenol blue) and then heated at 100 °C for 5 min. These samples were separated by 5–20% non-reducing SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were incubated for 1 h with Blocking One (Nacalai Tesque) to block nonspecific binding. The membrane was then incubated for 2 h at room temperature with an anti-GAPDH monoclonal antibody (1:300) in 10% Blocking One-PBST (0.05% Tween 20 and 0.02% NaN3 in PBS) followed by incubation for 1 h at room temperature with a peroxidase-conjugated affinity-purified secondary antibody (Invitrogen). The signals were detected using both SuperSignal West Pico chemiluminescent substrate (GE Healthcare) and LAS3000 (Fujifilm, Tokyo). To assess the purity of mitochondria isolated from cells, Western blotting was performed using an anti-CIV monoclonal antibody (1:1000, a mitochondrial marker), an anti-histone H2B polyclonal antibody (1:5000, a nuclear marker), or an anti-TPI polyclonal antibody (1:1000, a cytosolic marker); membranes were also blotted with an anti-GAPDH antibody.</p><!><p>Immunofluorescence we performed as described previously (4) with minor modifications. For mitochondria staining, cells were treated with MitoTracker Red CMXRos at 250 nm (Life Technologies) for 30 min at 37 °C with 5% CO2. Cells were then washed with PBS and fixed with 4% paraformaldehyde in PBS, pH 7.4, for 10 min at room temperature. The cells were permeabilized for 5 min with PBS containing 0.1% Triton X-100 and incubated with 10% BSA in PBS for 1 h at room temperature. The cells were incubated overnight at 4 °C with an anti-GAPDH (1:1000), anti-AIF (1:10,000), or anti-cyt c (1:200) antibodies in 10% Blocking One-PBST. After three washes with PBST, the specific signal was visualized by staining the cells with an Alexa Fluor 568-conjugated secondary antibody (1:1000; Invitrogen) using a confocal scanning microscope (C1si-TE2000-E, Nikon).</p><!><p>The pBAD-HisA vector carrying WT- or C152A-GAPDH cDNA was transformed into GAP(−) Escherichia coli strain W3CG (60). Expression and purification of these recombinant GAPDH proteins were carried out as described previously (14, 15). Protein concentrations were determined spectrophotometrically assuming ϵ0.1% at 280 nm = 1.0.</p><!><p>The aggregates of GAPDH were prepared as described in the published protocols (24). Briefly, purified recombinant WT-GAPDH (0.3 mg/ml) with or without C152A-GAPDH (0.75 mg/ml) was treated for 24 h with 100 μm NOR3 ((±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide) in G2′ buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, and 5% glycerol). The prepared aggregates were applied immediately to various experiments in the present study.</p><!><p>Liver mitochondria from mice (C57BL/6J males, 20–30 g, SLC Japan) were isolated as reported previously (40) with some modifications. Briefly, the liver was diced and then homogenized in solution A, pH 7.4 (10 mm Tris-HCl, 0.25 m sucrose, and 0.1 mm EDTA). After centrifugation at 80 × g for 7 min, the supernatant was layered on the same amount of solution B, pH 7.4, containing 10 mm Tris-HCl, 0.35 m sucrose, and 0.1 mm EDTA and centrifuged at 700 × g for 10 min. The supernatant was centrifuged at 7000 × g for 10 min. The pellet was resuspended in solution C, pH 7.4 (10 mm Tris-HCl and 0.25 m sucrose), and centrifuged again at 7000 × g for 10 min. The final mitochondrial pellet was resuspended in solution C. The solution containing the purified mitochondria was used immediately.</p><!><p>Isolated mitochondria were treated with aggregates of GAPDH for 30 min at room temperature and then centrifuged at 7000 × g for 10 min. The pellet containing the mitochondria was fixed in 2.5% buffered glutaraldehyde and postfixed in 1% buffered osmium tetroxide. The samples were gradually dehydrated and then embedded in epoxy resin for 48 h at 60 °C. Thin sections (80 nm) stained with uranyl acetate and lead citrate were examined with an electron microscope (Hitachi H-7500) at 75 kV. A mitochondrial cross-sectional area was quantified by using ImageJ software (National Institutes of Health, Bethesda, MD).</p><!><p>An assay solution (50 mm triethanolamine, pH 8.9, 0.2 mm EDTA, and 50 mm K2PO4) was mixed with 1 mm NAD+ and 10 μg of sample. The enzyme activity was initiated by the addition of 2 mm glyceraldehyde-3-phosphate. GAPDH activity was measured for 1 min at 25 °C via the change in absorbance at 340 nm using a VERSAmax microplate reader (Molecular Devices, Tokyo).</p><!><p>The mitochondrial swelling assay was carried out as described previously (61). The isolated mitochondria were diluted at 0.2 mg/ml in buffer M, pH 7.4 (10 mm Tris-HCl, 10 mm Mops, 5 mm succinate, 200 mm sucrose, 1 mm Pi, 10 μm EGTA, and 2 μm rotenone). After adding aggregates of GAPDH, mitochondrial swelling was measured by the decrease in absorbance at 540 nm for 150 min. The mitochondrial depolarization assay was performed as described previously (62). The isolated mitochondria were diluted at 0.2 mg/ml in buffer M and mixed with Rho 123 (5 μm). After a 5-min incubation, aggregates of GAPDH were added, and the fluorescence (excitation wavelength, 485 nm; emission wavelength, 535 nm) was measured using an ARBO multilabel plate reader (PerkinElmer Life Sciences). CsA (1 μm) was added 2 min before treatment with GAPDH aggregates for PTP inhibition.</p><!><p>Cells were treated with or without 200 μm NOC18 for 48 h, washed twice with PBS, and then incubated for 10 min in ice-cold hypotonic buffer (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 0.05% Nonidet P-40, 1 mm PMSF, and protease inhibitor). CsA (1 μm) was added 1 h before treatment with NOC18 for PTP inhibition. The lysates were centrifuged at 3200 × g for 5 min, and the supernatants and pellets were collected. The supernatants were further centrifuged at 100,000 × g for 60 min, and these supernatants were obtained as the cytosolic fraction. The pellets were suspended in isotonic buffer (10 mm HEPES-KOH, pH 7.5, 25 mm NaCl, 300 mm sucrose, 1 mm PMSF, and protease inhibitor), washed twice followed by centrifugation at 3200 × g for 1 min, and resuspended in the buffer. After the addition of hypertonic buffer (50 mm HEPES-KOH, pH 7.5, 400 mm KCl, 3 mm MgCl2, 1 mm EDTA, 1 mm DTT, 0.5% Triton X-100, 0.05% Nonidet P-40, 0.2 mm PMSF, and protease inhibitor), the pellets were sonicated for 30 s and obtained as the nuclear fraction. Western blotting was performed as described above using polyclonal anti-AIF (1:10,000) and monoclonal anti-cyt c (1:500) antibodies. The membranes were also reprobed with an anti-TPI antibody (1:4000) as a cytosolic marker or an anti-histone H2B polyclonal antibody (1:5000) as a nuclear marker.</p><!><p>The apoptosis assay was conducted using the MEBCYTO apoptosis kit (MBL, Nagoya, Japan) according to the manufacturer's instructions with minor modifications. In brief, cells were washed and resuspended in binding buffer. Hoechst 33342 (2 μg/ml), annexin V-FITC, and PI were added to the cell suspension, and then the mixture was incubated for 15 min in the dark at room temperature. Thereafter, the suspension was analyzed using a confocal scanning microscope (C1si-TE2000-E, Nikon). Quantitative analysis was performed by counting more than 700 cells in each examination.</p><!><p>This assay was performed as described previously (63) with minor modifications. Proteasome activity was detected by hydrolysis of the fluorogenic peptide Boc-LRR-MCA (for trypsin-like protease activity; Peptide Institute, Inc.) or Suc-LLVY-MCA (for chymotrypsin-like protease activity; Peptide Institute, Inc.). Briefly, cells were washed twice with PBS and lysed by freezing for 15 min at −80 °C and thawing for 15 min at 37 °C in lysis buffer (20 mm Tris-HCl, pH 7.4, 0.1 mm EDTA, 1 mm 2-mercaptoethanol, 5 mm ATP, 20% glycerol, and 0.04% Nonidet P-40). Freeze-thaw cycles were repeated four times, and lysates were centrifuged at 13,000 × g for 15 min at 4 °C. Protein concentrations of the samples were determined by the Bradford assay. The reaction mixture contained 50 mm HEPES-KOH, pH 8.0, 5 mm EGTA, and 100 μm Boc-LRR-MCA or Suc-LLVY-MCA. The reaction was performed at 37 °C for 60 min and stopped by the addition of 1% SDS. The fluorescence was detected by an ARBO multilabel plate reader (PerkinElmer Life Sciences) at an excitation wavelength of 380 nm and emission wavelength of 440 nm.</p><!><p>Cells were washed twice with PBS and lysed in buffer (50 mm Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, 1 mm PMSF, 100 μm sodium vanadate, 1 mm sodium fluoride, and protease inhibitor mixture). After rotating for 30 min at 4 °C, the lysates were centrifuged at 20,400 × g for 15 min at 4 °C, and the supernatants were subjected to Western blotting as described above using monoclonal anti-KDEL (GRP78), polyclonal anti-CHOP, and monoclonal anti-β actin (internal control) antibodies.</p><!><p>For the detection of LC3, cells were lysed in lysis buffer (50 mm Tris-HCl, pH 7.5, 1% Triton X-100, 150 mm NaCl, and protease inhibitor) and centrifuged at 16,400 × g for 20 min. The supernatants were subjected to SDS-PAGE and transferred to PVDF membranes. Western blotting was carried out with anti-LC3 (1:1000) (kindly supplied by Dr. Matsuzawa, Osaka Prefecture University) and anti-GAPDH antibodies. For MDC staining, the cells were treated with either vehicle or NOC18 and then stained with 200 μm MDC for 45 min. The cells were washed with PBS, and fluorescence was detected as described for cell immunofluorescence.</p><!><p>All data are presented as the mean ± S.D. of independent experiments as indicated (n) in each figure legend (Figs. 1–9). For statistical analysis, two or multiple groups were compared with unpaired Student's t tests or Dunnett's multiple tests after one-way analysis of variance, respectively.</p><!><p>H. N. designed the study. M. I., T. K., A. K., N. H., R. Y., and H. N. performed the biochemical and cell-based studies. M. I., T. I., and M. K. performed the transmission electron microscopy. M. I., T. K., H. N., A. K., Y. A., and R. Y. analyzed the data. H. N. and M. I. wrote the paper, and H. N. and T. T. supervised the study.</p><!><p>This work was supported in part by Grants-in-aid 22580339, 25450428, and 16H05029 (to H. N.), JSPS KAKENHI Grant 15J05838 (to M. I.) from the Japan Society for the Promotion of Science, and Grants-in-aid AS232Z02185G and AS242Z02311Q for Exploratory Research in A-STEP. The authors declare that they have no conflicts of interest with the contents of this article.</p><p>This article contains supplemental Figs. S1 and S2 and accompanying references.</p><p>endoplasmic reticulum</p><p>3-methyladfenine</p><p>apoptosis-inducing factor</p><p>C/EBP homologous protein</p><p>cytochrome c oxidase complex IV</p><p>cyclosporin A</p><p>cytochrome c</p><p>doxycycline</p><p>glucose-regulated protein 78</p><p>light chain 3B</p><p>monodansylcadaverine</p><p>1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene</p><p>propidium iodide</p><p>permeability transition pore</p><p>rhodamine 123 (6-amino-9-[2-(methoxycarbonyl)phenyl]-3H-xanthen-3-iminium chloride)</p><p>S-nitrosothiol</p><p>triosephosphate isomerase.</p>

PubMed Open Access

Evaluation of 3′-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides

Chemically modified oligonucleotides have advanced as important therapeutic tools as reflected by the recent advent of mRNA vaccines and the FDA-approval of various siRNA and antisense oligonucleotides. These sequences are typically accessed by solid-phase synthesis which despite numerous advantages is restricted to short sequences and displays a limited tolerance to functional groups. Controlled enzymatic synthesis is an emerging alternative synthetic methodology that circumvents the limitations of traditional solid-phase synthesis. So far, most approaches strived to improve controlled enzymatic synthesis of canonical DNA and no potential routes to access xenonucleic acids (XNAs) have been reported. In this context, we have investigated the possibility of using phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides. Phosphate is ubiquitously employed in natural systems and we demonstrate that this group displays most characteristics required for controlled enzymatic synthesis. We have devised robust synthetic pathways leading to these challenging compounds and we have discovered a hitherto unknown phosphatase activity of various DNA polymerases. These findings open up directions for the design of protected DNA and XNA nucleoside triphosphates for controlled enzymatic synthesis of chemically modified nucleic acids.

evaluation_of_3′-phosphate_as_a_transient_protecting_group_for_controlled_enzymatic_synthesis_of_dna

<!>Results<!>Discussion<!>Conclusions<!>Methods

<p>X enonucleic acids (XNAs) are synthetic genetic polymers that differ from canonical nucleic acids mainly by the chemical composition of their sugar, phosphate, and nucleobase moieties [1][2][3][4][5] . The presence of chemical modifications on the scaffold of XNAs endows these biopolymers with enhanced properties compared to natural DNA and RNA. For instance, the presence of modified sugar units massively enhances their resistance against nuclease-mediated degradation which is an important prerequisite for the development of therapeutic oligonucleotides [6][7][8][9][10] . Similarly, the presence of additional functional groups on nucleobases or the installation of unnatural base pairs improve the binding and catalytic properties of nucleic acids [11][12][13][14][15][16][17][18][19][20] . So far, synthetic access to XNA oligonucleotides is granted by two different approaches: (i) automated solid-phase synthesis using phosphoramidite building blocks and (ii) polymerase-mediated synthesis with modified nucleoside triphosphates (dN*TPs). While the first approach permits to produce larger amounts of XNA oligonucleotides 3,21 it is limited in terms of size (less than 100 nucleotides) and functional group tolerance 6,22 . On the other hand, the chemoenzymatic method grants access to oligonucleotides of virtually any length 23 and permits in vitro selection experiments to identify XNA aptamers and XNAzymes 20,[24][25][26][27][28][29] . However, this method also requires the use of specially engineered polymerases that are capable of copying DNA templates into XNA and then back into DNA [30][31][32] and all sites will contain the same type of modification. Recently, controlled enzymatic synthesis of DNA, a hybrid method combining elements of both approaches, has emerged and is raising increased attention. In this approach, nucleoside triphosphates are equipped with temporary 3′-protecting groups that can be removed after incorporation into a solid-phase bound primer sequence by a template dependent or independent DNA polymerase [33][34][35][36][37][38] . So far, most efforts focused on using the template-independent DNA polymerase Terminal deoxynucleotidyl Transferase (TdT) in conjunction with small, reversible protecting groups such as aminoalkoxyl 39 or 3′-O-azidomethylene 40 placed on the 3′hydroxyl moiety of DNA nucleoside triphosphates (dNTPs). While this strategy culminated in the launch of a prototype of enzymatic DNA synthesizer by the biotechnology company DNA Script 41 , controlled enzymatic synthesis is still mainly restricted to rather short oligonucleotides 42 and to deoxyribose chemistry exclusively. Herein, we have explored the possibility of expanding this method to the synthesis of XNA oligonucleotides. To do so, we have evaluated the use of 3'-phosphate as a simple, biocompatible protecting group for the controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides.</p><!><p>Rationale and design. The design of a reversible protecting group for controlled DNA and XNA synthesis involves a finely tuned balance between multiple factors. Indeed, polymerases have evolved as finely tuned enzymes capable of specifically recognizing canonical dNTPs or NTPs as substrates and to repel nucleotides with altered sugar moieties including those equipped with functional groups appended on the 2′/3′-OH groups 39,43 . Hence, the protecting group must be a rather small, preferably hydrophilic chemical entity that ensures substrate recognition by the polymerase and does not compromise its incorporation into DNA. In addition, the protecting group must be stable both upon storage of the nucleotide in buffered solution and during the polymerasemediated catalytic step so as to prevent the simultaneous incorporation of multiple nucleotides. Concomitantly, cleavage of the protecting group should proceed in high yields under mild conditions so as not to damage the growing DNA/XNA chain and to permit synthesis of longer oligonucleotides. The installation of a 3′-phosphate group fulfills most of these criteria since it is not a bulky, hydrophilic group that should be stable to hydrolysis under storage and synthesis and can easily be removed by the action of phosphatases. In order to evaluate the possibility of using a 3′-phosphate group to block the addition of nucleotides by polymerases we carried out primer extension (PEX) reactions with a 3′-phosphorylated primer with 10 different DNA polymerases and unmodified DNA dNTPs. Using a 31 nucleotide long template T1 and a 15 nucleotide long, 5′-FAM-labeled primer P1 equipped with a 3′-phosphate moiety 44 (see Supporting Information for sequence composition), all the reactions with the exception of those carried out with Therminator led to a negligible (i.e. <10% conversion) extension of the primer to full length or truncated products (Fig. 1). Importantly, treatment of primer P1 with phosphatases such as the FastAP thermosensitive alkaline phosphatase allowed removal of the 3′-phosphate protecting group and facilitated polymerasemediated DNA synthesis (Supplementary Fig. 1). Similar results were obtained with the TdT polymerase where the 3′-phosphorylated primer P1 prevented the polymerase from adding dT nucleotides and treatment with FastAP thermosensitive alkaline phosphatase restored the tailing reaction capacity of the TdT (Supplementary Fig. 2). Lastly, we performed an Autodock simulation study using the reported X-ray structure of the ternary complex of mouse TdT with ssDNA and an incoming nucleotide (PDB 4I27). In each analysis, we replaced the incoming nucleotide with either 3′-phosphate LNA-TTP or 3′-phosphate-dTTP. This analysis revealed that both modified nucleotides were rather well tolerated within the active site of the TdT polymerase with favorable free energies (−16.94 kcal/mol and −17.40 kcal/mol for the protected dTTP and LNA-TTP, respectively; see Supplementary Figs. 3 and 4) comparable to that of unprotected LNA-TTP 44 . Taken together, these initial experiments suggest that the 3′-phosphate group can efficiently block DNA synthesis, can be removed by the action of phosphatases, and 3′-phosphorlyated dNTPs appear to be rather well tolerated within the active site of certain DNA polymerases at least according to docking experiments.</p><p>Synthesis of 3′-phosphate-dTTP 5 and 3′-phosphate-LNA-TTP 10. We next synthesized the 3′-phosphorylated versions of dTTP (3′-phos-dTTP 5) and LNA-TTP (3′-phos-LNA-TTP 10) to assess whether these modified nucleotides are compatible with controlled enzymatic DNA and XNA synthesis (Fig. 2 and Supplementary Figs. 46-69). To do so, we envisioned a common synthetic pathway that involved first conversion of the commercially available dT phosphoramidite 1 or the known LNA dT phosphoramidite 6 45,46 to the corresponding H-phosphonates 2 and 7 using ETT as activator [47][48][49] . H-phosphonates 2 and 7 were then oxidized to the corresponding P(V) containing nucleotides with iodine under typical oxidation conditions used in solidphase DNA synthesis. The DMTr masking groups of 3 and 8 were then removed under acidic conditions and the deprotected nucleoside analogues were converted to the expected dN*TPs 5 and 10 by application of the 4 step one pot method developed by Ludwig and Eckstein 50 .</p><p>Biochemical characterization of 3′-phosphorylated nucleotides 5 and 10. With both 3′-phosphorylated nucleotides at hand, we set out to evaluate their substrate acceptance by DNA polymerases under PEX reaction conditions. To do so, we carried out PEX reactions using the P1/T1 primer/template system along with 10 different DNA polymerases and with both 3′-phosphorylated nucleotide analogs (Fig. 3). When PEX reactions were conducted with Taq, the expected n + 3 product (corresponding to the addition of a dA, a dC, and one phosphorylated nucleotide) formed in moderate yields (~50%) in the presence of 3′-phos-dTTP 5 and 3′-phos-LNA-TTP 10, which could be optimized to complete conversion of the primer to n + 3 product (Supplementary Fig. 5). Unexpectedly, all other polymerases extended the primer further and generated 22 nucleotide long oligonucleotides corresponding to n + 7 products. Reactions conducted with Therminator even led to the formation of full length products. Intrigued by these results, we analyzed the products stemming from the PEX reactions conducted with the combination of Taq polymerase and 3′-phos-dTTP 5 as well as that with Therminator and 3′-phos-LNA-TTP 10 by LCMS (Table 1, Supplementary Figs. 23-28, and Supplementary Note 1) using established protocols for products stemming from PEX reactions with natural and modified nucleotides 51,52 . This analysis clearly revealed that 1. no phosphorylated nucleotide was incorporated by polymerases and that 2. dA nucleotides were misincorporated opposite templating dAs instead of the modified triphosphates. Such a preference for dAMP misincorporation was further demonstrated when PEX reactions were performed in the presence of all natural dNTPs except for dTTP (Supplementary Fig. 6). Such a behavior was previously observed for the highly modified XNA nucleotide 7′,5′-bc-DNA since incorporation of the modified nucleotide by DNA polymerases proceeded with much lower efficiency than misincorporation of dAMP opposite templating dT residues 53 . Based on these considerations we next wondered if single incorporations might be observed when the 3′-phosphorylated analogs were used in the absence of competitors such as dATP. Hence, we carried out PEX reactions with template T2 that contains a stretch of dA nucleotides immediately 3′-downstream of the corresponding primer P1 as well as with template T3 which was designed as a universal template for controlled DNA synthesis 34 .</p><p>The PEX reactions carried out with dN*TPs 5 and 10 individually and template T2 are shown in Fig. 4. Analysis of the reaction products by gel electrophoresis revealed that both nucleotides were seemingly well accepted by a number of DNA polymerases since bands corresponding to n + 1 and n + 2 and sometimes even n + 3 and n + 4 products could be observed with both nucleotides with the polymerases HemoKlenTaq, Bst, Therminator, Vent (exo − ), and Kf (exo − ). Similar results were obtained when the universal template T3 was used instead of T2 (Supplementary Fig. 7). Here as well, we performed an LCMS analysis of the PEX reaction products obtained with the P1/T3 system in order to try to understand the origin of these multiple incorporation events. The results obtained with the n + 1 and n + 2 products with both modified nucleotides are summarized in Table 2 (also see Supplementary Figs. 29-40).</p><p>This analysis clearly reveals that both modified nucleotides are successfully incorporated when no competitor such as dATP is present. On the other hand, all observed products correspond to the addition of one or two nucleotides onto primer P1 but without the presence of the 3′-phosphate protecting group. These results suggest that both A-family (e.g. Taq) and B-family (e.g. Vent (exo − )) DNA polymerases are capable of removing the 3′phosphate protecting group either at the level of the incoming nucleotide or once installed on the extended primer. We next questioned whether the 3′-phosphate protected nucleotides are accepted as substrates by other polymerase families and whether the phosphate protecting group is also removed by these polymerases. To do so, we performed template-independent PEX reactions using the X-family DNA polymerase TdT along with the 3′-protected nucleotides 5 and 10. In addition, we supplemented the reaction mixtures with three different M 2+ cofactors since the metal preference of TdT is not very strict 33 . After significant optimization of the reaction conditions with 3′-phos-dTTP 5, ~50% conversion of 5′-FAM-labeled primer P2 (Supporting Information for sequence composition) 54 into the corresponding n + 1 product was observed in the presence of Mn 2+ alone or together with Mg 2+ (Fig. 5). On the other hand, 3′-phos-LNA-TTP 10 was not well accepted as a substrate by the TdT polymerase since very modest yields (10-20%) of n + 1 product formed even after long reaction times or when the feed ratio of monomers (i.e., modified triphosphates) to initiator (i.e., primer) was increased (Supplementary Fig. 8) 55 .</p><p>Collectively these results demonstrate that 3′-phosphate protected nucleotides are not very good substrates for DNA polymerases, particularly for family X polymerases such as the TdT. In the absence of competing nucleotides such as dATP, these nucleotides are readily incorporated into DNA by various family A and B polymerases but at the expense of an incomplete blocking activity of the 3′-phosphate group presumably due to the inherent esterase/phosphatase activity of several DNA polymerases. Certain DNA polymerases were recently shown to display an esterase activity (see Discussion) and hence a phosphatase activity is not totally unexpected. Even though commercially available DNA polymerases are certified by the supplier to display less than 0.0001 unit of alkaline phosphatase activity (New England Biolabs), we have performed an MS analysis on Kf (exo − ) which confirmed the absence of any contaminants including phosphatases (Supplementary Figs. 89 and 90, Supplementary Table 1, Supplementary Note 2, and Supplementary Discussion).</p><p>Effect of charge: 3′-cyanoethyl-phosphate protecting group. We first hypothesized that the poor acceptance of nucleotides 5 and 10 as polymerase substrates might be ascribed to the presence of the negative charges on the 3′-phosphate moiety. Hence, we rationalized that an additional phosphoester bond on the 3′protecting group might reduce this negative charge and improve the substrate tolerance. Such an additional ester linkage is readily available if the β-cyanoethyl protecting group of the original phosphoramidites is not removed by cleavage with ammonia. Moreover, docking experiments revealed that dTTP and LNA-TTP equipped with 3′-β-cyanophosphate groups fitted well into the active site of the TdT polymerase (Supplementary Figs. 9 and 10). The free energy for the docking of 3′-β-cyanophosphate-dTTP into the active site of the TdT was comparable to that of the corresponding 3-phosphate nucleotide (−16.65 kcal/mol) while a much more favorable free energy was obtained with 3′β-cyanophosphate-LNA-TTP (−18.18 kcal/mol). We thus converted analogues 4 and 9 into the corresponding triphosphates 11 and 12 by the application of the Ludwig Eckstein protocol (Fig. 6 and Supplementary Figs. 70-76).</p><p>With nucleotides 11 and 12 at hand, we evaluated their substrate capacity for DNA polymerases under PEX reactions with templates T1 and T2 as well as the possibility of using these analogs for TdT-mediated extension reactions. Gel analysis of all the products obtained from PEX reactions revealed that both nucleotides acted as poor substrates for DNA polymerases (Supplementary Figs. 11-14). Indeed, we either observed only very little n + 1 formation or multiple incorporation events were detected at longer reaction times, presumably due to the loss of the β-cyanoethyl protecting group caused by β-elimination in the lower pH of the polymerase buffers. On the other hand, highly contrasting results were obtained when both nucleotides were assayed with the TdT polymerase. Indeed, while the TdT polymerase did not accept the blocked canonical nucleotide 11 (Supplementary Fig. 15), large product distributions were observed when LNA nucleotide 12 was used as substrate (Fig. 7). These results are surprising because i) LNAs are poor substrates for the TdT and usually terminate synthesis after the addition of a single nucleotide 44,56 ; (ii) nucleotide 11 is not recognized as a substrate by the TdT and the primer is not extended by the polymerase; iii) the protecting group is removed either by the polymerase or in the reaction medium.</p><p>In order to shed some light into these results, we first performed TdT reactions with nucleotide 11 followed by the addition of 3′-unblocked LNA-TTP (Supplementary Fig. 16). This experiment clearly revealed that only a single LNA nucleotide was incorporated by the polymerase, suggesting that nucleotide 11 was not recognized by the enzyme. Next, we analyzed the products stemming from the TdT-mediated tailing reaction in conjunction with LNA nucleotide 12 by LCMS (Table 3 and Supplementary Figs. 43-S45). This analysis reveals that the intermediate bands as well as the n i and n i+1 products correspond to different chemical entities. In particular, bands corresponding to single or multiple addition events consist of the primer with one or multiple dehydrated LNA nucleotides devoid of any protecting groups. Such dehydration events have been observed in MS analysis of modified nucleotides 57 . In addition, bands that run between these bands correspond to similar species albeit with an additional Δm/z of 15 compared to the parent bands. Such a Δm/z is typically observed with misincorporation events (e.g. incorporation of dG instead of dA opposite templating dT or dC instead of dT opposite dA) under standard PEX reaction conditions 58 . However, under our experimental conditions, only modified triphosphate 12 was present as substrate. Moreover, similar double-banding events such as that displayed in Fig. 7 have already been described in the past for TdT primer extension reactions carried out in conjunction with sugar and 5′-phosphate-modified nucleotides 59 . This gel pattern was ascribed to the capacity of TdT to phosphorylate (and phosphonylate) oligonucleotides. While the double-banding pattern appears similar, the LCMS analysis of products does not fit with such a phosphorylation event. Such a Δm/z might potentially be connected to deprotection of the β-cyanoethyl moiety and the concomitant addition of the resulting acrylonitrile on a nucleobase. Hence, in order to shed more light into the nature of these products we first analyzed the stability of the cyanoethyl group on nucleotide 11 in a TdT-mediated reaction -19). This analysis revealed that even after 12 h of incubation, the chemical integrity of nucleotide 11 was not altered and no loss of the cyanoethyl group could be detected under these conditions. On the other hand, when LNA-TTP 44 was incubated with acrylonitrile prior to the extension reaction, only the expected n + 1 products could be observed suggesting that the emergence of additional products might arise via a different, yet unidentified mechanism (Supplementary Fig. 20).</p><p>Enhancing resistance against hydrolysis: 3′-thiophosphate group. The addition of an additional cyanoethyl moiety reduced the charge present on the 3′-phosphate blocking groups but Table 2 Summary of the results from the LCMS analysis of the PEX reaction products obtained on the P1/T3 system and with modified nucleotides. introduced a steric bulk that precludes efficient incorporation of the resulting nucleotides by most polymerases. Hence, we questioned whether a minimal perturbation of the phosphate moiety such as the substitution of an oxygen moiety by a sulfur atom could improve the substrate capacity of 3'-phosphate-modified LNA nucleotides. We rationalized that the introduction of a sulfur atom could decrease the capacity of polymerases at hydrolyzing the phosphate moiety since reaction at P = S centers is slower than for the native P = O centers 60,61 and concentrating the negative charge on sulfur could increase interactions with polymerases 62 . Docking experiments comforted these assumptions since the sulfur atom is predicted to interact mainly with an arginine of the active site of the TdT and the overall free energy is very favorable (−17.50 kcal/mol; see Supplementary Fig. 21). Synthesis of the 3′-thiophosphate-bearing nucleotide 15 is highlighted in Fig. 8 and makes use of our recently developed method for the synthesis of thiophosphates with the Beaucage reagent 44 . Briefly, H-phosphonate 7 (Fig. 2) is oxidized to the corresponding P(V) nucleotide 13 using the Beaucage reagent. After deprotection of the DMTr group, the 3′-phosphorothioate nucleotide 14 is converted to the corresponding 5′-triphosphate using the Ludwig-Eckstein approach (Supplementary Figs. 77-88).</p><p>The substrate acceptance of nucleotide 15 for DNA polymerases was investigated in PEX reactions and TdT-mediated tailing reactions (Fig. 9). Clearly, the presence of a 3′-thiophosphate moiety does not improve the substrate acceptance by polymerases since a similar product distribution as with LNA nucleotide 10 (Fig. 3) is observed during PEX reactions with primer P1 and template T1 (Fig. 9A). With the TdT, formation of the expected n + 1 product resulted but in low yields (~20%) and longer reaction times led to the appearance of additional bands, presumably stemming from hydrolytic degradation of the primer (Fig. 9B). These results suggest that the presence of a sulfur atom on the 3′-phosphate moiety does not improve the substrate acceptance by polymerases since misincorporation events might be favored even though the predicted hydrolysis of the protecting group might be reduced. Next, we synthesized nucleotide 16 (Fig. 8) which presents both a sulfur and a βcyanoethyl moiety on the terminal 3′-phosphate group in order to evaluate whether the combination of a sulfur atom and a reduction of the negative charge could improve the incorporation efficiency. However, similar PEX reactions conducted with primer P1 and template T1 (Supplementary Fig. 22) and with the TdT (Supplementary Fig. 23) did only show marginal improvements compared to the incorporation efficiency of the parent compound 15. Interestingly, the presence of the β-cyanoethyl moiety did not lead to multiple incorporation events when the TdT was used as polymerase as was the case for nucleotide 12 that has a P = O center rather than a P = S. Docking experiments reflect these results since a lower free energy (−16.81 kcal/mol) was calculated and unfavorable positioning of the 3′-protecting group within the active site of the polymerase were detected (Supplementary Fig. 24).</p><!><p>Controlled enzymatic synthesis of DNA, RNA, and XNAs represents an interesting and versatile alternative to chemical, phosphoramidite-based synthesis since in principle it is devoid of sequence length limitations and should be more tolerant to chemical modifications on nucleotides and oligonucleotides. This approach would be highly beneficial in a number of practical applications including storage of digital information [63][64][65][66] , assembly of synthetic genes and genomes 67,68 , or functional RNA oligonucleotides 69 . However, despite recent progress and increased interest in this methodology, no universal blocking group has been identified yet that allows synthesis of longer stretches of nucleic acids, particularly of XNAs. This difficulty resides in a delicate balance between steric bulk, robustness, and lability of a protecting group which is required to ensure substrate recognition of nucleotides by polymerases, efficient incorporation into oligonucleotides, and high yielding coupling and deprotection steps. In this context, we have explored the possibility of using 3′-phosphate as a temporary protecting group. Indeed, phosphate is ubiquitously used in nature for transient protection/ modifications of proteins but also of nucleotides and oligonucleotides. Phosphate moieties are robust but can easily be removed by phosphatases and do not introduce a massive steric bulk into scaffolds. Nucleotides bearing 3′-phosphate moieties, however, are poor substrates for A-and B-family DNA polymerases since misincorporation of dAMP moieties is favored to incorporation of such modified nucleotides. This poor substrate acceptance by polymerases might be ascribed to the presence of two negative charges-even though partially masked by interaction with mono-or divalent metal cations or by interactions with residues of side chains of the active sites of polymerases. A similar accumulation of negative charge at the 3′-end of nucleotides might also explain the inhibitory effect of magic spot nucleotides or alarmones (i.e. guanosine-3′,5′-bis(diphosphate) ppGpp and guanosine-3′-diphosphate-5′-triphosphate pppGpp) even though these compounds have never been assayed in conjunction with DNA polymerases 70,71 .</p><p>When reaction mixtures were supplemented with 3′-phosphate containing nucleotides alone, multiple incorporation events were observed which results from abstraction of the protecting group. Since 3′-phosphorylated primers cannot be extended by polymerases, we ascribe these multiple incorporation events to a moonlighting, phosphatase activity of polymerases directly at the level of the incoming, modified nucleotides. This observation is not totally unexpected since various polymerases including HIV-RT, Sequenase 72 , an exonuclease-deficient variant of the archaeal B-family 9°N DNA polymerase 73 , and the large fragment of the A-family DNA polymerase from Bacillus stearothermophilus (BF) 74 possess an efficient 3′-esterase activity once the ester group is installed on the extended primer. Recently, DNA polymerase I fragment (Klenow) was shown to possess a phosphatase activity at the level of nucleotides but this activity consisted in the removal of one or two phosphate groups from 5′-triphosphate entities and only in the strict presence of RNA 75,76 . Lastly, some DNA polymerases (mainly belonging to family X polymerases) such as involved in repair pathways recruit Polymerase Histidinol Phosphatase (PHP) domains to mediate phosphatase activity 77 .</p><p>While additional work will be necessary to pinpoint the site involved in such activity and to unravel its mechanism, this phosphatase activity of polymerases is unprecedented and further underscores the capacity of polymerases to act as enzymes with promiscuous activities.</p><!><p>Controlled enzymatic synthesis represents an alluring alternative to traditional synthetic methods for the generation of wild type and modified oligonucleotides. While intense research has been dedicated to the development of methods and protecting groups suitable for DNA synthesis, little or no efforts have been devoted to similar strategies but for RNA or XNAs. In this context, we have explored the possibility of using phosphate as a transient 3′blocking group for controlled enzymatic synthesis of DNA and LNA containing oligonucleotides. While this protecting group does not appear suitable for our approach despite meeting most of the required criteria, an unexpected and unprecedented moonlighting activity of various family A and B DNA polymerases was discovered. These results will allow us to refine the design and the chemical nature of other 3′-protecting groups to be explored for the controlled synthesis of XNA oligonucleotides and might have repercussions in understanding the mechanism of alarmones and the effect of phosphorylation of nucleotides in complex systems.</p><!><p>General protocol of TdT-mediated tailing reactions. Primer P2 (20 pmol) is incubated with the modified nucleoside triphosphate (200 µM) with a suitable metal cofactor (0.25 mM Co 2+ , 1 mM Mn 2+ , or 1 mM Mg 2+ ) and the TdT polymerase (10 U) in 1X reaction buffer (supplied with the polymerase; 10 µL final volume) at 37 °C for given reaction times. The reaction mixtures were then purified by Nucleospin columns and quenched by the addition of an equal volume of loading buffer (formamide (70%), ethylenediaminetetraacetic acid (EDTA, 50 mm), bromophenol (0.1%), xylene cyanol (0.1%)). The reaction products were then resolved by electrophoresis (PAGE 20%) and visualized by phosphorimager analysis.</p><p>General procedure for primer extension reactions. The template (15 pmol) was annealed to its complementary primer (10 pmol) by heating to 95 °C and slowly (over 30 min) cooling down to room temperature. The annealed oligonucleotides were then supplemented with modified and/or natural dNTPs (all 200 µM final concentrations) and polymerase (2 U) in 1X reaction buffer. The reaction mixtures were then incubated at the recommended temperature for given amounts of time. The reaction mixtures were then purified by Nucleospin columns and quenched by the addition of an equal volume of loading buffer (formamide (70%), ethylenediaminetetraacetic acid (EDTA, 50 mm), bromophenol (0.1%), xylene cyanol (0.1%)). The reaction products were then resolved by electrophoresis (PAGE 20%) and visualized by phosphorimager analysis.</p><p>Chemical syntheses. Detailed protocols for the synthesis of all nucleoside and nucleotide analogs can be found in the Supporting Information of this article.</p><p>Docking experiments. AutoDock version 4.2 was used for the docking simulation 78 . The TdT enzyme file was prepared using published coordinates (PDB 4I27). The magnesium atom was retained within the protein structure. A charge of +2 and a solvation value of −30 were manually assigned to the Mg atom. The molecules files were built on Biovia Discovery Studio ® 4.5 and saved as pdb files. The docking area was assigned visually around the presumed active site. A grid of 40 Å x 40 Å x 40 Å with 0.497 Å spacing was calculated around the docking area using AutoGrid. We selected the Lamarckian genetic algorithm (LGA) for ligand conformational searching, which evaluates a population of possible docking solutions and propagates the most successful individual solution from each generation into the subsequent generation of possible solutions.</p><p>For each compound, the docking parameters were as follows: trial of 20 dockings, population size of 150, random starting position and conformation, translation step ranges of 1.5 Å, rotation step ranges of 35°, elitism of 1, mutation rate of 0.02, crossover rate of 0.8, local search rate of 0.06 and 2,500,000 energy evaluations. The docking method was first evaluated by redocking the corresponding ligand of the PDB structure and then docking of the molecules of interest in the TdT active site. The conformation of the obtained results was inspected and compared to the literature and crystal structures. The docking results from each of the compounds were clustered on the basis of the root-mean-square deviation (rmsd) of the Cartesian coordinates of the atoms and were ranked on the basis of free energy of binding. The top-ranked compounds were visually inspected for correct chemical geometry.</p>

Nature Communications Chemistry

Substrate Protection in Controlled Enzymatic Transformation of Peptides and Proteins

AbstractProteins are involved in practically every single biological process. The many enzymes involved in their synthesis, cleavage, and posttranslational modification (PTM) carry out highly specific tasks with no usage of protecting groups. Yet, the chemists’ strategy of protection/deprotection potentially can be highly useful, for example, when a specific biochemical reaction catalyzed by a broad‐specificity enzyme needs to be inhibited, during infection of cells by enveloped viruses, in the invasion and spread of cancer cells, and upon mechanistic investigation of signal‐transduction pathways. Doing so requires highly specific binding of peptide substrates in aqueous solution with biologically competitive affinities. Recent development of peptide‐imprinted cross‐linked micelles allows such protection and affords previously impossible ways of manipulating peptides and proteins in enzymatic transformations.

substrate_protection_in_controlled_enzymatic_transformation_of_peptides_and_proteins

<!>Introduction<!>Molecular Recognition of Peptides<!>Micellar Imprinting of Peptides<!><!>Micellar Imprinting of Peptides<!><!>Micellar Imprinting of Peptides<!><!>Micellar Imprinting of Peptides<!>Sequence‐Selective Protection of Peptides by MINPs in Enzymatic Reactions<!><!>Sequence‐Selective Protection of Peptides by MINPs in Enzymatic Reactions<!><!>Sequence‐Selective Protection of Peptides by MINPs in Enzymatic Reactions<!><!>Sequence‐Selective Protection of Peptides by MINPs in Enzymatic Reactions<!>Conclusions and Outlook<!>Conflict of interest

<p>Y. Zhao, ChemBioChem 2021, 22, 2680.</p><!><p>Enzymatic efficiency and selectivity represent the ultimate goals of chemists who seek to develop catalysts for their interested reactions. Indeed, under largely ambient conditions in neutral aqueous solutions, enzymes hydrolyze particular amide bonds, selectively oxidize hydrocarbons, convert nitrogen into ammonia, and perform all kinds of transformations vital to the biological world.</p><p>The high selectivity of enzymatic catalysis allows cells to carry out desired biochemical transformations from exceedingly complex mixtures without usage of any protecting groups. Glycosyltransferases and glycosidases, for example, effortlessly make complex glycans and cleave them at specific locations.[1] In contrast, to synthesize even relatively simple glycans, chemists generally have to employ extensive protective/deprotective chemistry to deal with the many hydroxyl groups on the sugar building blocks that have little or no difference in intrinsic reactivity.[2] Only in this year of 2021, synthetic catalysts appeared in the literature that could hydrolyze oligo‐ and polysaccharides with a reasonable level of selectivity.[3]</p><p>Protective groups have been an indispensable tool in modern organic chemistry, not only in the synthesis of biomolecules such as carbohydrates, peptides, and nucleic acids full of degenerate functional groups, but also in total synthesis of almost any complex, multifunctional molecules.[4] Whenever chemists want to perform a chemical reaction that has compatibility issues with existing functional groups in the molecule, a straightforward and often the most reliable method is to protect the incompatible groups prior to the reaction and deprotect them at a suitable stage later on.</p><p>It seems, with the abundance of highly selective and even substrate‐specific enzymes, protection/deprotection is neither necessary nor useful in biology. However, this is not the case, at least when it comes for researchers to intervene and interrogate certain biological processes.</p><p>A good example is in the proteolysis of peptides and proteins. Cancer cells rely on over‐expressed proteases during their invasion and spread because of the need to remodel tissues.[5] Since the same proteases may be used by normal cells to perform their cellular functions, traditional protease inhibitors tend to have high toxicity. Many enveloped viruses depend on a critical proteolytic activation step in their cellular infection including coronavirus,[6] HIV‐1,[7] and influenza virus A.[7] Selective inhibition of a specific proteolytic reaction instead of all proteolysis is again vital to the antiviral treatment. Antibodies can be used to block proteolytic cleavage sites on proteins[8] but they are expensive and fragile molecules made of polypeptides, which are susceptible to broad‐specificity proteases themselves.</p><p>One other example is in the post posttranslational modification (PTM) of proteins. Kinases catalyze phosphorylation of proteins, a reaction critical to numerous processes in cell signaling, regulation, and development.[9] However, a vast number of potential phosphorylation sites exist in a cell, ∼700,000 by one estimation.[9b] Even if most of these sites are buried and kinases have their own preferred substrates, a cell still has a large number of substrates for a given kinase.[9] Traditional enzymatic inhibition is again facing a problem in this case, because unintended consequences will emerge when a multisubstrate kinase is shut down.[10]</p><p>A long‐recognized solution to the above problems lies in the selective inhibition of the peptide or protein substrates.[11] If a particular substrate of a protease or kinase can be selectively protected from the enzyme, one would be able to shut down a specific biological reaction with high precision. Such protection can help researchers understand the biological ramifications of the masked biological reaction, useful in mechanistic biology and also potentially in disease treatment.[8] Over the years, a few research groups have reported protection of peptides from chemical or enzymatic reactions, mainly using small‐molecule synthetic receptors. The reactions involved include proteolysis,[11, 12] acetylation of lysine side chains,[13] tyrosine phosphorylation,[14] and demethylation of methylated lysine side chains.[15]</p><!><p>The scarcity of peptide protection in the literature points to a great need in peptide recognition, especially of complex biological peptides. To protect a peptide sequence from its enzymatic catalyst, one needs a receptor to bind the peptide with high affinity and selectivity in aqueous solution. Supramolecular chemistry in the last several decades largely have stayed in organic solvents where directional noncovalent forces such as hydrogen bonds are effective.[16] Although examples of strong synthetic receptors in aqueous solution exist,[17] they are exceptions rather than rules and a general strategy for effective molecular recognition of complex biological molecules in water is missing.[18] For peptides, a particular challenge is in the distinction of the 20 possible side chains of a peptide, some of which differ minutely. Leucine (L) and isoleucine (I), for example, differ in the position of a single methyl group by one carbon. Glutamic acid (E) has one extra methylene than aspartic acid (D), and tyrosine (Y) has one extra hydroxyl in comparison to phenylalanine (F).</p><p>Chemists have developed many scaffolds to build peptide receptors,[19] often focusing on specific residues with good supramolecular handles such as acidic and basic amino acids.[20] Tryptophan (W) and phenylalanine are also popular targets because their aromatic side chains can fit into appropriate macrocycles[21] such as cyclodextrins,[22] cucurbiturils,[23] or self‐assembled nanocages.[24] Other interesting platforms include molecular tweezers and clips,[25] pseudopeptidic cages,[26] and gold nanoparticles that can be functionalized on the surface.[27]</p><p>Principles of complementarity and preorganization are the central dogma of supramolecular chemistry.[16, 28] It is impractical, however, to apply them in peptide recognition with a molecularly synthesized receptor. This is because, to bind a guest with multipoint noncovalent interactions, the host generally is larger than the guest and needs to possess a complementary, often concave‐shaped binding interface. For a biological peptide with 10 to 20 amino acid residues that have subtly different side chains, a complementary host, if constructed step‐by‐step, would be too difficult to design and synthesize.</p><p>A potential solution to the above problem comes from molecular imprinting, a simple and powerful method to create guest‐complementary binding sites in a cross‐linked polymer network.[29] The method involves formation of a covalent or noncovalent complex between a template (often the guest molecule itself) and polymerizable functional monomers (FMs) in the presence of a large amount of a cross‐linker. Polymerization fixes the FMs around the template molecules in the polymer network. Cleavage of the covalent bonds between the FMs and the templates or, in the case of noncovalent imprinting, washing off the noncovalently trapped templates leaves behind "imprints" or guest‐shaped voids in the polymer. The FMs turn into binding groups in the imprinted sites during polymerization and can increase the selectivity and binding affinity for the template molecules during rebinding.</p><p>Many molecularly imprinted polymers (MIPs) have been created for peptides since the conception of the technique.[30] One of the earliest examples of noncovalent imprinting used amino acid derivatives as templates.[31] Traditional MIPs are insoluble polymeric materials. Nonetheless, when prepared under precipitation polymerization, MIP nanoparticles, 10–100 nm in size, are obtained that have great biological compatibility.[32] Materials imprinted against mellittin (the major component of bee venom) in this way could be used to remove the toxin from the bloodstream of living mice.[33] MIP nanoparticles can be prepared for hydrophilic peptides as well, if they are first functionalized with a fatty acid acyl chain and anchored at the interface of inverse microemulsion.[34] One other way to produce water‐soluble imprinted materials is to perform imprinting on the surface of (diacetylene‐containing) vesicles, which after polymerization could report the binding event by fluorescence.[35]</p><!><p>To selectively protect a peptide under many biological settings, a 30–100 nm nanoparticle is probably still too large. Often times, it is a specific sequence of a long peptide to be protected in an enzymatic reaction, and the remaining peptide sequences could be part of a protein tertiary structure. Other times, a long peptide has several reaction sites and a specific site is to be protected while others remain accessible to their enzymatic catalysts. For all these situations, a high precision of protection is required that demands a peptide protector much smaller in size.</p><p>Our group in 2013 developed a method of molecular imprinting within doubly cross‐linked surfactant micelles.[36] The so‐called molecularly imprinted nanoparticles (MINPs) are ∼5 nm in diameter with surface ligands and ∼4 nm without. They are, hence, similar to a medium‐sized protein in dimension and quite a bit smaller than typical antibodies (∼10 nm). Their surface charge can be tuned by different types of cross‐linkable surfactants.[37] Micellar imprinting was first used to create selective receptors for bile salt derivatives and then quickly expanded to a wide range of biologically interesting small molecules/drugs,[36, 37, 38] carbohydrates,[39] and peptides,[40] all in water. Most recently, they are converted into artificial enzymes to catalyze a range of chemical reactions.[3, 41]</p><p>As shown in Scheme 1, micellar imprinting starts with spontaneous formation of micelles using a cross‐linkable surfactant (1) in the presence of the interested peptide as the template molecule, divinylbenzene (DVB, a free‐radical cross‐linker), and a small amount of 2,2‐dimethoxy‐2‐phenylaceto‐phenone (DMPA, a photo initiator). The surface of the micelle is covered with a dense layer of alkyne groups from tripropargylammonium headgroup of the surfactant, and can be cross‐linked by diazide 2 in the presence of Cu(I) catalysts via the highly efficient click reaction. Another round of click reaction with monoazide 3 installs a layer of hydrophilic ligands on the surface of the micelle. (The sugar‐derived surface ligands are highly hydrophilic but insoluble in organic solvents such as acetone and methanol, and thus help the isolation and purification of the final MINPs.)</p><!><p>Preparation of peptide‐binding MINP from molecular imprinting of a cross‐linked micelle, with a schematic representation of the cross‐linked structure containing WDR bound by polymerized FMs. (Reprinted with permission from Ref. [51]. Copyright 2021, the American Chemical Society.)</p><!><p>Micelles are highly dynamic, with diffusion‐controlled intermicellar exchange of surfactants.[42] Covalently tethered on the surface, the surface‐cross‐linked micelle (SCM) becomes a nanoconfined space for molecular imprinting, as UV irradiation initiates free‐radical polymerization/cross‐linking around the template molecule in the micellar core.[43] The nanoconfinement is found to be extremely important to the large imprinting factors obtained from micellar imprinting (often in the hundreds[39d, 41c] and sometimes up to 10,000[40e]). In addition, MINPs can easily detect the addition,[43] removal,[43] and shift[40a] of a single methyl (or methylene) group in the guest binding.</p><p>For peptide binding, we initially focused on those rich in hydrophobic amino acids because they have a strong driving force to enter the micelle.[40a] Our reasoning was that the hydrophobic side chains of amino acids have different degrees of hydrophobicity. For common hydrophobic amino acids, their side chains – shown schematically as blue shapes in Scheme 1 – differ in size, shape, and hydrophobicity. Thus, a "hydrophobic code" exists for each peptide that describes the number, size, shape, and distribution of its hydrophobic side chains.</p><p>Micellar imprinting, indeed, was found to create a complementary array of hydrophobic indentations or "dimples" on the cross‐linked micelles, essentially "encoding" the MINPs with supramolecular information to match the hydrophobic "code" of the peptide. These imprinted hydrophobic "dimples" turned out highly discriminating in their binding, to the point that the shift of a single methyl in leucine and isoleucine in isomeric di‐ and tripeptides could be distinguished, as well as phenylalanine and tyrosine.[40a] The binding was also highly selective. When 5 MINPs were created for 5 biological peptides, negligible cross‐reactivity was observed when a particular peptide was titrated with the 4 nonmatching MINPs or, conversely, a particular MINP with the 4 nonmatching peptides.</p><p>Specific FMs (4–6) can be included in the MINP preparation to further improve the binding. They generally contain a polymerizable vinyl group and a molecular recognition motif targeting specific functional groups on the peptide (see the schematic representation of WDR bound by polymerized FMs in Scheme 1). FM 4, for example, binds carboxylic acids through the hydrogen bond‐reinforced thiouronium–carboxylate salt bridge.[40c] FM 5, with abundant hydrogen‐bond acceptors in the structure, prefers the guanidinium side chain of arginine.[40b] FMs 6 is selective for the amino group on the side chain of lysine and also on the N‐terminus.[40d] With these FMs, we can target the hydrophobic, acidic, and basic groups of a peptide simultaneously, greatly enhancing both the binding selectivity and affinity of the MINP.[40d] The functionalized MINPs have been shown to distinguish closely related hydrophilic residues such as aspartic acid/glutamic acid and lysine/arginine.</p><p>One might be surprised by how well these hydrogen‐bonded FMs work in MINP formation and binding. The mechanism is the same as how proteins and nucleic acids use these noncovalent forces in water – i. e., in a relatively nonpolar microenvironment where water is largely excluded. Although hydrogen‐bonds are weakened by strong solvent competition in an aqueous solution, they are much stronger in the hydrophobic core of a micelle.[44]</p><p>Most recently, we discovered that, instead of specially designed FMs, commercially available amide‐containing cross‐linkers such as N,N′‐methylene‐bisacrylamide (MBAm) can be used instead of DVB during micellar imprinting (Figure 1).[40e] The radical initiator (DMPA), being hydrophobic, strongly prefers to reside within the nonpolar core of the micelle. Once the initiating radical reacts with the methacrylate of the cross‐linkable surfactant (1) inside the SCM, the propagating radical is covalently attached to the micellar core and can polymerize only the MBAm molecules diffused to the palisade layer of the micelle. As a result, a belt of hydrogen‐bonding groups is formed near the surfactant/water interface, around the peptide template residing in the same area by its amphiphilicity.</p><!><p>Structures of peptide templates used in the synthesis of MBAm‐functionalized MINPs.</p><!><p>When we compared the binding properties of MINPs prepared with DVB (our normal core‐cross‐linker) plus FMs and those prepared with MBAm (essentially as a hydrogen‐bonding functional cross‐linker), the MBAm‐cross‐linked MINPs were pleasantly found to outperform the DBV‐cross‐linked, functionalized MINPs consistently (Table 1). The binding constants for a number of complex biological peptides (7–12 in Figure 1) ranged from 60 to 90×105 M−1, corresponding to 110–170 nM of binding affinity. Excellent binding selectivity was also observed (Figure 2).[40e]</p><!><p>Binding data for biological peptides 7–12 by MINPs prepared with DVB and FMs, and by MINPs prepared with MBAm without FMs.[a]</p><p>Entry</p><p>Template</p><p>Cross‐linker</p><p>Ka [×105 M−1]</p><p>−ΔG [kcal/mol]</p><p>N [b]</p><p>1</p><p>7</p><p>DVB</p><p>34.4±1.73</p><p>8.91</p><p>0.9±0.1</p><p>2</p><p>MBAm</p><p>62.2±2.32</p><p>9.26</p><p>0.9±0.1</p><p>3</p><p>8</p><p>DVB</p><p>45.3±2.85</p><p>9.07</p><p>1.1±0.1</p><p>4</p><p>MBAm</p><p>67.50±2.66</p><p>9.31</p><p>1.1±0.1</p><p>5</p><p>9</p><p>DVB</p><p>59.2±0.31</p><p>9.23</p><p>1.1±0.1</p><p>6</p><p>MBAm</p><p>73.10±2.47</p><p>9.36</p><p>1.2±0.1</p><p>7</p><p>10</p><p>DVB</p><p>82.3±2.29</p><p>9.43</p><p>0.9±0.1</p><p>8</p><p>MBAm</p><p>89.10±2.47</p><p>9.47</p><p>1.1±0.1</p><p>9</p><p>11</p><p>DVB</p><p>66.4±2.65</p><p>9.30</p><p>0.8±0.1</p><p>10</p><p>MBAm</p><p>72.50±1.27</p><p>9.35</p><p>0.9±0.1</p><p>11</p><p>12</p><p>DVB</p><p>53.40±1.84</p><p>9.17</p><p>1.1±0.1</p><p>12</p><p>MBAm</p><p>66.20±3.36</p><p>9.30</p><p>1.0±0.1</p><p>[a] The titrations were performed in HEPES buffer (10 mM, pH 7.4) in duplicates at 298 K and the errors between the runs were <10 %. For MINPs prepared with FMs, the following stoichiometry was used in the formulation: 1.5 : 1 for 4/carboxylate, 1 : 1 for 6/amine, and 1 : 1 for 5/arginine. [b] N is the number of binding sites per nanoparticle determined by isothermal titration calorimetry (ITC). (Reprinted with permission from Ref. [40e]. Copyright 2020, the American Chemical Society.)</p><p>(a) ITC titration of peptides 7–12 to (a) MINP(8) and (b) MINP(11), showing only the desired peptide bound by the MINP. [MINP]=5.0 μM. [peptide]=75 μM in 10 mM HEPES buffer. The MINPs were prepared with 1 : 1 [1]/[MBAm]. (Reprinted with permission from Ref. [40e]. Copyright 2020, the American Chemical Society.)</p><!><p>MINP contains hydrogen‐bonding groups including triazole, hydroxyl, and ester. Although these "background" interactions cannot be defined as precisely as the supramolecular "codes" defined by the specifically shaped hydrophobic dimples and the specially designed FMs, they are expected to be optimized to some extent during the imprinting process, for both the peptide backbone and side chains. These secondary interactions also can play important roles, evident from the binding of peptides containing glycine that lacks a side chain.[40a, 45]</p><!><p>The nanodimension of MINPs and their strong and selective bindings for complex biological peptides bode well for their usage as protective agents for peptides. The Michaelis constants for common proteases are in the submillimolar to millimolar range[46] and those for kinases range from micromolar to millimolar.[47] The 100–200 nanomolar binding affinities (sometimes as low as 20 nM) of MINPs for hydrophobic and hydrophilic biological peptides[40] suggest that selective binding should be totally achievable.</p><p>Our first model peptide for protected proteolysis was Angiotensin III (A‐III, RVYIHPF),[48] cleavable by two common endopeptidases ‐trypsin after arginine (R) and by chymotrypsin after tyrosine (Y). LCMS analysis showed that MINP(A), i. e., MINP prepared with A‐III as the template, suppressed the proteolysis of the peptide to ≤10 % during a period of 2 h at 1 equiv. in the trypsin proteolysis and 2 equiv. in the chymotrypsin proteolysis. Nonimprinted nanoparticles (NINPs) prepared without templates only slowed down the reaction slightly. A strong correlation between binding and protection was observed when MINPs targeting the first 4, 5, and 6 amino acids of the N‐, and C‐terminal sequences were used for the protection. The protection factor, defined as the ratio between the yield in buffer at 2 h and the yield in the presence of the MINP, showed a linear relationship to the binding free energy.</p><p>Interestingly, the proteolytic yield of A‐III in the presence of MINP(A) fitted well to a 1 : 1 binding isotherm against the MINP concentration. The apparent "binding constant" obtained for trypsin inhibition (Ka =(2.35±0.31)×107 M−1) was quite close to the actual binding constant determined by ITC (Ka =(1.89±0.13)×107 M−1), suggesting the protection happened almost strictly with a 1 : 1 stoichiometry. Although the protection‐based "binding constant" for chymotrypsin was a few times lower than the ITC‐determined value, a strong binding–protection correlation was still observed. MINP protection was also found to work well for hydrophilic peptides (LRRASLG, PAGYLRRASVAQLT, and TGHGLRRSSKFCLK), if suitable FMs were used in the MINP preparation.</p><p>β‐Amyloid peptides are released through proteolysis and implicated in Alzheimer's disease.[49] We decided to use Aβ1–28 to demonstrate selective protection of a fragment of a long peptide because it contains two cleavable sites by trypsin – arginine at AA5 and lysine at AA16 (marked in green in Figure 3). Two MINPs, MINP(β1–14) and MINP(β15–28), were prepared, targeting the first and second halves of the parent peptide. ITC showed that the two MINPs bound the parent peptide strongly in pH 7.4 phosphate buffer, with K a=1.97×107 and 3.06×107 M−1, respectively.</p><!><p>Product distribution curves in the trypsin digestion of Aβ1–28 in buffer (a) and in the presence of 1 equiv. NINP (b), MINP(β1–14) (c), and MINP(β15–28) (d). (Reprinted with permission from Ref. [48]. Copyright 2021, Wiley‐VCH.)</p><!><p>In the phosphate buffer (Figure 3a) or in the presence of NINPs (Figure 3b), trypsin hydrolyzed Aβ1–28 to afford the expected peptide products (13–15). In addition, two peptides (16 and 17), with only the arginine or lysine cleaved, showed transiently in the first 2 h of reaction time. NINPs slowed down the proteolysis somewhat but the product distribution curves were similar in shape as those in the buffer.</p><p>A totally different product distribution was obtained when Aβ1–28 was treated with trypsin in the presence of 1 equiv. MINP(β1–14) or MINP(β15–28). The formerly transiently observed 16 (Figure 3c) and 17 (Figure 3d) were produced continually depending on which MINP protector was employed.</p><p>MINP protection did slow down the proteolysis of the exposed site, especially if the site was close to the protected sequence. For example, lysine 16 in Aβ1–28 was only two residues away from the protected sequence of Aβ1–14; its (selective) proteolysis in the presence of MINP(β1–14) took ∼24 h to complete (Figure 3c), instead of 4 h in buffer (Figure 3a) and 6 h with NINP (Figure 3b). Arginine 5, on the other hand, was 9 residues away from Aβ15–28 bound by MINP(β15–28) and its (selective) hydrolysis in Aβ1–28 took approximately 12 h (Figure 3d).</p><p>For MINP(A), MINP(β1–14), and MINP(β15–28), the nontemplating peptides showed very low cross‐reactivities (0.06–0.13 %) in the binding. This feature allowed us to carry out more advanced protections, using a 2 : 1 mixture of A‐III and Aβ1–28 for a proof of concept. Without any protector, the peptide mixture were digested by trypsin to afford peptides 13–15, as well as 18 from A‐III (Figure 4a). One equivalent of MINP(A) largely suppressed the proteolysis of A‐III, while Aβ1–28 hydrolyzed (Figure 4b). If MINP(β15–28) was used together with MINP(A), Aβ1–28 underwent the anticipated selective cleavage after arginine 5 to afford 13 and 17 while A‐III stayed largely intact (Figure 4c). Most interestingly, MINP(β1–14) and MINP(β15–28) could shied the long Aβ1–28 together: after 4 h of reaction time, nearly 90 % of A‐III hydrolyzed in the mixture while Aβ1–28 persisted (Figure 4d). ITC confirmed that the long peptide indeed could bind both MINPs simultaneously, although the binding constants were several times lower than those measured with only one MINP, suggesting some steric/electrostatic repulsion existed when two MINPs came together to bind one long peptide.</p><!><p>HPLC chromatograms of trypsin digestion of a 2 : 1 mixture of Angiotensin III and Aβ1–28 by trypsin (a) without any protection, and in the presence of (b) MINP(A), (c) MINP(A) & MINP(β15–28), and (d) MINP(β1–14) & MINP(β15–28). Reaction time was 4 h except in (c) which required 12 h for the selective hydrolysis of Aβ1–28. (Reprinted with permission from Ref. [48]. Copyright 2021, Wiley‐VCH.)</p><!><p>Because the inhibition of the enzymatic reaction is driven strictly by selective binding, we expect that, anytime a peptide is bound more strongly by an MINP receptor than its enzyme catalyst, the enzymatic reaction can be inhibited. The prediction was confirmed recently in selective phosphorylation of peptide mixtures by cyclic AMP‐dependent protein kinase (PKA), an enzyme with over 100 physiological substrates.[50] A particular challenge in controlled phosphorylation is that different substrates of a kinase generally have very similar or even identical "consensus motifs" surrounding the phosphorylation sites.[50b] PKA, for example, phosphorylates peptides with an RRXS motif (X=a variable amino acid). Yet, MINP was able to control the PKA‐catalyzed phosphorylation of Kemptide (LRRASLG), β2‐adrenergic receptor peptide (TGHGLRRSSKFCLK), pyruvate kinase peptide (PAGYLRRASVAQLT), and cardiac myosin binding protein‐C peptide (FRRTSLAGGGRRISDSHE) completely.[51] Note that Kemptide and pyruvate kinase peptide share identical consensus motifs, even the leucine (L) in front of the recognition motif. For cardiac myosin‐binding protein‐C peptide, selective protection of a fragment of the long peptide and cooperative protection of the entire sequence by two MINPs were both achieved.</p><p>Biological phosphorylation frequently occurs within a protein complex. One such example is the phosphotransfer step in the activation of the proline‐rich tyrosine kinase 2 (Pyk2), a regulator of leukocyte motility, bone remodeling, and neuronal development.[52] As shown in Figure 5a, the Pyk2 activation occurs when tyrosine Y402 in the linker between the regulatory FERM and the kinase domain is autophosphorylated.[53] The intramolecular nature of the reaction makes it even more challenging to protect the substrate because MINP binding will have to compete with intramolecular protein–protein interactions.</p><!><p>(a) Domain organization of Pyk2 and structural model depicting the Pyk2 FERM (PDB 4eku) and kinase (PDB 3fzp) aligned to the FAK FERM‐kinase (PDB 2j0j). The FAK FERM‐kinase linker is superimposed (yellow) to illustrate putative MINP binding sites. (b) Inhibition of Pyk2 autophosphorylation by MINPs at 1 : 1, 1 : 3, and 1 : 6 protein/MINP ratios, with the NINP as the control. [Pyk2]=1.0 μM. (Reprinted with permission from Ref. [51]. Copyright 2021, the American Chemical Society.)</p><!><p>Since we can prepare MINPs conveniently to target different sections of a long peptide, we can essentially "scan" the linker by different MINPs and observe how the MINP binding affects the phosphorylation. Interestingly, when three MINPs targeting AA373–383, 388–398, and 400–411 of the linker, were added to the protein complex and ATP mixture, MINP(19 a) and MINP(19 b) turned out significantly more potent than MINP(19 c) in the inhibition of the autophosphorylation (Figure 5b), even though it was MINP(19 c) that directly impinged on Y402 in its binding. This unusual behavior might have resulted from a lower accessibility of 19 c sequence, since evidence exists that suggests the autophosphorylation site could be part of an abbreviated β sheet.[54]</p><!><p>MINPs have a remarkable ability to bind complex biological peptides in aqueous solution. With appropriate functional monomers[40d] and/or cross‐linkers,[40e] they can frequently achieve tens of nanomolar binding affinities for peptides with 10–20 amino acid residues. Their ability to distinguish closely related residues including leucine/isoleucine,[40a] phenylalanine/tyrosine,[40a] glutamic acid/aspartic acid,[40c] and lysine/arginine[40b] makes them an extremely attractive class of materials for biological applications. Once the cross‐linkable surfactant, cross‐linker, and templates are available, their preparation takes less than 2 days and purification requires nothing other than precipitation and washing.</p><p>Because most enzymes bind their substrates with millimolar to micromolar affinities, MINPs are expected to compete effectively with many enzymes in the binding and, in turn, to shield their peptide substrates from enzymatic actions. Controlled proteolysis and phosphorylation are just examples chosen to illustrate the power of substrate protection, which should be quite general. Biology historically has been a great source of inspiration to chemists in their development of methods to recognize, transport, and transform molecules. Protection/deprotection, on the other hand, is largely an invention of chemists for the synthesis of complex organic molecules. Maybe, the strategy now is ready to find its way back into biology, as a way to return the favor.</p><!><p>The authors declare no conflict of interest.</p>

PubMed Open Access

Iron detection and remediation with a functionalized porous polymer applied to environmental water samples

Iron is one of the most abundant elements in the environment and in the human body. As an essential nutrient, iron homeostasis is tightly regulated, and iron dysregulation is implicated in numerous pathologies, including neurodegenerative diseases, atherosclerosis, and diabetes. Endogenous iron pool concentrations are directly linked to iron ion uptake from environmental sources such as drinking water, providing motivation for developing new technologies for assessing iron(II) and iron(III) levels in water. However, conventional methods for measuring aqueous iron pools remain laborious and costly and often require sophisticated equipment and/or additional processing steps to remove the iron ions from the original environmental source. We now report a simplified and accurate chemical platform for capturing and quantifying the iron present in aqueous samples through use of a post-synthetically modified porous aromatic framework (PAF). The ether/thioether-functionalized network polymer, PAF-1-ET, exhibits high selectivity for the uptake of iron(II) and iron(III) over other physiologically and environmentally relevant metal ions. Mössbauer spectroscopy, XANES, and EXAFS measurements provide evidence to support iron(III) coordination to oxygen-based ligands within the material. The polymer is further successfully employed to adsorb and remove iron ions from groundwater, including field sources in West Bengal, India. Combined with an 8-hydroxyquinoline colorimetric indicator, PAF-1-ET enables the simple and direct determination of the iron(II) and iron(III) ion concentrations in these samples, providing a starting point for the design and use of molecularly-functionalized porous materials for potential dual detection and remediation applications.

iron_detection_and_remediation_with_a_functionalized_porous_polymer_applied_to_environmental_water_s

Introduction<!>General methods<!>Synthesis of PAF-1-ET<!>NMR experiments.<!>Metal ion adsorption in PAF-1-ET<!>Mössbauer experiments<!>X-ray absorption measurements<!>Synthesis and characterization<!>Selectivity and kinetics of iron uptake<!>Spectroscopic characterization<!>Iron coordination in PAF-1-ET<!>Iron capture and detection in synthetic and environmental water samples<!>Conclusions

<p>Iron is the fourth most abundant element in the earth's crust and the most abundant transition metal in the human body. 1 It is required for sustaining a range of physiological processes such as electron transfer, oxygen transport, respiration, and gene expression, [2][3][4][5] and iron deficiency leads to anemia. 6 However, excess iron can increase production of reactive oxygen species, resulting in oxidative stress cascades that lead to lipid oxidation and DNA damage. [2][3][4]7,8 Aberrant iron accumulation is implicated in aging and in several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. 6,[9][10][11][12][13][14] In this context, chronic exposure to elevated iron levels in common drinking water is a potential contributor to abnormal iron accumulation. The World Health Organization (WHO) recommends an upper limit of 0.3 mg/L for iron ions in drinking water. 15 Unfortunately, the reported iron ion concentrations in drinking water sources can vary over several orders of magnitude-for instance from 0.007 to 33.6 mg/L in West Bengal 16 or from undetectable amounts to 950 mg/L based on Groundwater Ambient Monitoring and Assessment data from the San Francisco Bay (see Supporting Information). Because traditional methods for iron(II) and iron(III) detection require expensive instrumentation, such as inductively coupled plasma mass spectrometry or atomic absorption spectroscopy, 17,18 it remains a challenge to rapidly and inexpensively screen drinking water for quantities of iron and other metal ion contaminants, particularly in developing countries and other lower-resource environments. 11,16,17,[19][20][21][22][23][24] To meet this challenge, we sought to develop a chemical strategy that would enable simultaneous detection and removal of both iron(II) and iron(III) ions from drinking water and other environmental and biomedical samples, with high selectivity over other metal ion contaminants. In particular, we This journal is © The Royal Society of Chemistry 20xx</p><p>Please do not adjust margins Please do not adjust margins envisioned a robust, molecularly-tailored solid-state adsorbent that would efficiently capture and remove iron ions from a complex water sample obtained in the field while also permitting a quantitative measure of the iron concentration. We chose to investigate porous aromatic frameworks (PAFs) for this dual remediation and quantitative detection function, owing to their high chemical and thermal stability-particularly in aqueous and biological samples-and because of their ability to be functionalized in a molecular fashion. [25][26][27][28][29][30][31][32][33][34][35][36][37] The polymer PAF-1 is one such material that exhibits a high Brunauer-Emmett-Teller (BET) surface area 38 of up to 5600 m 2 /g and is readily functionalized through post-synthetic modification. [38][39][40][41][42][43] Indeed, we recently reported a thioetherfunctionalized variant of this porous polymer, PAF-1-SMe, as a platform for selective copper ion capture from biofluid samples, ultimately as a diagnostic tool for Wilson's disease. 42 Here, we present the synthesis of an iron-selective porous polymer via ether-thioether (ET) functionalization of PAF-1. The polymer PAF-1-ET (Figure 1a) exhibits highly selective iron(II) and iron(III) ion uptake over competing metal ions in laboratory and field water samples. The combination of this polymer with 8-hydroxyquinoline indicator enables rapid and quantitative monitoring of iron levels with a simple colorimetric assay. We highlight the potential utility of this method for remediation and screening of synthetic groundwater as well as field samples of drinking water collected from West Bengal, India. This work provides a starting point for the development of new porous polymers for simplified, accurate, and rapid diagnostic and remediation applications without the need for bulky and expensive instrumentation.</p><!><p>All reactions were performed under a nitrogen or argon atmosphere and in dry solvents, unless otherwise stated. Tetrakis(4-bromophenyl)methane was purchased from TCI America as a pale yellow powder. All other starting materials and reagents were purchased from Sigma-Aldrich. Nitrogen adsorption isotherms were measured using a Micromeritics ASAP 2020 or 2420 instrument. Samples were transferred to a pre-weighed glass analysis tube that was capped with a Transeal and then evacuated on the ASAP until the outgas rate was less than 3 µbar/min. Ultrahigh-purity grade (99.999%) nitrogen was used for gas adsorption measurements. Nitrogen isotherms were obtained using a 77 K liquid-N2 bath and were used to determine the surface areas and pore volumes using the Micromeritics software, assuming a value of 16.2 Å 2 for the molecular cross-sectional area of N2. Infrared spectra were obtained on a Perkin-Elmer Spectrum 100 Optica FTIR spectrometer furnished with an attenuated total reflectance accessory. Thermal gravimetric analysis data were collected at a ramp rate of 5 °C/min under flowing nitrogen using a TA Instruments TGA Q5000. Scanning electron microscopy (SEM) samples were prepared by dispersing fine polymer powders into methanol and drop casting onto silicon chips. To dissipate charge, the samples were sputter coated with approximately 3 nm of Au (Denton Vacuum). Polymers were imaged at 5 keV and 12 μA by field emission SEM (JEOL FSM6430). Elemental analyses (C, H, N, S) were obtained from the Microanalytical Laboratory at the University of California, Berkeley. Elemental analysis for chlorine was performed at Galbraith Laboratories. UV-Vis spectroscopic measurements were performed in 100 mM HEPES buffer (pH 6.7). Absorption spectra were recorded using a Varian Cary 50 spectrophotometer, and samples for absorption measurements were prepared in 1 × 0.5 cm quartz cuvettes (1.4-mL, Starna). Inductively coupled plasma-mass spectrometry (ICP-MS) was performed on samples that had been diluted into 2% nitric acid (made freshly from concentrated nitric acid [BDH Aristar Ultra] and Milli-Q water) containing 20 µg/L Ga internal standard (Inorganic Ventures, Christiansburg, VA). The samples were analyzed on a ThermoFisher iCAP-Qc ICP-MS in Kinetic Energy Discrimination mode against a calibration curve of known metal concentrations (made from CMS-5, Inorganic Ventures, Christiansburg, VA). Lowtemperature X-band EPR spectra were recorded using a Varian E109 EPR spectrometer equipped with a Model 102 microwave bridge. Sample temperature was maintained at 8 K by using an Air Products LTR liquid helium cryostat. The following spectrometer conditions were used: microwave frequency, 9.22 GHz; field modulation amplitude, 32 G at 100 kHz, and a microwave power of 20 mW.</p><!><p>2-(methylthio)ethan-1-ol (1.83 mL, 0.021 mol) and 3 equiv. of NaH (1.5 g, 0.063 mol) were mixed with toluene (100 mL) in a 250 mL Schlenk flask under N2. After 5 min, freshly-prepared PAF-1-CH2Cl (260 mg, see the Supporting Information) was added, and the mixture was stirred at 90 °C for 3 days. The resulting solid was collected, washed sequentially with 100 mL each of H2O, ethanol, CHCl3, and THF, and dried in a vacuum oven at 150 °C to produce PAF-1-ET as an off-white powder. Calc. for C32.5H34O2S2 (%): C 74.96, H 6.58, S 12.31, Cl 0.00; observed: C 74.89, H 5.08, S 5.50, Cl 1.97. Based on the sulfur elemental analysis, this preparation resulted in 45% substitution with 2-(methylthio)ethan-1-ol. § Solid-state 13 C NMR NMR sample preparation. Samples of PAF-1-ET (35 mg) and PAF-1-CH2Cl (20 mg) were dried at 100 °C for 3 h before data collection. Iron(III)-loaded PAF-1-ET was prepared by stirring PAF-1-ET (50 mg) at room temperature overnight in a solution of FeCl3 (10 mL, 100 mg/L) dissolved in 100 mM HEPES buffer (pH = 6.7) with 2 equiv. of citric acid. The filtered iron(III)-PAF-1-ET was washed with Milli-Q H2O, ethanol, dichloromethane, and THF (50 mL each) and dried at 100 °C for 3 h before data collection.</p><!><p>All experiments were conducted at a 13 C frequency of 75.5 MHz using a Tecmag Discovery spectrometer equipped with a 7.05 T magnet and a Chemagnetics 4 mm HX CP/MAS probe (magic-angle spinning rate of 10 kHz). Crosspolarization from 1 H was used when acquiring spectra for PAF-1-Please do not adjust margins Please do not adjust margins CH2Cl and PAF-1-ET. The Hartmann-Hahn condition 43 for crosspolarization experiments was obtained on solid adamantane, which is also a secondary 13 C chemical shift reference (the methylene signal of adamantane was set to 38.48 ppm relative to TMS). The PAF-1-CH2Cl spectrum was collected using a CP contact time of 10 ms and a pulse delay of 4 s. A two-pulse phase modulation (TPPM) proton decoupling scheme was used, with a TPPM angle of 15 degrees and decoupling field strength of ~60 kHz. The spectrum for PAF-1-ET was obtained using a contact time of 1 ms and a pulse delay of 4 s. Direct polarization (1 s pulse delay) was used to collect the spectrum for iron(III)-loaded PAF-1-ET. Continuous wave proton decoupling was used for both the PAF-1-ET and iron(III)-PAF-1-ET spectra, with a decoupling field strength of ~60 kHz.</p><!><p>Iron adsorption measurements. Samples of PAF-1-ET (2.0 mg) were added to conical tubes containing 5 mL of (NH4)2Fe(SO4)2•6H2O (dissolved in 100 mM HEPES buffer, pH 6.7) with concentrations ranging from 10 −3 -240 mg/L. Each mixture was capped under air and stored in a shaker at room temperature overnight. Each solution was subsequently filtered through a 0.45-µm membrane filter, and the filtrates were analyzed by ICP-MS to determine the residual iron content. Iron uptake (initial−residual iron concentration) data was fit using a Langmuir model given by: where qe is the adsorption capacity (mg/g), Ce is the equilibrium iron ion concentration (mg/L), qsat is the adsorption saturation capacity (mg/g), and KL is the Langmuir constant (L/mg), which is related to the binding affinity of the adsorption site. Langmuir fits are shown in Figure 2a, and fit parameters are provided in Table S1.</p><p>Metal ion adsorption selectivity studies. Samples of PAF-1-ET (2 mg) were added to conical tubes containing aqueous solutions of NaCl, KCl, MgCl2, CaCl2, MnCl2, (NH4)2Fe(SO4)2•6H2O, FeCl3, CoCl2, NiCl2, CuCl2, or ZnCl2 at initial concentrations of 0.3, 2, or 20 mg/L in 100 mM HEPES buffer (pH 6.7). Iron(II) samples were prepared and stored under anaerobic conditions until analysis by ICP-MS. In the case of iron(III), one equivalent of citric acid was also added to the samples to prevent Fe(OH)3 precipitation. The slurries were stored in a shaker at room temperature overnight and then filtered through a 0.45-µm membrane. The filtrates were analyzed using ICP-MS, and the amount of metal ion adsorbed was calculated by subtracting the residual iron concentration from the initial iron concentration. The distribution coefficient, Kd, for each metal ion was determined as described in Section 3 of the Supporting Information.</p><p>Iron adsorption kinetics. An Erlenmeyer flask containing 2 mg of PAF-1-ET was charged with a solution of (NH4)2Fe(SO4)2•6H2O (10 mL, 10.2 ppm) in 100 mM HEPES buffer (pH = 6.7) and 1 equiv. of citric acid. The mixture was stirred at room temperature for 8 h. During this period, aliquots of the mixture were filtered at intervals through a 0.45-µm membrane. The filtrates were analyzed using ICP-MS to determine the iron ion concentration. The amount of iron adsorbed by PAF-1-ET was calculated by subtracting the residual from the initial iron concentration. The adsorption data were fit with the pseudo-second-order kinetic model (see Figure S7):</p><p>where k is the pseudo-second-order rate constant (g/(mg min)) and qe is the amount of iron adsorbed at equilibrium (mg/g).</p><!><p>A sample of PAF-1-ET (~50 mg) was added to an aqueous solution of 57 FeCl3 (50 mg/L, see the Supporting Information), and the mixture was stirred overnight at room temperature under N2. The resulting 57 Fe(III)-loaded PAF-1-ET was collected, washed with warm H2O (100 mL) and CHCl3 (100 mL), and then dried in a vacuum oven at 150 °C to yield a white powder. Mössbauer spectra were obtained between 5 and 300 K with a SEE Co. Mössbauer spectrometer equipped with a Co-57 source in Rh matrix. Reported isomer shifts are given relative to α-iron at 295 K. The spectral absorber was prepared in air by packing the sample into a 1.27 cm diameter Nylon washer before transferring to the spectrometer, where the absorber was always maintained under a He atmosphere. See the Supporting Information (Section 6) for full measurement details.</p><!><p>X-ray absorption spectra were collected at the Stanford synchrotron radiation light source on beamline 9-3 with ring storage conditions of 3.0 GeV and 500 mA. The iron K-edge absorption spectra of the PAF samples, packed in 0.5 mm thick aluminum sample holders with Kapton film windows, were recorded at room temperature. Reference compounds were analyzed after dilution with boron nitride. The spectral data were collected in transmission mode for Fe, Fe2O3, and FeO and in fluorescence mode for PAF-1-ET with a 100-element Ge monolithic solid-state detector from Canberra. The incident radiation was monochromatized using a Si(220) double crystal monochromator, which was detuned to 50% of flux maximum at the iron K-edge to minimize the higher harmonics and reduce X-ray flux. A harmonic rejection mirror was used to further reduce the contamination from higher harmonics radiation. The incident and transmitted X-ray intensities were monitored with N2filled ion chambers. An iron foil spectrum was concomitantly recorded for energy calibration where the first inflection point was assigned to 7111.2 eV. Even at the low X-ray flux density used, a slight photoreduction of PAF-1-ET was observed even after two scans at a given sample position. As a consequence, the spectral data were collected at multiple spots, and only the first two scans at each position were used for averaging the spectral data over multiple positions. Data reduction was carried out with the SamView software obtained from SixPack software. ‡ Athena software, Demeter version 0.9.25 45 was used for data averaging and removal of the This journal is © The Royal Society of Chemistry 20xx</p><p>Please do not adjust margins Please do not adjust margins pre-edge and post-edge background absorption. A five-domain cubic spline was used to remove low-frequency background in kspace. The resulting k-space data, as k 3 χ(k), was then Fourier transformed into r-space over a k range of 3.46 to 10.52 Å −1 and used for the extended X-ray absorption fine structure (EXAFS) refinement. The EXAFS fitting was carried out using the Artemis software 45 with phase and amplitude functions obtained from FEFF, version 6. 46 The average bond distance between the iron and scattering atom (R) and the mean square displacement of the bond distance (σ 2 ) were allowed to vary, while N, the number of atoms in the shell, was systematically varied in integral steps. The value of E0, the energy of the zero value of the photoelectron wave vector k, was also varied but constrained to a common value for every shell in a given fit. The value for S0 2 , the amplitude reduction factor, was extracted from the fit of the Fe foil data and was fixed at 0.95 during all other fits. The best fit parameters for EXAFS fits are given in Table S3.</p><!><p>Building upon the design of the thioether-functionalized polymer PAF-1-SMe for selective copper ion capture 42 and the related PAF-1-SH for mercury ion adsorption, 40 we initially synthesized a variety of PAF-1 derivative scaffolds. Subsequent testing for metal ion uptake from aqueous solutions showed that the variant with an extended ether-thioether pendant, referred to here as PAF-1-ET, is an effective material for selective capture of both iron(II) and iron(III). This work complements our recent efforts to develop activity-based fluorescent probes for iron(II). [47][48][49][50][51][52] The polymer PAF-1-ET was synthesized in three steps, starting with the synthesis of parent PAF-1 and PAF-1-CH2Cl (see the Supporting Information) 40 followed by treatment of the latter with 2-(methylthio)ethan-1-ol to yield PAF-1-ET (Figure 1a). Elemental analysis revealed a decrease in chlorine content from 13.60% in PAF-1-CH2Cl to 1.97±1.5% in PAF-1-ET, and the sulfur content of the latter was found to be 5.50±0.82%-corresponding to grafting of 45±6.7% of the phenyl groups in PAF-1-ET or a loading of 1.7 mmol/g. In the IR spectrum of PAF-1-ET, the absence of a peak at 1270 cm −1 (assigned to the C-H wagging mode of -CH2Cl in PAF-1-CH2Cl) further confirmed functionalization (Figure S2). Solid-state 1 H− 13 C cross-polarization magic angle spinning NMR spectroscopy revealed new 13 C chemical shifts at 73, 48, 39, and 17 ppm for PAF-1-ET, arising from the ether-thioether groups, and the absence of a shift at 43 ppm corresponding to Please do not adjust margins Please do not adjust margins the -CH2Cl groups of PAF-1-CH2Cl (Figure 1b). Nitrogen adsorption isotherms obtained at 77 K revealed that PAF-1-ET retains permanent porosity with a high BET surface area of 1500±420 m 2 /g (Figure 1c; error determined by measuring the N2 adsorption of four different PAF-1-ET samples). The average pore size distribution obtained from the adsorption isotherms was found to be <10 Å for PAF-1-ET, which is smaller than the average value of 12 Å for PAF-1 and supports incorporation of the ether-thioether groups (see Figure S3).</p><!><p>Equilibrium iron(III) isothermal adsorption data were collected for PAF-1-ET and PAF-1-CH2Cl over aqueous ion concentrations ranging from 1 µg/L to 300 mg/L, and these data were fit using the Langmuir model 29 to assess framework saturation capacities and binding affinities for iron(III) (Figure 2a and Table S1). Notably, PAF-1-ET exhibited much higher iron(III) uptake than PAF-1-CH2Cl over the entire concentration range and a binding affinity twice that of PAF-1-CH2Cl. At saturation, the maximum adsorption capacity (qsat) of PAF-1-ET was found to be 105(4) mg/g, which corresponds to an uptake of 1.8 mmol of iron(III) per gram of material-nearly three times the capacity of PAF-1-CH2Cl (37(2) mg/g). Based on the maximum adsorption capacity of PAF-1-ET, the ET:iron ratio is at least 1.1. We also collected adsorption data using iron(III) chloride, Please do not adjust margins Please do not adjust margins iron(III) sulfate hydrate, or ammonium iron(III) citrate to investigate the effect of the counterion on iron uptake in PAF-1-ET. For all salts, PAF-1-ET showed comparable iron uptake at low and high iron concentrations (see the Supporting Information, Section 4).</p><p>Importantly, PAF-1-ET also exhibited high selectivity for the adsorption of iron(II) and iron(III) ions over other biologicallyrelevant metal ions at initial concentrations of 0.3, 2, and 20 mg/L (Figure 2b). For example, the distribution coefficient, Kd, for 10 mg/L iron(II) in pH = 6.7 HEPES buffer was found to be 2.6(7) × 10 4 mL/g, over an order of magnitude greater than the Kd values for 10 mg/L of Na + , K + , Mg 2+ , Ca 2+ , Cu 2+ , and Zn 2+ (600, 120, 180, 770, 3300, and 38 mL/g, respectively). Given this exceptional performance, PAF-1-ET should be useful for iron from a variety of water samples.</p><p>The concentration dependence of iron(III) uptake by PAF-1-ET was also evaluated by examining various pH 6.7 aqueous solutions in HEPES buffer, and it was found that the adsorbed amount increases with increasing ion concentration in solution (Figure 3a). In order to develop a colorimetric assay for detection of the adsorbed iron(III), we evaluated the ability of 8-hydroxyquinoline to bind iron(III) captured within the porous framework. Upon binding free iron(III), 8-hydroxyquinoline undergoes a distinct change from colorless (315 nm absorption, ε = 1.95 × 10 3 M −1 cm −1 ) to blue-green (460 and 560 nm absorption, ε = 750 M −1 cm −1 at 460 nm), which is distinctive of an iron(III) 8-hydroxyquinoline complex. [53][54][55][56] Thus, successful binding of iron(III) within PAF-1-ET by 8hydroxyquinoline should permit a facile and quantitative determination of the quantity adsorbed. To confirm this capability, a 1 mM solution of 8-hydroxyquinoline in dimethyl sulfoxide was added to dried samples of PAF-1-ET that had been exposed to the aqueous iron samples in Figure 3a. Gratifyingly, in the presence of these samples, the 8hydroxyquinoline absorption spectra exhibited two new peaks at 460 and 560 nm (Figure 3b), indicative of iron(III) complex formation. The calculated amount of iron(III) adsorbed by PAF-1-ET-based on the 460 nm absorbance peak for the highest sample concentration-correlated well with amounts determined directly via ICP-MS (Figure 3c).</p><!><p>To obtain additional insight into the nature of the interactions between adsorbed iron(III) and the framework functional groups, 57 Fe Mössbauer spectra were collected between 5 and 300 K. Representative spectra at 100 and 5 K are shown in Figure 4a and b (see also Figure S9 and Table S2). At all temperatures, the spectral fits indicated the predominant presence of paramagnetic, high-spin iron(III) adsorbed within PAF-1-ET (red lines), with no evidence of long-range magnetic order. A small constrained component (green lines, 9% by area) was also present in all data, likely due to residual highspin iron(II) from sample preparation. The spectra were found to be very similar between 50 and 300 K, with a predominant bimodal distribution of quadrupole splittings, ΔEQ, between 0.6 and 1.0 mm/s, centered about a unique high-spin iron(III) isomer shift, δ, of 0.385(2) mm/s at 300 K (0.507(1) mm/s at 50 K). These values are consistent with an iron(III) ion residing in a pseudooctahedral coordination with a distribution of nearneighbor oxygen environments, a conclusion that is consistent with the extended X-ray absorption fine structure (EXAFS) data below. Interestingly, upon cooling to 20 K and below, some of the highly dispersed iron(III) ions adsorbed in PAF-1-ET exhibited initial evidence for slow paramagnetic relaxation on the Mössbauer timescale (~10 -8 s), with predominant hyperfine fields of 45 T (17.6(6)% area), 46.6(1) T (50.4(6)% area), and 46.6(1) T (58.8(4)% area), respectively. Please do not adjust margins Please do not adjust margins Iron K-edge X-ray absorption spectroscopy was used to investigate the local coordination environment of the adsorbed iron. The X-ray absorption near edge structure (XANES) spectrum of iron(III) adsorbed in PAF-1-ET is shown in the inset of Figure 4c, along with Fe2O3, FeO, and Fe foil for reference. The rising edge energy of the sample aligns well with that of Fe2O3, supporting the presence of iron(III). Figure 4c shows the k 3 -weighted EXAFS data for iron(III) adsorbed PAF-1-ET in r-space along with the best fit (see Table S3). The horizontal axis represents the apparent distance R′, which is shorter than the actual distance by ~0.5 Å due to a phase shift. For iron(III) adsorbed PAF-1-ET, 57 the best two-shell fit was achieved with a coordination environment of six oxygen atoms at a distance of 2.00(1) Å and 12 carbon atoms at a distance of 3.06(4) Å.</p><p>We also obtained the 13 C NMR spectrum of iron(III)-loaded PAF-1-ET to compare with that of PAF-1-ET. The coordination of paramagnetic iron(III) resulted in severe peak broadening in addition to an overall shift in the peaks observed for PAF-1-ET (Figure S11). [58][59][60] The benzene ring resonances between 148-132 ppm for PAF-1-ET shifted to 147-140 ppm in the spectrum of iron(III)-loaded PAF-1-ET, and all peaks corresponding to the ET functional groups of PAF-1-ET (73, 48, 39, and 17 ppm) shifted upfield in iron(III)-loaded PAF-1-ET (to ~34-5 ppm). The more dramatic shift in the ether-thioether peaks provides additional evidence that this group is indeed bound to iron(III).</p><!><p>To investigate possible iron coordination environments within PAF-1-ET, we used the program Materials Studio to generate a hypothetical portion of the PAF-1-ET structure, featuring one iron ion within a single diamond net. Based on the EXAFS data, it was assumed that a total of six oxygen atoms-and no sulfur atoms-coordinate to the iron. A monodentate sulfate anion was always included at one of the coordination sites for charge balance, and the remaining sites were coordinated by water and the ET oxygen atoms. The modeling revealed that up to three oxygen atoms from three different functional groups (ET:Fe = 2.9) can coordinate to the same iron ion at the low loading observed for genuine groundwater samples, provided that these groups are located at the 2-, 2′-, and 3-positions of two adjacent biphenyl groups (Figure S12). Importantly, the modeling also showed that the ET group flexibility may enable two groups to bind the same iron ion, regardless of their respective positions on a biphenyl unit (Figure S12a-c). Based on the maximum iron(III) adsorption capacity data, for high iron loading (ET:Fe = 1.1), it is presumed that only a single ET group can bind to the iron. In this case, a coordinated water molecule might participate in a weak CH-π interaction with a benzene ring that stabilizes the iron ions (Figure S13), a hypothesis supported by the benzene ring shift in the 13 C NMR spectrum of iron(III)-loaded PAF-1-ET (Figure S11).</p><p>To investigate the importance of the ether-thioether orientation and pore environment in PAF-1-ET for iron(III) uptake, we prepared a series of related porous polymers and evaluated their iron adsorption properties. The first of these Please do not adjust margins Please do not adjust margins polymers, PAF-1-TE, was synthesized from PAF-1-CH2Cl using 2-methoxyethane-1-thiol, yielding a material analogous to PAF-1-ET but featuring interchanged positions for the pendant oxygen and sulfur moieties. We also prepared etherfunctionalized porous polymers PAF-1-OMe and -Ethoxy (Figure S14) and a linear polysulfone polymer functionalized with the ether-thioether ligand, PSF-ET (Figure S15). When exposed to a solution of FeCl3 (20 mg/L) dissolved in 100 mM HEPES buffer (pH = 6.7) with one equivalent of citric acid, each of the derivative polymers exhibited significantly lower iron(III) uptake than PAF-1-ET (Figure S16). The much lower uptake of PAF-1-TE, -OMe, and -Ethoxy suggests that both the position of oxygen and the presence of sulfur are crucial for iron adsorption, while the low uptake of PSF-ET emphasizes the importance of a compact pore environment in tandem with the ET functionality.</p><!><p>To verify the detection capability of PAF-1-ET when exposed to iron(III) sources from different regions, synthetic groundwater was prepared according to the Gadgil 61 procedure with iron(III) concentrations of 1.8, 4.7, 6.7, and 37 mg/L. The polymer was also used to treat genuine groundwater samples collected in West Bengal, India, reported to contain 14 mg/L of iron(III) ions (Figure 5a). 16 Notably, PAF-1-ET adsorbed between ~41 and 91% of the iron(III) in the synthetic groundwater samples, from initial concentrations between 37 and 1.8 mg/L, respectively. In the presence of PAF-1-ET, the concentration of iron(III) in the genuine groundwater decreased with time as given by the expression y = Ae −t /t0 + C, where y is the detected amount of iron(III), A is a scale factor, C is a constant, t0 is the decay time, and t is the elapsed time. The best fit shown in the inset to Figure 5b corresponds to A = 9.2(3) mg/L, C = 4.1(1) mg/L, and t0 = 12(1) min. In other words, within 24 min PAF-1-ET captured 72% of the iron(III) ions and was essentially saturated after ~36 min, such that the iron(III) concentration in the genuine groundwater reached a constant value of ~3.92 mg/L.</p><p>The final concentration in the genuine groundwater sample is higher than that recommended for safe drinking water by the WHO (0.3 mg/L), and the same is true for the synthetic groundwater solution with an initial concentration of 37 mg/L. However, the saturation capacity of PAF-1-ET suggests that the framework is capable of reducing the iron(III) content in both these solutions to levels lower than 0.3 mg/L. It is likely that at higher iron(III) concentrations, precipitation of Fe(OH)3 within the pores blocks some of the accessible coordination sites and reduces the effective capacity. Even still, PAF-1-ET functions exceptionally well in the removal of iron from solutions with relatively low initial concentrations-indeed, the framework was able to reduce the iron(III) ion content to safe drinking levels for solutions with initial iron concentrations of 1.8, 4.7, and 6.7 mg/L. For the effective treatment of water containing higher iron ion concentrations, it may be necessary to use larger quantities of citric acid to prevent Fe(OH)3 precipitation.</p><p>Analysis of the PAF-1-ET samples using an 8hydroxyquinoline assay revealed an increase in absorbance at 460 and 560 nm with increasing iron(III) concentration, as associated with the original water samples (Figure 5b). The iron(III) concentrations calculated from the absorption at 460 nm were again in good agreement with those determined from direct ICP-MS measurements (Figure 5c). Finally, using the three-sigma method (3σ/k) [62][63][64] the iron(III) detection limit for the PAF-1-ET and 8-hydroxyquinoline assay was determined to be 150 µg/L (see Figure S17 and the Supporting Information for details). Importantly, PAF-1-ET retains structural integrity and porosity following the addition of 8hydroxyquinoline and can be cycled at least three times without noticeable loss of adsorption capacity (Figure S18).</p><!><p>We have demonstrated that the ether-thioetherfunctionalized porous aromatic framework PAF-1-ET is capable of selective and efficient iron ion uptake and removal from both synthetic water and environmental groundwater. In this material, captured iron(III) is preferentially bound by oxygen in a pseudooctahedral coordination environment, as confirmed by Mössbauer and X-ray absorption spectroscopy characterization. The introduction of oxygen functionality within the framework is thus responsible for a shift to iron ion selectivity from our previously reported copper-selective thioether-functionalized material, PAF-1-SMe. 42 Finally, the combination of PAF-1-ET with 8-hydroxyquinoline as a colorimetric indicator provides an efficient and accurate tool for directly determining the iron ion concentrations from groundwater samples, with minimal processing and equipment needs.</p><p>Notes and references ‡ http://www.sams-xrays.com/sixpack. § Less than 100% loading of 2-(methylthio)-ethan-1-ol loading might be attributed to the reactivity of sodium hydride with benzyl chloride and the conversion of some of the -CH2Cl groups to other functionalities such as methyl, or the linkage of these groups to form polymeric 1,2-diphenylethane (see S. Bank, M. C. Prislopski, J. Chem. Soc. Chem. Commun., 1970, 0, 1624; M. I. Watkins, G. A. Olah, J. Am. Chem. Soc., 1981, 103, 6566; and Y. Yuan, Y. Bian. Appl. Organomet. Chem., 2008, 22, 15.)</p>

ChemRxiv

PreSS/MD: Predictor of Skin Sensitization Caused by Chemicals Leaching from Medical Devices

Safety evaluation for medical devices includes the toxicity assessment of chemicals used in device manufacturing, cleansing and/or sterilization that may leach into a patient. According to international standards on biocompatibility assessments (ISO 10993), chemicals that could be released from medical devices should be evaluated for their potential to induce skin sensitization/allergenicity, and one of the commonly used approaches is the guinea pig maximization test (GPMT). However, there is growing trend in regulatory science to move away from costly animal assays to employing New Approach Methodologies including computational methods. Herein, we developed a new computational tool for rapid and accurate prediction of the GPMT outcome that we named PreSS/MD (Predictor of Skin Sensitization for Medical Devices).To enable model development, we (i) collected, curated, and integrated the largest publicly available dataset for GPMT; (ii) succeeded in developing externally predictive (balanced accuracy of 70-74% as evaluated by both 5-fold external cross-validation and testing of novel compounds) Quantitative Structure-Activity Relationships (QSAR) models for GPMT using machine learning algorithms, including Deep Learning; and (iii) developed a publicly accessible web portal integrating PreSS/MD models that enables the prediction of GPMT outcomes for any molecules using. We expect that PreSS/MD will be used by both researchers and regulatory agencies to support safety assessment for medical devices and help replace, reduce or refine the use of animals in toxicity testing. PreSS/MD is freely available at https://pressmd.mml.unc.edu/.

press/md:_predictor_of_skin_sensitization_caused_by_chemicals_leaching_from_medical_devices

Introduction<!>Materials and Methods<!>European Chemical Agency (ECHA) dataset<!>Literature<!>Combined GPMT data from ECHA and the literature<!>Case studies sets<!>Data curation<!>QSAR modeling<!>Mechanistic interpretation of QSAR models<!>Model implementation<!>QSAR models for predicting skin sensitization using GPMT data<!>PreSS/MD usability<!>Case studies<!>An alternative to animal testing for skin sensitization for medical devices<!>Discussion and conclusions.

<p>Sensitization is a toxicological endpoint associated with the ability of an offending chemical to cause or elicit an allergic response in some people following repeated exposures to the allergen. 1,2 Traditionally, assessing the sensitization potential for a chemical or material has relied on the use of animal models. The guinea pig maximization test (GPMT) of Magnusson and Kligman 3 and the Buehler test 4 have been predominantly used methods for more than five-decades since their original development. 3,4 Alternative assays, such as the murine Local Lymph Node Assay (LLNA), have been employed for assessing skin sensitization as well. However, more recently, regulatory agencies have been supporting the development of alternative in vitro and in chemico methods that could help reduce, refine or replace testing in animals without compromising the acceptable standards for the identification of sensitizers. 5,6 Medical devices encompass a vast array of products intended to treat patients or diagnose diseases or other health-compromising conditions. 7 For marketing in the United States, the Food and Drug Administration (FDA) has set the definition of a medical device in Section 201(h) of the Food, Drug, and Cosmetic Act. 8 Medical devices require a pre-market biocompatibility assessment described in Guidance for Industry and FDA Staff on Use of International Standard ISO 10993-1, Biological evaluation of medical devices -Part 1: Evaluation and testing within a risk management process. 9 Many medical devices, such as implants and glucose meters, contain chemicals that may leach and cause toxicity. [10][11][12] Depending on the type and the duration of the contact with the body, a device may be evaluated for its biocompatibility, including the potential to produce localized sensitization responses. 13 Pre-market submissions for medical devices address sensitization potential with data gathered primarily with the GPMT or Buehler tests as recommended by the International Organization for Standardization (ISO) standard 10993 Part 10. 9 In the last several years, both our [14][15][16] and other 17,18 groups have developed computational models for predicting the sensitizing activity of chemicals in LLNA. In an effort to modernize the evaluation of medical devices potential for causing skin sensitization and help reduce in vivo animal testing, we embarked on the development of a unique open-source computational tool and web app that we named PreSS/MD (Predictor of Skin Sensitization caused by Medical Devices).</p><p>We envisioned a context of use where this tool can be employed to assess the skin sensitization potential of medical devices, to supplement and, potentially, replace the experimental assessments such as animal-based tests currently accepted for regulatory submissions of medical devices. To achieve this goal, we (i) collected, curated, and integrated the largest publicly available dataset for GPMT; (ii) developed and externally validated QSAR models to predict GPMT; and (iii) incorporated GMPT models into the PreSS/MD web portal to help evaluate the skin sensitization potential for medical devices.</p><!><p>The workflow employed in the study is depicted in Figure 1.</p><!><p>Experimental animal data on skin sensitization evaluated with the Guinea Pig Maximization Test (GPMT) were retrieved from the ECHA study results database (https://iuclid6.echa.europa.eu/reach-study-results). Unfortunately, there were numerous problems with the collected raw data. For instance, many numerical data were represented as string variables, the units of measurements were not standardized through the datasets, and there were many "free text" data. Therefore, we extensively cleaned and standardized all the data and converted measurements to the same units in each dataset. We also used regex expressions to find essential features for the database that were described in text format; this was key to classifying endpoints into GHS hazard categories. Following this laborious data preparation and standardization, we performed both chemical and biological data curation. After removing inconsistent data and non-modelable compounds (see Data Curation section), 1,023 out of the original 5,727 data points were kept. Among 23 duplicate chemical pairs in the dataset, biological annotations for 20 of them were concordant and for three, were discordant, i.e., duplicative compounds had different annotated classifications (sensitizer vs. non-sensitizer). All the discordant replicates and one of each concordant replicate were removed. The final dataset comprised 995 unique chemical compounds, including 247 sensitizers and 748 non-sensitizers.</p><!><p>We also collected GPMT skin sensitization experimental data from the scientific literature. [19][20][21][22][23] After removing mixtures, inorganics, and counter ions, 701 out of the original 745 data points were kept. Only one pair of duplicates showed biological annotation disagreement among 221 chemicals with more than one data point in the dataset. The discordant replicates were removed and only one data point for each concordant replicate was kept. Thus, the final dataset had 374 unique chemical compounds, including 173 sensitizers and 201 non-sensitizers.</p><!><p>We merged the curated data from ECHA and the research literature and examined the content of this combined data. There were 41 pairs of replicates between these two data sets, and the sensitization potential of only six of these pairs was annotated differently. These discordant records were removed, and only one record for each concordant pair of duplicates was kept. The merged data set had 1322 unique compounds including 432 sensitizers and 890 non-sensitizers, i.e., it was imbalanced with the ratio of sensitizers to non-sensitizers of approximately 1:2.</p><!><p>An additional literature search executed identified nine new compounds with GPMT data that were not part of the training set used for model development. These compounds were standardized and used as an additional validation set. We also collected the 474 compounds available in the Extractables and Leachables Safety Information Exchange (ELSIE) Database 24 After the removal of inorganics, mixtures, and duplicates, 415 compounds remained. We found that 102 compounds were present on our GPMT list and 313 unique compounds were kept for model evaluation.</p><!><p>Datasets were thoroughly curated following the workflows developed by us earlier. 25 . First, we performed chemical structure curation and removed mixtures, inorganics, and organometallic compounds, cleaned and neutralized salts, normalized the specific chemotypes, and applied the special treatment to chemicals with multiple replicated records as follows: (i) when replicated records presented the same binary outcome, only one record was kept; (ii) when a majority of replicated records presented the same binary outcome and one had different binary outcome, only one record with the agreeing binary outcome was kept, (iii) when replicated records had different binary outcomes, all of them were removed. All the curated data are available in Supplementary Material.</p><!><p>The modelability index (MODI) 26 was calculated to estimate the feasibility of obtaining predictive QSAR models. We developed our models following the best practices of QSAR modeling. 27 The models were developed using open-source chemical descriptors based on ECFP4like circular fingerprints with 2048 bits and an atom radius of 2 (Morgan2) calculated in RDKit 28 .</p><p>Machine learning approaches included Support Vector Machine (SVM) 29 , Random Forest (RF), 30 and Light Gradient Boosting Machines (lightGBM) algorithms implemented in Scikit-learn. 31 All models were optimized using a Bayesian approach implemented in Scikit-Optimize v.0.7.4. 32 The details of hyperparameters explored in this work are available in the Supporting Information. The Bayesian optimization may be defined as follows (Equation 1):</p><p>where, xi is the ith sample, and 𝑓(xi) is the observation of the objective function at xi. The observations D1:t = {x1:t, 𝑓(x1:t)} are accumulated. The prior distribution is combined with the likelihood function P(D1:t|𝑓) of overserving D 1:𝑡 given model 𝑓 multiplied by the prior probability of P(𝑓). In doing so, Bayesian optimization finds hyperparameters that maximize the objective function (G-mean score) by building a surrogate function (probabilistic model) based on past evaluation hyperparameters of the objective. 32,33 The geometric (G)-mean was selected as the scorer since it measures the balance between classification performances on both the majority (non-toxic) and minority (toxic) classes.</p><p>The QSAR models employing deep learning were developed using Keras The predictivity of the models were assessed by the Equations 2-7:</p><p>Balanced accuracy:</p><p>Sensitivity:</p><p>Specificity:</p><p>Positive Predictive Value (PPV):</p><p>Negative Predictive Value (NPV):</p><p>Kappa 𝐾𝑎𝑝𝑝𝑎 = 2×(𝑇𝑃×𝑇𝑁−𝐹𝑁×𝐹𝑃) (𝑇𝑃+𝐹𝑃)×(𝐹𝑃+𝑇𝑁)+(𝑇𝑃+𝐹𝑁)+(𝐹𝑁+𝑇𝑁) (7) where TP are the true positives, FP are the false positives, TN are the true negatives, and FN are the false negatives.</p><!><p>Maps of predicted fragment contribution 34,35 were generated from the QSAR models to help identify and visualize the substructure(s) predicted to provide significant contribution to the skin sensitization potential. Here, the contribution of an atom is estimated by a contribution difference obtained when the associated bits in the fingerprint corresponding to the atom are removed. Then, the normalized contributions were used to color-code the atoms in a topographylike map, in which green indicates negative contribution for toxicity (i.e., skin sensitization reduces when the atom is absent), and magenta indicating a positive contribution for toxicity (i.e., skin sensitization increases when the atom is present). 35</p><!><p>The PreSS/MD web app was implemented on an Ubuntu Server. The app is coded using Flask (http://flask.pocoo.org), uWSGI (https://uwsgi-docs.readthedocs.org), Nginx (http://nginx.org), Python (https://www.python.org), RDKit (http://www.rdkit.org), scikit-learn (http://scikit-learn.org), and JavaScript (http://www.ecma-international.org). PreSS/MD also includes the JSME molecule editor written in JavaScript, 36 supported by the most popular web browsers. Java or Flash plugins are not required to use the app.</p><!><p>High values of MODI (≥0.7) allowed us to expect that robust and predictive QSAR models could be developed for this dataset. The statistical characteristics of the skin sensitization models built and validated using GPMT data are shown in Table 3. The machine learning models built using RF, SVM, lightGBM, and Deep Learning were able to predict the external set with balanced accuracy of 73%, 74%, 70%, and 72%, respectively.</p><!><p>PreSS/MD has an intuitive user interface (Figure 2). The user may draw a molecule of interest or directly paste the query chemical structure's SMILES string in the "molecular editor" potential. These predictions are followed by the prediction's confidence, which is estimated by the ratio of predictions made by internal models, 30 the applicability domain (AD), and the maps of predicted fragment contribution.</p><!><p>As an example of a practical application, we tested PreSS/MD by employing it to predict the skin sensitization potential of nine medical device ingredients identified internally at the FDA with discordant data between GPMT and human clinical data. We compared this list with the dataset used to build our models and found that all these compounds were new and were not included in the original dataset. Therefore, we performed a blind prediction using the PreSS/MD to predict the skin sensitization potential of these nine compounds. The predicted results are shown in Table 4. PreSS/MD correctly predicted six out of nine compounds (balanced accuracy of 65%, sensitivity of 80%, specificity of 50%, PPV of 66% and NPV of 66%). Although the evaluation of these nine compounds presented low specificity, the NPV indicates the probability of predicted non-sensitizer being truly non-sensitizers is high as 66%. In addition to these nine compounds with GPMT data, we exploited our models to predict a list of 474 chemicals known to leach from MD. After the removal of inorganics, mixtures, and duplicates, 415 compounds remained, and we found that 102 compounds were present in our curated GPMT list. Out of the 313 remaining compounds, our models predicted 98 compounds as sensitizers in the GPMT assay and 215 as non-sensitizers. We analyzed this list's overlap with the expanded skin sensitization dataset of human, LLNA, and three non-animal assays (DPRA, KeratinoSens, and h-CLAT) data described in our previous paper. 14 Out of 313 chemicals, we found that 34 had experimental data in one of the skin sensitization assays. Table 3 shows the concordance of the predicted values using PreSS/MD and the skin sensitization potential available from experimental assays. Although the pool of compounds was small, the results show a high concordance with all assays. This high concordance suggests that integration of PreSS/MD models with non-animal methods, such as DPRA, KeratinoSens, and h-CLAT may be complementary to assess skin sensitization. The use of GPMT to predict human skin sensitization.</p><p>Previously, we analyzed the correlation of LLNA and Human skin sensitization data to understand how valuable the animal model is for determining risk assessment. 37 As GPMT is still being used to check the sensitization potential of leachable chemicals from medical devices, 9 we decided to conduct a similar analysis we reported before, when comparing LLNA vs. human data. 37 Here we compared the overlap between the 1322 compounds with GPMT data and the 138 compounds with human data we previously reported elsewhere. 14 As seen in Table 4, 109 compounds were both tested in GPMT and had human clinical data. In total, 46 compounds were sensitizers in both tests and 41 compounds were classified as non-sensitizers in both tests, while 22 disagreed in classification. Therefore, our analysis has shown that the accuracy of using GPMT to predict human skin sensitization is estimated to have the balanced accuracy of 80%, sensitivity of 85%, PPV of 77%, specificity of 74%, and NPV of 84%. Out of the 112 compounds shown in Table 4, 14 compounds were labeled to be leaching from medical devices in the ELSIE dataset.</p><p>Of these there were 9 sensitizers and 5 non-sensitizers with human data. All the non-sensitizers in humans were also non-sensitizers in GPMT and only one sensitizer in humans was labeled as a non-sensitizer in GPMT.</p><p>Given the small number of compounds with known experimental values from both GPMT and humans, we decided to apply our previously developed QSAR models of human data 16 to the remaining 1210 compounds with GPMT data lacking human data. The use of QSAR-imputed human data allowed us to examine the possible relationships between the two endpoints for a much larger set of compounds. 38 found that GPMT had sensitivity of 70% and specificity of 100%. However, the data analyzed was much smaller, with 57 chemicals and only 3 non-sensitizers. Variability of the GPMT has been documented as dependent on the total number of animals, dosage, and grade patterns of the sensitization response considered in the test. 39 Within the extensive data collected in this work, GPMT data showed high reproducibility. In the ECHA dataset, only three pairs of compounds out of 23 duplicate chemicals had discordant annotations. The data collected from the literature had only one pair of duplicates with discordant annotations among 221 chemicals. Finally, there were 41 pairs of replicates between these two data sets, and the sensitization potential was different for only six of these pairs. Conversely, human tests show high inter-individual variability, especially for compounds tested at a high dose, which can show weak sensitization rates in the tested populations. 40 In our previous analysis, 14 we found the accuracy of LLNA to predict Human skin sensitization was estimated to have a balanced accuracy of 68%, sensitivity of 84%, and specificity of 52%. The low specificity means that LLNA is oversensitive to predict human skin sensitization, i.e., more compounds tend to be skin sensitizers in mice than in humans. Conversely, GMPT showed higher concordance with human data, with specificity as high as 75%.</p><!><p>The GPMT was first published in 1969 3 and was considered the preferred animal method to assess skin sensitization caused by chemicals for decades. In 1989, the LLNA was first described. 41 Since then, it underwent multiple evaluations and refinements, becoming the preferred animal testing for skin sensitization after the publication of the Organisation for Economic Cooperation and Development (OECD) Testing Guideline No. 429. 42 However, international standards (ISO 10993) 43 still recommend the evaluation of chemicals released from MD for skin sensitization/allergenicity potential using the Guinea Pig Maximization Test (GPMT). 6 Recently, Svobodová et al. 44 evaluated the sensitization potential of chemicals present in MD using a combination of in chemico (DPRA) and in vitro (LuSens) methods in comparison with the LLNA method and suggested a testing strategy for the safety assessment of medical device extracts. The authors reported an overall concordance of 63.9-82.5% between LLNA and DPRA and 80-85.4% between LLNA and LuSens. Unfortunately, no sensitivity and specificity were reported. The results shown in Table 4 of this study reveal that there is a high concordance between GPMT and human data, which is in contrast with our previous findings showing that LLNA tends to be oversensitive as compared to the human response. 14,37 Although GPMT shows a higher concordance with human data than the LLNA, it is important to note that GPMT requires the sacrifice of several animals 45 for each tested chemicals and, therefore, better approaches need to become available soon. Recently, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) published a Strategic Roadmap, 1 calling for the development of alternative approaches to reduce animal testing of chemical and medical agents. Thus, there is an expressed need to modernize the safety evaluation of MD using alternative methods, shorten the regulatory review time, and ultimately bring safer devices to the market faster.</p><p>QSAR models developed in this study and implemented in the PreSS/MD web app showed balanced accuracy of 70-74%. Although our analysis of replicates identified only six out of 41 replicated entries to disagree, a previous study has shown that dose, number of animals, and response pattern may influence in the outcome, which is evaluated by a specialist. Therefore, considering the absence of state-of-the-art predictors of GPMT as well as the variability of the assay, we suggest these models can be used to reduce the use of GPMT when used within integrated testing strategies. Moreover, since GPMT has shown higher concordance to human data than the LLNA, we suggest that QSAR models based on GPMT are more appropriate than running GPMT to assess the response to chemicals in humans.</p><!><p>Previously, our group has developed the first QSAR models for skin sensitization based on human data. 37 Later, we employed an innovative approach using human, LLNA, and three validated non-animal assays within a Bayesian model to predict the human response. 14 This model showed higher accuracy in predicting the human response than the model built using only human data. 14 These models were implemented in a newer version of the Pred-Skin web app. 16 Since the publication of the OECD Testing Guideline No. 429, 42 LLNA has been regarded as the preferred animal test for evaluating skin sensitization. However, the GPMT is still required for the approval of MD. For this reason, we decided to develop a separate skin sensitization web application focusing on the safety evaluation of these devices.</p><p>In order to apply in silico methods to predict the toxicity of MD, it is essential to note that a cornerstone in any safety evaluation of FDA-regulated products is an exposure assessment focused on actual conditions of use. Traditional methods to estimate exposure do not apply to all MD. Consequently, the medical device regulatory framework has implemented a chemical characterization and subsequent toxicological risk assessment approach. The chemical characterization involves identifying the device's component or determining chemicals that might leach into a patient during use and corresponding quantities. 46 Toxicologists use this information to conduct a risk assessment to ascertain whether any of the leachable chemicals might pose a health risk to patients at the doses quantitated. Both the chemical characterization and toxicological risk assessment for MD are generally done as recommended by the ISO standard 10993 Parts 18 and 17, respectively. PreSS/MD can predict potential leachable compounds submitted for regulatory pre-market consideration.</p><p>In summary, in this contribution we described the development of PreSS/MD, a web application to predict the skin sensitization potential of chemicals based on GPMT. This tool is the first publicly available tool based on this assay. Although non-animal assays have been explored to evaluate the potential skin sensitization effects of chemical hazards, 2 animals are still required by regulatory agencies to evaluate MD. Our results here show that GPMT has a good correlation with human data, which is higher than the murine LLNA. However, although the use of guinea pigs is justified as their response to various skin sensitizers is similar to humans, interpretation of these assays' results requires unique expertise. 47 Moreover, the use of guinea pigs raises moral and ethical concerns, defying the principle of the 3Rs -Replacement, Refinement, and Reductionwhose goal is to identify alternative methods that utilize phylogenetically lower species, reduce the number, and refine the use of animals to lessen pain and distress. 1,48 Therefore, there is an imperative need to replace these assays. Our results show that the historical and publicly available GPMT data is sufficient to generate predictive and robust in silico models using machine learning approaches. The PreSS/MD web application fulfills an unmet need to help modernize the evaluation of skin sensitization for MD to reduce the need for animal testing. These models can be employed within integrated testing strategies to provide a weight of evidence of the sensitization potential of chemicals leaching from MD without requiring further animal tests. Moreover, we expect that the models developed in this study are applicable to estimate the toxicity of other industrial chemicals. 49 The PreSS/MD web application is publicly available at https://pressmd.mml.unc.edu/.</p>

ChemRxiv

Aluminum Doping Effects on Interface Depletion Width of Low Temperature Processed ZnO Electron Transport Layer-Based Perovskite Solar Cells

Rapid improvement in efficiency and stabilities of perovskite solar cells (PSCs) is an indication of its prime role for future energy demands. Various research has been carried out to improve efficiency including reducing the exciton recombination and enhancement of electron mobilities within cells by using electron transport material (ETM). In the present research, electrical, optical, and depletion width reduction properties of low temperature processed ZnO electron transport layer-based perovskite solar cells are studied. The ZnO thin films vary with the concentration of Al doping, and improvement of optical transmission percentage up to 80% for doped samples is confirmed by optical analysis. Reduction in electrical resistance for 1% Al concentration and maximum conductivity 11,697.41 (1/Ω-cm) among the prepared samples and carrier concentration 1.06×1022 cm−3 were corroborated by Hall effect measurements. Systematic impedance spectroscopy of perovskite devices with synthesized ETM is presented in the study, while the depletion width reduction is observed by Mott Schottky curves. IV measurements of the device and the interfacial charge transfer between the absorber layer of methylammonium lead iodide and ETM have also been elaborated on interface electronic characteristics.

aluminum_doping_effects_on_interface_depletion_width_of_low_temperature_processed_zno_electron_trans

Introduction<!>Experimentation<!>Characterization<!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!>Device Fabrication and Characterization<!><!>Device Fabrication and Characterization<!><!>Device Fabrication and Characterization<!><!>Device Fabrication and Characterization<!><!>Device Fabrication and Characterization<!><!>Conclusion<!>Data Availability Statement<!>Author Contributions<!>Conflict of Interest<!>Publisher’s Note<!>Supplementary Material<!>

<p>To cope with recent energy demands, the role of solar cells is attaining much attention (Iqbal et al., 2012; Tsarev et al., 2021; Ying et al., 2021). The need of the hour is to shift towards sustainable and green energy products (Midilli et al., 2006). Due to the increased human population and growth in energy consumption, the world's energy resources are depleting day by day. The dependence on fossil fuels like natural oil and coal gas is exceeding by an alarming rate (Höök and Tang, 2013; Russo, 2021; Zipper et al., 2021). Also, the burning of these materials produces gases like CO2, SOx, and NOx which are not only causing pollution in the environment but also threatening the human race's survival by causing global warming and greenhouse effects (Raval and Ramanathan, 1989). Green energy production is the cardinal matter in the context of natural resources preservation which opens up the avenues for synthesis of ubiquitous potential materials for energy harvesting applications (Balaprakash et al., 2018). Nanotechnology is arguably the technology of century (Roco, 2011): Few notable properties that might change or behave differently for nanomaterials than bulk materials are optical (Djurisic and Leung, 2006), electrical, magnetic (Slonczewski, 1961; Adnan et al., 2021; Rabbani et al., 2021), mechanical strength, charge storage capacity, biological activities (Usman et al., 2020), fluorescence, magnetic permeability, chemical reactivity (Tao, 2008), and melting points.</p><p>Many materials are being exploited which do not only provide renewable energy but are also cost-effective (Dincer, 2000; Raza et al., 2020; Usman et al., 2021). The need of the hour is to prepare environment-friendly and green products so that these energy harvesting devices do not pollute the environment. Since then, many materials have been synthesized, and many devices have been fabricated like solar cells (Yamaguchi et al., 2005), wind energy, fuel cells, capacitors, and supercapacitors which not only can produce renewable energy but also can store energy for a long power run. The increased usage of energy and reliance on portable energy devices is demanding increased energy and power density.</p><p>Perovskites were first effectively used in solid-state solar cells in 2012 (Yamaguchi et al., 2005); these types of solar cells contain perovskite structured material such as hybrid organic and inorganic material as an absorber layer for photovoltaics properties (Ramanathan et al., 2003; Qiao et al., 2019; Liu et al., 2020). Perovskite material is used as a photon harvesting material, whilst for charge transportation, different materials are used. At the interface of charge transport material and perovskite, the separation of exciton occurs. For electron transport, material only permits electrons and blocks the holes and vice versa for hole transport materials (Lin et al., 2018); these layers of material are also termed as ETL and HTL, respectively. The perovskite solar cells are a contender for the lead role of energy conversion devices of future generations. Their promising energy conversion feature with the low-cost manufacturing made them the most persuaded topic of research and exploring the new materials for electron transport layer/materials is advancing over time.</p><p>These materials are specifically used as electron extractors from the perovskite absorber materials. The basic principle which is being applied is bandgap alignment, i.e., the conduction and valance band in the case of inorganic semiconductors, while HOMO and LUMO of organic materials are as such that electron travels across the high potential, while Holes are being blocked. Historically, the rapid increment in the efficiency for the perovskite-based solar cells somewhat depends on the higher transfer of electron transport materials which imitates the purpose of exploring the new, efficient, and cost-effective electron transport materials; the key factors which might be the priority to use some materials for the electron transporting layer are higher electron mobility, high surface area, compatible bandgap; a sufficient energy band gap is preferable which restricts the electron-hole recombination.</p><p>These are compounds have metallic and oxide ions, and metal oxides are basic in chemical natures (Mann and Watson, 1958). Metal oxides have been widely studied for their properties spanning from electrical conductivity (Shahid et al., 2016) and catalytic activity (Iqbal et al., 2012) to biological activities (Riaz et al., 2019); their abundance is one of the most significant factors to consider the metal oxides for applications.</p><p>Many metal oxides such as ZnO (Gupta, 1990), TiO2 (Mughal et al., 2019), SnO2, and ZnSO4 have been utilized as an electron transport layer for better extraction of charge (Akimoto, 2012). Also, various research groups have studied core-shell nanoparticles TiO2/MgO (Karuppuchamy and Brundha, 2015), Al2O3/ZnO, and WO3/TiO2 for charge blockage, and enhanced charge recombination resistance by such material which is an insulator with effective charge deployment and charge transports. The state-of-the-art PSCs employed mesoporous metal oxide scaffolds, mostly TiO2, which requires a high-temperature annealing process (≈500 °C) which limits their use for flexible substrates, while the next widely studies ETL is SnO2 which has oxidation and morphological issues. So, to reduce the mentioned issues, the ZnO-based ETLs are studied in this literature.</p><p>Zinc oxide has 60 meV exciton binding energy (Aftab Akram et al., 2016) and fast electron mobility. ZnO is a semiconductor material with a direct bandgap (3.11–3.36 eV), while its higher exciton binding energy makes it ideal for several purposes in different fields such as its nanoparticle-based films for solar cell, light-emitting diodes, spintronics devices, and sensors for gas detection. The lattice parameters of ZnO are as follows; unit cell area = 3.24 Å and lattice constant = 5.1 Å. Their ratio (c/a ∼1.60) is optimum value for hexagonal unit cell c/a = 1.633. Its bonding is ionic in nature (Zn2+–O2−) with ionic radii of 0.75 Å for Zn2+ and 1.30 Å for O2−. Crystal structures of zinc oxide are rock salt (Razavi-Khosroshahi et al., 2017), zinc blende, and wurtzite. The preferable crystal structure for better electrical properties is wurtzite.</p><p>It is also stated that the effect on electrical resistivity by surface modification of ZnO and doped ZnO-based ETL is observed, which also incorporated the bandgap tuning, enhancement of optical transmission, and the effect of surface morphology on other factors such as light-harvesting of absorber material and electron transport.</p><!><p>For the substrate soda-lime glass and FTO coated glass was used. Initially, the fluorine doped tin oxide coated glass slides were rinsed with soap water several times, then in bath sonicator treated with acetone, ethanol, and deionized water for 15 min respectively and dried after each washing. To attain better charge conductivity and for compact films adherence, the FTOs were annealed at 150°C for 15 min (Salam et al., 2013).</p><p>The sol-gel method was brought into consideration for the synthesis of ZnO. Solution (0.2 mol) of zinc acetate hexahydrate (Zn(CH3COO).6H2O, Sigma-Aldrich >99.0%) into iso-propanol was reported in literature (Salam et al., 2011). The continuous stirring was done for homogenous mixing. After the complete dissolution of precursor, monoethonalamine was added dropwise for stabilizing of reaction. It acts as a capping agent to ZnO nanoparticles in solution. After this, solution was kept for ageing for 24 h at room temperature. Afterward, the aged solution was used for thin-film formation.</p><p>For aluminum doping into zinc oxide, two solutions were prepared of 0.2 M aluminum chloride di-hydrate in ethanol and 0.2 M zinc acetate hexahydrate in isopropanol. For doping appropriate volume of aluminum solution into zinc solution, stirring was done for the complete mixture. Afterward, samples were labeled as zinc oxide is i-ZnO, 1 at% Al-doped ZnO as 1% Al, 2 at% Al-doped ZnO as 2% Al, and 3 at% Al-doped ZnO as 3% Al and annealed at 150 °C for 1 h.</p><!><p>The morphology and mapping images of synthesized material were obtained using a scanning electron microscope (SEM JSM6490, JEOL). The XRD patterns of samples were recorded using an X-ray diffractometer (Bruker D8 ADVANCE) with Cu Kα (λ = 1.5406 Å) radiation. The lattice constants of as-prepared films were calculated by X'Pert HighScore software. Ultraviolet-visible (UV-vis) absorption spectra of various ZnO/AZO films were characterized on Jenway UV-73 Series spectrophotometer in the wavelength range of 350–800 nm. The J-V curves were recorded by a solar simulator developed by SCEINCETECH Co., Ltd. under AM 1.5G simulated solar illumination (100 mW/cm). The electrochemical characterization was carried on Potentiostate Biologic VSP.</p><!><p>The identification of crystal structure and study of crystallographic planes were carried out by X-ray diffraction (XRD). Figure 1 represents the XRD patterns for undoped ZnO and Al-doped ZnO nanomaterials. The interpretation and analysis of patterns were carried out with scrupulous attention and gingerly as it is the foremost technique for confirmation of doping. From the analysis of XRD data, the hexagonal wurtzite crystal structure having space group P63mc of ZnO was confirmed and matched with JCPDS 01-089-1397 (Suwanboon et al., 2008). In addition to this, XRD patterns of Al-doped ZnO at various concentrations affirm that no change in the wurtzite crystal structure occurs as there were no additional peaks. Upon details analysis, by addition of Al in ZnO, a left volumetric shift among peaks was observed. In XRD patterns for Al doping, peaks are shifting toward left i.e., θ is decreasing which suggests an increase in d spacing between planes. For further detailed structural studies of ZnO lattice, constant values were estimated; by using unit cell software, a was 3.2529Å and c was 5.21299Å, while entire cell volume was found 47.7726 Å for 1% Al-doped sample. As ionic radii of Al3+ is 0.53Å which is fewer than ionic radii of Zn2+ which is 0.74Å so substitutional doping of Al3+ in ZnO produces shrinkage of the lattice which is because of left volumetric change was found in XRD patterns. For spin-coated films, this gives crystallite size value in the range of 16–20 nm. The crystallite size for intrinsic zinc oxide film is found 18 nm approximately, for 1% Al-doped slightly changes to 20 nm, while for 2% Al-doped found 22 nm and 3% Al-doped it is estimated in 18–20 nm range and shown in Table 1. In Figure 1, the XRD patterns for intrinsic ZnO are shown. These XRD patterns confirm the hexagonal crystal structure having 100 planes at 2 θ= 31.02, 002 planes at 33.75, 101 planes at 35.6075, 102 planes at 46.98, 110 planes at 55.98, while 103 planes at 62.105.</p><!><p>XRD spectra of ZnO and Al doped ZnO nanomaterials.</p><p>Average Crystallite size of samples.</p><!><p>Characterization of atomic vibrational study of Al-doped ZnO is done by FTIR. In Supplementary Figure S1, it has been observed that for intrinsic ZnO, 1% Al-doped and 2% Al-doped weak absorption spectra at wavenumber 398 cm−1, 406 cm−1, and 414 cm−1 are detected which are referring to Zn-O-Zn. Also, there are stretching at 1383 cm−1 because of the stretching vibration of Zn-O-Zn interfered by Al atoms. Moreover, asymmetric vibration of the Zn-O-Zn bond varies because of uptake of Al-O-Al (Balaprakash et al., 2018). For ZnO FTIR spectra, spectra are matched by reference, and it was found that weak absorption spectra at wavenumber 398cm−1, 406cm−1, and 414 cm−1 are detected which are referring to Zn-O-Zn. The FTIR spectra of 1% Al-doped is showing peaks 398 cm−1, 406 cm−1, and 414 cm−1 which are because of Zn-O-Zn absorption spectra, while the extra peak at 1383 cm−1 which is because of stretching vibration of Zn-O-Zn interfered by Al atoms which are residing in lattice (Bai et al., 2013).</p><p>Similarly, the FTIR spectra of 2% Al-doped is showing peaks 398 cm−1, 406 cm−1, and 414 cm−1 which are because of Zn-O-Zn absorption spectra, while the extra peak at 1383 cm−1 is because of stretching vibration of Zn-O-Zn interfered by Al atoms which are residing in the lattice.</p><p>Also, further doping has no significant influence on the spectra; for 3% Al-doped ZnO FTIR spectra are showing peaks 238 cm−1, 298 cm−1, and 414 cm−1 which are because of Zn-O-Zn absorption spectra, while the extra peak at 1383 cm−1 and 1621 cm−1 is because of stretching vibration of Zn-O-Zn interfered by Al atoms which are residing in the lattice.</p><p>For the analysis of surface modification, scanning electron microscopy (SEM) is used. The top view SEM images of ZnO, 1%Al, 2% Al, and 3% Al thin films are shown in Figure 2. The SEM analysis suggests the film uniformity and smoothness are gradually improved for 1 and 2% of Al doping concentration in ZnO. These properties render to the synthesis technique and parameters of film thickness; further, films are spin-coated and parameters like spin speed and sample loading are kept almost constant. The images show a relatively fully covered area. The EDS spectra of doped samples are shown in Supplementary Figure S5.</p><!><p>Top view SEM images (A) ZnO thin film, (B) 1% Al doped ZnO, (C) 2% Al doped ZnO d) 3% Al doped ZnO.</p><!><p>For morphological analysis and roughness measurements, AFM images of the sample were obtained, and 3D AFM images are shown in Figure 3. For ZnO, it can be interpreted by the image that nanoparticles are homogenously distributed in films. Furthermore, film thickness studies show uniformity across all scanned areas. Root mean square roughness was estimated 10.4 nm. For 1% Al-doped ZnO film smoothness is evident from the results. Also, in some areas, there is a slight agglomeration of particles which might be because of fewer nucleation sites available and time of reaction as reported in the literature (Salam et al., 2013). The root means square roughness found to be 7.2 nm which decreasing with doping. Similarly, for 2% Al & 3% Al, no such differences are observed from the first two samples except for roughness increasing trend and for 3% Al particles are more agglomerated and film uniformity is slightly disturbed. The surface roughness for pure ZnO is 1.53 nm which is corroborated by XRD results as crystallite size was calculated by XRD patterns is 18 nm which results in uniform particle size and nucleation at the interface is rapid which also helps in the uniformity of particle's distribution as low surface roughness. For 1%, 2%, and 3% Al-doped ZnO was found 2.43, 2.34, and 2.12 nm.</p><!><p>Three-dimensional AFM images of (A) ZnO, (B) 1% Al, (C) 2% Al, and (D) 3% Al.</p><!><p>All these results were in comparison of XRD results as crystallite size for 1% Al-doped ZnO was calculated by XRD patterns is 20 nm which is slightly higher than intrinsic ZnO which results in less uniform particle size and although nucleation at the interface is rapid which also helps in uniformity of particle's distribution but due to Al doping lattice shrinkage results in larger grains which causes surface defects and higher surface roughness. As the doping percentage increase, the surface morphology is changing, though the doping percentage is very less an increase in crystallite size results in broader particle size, this causes a slight variation in surface roughness, and it increases. Using AFM in contact mode, surface roughness was found 2.78 nm as crystallite size was calculated by XRD patterns is 28 nm which is slightly higher than intrinsic ZnO which results in less uniform particle size and although nucleation at the interface is rapid which also helps in uniformity of particle's distribution but due to Al doping lattice shrinkage results in larger grains which causes surface defects and higher surface roughness. Also, the other factors which involve the sol viscosity and sample treatment have played an important role in the film roughness, as sample preparation was carried out by spin coating which involves the spin speed, acceleration, and deceleration speed of spin coater. These factors have a significant effect on the surface morphology as well as the temperature of annealing has played a pivotal role in homogenous film formation. These roughness properties were corroborated by the electrical properties such as charger carrier concentration and resistivity.</p><p>Ultraviolet-visible (UV Vis) spectroscopic studies were carried at room temperature; the relation of transmittance in correspondence to wavelength is shown in Figure 4. It shows a change of transmittance at the Al doping. As the doping concentration increases, the transmittance follows the blue shift trend monotonically. However, upon further doping decrease of transmittance is evident and it follows the trends for further doping of aluminum (Djurisic and Leung, 2006). This is one of the traits of Al substitutional doping as it resides in Zn defects and reduces oxygen vacancy in the lattice which was initially causing the green and yellow emission (Mani Rahulan et al., 2019). For band gap measurements from Uv Vis Spectra tauc plot method was used and a bandgap for intrinsic Zinc oxide was found 3.11 eV; for 1% Al-doped zinc oxide it was slightly higher 3.20 eV, 2% Al 3.21 eV and for 3% Al it was 3.24 eV as Shown in Table 2. The change in bandgap arise as donor impurity was introduced in the lattice which donated electrons in the conduction band resulting shift of Fermi level towards conduction band. In the present case, the Fermi level shift towards conduction band termed as blue shift causes the increase in the bandgap of Al-doped ZnO which increases with uprising concentration of dopant.</p><!><p>Optical transmittance spectra of ZnO and Al doped ZnO thin films.</p><p>Energy Band gap of samples.</p><!><p>Electrical properties were measured by the DC Hall effect apparatus and shown in Figure 5. Swin system of 5300G magnetic field was employed in the system, testing was carried out at a 300 K temperature and dark. Hall effect measurements of prepared samples were carried out to study the electrical properties of materials; specifically, the establishment of electric current through prepared films, the effect of doping concentration on the three main parameters which defines the characteristics of electrical behaviors was evident and by far had a discreet difference. In Supplementary Table S1, sheet resistance (ohm/sq), resistivity (ohm-cm), conductivity (1/ohm-cm), carrier concentration (cm−3), and sheet carrier mobility (cm2/Vs) of ZnO undoped and Al-doped thin films are illustrated. Resistivity is directly proportional to sheet resistance by the following relation Rs=ρ/T (1)</p><!><p>Variation in resistivity, carrier mobility, and carrier concentration of ZnO films with Al doping.</p><!><p>Here, Rs is sheet resistance,  ρ is resistivity, and T is the thickness of the film. Thickness was measured by EM which was found in the nanometer range. Similarly, conductivity is reciprocal of resistivity. α=1/ρ (2)</p><p>Here α is conductivity which varies inversely with resistivity. It is noted that electrical properties depend on carrier concentration and sheet resistance (Manifacier et al., 1979). Both properties are interlinked with Al doping, in which Al3+ resides on substitutional sites of Zn2+, Zn, and Al interstitial atoms and oxygen vacancies. For Al doping, the change in resistivity is drastic.</p><p>For intrinsic ZnO, it has a value of 5.387 × 10−1 (ohm-cm) which decreases to an appreciable value of 7.13 × 10−5(ohm-cm) for 1% Al doping. This is because Al3+ replaces Zn2+ in lattice sites which ultimately increases carrier concentration.</p><p>Upon further Al doping in ZnO, slight increase in resistivity is observed with increasing doping concentration. One the reason which might be suggested for this phenomenon is that Al has limited solubility in ZnO and, upon further doping, Al3+ might form Al2O3 and ZnAl2O4 which are non-conductive in nature and accumulate at grain boundaries causing high resistivity.</p><p>In present work that electrical properties such as resistivity and carrier concentration for Al-doped ZnO thin films were reported, the properties were improved from previously reported values because of pretreatment, improvement in film surface defects control, and several time bettered results are obtained by pre-treating substrate at 150°C for initiation of nucleation at sites and better film growth. Similarly, annealing of samples was carried out in an inert atmosphere which helped in the reduction of oxygen vacancies and fewer impurities.</p><p>The sheet resistance of the sample denoted by Rs is determined by measuring characteristic resistance RA and RB with the four-probe method by the placement of the sample in the sample holder and connecting the four edges (Chwang et al., 1974). Van der Pauw equation for the relation of parameters is given below (Look and Molnar, 1997) exp(−ρ RA/Rs)+exp(−ρ RB/RR)=1 (3) Also, charge carrier densities and charge mobilities are determined by this technique by applying a series of voltage measurements across the test specimen, while the current and magnetic field constant are kept constant.</p><p>Samples were prepared in a 1 × 1 cm2 area and tested in the same size in the Hall effect measurement apparatus. Sheet resistance for intrinsic ZnO is 5.986 × 104 Ω/sq which is reduced to 8.07 Ω/sq for 1% Al doping, as by doping the Al3+helps in the supply of more charge carriers.</p><p>Further, for 2% Al sheet resistance is increased slightly to 8.57 Ω/sq and with 3% al doping sheet resistance now starting to increase up to 8.91 Ω/sq which might be because of Al atoms which are excessive and doping limit is complete so they tend to make aluminates which are insulator in nature and causing of increasing the sheet resistance (Salam et al., 2011).</p><!><p>For solar cell fabrication, FTO/glass was etched with zinc powder and HCL to obtain the required patterns and properly rinsed with detergent, DI water, acetone, and isopropyl alcohol respectively with a duration of 10 min each and. The solar cell structure was planar in which afterwards the washing patterned FTO/glass substrates were coated with ETL followed by absorber material and HTL as systematic is shown in Figure 6. ZnO,1% Al, and 2% Al films were coated on patterned FTO via spin coating. These films primarily act as ETL. On the ETL layer, CH3NH3PbI3 via two-step method acts as an absorber layer for solar cell: synthesized via hot injection method was spin-coated and Spiro-OMeTAD was used as HTL.</p><!><p>(A) Systematic representation of fabrication of device. (B) Energy band diagram of device.</p><!><p>The morphological analysis of the fabricated device is carried out by SEM. In Figure 7 SEM images are shown. These figures illustrate when the perovskite layer is deposited on ZnO. It has smaller grains and slight pin holes attributed to the nature of ZnO. Similarly with the addition of dopant slight change in grain size and smooth film morphology is observed. The cross-section image of the champion device is shown in Supplementary Figure S2.</p><!><p>SEM top view of perovskite layer deposited on (A) ZnO ETL, (B) 1% Al, and (C) 2% Al.</p><!><p>The solar cell testing is carried out by using Science Tech. Solar cell testing mode is used for IV curves measurements, under light using the lamp having power100 mW. The cell area was 1 × 1 cm2. Figure 8 shows the device performance of the fabricated solar cells which are control samples of ZnO based ETL and Al-doped ZnO as ETLS. It shows the champion cell contains the 1% Al as ETL, having the PCE of 9.60% with Jsc, Voc, and FF of 12.80 mA/cm2, 0.887 V, and 84.36%, respectively, which has been greater than previously reported single layer by Mahmood et al. (2014) and almost equal to double layer which is 10% efficient. Also, as reported by Zhang et al. (2015) electrodeposited ETL has produced efficiencies about 11%. As Liu and Kelly (2014) have reported and developed ZnO nanoparticle electron-transport layers for CH3NH3PbI3-based solar cells, they have demonstrated that neither effect of a mesoporous scaffold nor any high-temperature processing steps has a significant impact to achieve PCEs as high as 15.7%. This is expected to simplify dramatically the device fabrication procedures for such devices, but at the same time maintain or improve their already high device efficiency the present study involving the effect on interface depletion width of perovskite and electron transport layer and reduction in width as corroborated by the MS data with aluminum doping plays a vital role to understand the mechanism of exciton carrier's movement within the cell.</p><!><p>IV characteristics of fabricated cells.</p><!><p>For attaining a depth understanding of device performance in terms of recombination, interfacial charge transfer phenomena, and carrier transport within the device, we have performed electrochemical studies such as electrochemical impedance spectroscopy (EIS) and Mott-Schottky characterization of the complete device. It has been ensured that the electrical response is attained shortly after the fabrication of the device and not of the degraded device. Also, the device has retained the photovoltaics performance identical before and after the electrochemical analysis which asserts that these analyses are not influenced by the degradation of the device.</p><p>Figure 9 shows the Nyquist plot for perovskite devices with ZnO, 1% Als, and 2% Al ETL performed at 700 mV under dark. From the amplified view of the high-frequency portion of the curves, we observe a small radius arc in high frequency which is followed by a larger radius arc for each device. Our experimental data fit nicely with a previously reported model (Liu et al., 2014) that used ZnO nanostructures as ETL in a perovskite solar device. The equivalent circuit consists of a series combination of one parallel R-C element and another parallel R-CPE (constant phase element) element combined with a series resistance, RS. In Table 3, RC, CC, RRec, CPE, and RS denote contact resistance and capacitance at ETL/perovskite or HTL/perovskite interface, recombination resistance, constant phase element originating from heterogeneity, and resistance incorporating metal contact and wire respectively. In the present work, the Spiro-oMetad is used as HTL, and metal contact resistance for all devices is almost identical 10 Ωcm2. The value for other parameters obtained from EIS data is given in Table 3.</p><!><p>(A) Nyquist plot of perovskite devices and Mott Schottky curve at 10 kHz frequency for perovskite device with (B) ZnO ETL, (C) 1% Al ETL, and (D) 2% Al ETL.</p><p>Electrical properties of samples.</p><!><p>There is a profound impact of flat-band potential and depletion width on device ultimate output. We can relate the device performance to these key device parameters with the information retrieved from the Mott-Schottky plot under dark conditions. The x-axis intercept of the extrapolated linear section of the Mott-Schottky curve gives the flat-band potential of the device, while the charge carrier density or doping density can be found from the slope of the curve using the following Eq. 4 N=(Vfb−V)C2qA2ϵ (4)</p><p>Here V, Vfb, C, A, q, and ε are applied bias, flat-band potential junction capacitance appearing due to the modulation in depletion width, active device surface area, elementary charge, and permittivity for perovskite respectively. The p-doped behavior of perovskite is evident (Bisquert and Garcia-Belmonte, 2011; Guerrero et al., 2016) from the Mott-Schottky curve lines. Since the dipping two-step fabrication technique of perovskite does not involve any annealing, the observation of p-type behavior from Mott-Schottky analysis complies with the report from the earlier literature (Mahmud et al., 2017). The perovskite layer tends to form P-N junction with adjacent ZnO and AZO layers, and Mott Schottky's behavior should be contributed from both ETL and perovskite (Guerrero et al., 2014). So, the flat potential can be expressed as Vfb = Vfb (perovskite)+Vfb (ZnO). Figures 9B,C,D show the flat band potential and charge carrier density of the device obtained from the Mott Schottky curve. The overall flat band potential for the 1% champion device is 0.67 V, and the overall charge carrier density for the device was found 3. 60 × 1016. The flat band potential for ZnO, and 2% Al devices was found 0.55 and 0.60 V, respectively as shown in Table 4. Hence, all results obtained from different characterization techniques are consistent and show no anomaly, and bear testimony to the proper optimization of the Al-doped ZnO layer as an electron transport layer to be applied in methylammonium lead iodide perovskite Solar cell.</p><!><p>MS data of samples.</p><!><p>In conclusion, we have reported low temperature (<150°C) wet chemical processed ZnO and Al-doped ZnO thin films as an electron transport layer with methylammonium lead iodide perovskite solar cell. This synthesis protocol provides a relatively cheaper and faster method of fabrication of solar cells. We have found that 1% Al-doped ZnO provides the most suitable ETM, antireflective electron extracting layer suppressing the deep trap states resulting in the decrease in depletion region width of perovskite and AZO (Mahmud et al., 2017), rendering to relatively better performance of device up to 9.60%. In the present work, we had used impedance spectroscopy to study the useful information regarding carrier generation, recombination, charge transport, and extraction in a perovskite solar device with synthesized ETM. Also, low contact resistance found from the Nyquist plot and thinner depletion width owing to high flat-band potential and larger carrier density from the Mott-Schottky curve indicate conforming ETL/perovskite interface for most efficient charge extraction.</p><!><p>The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.</p><!><p>MAA and MI conceptualized the work. MA, MU, and SA synthesized the products. MAD, MU, and SJ performed the electrochemical testing. All authors contributed to the article and approved the submitted version.</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><!><p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p><!><p>The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2021.795291/full#supplementary-material</p><!><p>Click here for additional data file.</p>

PubMed Open Access

Minimal Detection of Nuclear Mutations in XP-V and Normal Cells Treated with Oxidative Stress Inducing Agents

Elevated levels of reactive oxygen species (ROS) can be induced by exposure to various chemicals and radiation. One type of damage in DNA produced by ROS is modification of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG). This particular alteration to the chemistry of the base can inhibit the replication fork and has been linked to mutagenesis, cancer and aging. In vitro studies have shown that the translesion synthesis polymerase, DNA polymerase \xce\xb7 (pol \xce\xb7), is able to efficiently bypass 8-oxoG in DNA. In this study we wanted to investigate the mutagenic effects of oxidative stress, and in particular 8-oxoG, in the presence and absence of pol \xce\xb7. We quantified levels of oxidative stress, 8-oxoG levels in DNA, and nuclear mutation rates. We found that most of the 8-oxoG detected were localized to the mitochondrial DNA, opposed to the nuclear DNA. We also saw a corresponding lack of mutations in a nuclear encoded gene. This suggests that oxidative stress\xe2\x80\x99 primary mutagenic effects are not predominantly on genomic DNA.

minimal_detection_of_nuclear_mutations_in_xp-v_and_normal_cells_treated_with_oxidative_stress_induci

INTRODUCTION<!>Cell lines, growth conditions and treatments protocols<!>Total Cellular ROS detection<!>Protein ROS detection<!>8-oxoG detection by alkaline gel<!>8-oxo-dG detection by 2D Mass Spectrometry<!>Mutation Frequency<!>Analysis of mutations at the ura3-29 locus after MBL dosing<!>Production of Cellular Oxidative Stress<!>Effects of oxidative stress - Protein Oxidation<!>8-oxoG detection by alkaline gel<!>8-oxoG detection by 2D mass spectrometry<!>Evaluation of Nuclear Mutations using the HPRT locus<!>Oxidative stress dependent Ura3-29 mutations in yeast cells<!>DISCUSSION<!>

<p>Oxidative stress is defined as an excess of reactive oxygen species (ROS) over antioxidants. ROS can occur as a consequence of normal respiration, as well as from external sources; it is a relevant challenge to normal homeostasis and human health. ROS takes many forms including the radical species superoxide anion (O2˙−) and hydroxyl radical (˙OH), in addition to such non-radicals including H2O2 [1–3]. ROS can cause cellular damage by attacking DNA directly, or indirectly, as well as damaging lipids and proteins. All of these processes can lead to various diseases, aging and cancer [1, 2].</p><p>One of the most abundant and mutagenic oxidative alterations is 7,8-dihydro-8-oxoguanine (8-oxoG), which can be formed by ROS directly attacking guanine bases within double stranded DNA. 8-oxoG can potentially Hoogsteen base pair with adenine, instead of forming a normal Watson-crick base pair with cytosine. This mispair causes a GC → TA transversion. Due to the structural similarity of 8-oxoG:A base pair to that of the T:A base pair, this mispair can be refractory to proof reading and possibly some repair mechanisms as well [4–6]. There are, however, multiple mechanisms to try and reduce the effects of 8-oxoG on the genome. One of these is 8-oxoguanine glycosylase (hOGG1) which is responsible for excising the damaged 8-oxoG base, thus initiating base excision repair. Additionally, there is human Mut T homolog (hMTH1) which hydrolyzes free 8-oxoG nucleotide triphosphate in the nucleotide pool and human Mut Y homolog (hMYH) which removes the mispaired A from the 8-oxoG:A pair [1]. The 8-oxoG remaining in the DNA during DNA replication is then able to inhibit progression of the replication fork and becomes a candidate for translesion synthesis (TLS) [7].</p><p>Studies have shown that the Y-family of DNA polymerases (pol) (DNA polymerase η, ι, κ and Rev 1) have some ability to bypass the damaged 8-oxoG base, usually inserting A [5, 8, 9]. In vitro lesion bypass assays and cell based plasmid replication assays have been used to evaluate pol η, showing it can by pass 8-oxoG, and that it does so with better efficiency than past normal guanines, but with very low fidelity [5, 10, 11]. For this reason, we decided to evaluate a cellular response of oxidative stress on nuclear DNA, in particular evaluating for effects of 8-oxoG in cells proficient in known DNA repair pathways (normal human fibroblast)and in cells deficient in functional pol η (Xeroderma pigmentosum variant (XP-V) cell). The goal was to evaluate the role of pol η in oxidative stress induced nuclear mutagenesis.</p><p>In order to evaluate for nuclear mutagenesis we chose to use the hypoxanthine-guanine phosphoribosyltransferase (HPRT) nuclear mutation assay with two chemicals which are known oxidative stress agents. Menadione (MD)is a general oxidative stress agent which affects complex I of the electron transport chain, causing the production of superoxide anion [12–14]. Methylene blue plus light (MBL), causes redox cycling and the production of superoxide. MBL has previously been shown to preferentially produce 8-oxoG in the treatment of plasmids [11, 15–17]. In addition to HPRT mutation rates, we also evaluated the level of oxidative stress and 8-oxoG generated during these treatments.</p><!><p>Cell lines used were GM02359-hTERT (XP-V strain XP115LO; referred throughout as XP-V), a pol η deficient line; and NHF1-hTERT (referred to as NHF), a normal fibroblast control line, both previously described [18–21]. Conditions for growth were as described previously [18]. Treatment conditions were determined by literature review in conjunction with preliminary cell viability studies. Menadione (USB Corporation, Cleveland, OH) was used at 125 µM. Stock solutions of 1 mM were made in HBSS (Sigma-Aldrich, Saint Louis, MO) and filtered sterilized with a 0.2 µm filter (Genesee Scientific, RTP, NC) and diluted immediately prior to use in HBSS. Cells were treated for 20 minutes at room temperature [12, 13, 22]. Methylene blue plus light treatments were completed as follows: methylene blue (Calbiochem, EMD Biosciences, La Jolla, CA) stocks of 1 mM were prepared in ppH2O and filter sterilized (0.2 µm).Cells were treated based on McBride et al and Lee and Pfeifer [11, 16]. Briefly, cell growth media was removed and replaced with 10 mM sodium phosphate buffer (pH 6.9) (Fischer-Scientific, Fair Lawn, NJ) containing deferoxaminemesylate (Calbiochem, EMD Biosciences, La Jolla, CA) at a concentration of 0.1 mM [11, 16]. Methylene blue was added to the phosphate buffer at a final concentration of 5 µM, then culture plates were exposed to an LED light (warm white 800 lumens, equivalence to 60W incandescent) at a distance of 18 inches for 15 minutes at room temperature [11, 15, 16]. For exposure to ultraviolet A radiation (UV-A), a dose of 400 mJ/cm2 was used, from a 360 nm monochromatic lamp (EN-280L; Spectroline). The setup is a dual 8-watt tube with a 2F082 filter. Fluence was determined by a SEL033 (#SEL0339663) detector from International Light technologies attached to a phototherapy UVA measurement system ILT1400SEL033. Ultraviolet B radiation (UV-B) was used at 10 mJ/cm2 as previously described [18]. Addition of caffeine for specific treatments were as previously described [18]. The positive control for the flow cytometry was t-butylhydroperoxide (tBHP) (Sigma-Aldrich, Saint Louis, MO) at final concentration of 1 mM, treated for 1 hour at 37°C, as recommended by the manufacturer.</p><!><p>Cells were plated at 0.5×106 cells per P10 plate (Genesee Scientific, RTP, NC), incubated for 24 hours and then treated (as described above). Immediately after treatment, cells were washed with HBSS (Sigma-Aldrich, Saint Louis, MO) and then 3 mL of HBSS was added to the plates. CM-H2DCFDA (DCF) (Gibco-Life Technologies, Grand Island, NY) dye was added to the HBSS to a final concentration of 1 µM (as pre-determined by preliminary background fluorescence studies). Plates were incubated for 30 minutes at 37°C in the presence of DCF. The cells were then washed again with HBSS and trypsinized, harvested in 5 mL polystyrene round-bottom tubes (BD Falcon, BD Biosciences: Franklin Lakes, NJ), washed again in HBSS, harvested and the cell pellet was brought up in 0.5 mL of HBSS and kept on ice in the dark until analysis by flow cytometry. Data was acquired on a Becton Dickinson LSRII cytometer (BD Biosciences, Franklin Lakes, NJ) using Diva 6.1 software. Data analysis was performed using FlowJo software.</p><!><p>Detection of oxidized protein was performed as previously described with slight modifications [23–27]. Cells were plated at 80% confluency and grown for 24 hours. Cells were then treated and collected at 0, 4 or 24 hours after treatment. Treatments were as described above. Cells were trypsinized, washed with HBSS, and cell pellets collected. Cell pellets were lysed as described previously [28]. An equal volume of 20% trichloroacetic acid (TCA) was added to the lysate, followed by vortexing and centrifugation at 800 g for 5 minutes. The supernatant was discarded and 250 µL of 2N hydrochloric acid (HCL) was added to re-suspend the pellet. Then 250 µL of 2,4-Dinitrophenylhadrazine (DNPH) (Sigma-Aldrich, Saint Louis, MO) in 2N HCL was added and the mixture was rotated for 1 hour at room temperature. 500 uL of 20% TCA was added and the tubes vortexed to mix. Protein pellets were harvested by centrifugation at 800 g for 5 min. Pellets were washed with ethanol:ethylacetate (1:1) three times. The pellet was then dried and resuspended in 8M urea. This mixture was centrifuged at 13,400 g for 5 minutes and the upper phase was collected. Protein concentrations were determined by Bio-Rad protein assay as described [28]. Samples (3 µg) were separated using 12% SDS-PAGE. Gels were wet transferred to nitrocellulose and Western blot analysis was performed. Antibodies used were used: Anti-DNP (D9656, Sigma-Aldrich, Saint Louis, MO); anti-β-actin (A5441). Imaging was on a Storm 865 (GE Life Science) using Cy5 labelled secondary antibodies:ECLPlex Goat anti rabbit Cy5 (PA45011V) and ECL Plex goat anti mouse Cy5 (PA45009V).</p><!><p>Replicative form plasmid DNA, M13mp18, from XL1 blue cells was purified by Qiagenminiprep kit. It was then treated with either: no treatment, methylene blue only, light only or methylene blue plus light. Samples were treated in 10 mM phosphate buffer, and underwent three ethanol precipitations to remove residual methylene blue. Two µg of treated plasmid was digested with FPG (New England Biolabs) under conditions recommended by the manufacturer. FPG was deactivated by heating for 10 minutes at 60°C, and samples were separated through a 1.5% alkaline gel as described [29].</p><!><p>5 × 106 cells were treated as described above and harvested for cell pellets at either 1 hour or 24 hours post treatment. Total cellular DNA was prepared from cells by the addition of lysis buffer (Qiagen 1045696) to which was added 20 mM 2,2,6,6-Tetramethylpiperidinooxy (Tempo) to prevent additional 8-oxoG production. Proteins were precipitated using neutralization solution (Qiagen 1045697). Samples were vortexed and centrifuged at ≥16,000 g for 15 minutes. Supernatant was collected and DNA/RNA precipitated with isopropanol. The pellet was resuspended in lysis/20 mM Tempo buffer, RNaseA (Sigma-Aldrich) was added and incubated for 30 minutes at 37°C. Protein precipitation solution was again added the sample was centrifuged at ≥ 16,000 g for 15 minutes, followed by a second isopropanol precipitation. DNA was resuspended in 30 µL of ppH2O with 1 mM Tempo. 2D chromatography was performed as in Boysen et al [30]. See Supplemental Figure 3 for description of mitochondrial DNA preparation. 2D mass spectrometry was performed by Leonard Collins at the University of North Carolina Biomarker Mass Spectrometry Facility.</p><!><p>The HPRT mutation assay was conducted as previously described [18–20].</p><!><p>The wild-type (proficient in both OGG1 and RAD30 (pol η) base yeast strain used is the same as generated in a previous report (7B-YUNI300;MATaCAN1his7-2leu2-Δ::kanMX ura3-29:agp1 trp1-289 ade2-1 lys2-ΔGG2899-2900) [31]. OGG1 and/or RAD30 were deleted separately or in combination as described previously [31, 32]. They are referred to here as OGG1 RAD30, ogg1Δ RAD30, OGG1 rad30Δ, and ogg1Δ rad30Δ. Each strain contains a non-functional ura3-29 locus (codon 86, TCT) which reverts to a functional state after GC → AT mutations. This change is suggestive of 8-oxoG dependent mutagenesis [33]. For one day treatments, cultures (10 ml) of each strain were grown from a single colony overnight at 30°C, 250 RPM in YPD. Cells were harvested by centrifugation, washed once in PBS, resuspended in 10 ml PBS, and methylene blue (Calbiochem, EMD Biosciences, La Jolla, CA) was added to final concentration of 5 µM. Cells were transferred to a P10 culture dish and exposed to an LED white light source as described above and previously [18]. Cells were then harvested and washed twice with 10 ml PBS. After the final wash, cells were re-suspended in 0.5 ml PBS and plated onto either complete synthetic media (CSM) or CSM-Uracil (CSM -Ura) to obtain total cell numbers and numbers of mutants at the URA3 locus, respectively. Mutation frequency was calculated as (Number of mutants/Total number of cells). Experiments were performed at least 3 times. For three day treatments, after the final wash a 100 µl aliquot of cells was added to 9.9 ml YPD and grown overnight at 30°C, 250 RPM. The dosing step was repeated on day 2 and again on day 3 before plating cells onto CSM/CSM -Ura as described above.</p><!><p>In order to verify that our treatments were creating oxidative stress, we performed flow cytometry using 2'–7' dichlorofluorescin (H2DCF) dye which becomes oxidized to the fluorescent dichlorodihydrofluorescein (DCF) in the presence of ROS [34]. Controls included cells which were both untreated and undyed, and cells that were untreated but dyed with H2DCF to give baseline values to compare against for increases in fluorescence caused by increased ROS. The positive control used was tert-butyl hydroperoxide (tBHP) at a dose of 1 mM for one hour at 37°C. Oxidative stress treatments evaluated included 125 µM MD, 5 µM MBL, 10 mJ/cm2 UV-B, and 400 mJ/cm2 UV-A. As expected, untreated, undyed cells displayed the lowest levels of fluorescence, and tBHP treated cells displayed the highest levels of fluorescence (Figure 1A and B; Table 1). The shift from left to right for untreated, dyed cells (Figure 1A and B, Clear curves; Supplementary Figures 1 and 2) indicates background fluorescence of the dye and/or basal levels of ROS. From this we see that there may be slightly different levels of ROS in the NHF compared to the XPV line under normal growth conditions, or that the cells have intrinsically different background fluorescence. The large increase in fluorescence caused by tBHP (Figure 1A and B, red curves) suggests a very high level of ROS production. This is consistent with the severe cytotoxicity observed by tBHP at this treatment level (data not shown). Surprisingly, despite using agents and doses that have been reported in the literature to cause oxidative stress, in our hands the treatments generated ROS at roughly the same levels as observed in untreated cells (Figure 1A and B, orange, green, purple curves). The exception to this is MBL, which in the NHF cells did cause a detectable increase in DCF fluorescence (Figure 1A). Although additional dyes to test for cell death were not done in conjunction with ROS staining, it seems likely that MBL treatment has affected cell survival, as suggested by the decrease in the percentage of the parent population for MBL compared to Untreated (Table 1). This holds true for both NHF and XPV cells, and is similar to the effect seen with tBHP exposure. This suggest that despite the overall FITC-A level (i.e. level of detected ROS) for MBL being only slightly elevated in NHF cells and somewhat lower in XP-V cells compared to untreated samples, the treatment is in fact affecting the cell population in other ways.</p><!><p>In addition to direct detection of ROS we were interested in evaluating possible effects of oxidative stress within the cells. First, we chose to look at protein oxidation using a Western blot assay. After increased ROS, an antibody that recognizes a DNP conjugate to carbonylated proteins will show increased numbers of bands and a streakier appearance of lanes. Consistent with the flow cytometric detection of ROS results, Figure 1D–G shows moderate increases in oxidized proteins after most of the treatments and time points. The most prominent increase in oxidized protein is after MBL treatment, which correlates well with the above described flow cytometry based data.</p><!><p>Replicative form DNA of M13mp18 bacteriophage was used as a model to test the efficacy of MBL in generating 8-oxoG. DNA was exposed to MBL treatment, purified, and then treated with FPG, a protein that cleaves 8-oxoG from the DNA, leaving an abasic site. Separation of the DNA in an alkaline gel causes strand breaks at abasic sites, producing an increase in smaller fragments, or smear of DNA instead of discrete bands (Figure 2A). Figure 2B shows that only in the presence of both MB and white light, rather than each treatment alone, are detectable levels of 8-oxoG generated. The smear evident in the right most lane, confirms that the methylene blue plus light treatment creates 8-oxoG in greatly elevated levels compared to no treatment.</p><!><p>While the above assay shows that MBL can cause 8-oxoG in DNA in solution, we were also interested in whether our treatments were capable of generating 8-oxoG in DNA within cells. To test for this, we collected DNA from cells after treatments and analyzed it using mass spectrometry. We dosed cells with 125 µM MD, 10 mJ/cm2 UV-B, 400mJ/cm2 UV-A or 5 µM MBL and collected DNA 1 hour post treatment. We also collected DNA 24 hours post treatment for the MBL dose. As shown in Table II and Figure 2, we were unable to detect elevated 8-oxoG levels with MD, UV-B or UV-A treatments. However, we did observe a roughly 10 fold increase in 8-oxoG from the MBL treatment. This was observed in both cell lines, suggesting a similar mechanism of formation. Additionally, 8-oxoG levels 24 hours after treatment show that repair of the damage is occurring, and that the rate is similar in both cell lines (Figure 2C). Due to the fact that menadione uses Complex I of the mitochondrial respiratory chain for reduction and is known to interfere with mitochondrial respiration, and that methylene blue causes redox cycling of NADPH and NADH within the mitochondria [12, 35], we also looked at 8-oxoG levels specifically in DNA fractions highly enriched for mitochondrial DNA (Supplementary Figure 3). As seen in Table II and Figure 2D, once separated out from the total cellular DNA, we observed that most of the detected 8-oxoG is localized in the mitochondrial DNA and not in nuclear DNA fractions.</p><!><p>The fidelity of pol η has been assessed in numerous biochemical experiments and studies. Many of these studies have confirmed pol η performs low fidelity bypass of 8-oxoG in vitro [5, 10, 11, 36–38], while other studies have suggested that this bypass is "error free" (for lack of a better term) [33, 39, 40]. In addition to biochemical experiments, pol η also has been evaluated using damaged plasmids transfected into cells. This work suggests that the presence of pol η suppresses mutagenesis [11]. With this background of biochemical and plasmid based data, we wanted to transition into a cell based assay to investigate whether the presence of pol η would affect nuclear mutagenesis rates after oxidative stress. Unexpectedly, we found that our oxidative treatments seemingly had no effect on nuclear DNA mutation frequencies, as measured by the HPRT assay (Table III). Untreated XP-V cells had a mutation frequency (MF) of 0.98×10−5. Treatment with menadione (MF 2.33×10−5; 2.4×) and MBL (MF = 1.01×10−5; 1.0×) gave nearly identical frequencies. Similarly, untreated NHF cells gave a MF value of 0.55×10−5, with menadione (1.65×10−5; 3×) and MBL (1.27×10−5; 2.4×) again causing very little difference. This is in comparison to our previously published work using environmentally relevant levels of UV-B(10 mJ/cm2) in which the MF of XPV cells was 25.8×10−5 (26× higher than untreated) and for NHF cells the MF was 8.56×10−5 (15.6× untreated) [18]. In addition we attempted alternative treatment protocols in an attempt to exacerbate the effects, in case a single, short oxidative stress inducing treatment was insufficient to generate damage/mutations at levels detectable in this assay. Repeated, low dose MD treatments caused cell death, regardless of the levels used. Repeated MBL treatments did not alter the mutation frequency, but did result in extremely low colony forming efficiencies (CFE). Treatments with H2O2 produced highly variable results (both within replicates and within different experiments) and were therefore unable to be used. We also attempted the addition of caffeine after MBL treatment (similar to as has been used to increase cytotoxicity effects of XP-V lines under UV-C and UV-B treatments) [18, 41, 42], but again this lowered the CFE (less than 1%) to a point that the mutation frequencies obtained were unreliable. These additional treatments are described in Supplementary Note I. In total, we were unable to find treatment levels/conditions that balanced survivability with mutations.</p><!><p>In order to further explore the effects of MBL in cells, we used a set of yeast strains that were deficient in OGG1, RAD30, or both. OGG1 is the sole 8-oxoG repair enzyme in S. cerevisiae cells, and RAD30 (pol η) is the only Y-family member with significant lesion bypass ability. It has previously been shown that spontaneous mutations at the Ura3-29 locus are increased in an ogg1 mutant strain, and that deletion of rad30 further increases this mutation frequency [33]. Here, we performed similar experiments, but instead dosed cells either once or on 3 consecutive days with 5 µM MBL (20 minute exposure). Consistent with our results described above, deletion of pol η alone did not affect the mutation frequency at a nuclear locus (Figure 3). We do see, however, that both deletion of either OGG1 alone or both OGG1 and RAD30 gives a large increase in the mutation frequency at the Ura3-29 locus. This is true after either a single exposure or 3 separate days of treatment. Determination of the sequence of functional Ura3 genes in mutants from the double knockout strain showed that they were all GC → AT changes (data not shown), as expected if errors when bypassing 8-oxoG were the cause. These data suggest that when increased levels of 8-oxoG are present, the mutagenic propensity of it are partially mitigated by pol η, at least in yeast. They also suggest that the repair of 8-oxoG is more important in preventing mutagenesis than lesion bypass; whether or not a similar phenomenon is occurring in human cells remains to be seen. It is important to keep in mind that human pol η has a much greater in vitro error rate when bypassing 8-oxoG as compared to yeast pol η [5].</p><!><p>Numerous studies have evaluated the role of human polymerases encountering oxidative lesions; including plasmid replication assays and in vitro biochemical studies. In addition, studies assessing oxidative stress cytotoxicity have been performed, but none have combined both. Therefore, we treated cells with MD and MBL, in hopes of gaining a broader understanding of lesion bypass after oxidative stress. We chose MD for its properties as a general oxidative stress agent, and MBL for its defined ability to produce 8-oxoG in DNA [15]. Additionally, UV-B and UV-A were used for their well-known mutagenic effects in conjunction with their general consideration as oxidative stressors.</p><p>First, we tested for oxidative stress by flow cytometry. Our treatments, despite being published as oxidative stress agents, showed relatively low levels of detectible ROS (Figure 1A–C). However, these particular doses were chosen for their relatively minor cytotoxic effects to allow for evaluation with the long term mutagenicity assay; higher levels of the treatments could have produced larger quantities of ROS, but, it would enhance cytotoxicity, reducing the effectiveness of the HPRT assay. Our MD and MBL treatments show clear signs of oxidative stress, as evident through production of oxidized proteins (Figure 1D–G; Table I), as well as detectible 8-oxoG lesions in DNA (Figure 2; Table II). Despite seeing effects of oxidative stress throughout the cell, our nuclear mutation frequencies (assayed at the HPRT locus) of these treatments were disappointingly low and not significantly different from untreated cells. Attempts to exacerbate oxidative stress to increase mutation levels (Supplementary Note 1) were unsuccessful. Despite our additional efforts, we were unable to find conditions that balanced longer term survivability with detection of mutations, with many possible contributing factors. The first possibility is that the oxidative stress treatments used here were not enough on their own to cause detectable mutations. Under more 'real world' conditions exposure to multiple different agents simultaneously is more realistic, including combined exposures of sunlight, chemicals within food or water, and other sources of stress. Another possibility suggested by our yeast data and other previously published results [5, 11, 33] is, a pol η deficiency on its' own is insufficient for 8-oxoG damage to cause mutations, and instead, there would need to be a concomitant deficiency in OGG1 or other repair factors to increase the 8-oxoG load. In addition, it is interesting to note that our MBL treatment caused 8-oxoG to be mainly localized in the mitochondria. MBL and MD both use the electron transport chain in order to create ROS, so it is perhaps not surprising to see effects in the mitochondria DNA; however it was unexpected to not see more general cellular effects caused by the increased ROS. This lack of effect could be due to the diffuse nature of ROS compared to a more direct damaging agent such as UV light. These results suggest that more research is needed to determine when the cells robust repair mechanisms become overwhelmed by ROS, therefore permitting TLS to bypass the DNA damage caused by ROS and creating the potential for mutagenicity. Lastly, it is entirely possible that the HPRT assay has insufficient sensitivity to detect low levels of mutations, and therefore another method may be more suited to detecting these changes; including the potential for deep sequencing [43].</p><!><p> STATEMENT OF AUTHOR CONTRIBUTIONS </p><p>K.N. Herman performed and designed experiments, analyzed data, and prepared the manuscript. S.M. Toffton provided technical assistance with experiments. S.D. McCulloch designed experiments, provided intellectual input, and made final edits to the manuscript. All authors approved the final manuscript. K.N. Herman and S.D. McCulloch had complete access to the study data.</p><p>All authors declare no conflicts of interest.</p>

PubMed Author Manuscript

Long Noncoding RNAs: Fresh Perspectives into the RNA world

Large scale mapping of transcriptomes has revealed significant levels of transcriptional activity within both unannotated and annotated regions of the genome. Interestingly, many of the novel transcripts demonstrate tissue-specific expression and some level of sequence conservation across species, but most have low protein-coding potential. Here we describe progress in identifying and characterizing long noncoding RNAs and review how these transcripts interact with other biological molecules to regulate diverse cellular processes. We also preview emerging techniques that will help advance the discovery and characterization of novel transcripts. Finally, we discuss the role of long non-coding RNAs in disease and therapeutics.

long_noncoding_rnas:_fresh_perspectives_into_the_rna_world

Pervasive transcription in mammalian genomes<!>lncRNAs: functional transcripts vs. biological noise<!>Mechanisms of lncRNA function<!>Chromatin modification by lncRNAs<!>Regulation of transcription initiation<!>Co- and Post-transcriptional regulation<!>Challenges in the lncRNA field<!>lncRNAs in disease: Xist as a model<!>Box 1: LncRNAs and therapeutics<!>Concluding remarks<!><!>Mechanisms for long noncoding RNA (lncRNA) function<!>Techniques for mapping RNA-protein interactions<!>

<p>A major advance in molecular biology over the past 25 years has been the discovery of and demonstration of function for long noncoding RNAs (lncRNAs). The maturation of high throughput genomic tools such as next generation sequencing and large scale tiling arrays has helped accelerate the pace of discovery. A series of projects involving either cDNA library sequencing [1, 2] or their hybridization to genomic arrays [3–7] provided comprehensive maps of the transcriptional landscapes within cells. The recent ENCODE project [8–10], the most comprehensive effort yet for surveying transcription in human cells, confirmed earlier reports of pervasive transcription in mammalian genomes. ENCODE performed RNA-seq across 15 cell lines and detected primary transcripts from a cumulative 75% of the human genome [9]. There appear to be over 9000 genomic loci which give rise to lncRNAs in human cells [10]. The noncoding RNA Expression Database (NRED) for mouse lncRNAs reported expression of about 3000 transcripts in six different biological contexts [11]. Their noncoding status is deduced from a lack of sequence homologies to known proteins, the absence of substantial open reading frames, codon substitution frequencies which deviate from that for protein coding regions [12, 13], and tandem mass spectrometry analysis [9, 14] which determine whether peptides corresponding to the RNA sequences of interest are represented. Noncoding transcripts seem to be concentrated within the cell nucleus, and are often expressed at significantly lower levels than coding RNA [9]. In addition, an increasing number of studies have begun to analyze the proportion of polyadenylated to non-polyadenylated transcripts making up the noncoding transcriptome. A significant component of the noncoding transcriptome, at least in human cells, seem to be non-polyadenylated [9, 15, 16] – an observation that could partly be explained by the discovery of polyA- transcripts emanating from many active enhancer elements in the human genome (enhancer RNAs, or eRNAs)[17–20].</p><p>The complex transcriptional landscape in mammalian cells, confounded by the lack of functional annotations for the majority of lncRNAs, has made it particularly challenging to classify novel noncoding transcripts. An arbitrarily selected cutoff of 100–200 nt is commonly used to broadly distinguish lncRNAs from 21–35 nt "small RNAs" such as microRNAs, Piwi-interacting RNAs (piRNAs), and small-interfering RNAs (siRNAs). However, such oversimplifications inevitably create difficulties in some cases, such as for the class of short RNAs which are transcribed very close to transcription start sites [6, 21, 22]. These transcripts usually fall below the 200 nt threshold but are biologically distinct from Ago-associating short transcripts, small-nuclear RNAs (snRNAs) or small-nucleolar RNAs (snoRNAs). LncRNA above the 200 nt threshold may be classified to reflect their locations relative to genomic elements. Transcriptional loci may (a) overlap with annotated gene bodies with transcription initiating from either exons or introns from the sense or antisense strands, (b) lie within cis-regulatory regions of genes as in the case of eRNAs or (c) lie in intergenic regions, giving rise to long intergenic noncoding RNAs (lincRNAs). These diverse classes of lncRNAs and their biological roles in the cell are the focus of this review. In any case, more biologically meaningful ways for classifying noncoding transcripts should be possible in the near future as we glean more insight into lncRNA mechanisms.</p><!><p>The biological relevance of pervasive transcription and their associated lncRNAs is a current topic of debate [23–26]. Their low sequence conservation across model organisms and low expression levels have led some to postulate that many lncRNAs could arise from low fidelity RNA polymerase (RNAP) activity [27] and that this spurious activity is of little significance. However, in-depth analyses of lncRNA sequences may suggest the contrary. First, promoter regions and splice sites of lncRNAs have a degree of sequence conservation comparable to that for protein-coding genes [10, 28]. Second, while sequence conservation along the length of lncRNAs may be lower than that for mRNA [10, 29], lncRNA function may not necessarily depend on strict sequence conservation, especially if only small segments of the lncRNA are in contact with proteins, or if conservation of secondary structures takes precedence over that of primary sequences [29, 30]. For example, the well-characterized Xist RNA harbors only short segments of conserved sequence, but is known to play a critical role in dosage compensation [31].</p><p>Information from RNA-seq performed by the ENCODE consortium also reported that the boundaries of many human genes need to be expanded to account for extensive transcriptional activities. This has correspondingly resulted in a more than three-fold decrease in length of intergenic regions. Annotated genes can express between 10 to 12 isoforms simultaneously, of which a significant portion are novel elements that are non protein-coding [9]. There is concern that such lncRNAs are merely extensions of the nearby coding transcripts. The distinction can be made by coupling RNA-seq or tiling array data to end capture assays such as CAGE (cap analysis of gene expression) for 5' ends [8], 3P-seq for 3' ends [30] or RNA paired end ditag (PET) sequencing to mark both ends. Intersection of CAGE and PET libraries with the lncRNA database revealed that a significant portion of lncRNAs has unique start and stop sites, suggesting that lncRNA transcription can occur independently [10]. Novel elements also covered a large majority of intronic sequences – raising the question of how distinctions between novel lncRNA and unprocessed nascent transcripts may be made. Apart from the presence of unique 5' and 3' ends, the fact that some intronic lncRNAs can be detected in cytoplasmic fractions of cell extracts also argues against their being nascent mRNA [5]. In fact, the prevalence of lncRNAs emanating within close vicinities of coding genes is likely to be a reflection of the cis-acting nature of some transcripts (as reviewed below), or of gene regulation elicited from transcriptional overlap (such as imprinting of Igf2r by the lncRNA Airn [32]).</p><p>The idea of regulation by transcriptional interference, such as that proposed for Airn, is a reminder that the function of some lncRNAs may hinge on the transcriptional process, rather than the RNA product. Experiments to distinguish between the two, such as that done for imprinted lncRNA Kcnq1ot1 [33], have not been systematically performed. These would be crucial experiments in many cases, such as for eRNAs, which are noncoding, predominantly non-polyadenylated transcripts originating from a subset of putative enhancer elements [18, 19, 34]. eRNA levels demonstrate strong correlation with transcriptional activities of corresponding coding genes, yet it is still unclear in many cases whether eRNA synthesis is important for enhancer/promoter activation and the eventual activation of target genes, or if eRNAs are merely by-products of active enhancers in close association with gene promoters and the basal transcriptional machinery. Recent work by Kraus and colleagues showed that inhibition of eRNA transcription via flavopiridol, an inhibitor of transcription elongation, has little impact on the establishment of epigenetic marks (e.g. H3K4me1) or loading of RNA polymerase II (RNAPII) and other coactivators (e.g. E1A binding protein p300 (EP300) and CREB binding protein (CREBBP)) at enhancers [20]. In addition, enhancer/promoter loopings were also largely unaffected in the absence of eRNAs [20]. This suggests that molecular features usually associated with enhancers can occur independently of eRNA synthesis. It is important to note that further experiments are needed to determine whether eRNAs contribute to other aspects of enhancer function and target gene expression since flavopiridol have effects beyond transcription elongation [20].</p><p>As we begin to appreciate the complexities of transcriptional activity in the genome, it is clear that the traditional concept of a gene needs to be redefined. Fundamental differences between mRNA and lncRNAs point to the inadequacies of applying rules used to assess mRNA function on other transcripts whose functions lie outside the realm of protein production. In addition, coding and noncoding transcripts emanating from overlapping genomic loci blurs the distinction between regulatory and protein-coding sequences. Future work in unraveling lncRNA function and how underlying genomic sequences contribute to function will be key to understanding the true nature of the genome.</p><!><p>LncRNAs have been implicated in the regulation of a diverse array of biological processes including dosage compensation [35], imprinting [33, 36], cell cycle control [37–39], development [30, 40], and gametogenesis [41]. The function of lncRNAs cannot currently be predicted from sequence information alone, unlike proteins which often have well-defined modular domains and whose functions may be deduced from those of related proteins. An emerging theme, however, is the capacity of lncRNAs to modulate gene expression, either through action in cis on neighboring genes [33, 35, 36, 42, 43] or action in trans regardless of gene location [20, 44].</p><!><p>A classic example of lncRNA-mediated chromatin modification comes from eutherian dosage compensation, a whole-chromosome silencing mechanism that depends on expression of Xist RNA [35]. Synthesis of Xist RNA from the future inactive X chromosome (Xi) during early development triggers large scale recruitment of Polycomb repressive complex 2 (PRC2) in cis to the chromosome, establishing facultative heterochromatin extensively marked by the repressive H3K27me3 modification [45]. Native RNA immunoprecipitation (RIP) of Enhancer of Zeste 2 (EZH2), the catalytic subunit of PRC2, has shown that Xist RNA interacts with PRC2 during X-chromosome inactivation (XCI) to initiate and spread chromosomal silencing [42]. This RNA-protein interaction is believed to involve the repeat A region within Xist [46]. In line with RIP data, another study reported that ectopic expression of Xist from an autosomal locus is sufficient for the deposition of H3K27me3 around the site of transgene integration, providing support for a direct role of Xist in PRC2 recruitment and H3K27me3 deposition [47]. Apart from Xist, PRC2 is found to complex with other lncRNAs such as Kcnq1ot1, antisense noncoding RNA in the INK4 locus (ANRIL), and HOX transcript antisense RNA (HOTAIR) [33, 39, 44]. In the case of HOTAIR, action occurs in trans [48, 49]. Together, these observations lend credibility to the hypothesis that lncRNAs play crucial roles in recruitment of chromatin-modifying complexes to appropriate genomic loci both in cis and in trans (Figure 1a). The dependency on lncRNAs (and their secondary structures) to target PRC2 may explain the many as yet unsuccessful searches for DNA-based polycomb responsive elements (PREs) in mammalian systems [50].</p><p>LncRNA partners for other chromatin-associated proteins have also been found. The H3K9 methyltransferase G9a, which has been implicated in imprinting, associates with the lncRNA Airn to mediate silencing of the Igf2r/Slc 22a2/Slc22a3 gene cluster on the paternal allele in the murine placenta [36] and with Kcnq1ot1 at the imprinted Kcnq1 domain [33]. In budding yeast, the IRT1 transcript recruits both Set2 (a histone methyltransferase) and Set3 (a histone acetylase) to the IME1 locus for regulation of gametogenesis [41]. Interactions of lncRNAs with proteins are by no means exclusive to repressive chromatin modifiers; Mixed-lineage leukemia (MLL), a component of the Trithorax complex which deposits activating methylation marks at H3K4, has been shown to be recruited by lncRNAs associated with homeotic genes such as Hoxb5/6as [51], Evx1as [51] and Mistral [52], gene activation of Xist has been shown to involve Jpx RNA-mediated eviction of a CCCTC-binding factor (CTCF) repressor [53], and promoter associated transcripts at rRNA genes associate with DNA methyltransferase 3b to mediate gene silencing [54].</p><p>These findings prompt the question of how lncRNAs achieve targeting to genomic loci with high specificities. Here, several hypotheses have been proposed [48]. Theoretically, lncRNAs may form triplexes with genomic DNA containing complementary sequences. Alternatively, targeting specificity may be achieved with favorable chromatin architectures. For example, lncRNAs could demonstrate preferential loading of chromatin modifiers to segments of the genome which are in close proximity. Chromosome conformation capture experiments have suggested this mechanism [55, 56]. The third possibility calls upon additional DNA binding factors to bridge the gap between lncRNAs and chromatin. In the case of Xist RNA, work by Jeon and Lee has revealed the role of the transcription factor Yin Yang 1 (YY1) in tethering Xist RNA to the X inactivation center (Xic) on Xi [47]. YY1 was found to bind both Xist DNA and RNA, and its depletion resulted in a loss of Xist loading on the Xi. These observations suggested that YY1 is the docking factor responsible for the cis-acting nature of Xist RNA.</p><!><p>The classical noncoding U1 snRNA, a component of the spliceosome, interacts with transcriptional initiation factor TFIIH to boost initiation rates of the basal transcriptional complex [57]. Novel lncRNAs have demonstrated similar capabilities, bypassing chromatin-modifying complexes to communicate directly with gene promoters, the basal transcriptional machinery, and transcription factors. These lncRNAs are usually synthesized from regulatory loci such as enhancers and promoters and act in cis to mediate rapid, sensitive, and localized transcriptional regulation. For example, the Evf2 lncRNA is transcribed from an ultraconserved enhancer at the Dlx5-6 gene cluster and forms a complex with the transcription factor Dlx2 to elicit activation of the Dlx gene cluster [58]. Depletion of Evf2 resulted in the decrease in GABAergic interneurons in the early postnatal hippocampus and dentate gyrus of mice [59]. Recent studies have uncovered more lncRNAs that function as transcriptional activators in both mice and humans [17, 18, 60]. Many of these transcripts are synthesized at enhancers and the majority influences the activity of enhancers, or help with the recruitment of protein factors to enhancers. For example, two lncRNAs highly expressed in aggressive prostate cancers bind to the androgen receptor (AR) to enhance AR loading at gene enhancers, even in the absence of AR ligands [61]. Activating lncRNAs were also found in association with Mediator, acting as cofactors which help to model chromatin architecture and enhance kinase activity [62]. The transcription of the noncoding transcripts at enhancers is also proposed to play a role in enhancer activation by mediating the deposition of H3K4 mono- and di-methylation [63].</p><p>The ability of lncRNAs to interact with both the basal transcriptional machinery and key regulatory sequences on the chromatin, possibly through RNA-DNA interactions, is demonstrated by the lncRNA transcribed from the minor promoter of the human dihydrofolate reductase (DHFR) gene [64] (Figure 1b). The noncoding transcript is proposed to form a triplex with the major DHFR promoter and bind to TFIIB to displace the pre-initiation complex from the DHFR locus, thereby blocking gene expression. The DHFR study presents a case where lncRNAs mediate gene repression by interfering with the activity of the basal transcriptional machinery. Similarly, murine B2 RNA and human Alu RNA, both of which are transcribed from short interspersed elements (SINEs), mediate repression of heat shock genes by binding to and deactivating RNAPII [65, 66]. Although these RNAs all bind the transcription initiation complex, they bear little resemblance to each other in sequence or structure [66]. Primary and/or secondary structures relevant for these interactions are currently of significant interest. Identification of more noncoding RNAs operating in the same manner should shed light on this question.</p><!><p>Co- and post-transcriptional processes such as splicing, transport, translation of mRNA, and subcellular localization of proteins may also be controlled by lncRNAs. Interaction of lncRNAs with primary coding transcripts can occlude splice junctions and result in production of alternative isoforms. Studies which look at the regulation of the transcription factor Zeb2, which has been implicated in epithelial-mesenchymal transitions (EMT) during embryogenesis and cancer transformation, have revealed that Zeb2 is regulated post-transcriptionally by its natural antisense transcript (NAT). The noncoding NAT, synthesized from the antisense strand of the Zeb2 promoter, shields an internal ribosome entry site (IRES) within the 5' UTR of Zeb2 from mRNA splicing, thereby allowing for increased rates of Zeb2 translation and driving EMT [67]. In the absence of antisense expression, the loss of the IRES results in significantly lower levels of Zeb2 protein (Figure 1c).</p><p>The expression of ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene implicated in brain function and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease [68], has also been found to be regulated by a lncRNA. The Uchl1 antisense transcript (Uchl1-AS), which partially overlaps with the 5' end of Uchl1 mRNA, is initially concentrated in the nucleus but translocates to the cytoplasm under conditions of cellular stress [69]. Once there, Uchl1-AS promotes translation of Uchl1 mRNA by enhancing polysome loading on the mRNA. mRNA levels remain unperturbed in the presence of increasing amounts of the antisense transcript, attesting to the participation of Uchl1-AS in regulating post-transcriptional processes. Characterization of Uchl1-AS revealed that the 5' overlap region, as well as a short interspersed nuclear element (SINE) B2 repeat element harbored within the transcript, are critical for lncRNA function [69]. How the two elements work together to increase polysome loading onto mRNA remains unknown. It is likely that some form of RNA duplex formation occurs at the overlapping region, perhaps inducing changes in the architecture of mRNA to allow for efficient translation. The requirement of a SINEB2 repeat element is intriguing because Xist RNA also consists of repeat regions that play critical roles in chromosome silencing [70]. A search through the mouse cDNA FANTOM3 database for antisense transcripts with similar properties (5' overlap and presence of SINEB2 repeat) identified another lncRNA at the Uxt gene that is also capable of eliciting increases in protein levels post-transcriptionally [69]. In addition, a survey of human lincRNAs also revealed an enrichment for transposable elements [71]. More in-depth analyses of RNA structure for a larger number of lncRNAs are needed to explore possible links between the presence of particular genomic elements (e.g. repeat sequences) and lncRNA function (discussed below).</p><!><p>While sequencing technologies have allowed for rapid discovery of lncRNAs, elucidating the biological roles of lncRNA in vivo remain challenging. The problem arises partly because novel lncRNAs seem to be governed by a set of rules distinct from that used by proteins. Numerous strategies have been employed by different groups to address the challenges, such that the set of functionally annotated lncRNAs is expanding very rapidly (well beyond the examples this review has been able to highlight) as evidenced by the establishment of several lncRNA databases to provide comprehensive documentation of sequence information, evolutionary conservation, expression profiles and functional evidence [11, 72]. Many groups turned to clues including the level and specificity of expression, or the chromatin state around a locus of transcription to focus on lncRNAs with potentially higher chances of biological relevance from the large pool of transcripts [73–75]. It is expected that biologically significant lncRNAs will be tightly regulated and be highly expressed only in appropriate contexts. Alternatively, the strategy of identifying lncRNAs which can perturb expression of specific genes makes follow up studies much more manageable, as demonstrated by the process through which the evolutionarily conserved lncRNA NRON was identified [76]. Here, work by Schultz and colleagues utilized RNAi strategies to screen for highly conserved noncoding transcripts which regulate the expression of NFAT, a transcription factor implicated in initiation of T-cell receptor mediated immune responses. NRON is believed to modulate the nuclear trafficking of NFAT by associating with well known nuclear import proteins, and depletion of NRON correlates with increased activity of NFAT [76]. Other strategies, such as narrowing down the search to specific lineage differentiation pathways, cell types, or to those regulated by proteins of interest simplify the search for biological function. These methodologies have enabled identification of lncRNAs with roles in adipocyte differentiation [77] and in cancer [37, 78, 79]</p><p>An alternative for lncRNA identification involves enriching for RNA in association with chromatin-bound proteins (e.g. chromatin modifiers) known to be important in gene regulation. Native RIP-seq, for example, has been used to define a transcriptome associated with PRC2, either directly or indirectly [80] (Figure 2). In addition, methodologies such as CLIP (CrossLinking and ImmunoPrecipitation [81, 82]) or its variant PAR-CLIP (PhotoActivable-Ribonucleoside-enhanced CrossLinking and ImmunoPrecipitation [83]) allow for identification of RNA directly bound to protein and potentially providing a short "footprint" suitable for identifying RNA motifs involved in protein binding (Figure 2). CLIP was used to identify an intronic transcript of the H3K4 methyltransferase SMYD3, with antiproliferative effects on cells when overexpressed [84]. Deep sequencing of CLIP products, however, remains a challenging procedure. RNA yields after immunoprecipitation and gel extraction are often very low, making the procedure especially sensitive to RNA contamination at each step of experimental manipulation. Further refinement of the procedure would be critical for establishing CLIP as one of the standard tools in studies of lncRNA-protein interactions.</p><p>Another novel approach for dissecting lncRNA function relies on mapping interaction sites between RNA and genomic DNA. This is especially helpful for understanding the mechanistic details for trans-acting lncRNAs for which target sites are located away from their site of synthesis. Two methods, CHART (capture hybridization analysis of RNA targets) and ChIRP (chromatin isolation by RNA purification) were recently employed to interrogate genome-wide localization sites of lncRNAs such as HOTAIR, the human telomerase RNA TERC, and dosage compensation factors such as Xist and the roX RNAs [48, 49]. These techniques should be broadly applicable to other lncRNAs.</p><p>In the RNA world, secondary and tertiary structures are crucial for specificity of interaction with proteins or other nucleic acids [85]. Such RNA structures therefore are expected to regulate the activity and function of lncRNAs, though our ability to map secondary and tertiary structures is presently rudimentary. Analysis of RNA structure of RepA, an internal transcript from the repeat A region of the Xist locus, demonstrated that a 28 nt stem-loop found within one of its repeat sequences bound directly to Ezh2 in vitro, suggesting that the stem-loop is likely to be important for its protein interaction [42, 70]. Repeat A, encompassing approximately 8 repeat units, has itself been proposed to demonstrate higher order structure by forming two larger stem-loops each consisting of 4 repeat units [86]. Importantly, full-length repeat A demonstrated higher affinity for the PRC2 component SUZ12 than truncated versions, suggesting that these larger stemloops are necessary for efficient recruitment of PRC2 to Xist RNA [86]. Interestingly, some promoter-associated transcripts of PRC2-repressed genes also demonstrate similar stem-loop structures [22]. In addition, Uchl1-AS and Uxt-AS, both of which regulate expression of their corresponding genes post-transcriptionally, harbor the SINEB2 repeat element, suggesting that higher order structures of repeat elements could contribute to lncRNA function. These studies highlight the need for more comprehensive mapping of RNA structures to identify lncRNA structural domains that may be relevant for biochemical interactions in the cell. High throughput techniques for lncRNA structural mapping [85] such as fragmentation sequencing (FRAG-seq) [87], SHAPE-seq (which utilizes selective 2' -hydroxyl acylation chemistry analyzed by primer extension) [88] and parallel analysis of RNA structure (PARS) [89] would be invaluable in this aspect.</p><!><p>lncRNAs are implicated in a variety of diseases, especially those involving genomic imprinting and cancer, underscoring their importance in maintaining cellular homeostasis. Transcripts associated with cancer including ANRIL (transcribed from the Ink4b (p15) – ARF (p14) – Ink4a (p16) tumor suppressor loci [39, 90, 91]), PCAT-1 (a pro-proliferation transcript upregulated in prostate cancer samples [79]), HOTAIR of the HOXC locus [92]) and MALAT1 (a prognostic marker of several cancer types [93]). Significantly, Xist RNA has now been directly implicated in human cancers. Since Xist maintains dosage compensation for ~1000 genes on the X chromosome, several of which are putative oncogenes (reviewed in [94]), it is possible that misregulation of Xist contributes to cancer phenotypes through aberrations in expression of X-linked oncogenes.</p><p>In line with this idea, cytogenetic studies of human breast, ovarian and cervical cancer samples since the 1950s have noted a high frequency of Barr body (the inactivated X chromosome) loss, particularly in more aggressive breast tumors (reviewed in [95]). Barr body loss was often concomitant with the acquisition of supernumerary active Xs (Xa) and down-regulation of XIST RNA. RNA expression profiling of sporadic basal-like cancers (BLC) which have lost Xi and XIST RNA also demonstrate overexpression of some X-linked genes [96]. These observations suggested that loss of XIST RNA could drive disease progression in these cancer types, possibly through reactivation of Xi.</p><p>Direct causality has now emerged from an in vivo study where deletion of Xist in the hematopoietic lineage resulted in the development of leukemia in mice with full penetrance [97]. Gene expression profiling over the course of disease progression revealed significant upregulation of X-linked genes, suggesting the possibility of X reactivation following Xist loss. This sensitivity of hematopoietic cells to Xist misregulation corroborates with previous work in which overexpression of Xist in mice results in lethal anemia due to defective hematopoiesis [98], and overexpression in a lymphoma cell model suppresses tumorigenicity [99]. Further studies are needed to determine whether the tumor suppressive properties of Xist extend to breast and ovarian tissues as well. In any case, these studies support the notion that XIST RNA could be useful as a therapeutic target in female cancers or establish XIST RNA as a diagnostic parameter for stages of tumor progression (Box 1).</p><!><p>While lncRNA targeting for therapeutic purposes is still in early stages of development, RNA-based drugs have been in development for more than two decades. A first such drug, fomivirsen, emerged in 1998 for treatment of cytomegalovirus-mediated eye infections [100]. RNA therapeutics often rely on the use of antisense oligonucleotides (ASO, which hybridize to complementary sequences and induce RNaseH-mediated RNA degradation) or siRNAs (which act through the RNAi machinery) to degrade mRNAs or microRNAs of interest, and feasibility for such methods has already been demonstrated for several drug targets currently in clinical trials. One ASO (Kynamro) was recently FDA-approved for hypercholesterolemia [101]. Many others are still in pre-clinical phase. For example, a proof-of-concept study showed that BDNF-AS, a ~1kb lncRNA transcribed antisense to the BDNF gene (brain-derived neurotrophic factor) could be a useful ASO drug target. Using intracerebroventricular delivery of chemically modified oligonucleotides, the group was able to knockdown BDNF-AS in the mouse brain to relieve BDNF repression and allow for increased neuronal proliferation [102]. This result speaks to the possibility of using antagonizing antisense transcripts for lncRNA depletion. However, given the length of some lncRNAs, it is possible that extensive secondary structures will restrict accessibility of antisense oligos to crucial parts of the transcript. This once again emphasizes the importance of structural mapping for lncRNAs.</p><p>Delivery also remains a major obstacle for some RNA-based therapeutics. Recent technical developments such as lipid nanoparticles may facilitate delivery of siRNAs [103], though toxicity has been a concern. Naked delivery of ASOs may also be effective for some tissues and organs, such as liver and kidney, though penetration into other tissues is currently problematic.</p><p>An alternative to the oligotherapeutics strategy could involve the use of small molecules to disrupt interactions of lncRNAs with proteins or DNA. In this case, structural motifs of lncRNAs which allow them to recruit chromatin modifiers, or to form triple helixes with DNA, could be targeted by small molecules, thereby rendering the lncRNA ineffective in perturbing gene expression. Chemical library screening, as have been done for microRNAs [104], might be useful in the identification of small molecule inhibitors of lncRNAs. This type of approach is currently in nascent stages.</p><!><p>The detection of pervasive transcription and the ensuing discovery of noncoding transcripts have redefined our understanding of how non-genic regions of the genome are involved in the regulation of gene expression profiles within a cell. While the field is still in the early days of assigning biological function to the thousands of transcripts detected, it is clear that lncRNAs add an important layer to the repertoire of regulatory mechanisms used by mammalian cells to modulate gene expression. As more lncRNAs are identified, it will become important to analyze lncRNA sequences and secondary structures to establish structure-function relationships that define mechanisms of lncRNA function. This would move the field forward by both speeding up the identification and discovery of more biologically relevant transcripts, as well as allow for a better understanding of how lncRNA perturbation can be utilized for locus-specific manipulation of gene expression for therapeutic purposes.</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><!><p>Characterization of lncRNA function has revealed the ability of these transcripts to regulate gene expression through chromatin remodeling, control of transcription initiation and post-transcriptional processing. (a) lncRNAs such as Xist, Kcnq1ot1, Airn and HOTAIR have been found to interact with chromatin remodeling proteins such as polycomb repressive complex 2 and G9a (represented in green) to mediate deposition of repressive chromatin marks. (b) lncRNAs can directly regulate messenger RNA synthesis at genomic loci by interacting with transcription factors (see text) or components of the basal transcriptional machinery. In the case of DHFR regulation, an upstream lncRNA transcribed from the minor promoter has been shown to bind both the major DHFR promoter as well as TFIIB, leading to displacement of TFIIB from the major promoter. (c) lncRNAs can regulate co-transcriptional processes such as RNA splicing (see text) and translation. The Uchl1-AS RNA is transcribed in times of cellular stress and acts to speed up translation of Uchl1 mRNA by enhancing polysome loading onto the mRNA in the cytoplasm. The mechanisms for this activity is not well understood, although it has been proposed that the SINEB2 repeat element could play a crucial role.</p><!><p>In both chromatin crosslinking and immunoprecipitation (CLIP) and UV RNA immunoprecipitation (UV-RIP), cells are irradiated with 254nm UV light, which crosslinks proteins and nucleic acids that are in close proximity. For PAR-CLIP, cells are incubated with 4-thiouracil and crosslinked at 360nm UV. In CLIP, cellular lysate is treated with RNAse after crosslinking to fragment RNA before performing immunoprecipitation. A 3' adaptor can be added to enriched RNA fragments (not necessary unless for downstream sequencing library preparation). RNA is end labeled with [γ32] p, following which the reaction is subjected to SDS-PAGE. Following transfer to nitrocellulose membranes, the appropriate region of the membrane is excised (dependent on size of protein probed) and treated with proteinase K to release RNA fragments. Denaturing conditions of SDS-PAGE ensures enrichment for RNA fragments in direct interaction with protein of interest. In UV-RIP, cell lysate is subjected to immunoprecipitation to enrich for RNA bound either directly or indirectly to the protein of interest. A high stringency salt wash further enriches for RNA bound both directly and indirectly to the protein of interest by removing uncrosslinked fragments. Proteinase K treatment releases bound RNA. The RNA fraction obtained from both CLIP and UV-RIP can be analyzed by RT-qPCR, array hybridization as well as high throughput sequencing.</p><!><p>Long noncoding RNAs (lncRNAs) provide additional layers of gene regulatory control</p><p>Emerging technologies will allow biological function of lncRNAs to be elucidated more efficiently</p><p>lncRNAs can be useful diagnostic markers or therapeutic tools for diseases</p>

PubMed Author Manuscript

Electronic, Magnetic, and Redox Properties and O2 Reactivity of Iron(II) and Nickel(II) o-Semiquinonate Complexes of a Tris(thioether) Ligand: Uncovering the Intradiol Cleaving Reactivity of an Iron(II) o-Semiquinonate Complex

The iron(II) semiquinonate character within the iron(III) catecholate species has been proposed by numerous studies to account for the O2 reactivity of intradiol catechol dioxygenases, but a well-characterized iron(II) semiquinonate species that exhibits intradiol cleaving reactivity has not yet been reported. In this study, a detailed electronic structure description of the first iron(II) o-semiquinonate complex, [PhTttBu]Fe(phenSQ) [PhTttBu = phenyltris(tert-butylthiomethyl)borate; phenSQ = 9,10-phenanthrenesemiquinonate; Wang et al. Chem. Commun. 2014, 50, 5871\xe2\x80\x935873], was generated through a combination of electronic and M\xc3\xb6ssbauer spectroscopies, SQUID magnetometry, and density functional theory (DFT) calculations. [PhTttBu]Fe(phenSQ) reacts with O2 to generate an intradiol cleavage product, diphenic anhydride, in 16% yield. To assess the dependence of the intradiol reactivity on the identity of the metal ion, the nickel analogue, [PhTttBu]Ni(phenSQ), and its derivative, [PhTttBu]Ni(3,5-DBSQ) (3,5-DBSQ = 3,5-di-tert-butyl-1,2-semiquinonate), were prepared and characterized by X-ray crystallography, mass spectrometry, 1H NMR and electronic spectroscopies, and SQUID magnetometry. DFT calculations, evaluated on the basis of the experimental data, support the electronic structure descriptions of [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) as high-spin nickel(II) complexes with antiferromagnetically coupled semiquinonate ligands. Unlike its iron counterpart, [PhTttBu]Ni(phenSQ) decomposes slowly in an O2 atmosphere to generate 14% phenanthrenequinone with a negligible amount of diphenic anhydride. [PhTttBu]Ni(3,5-DBSQ) does not react with O2. This dramatic effect of the metal-ion identity supports the hypothesis that a metal(III) alkylperoxo species serves as an intermediate in the intradiol cleaving reactions. The redox properties of all three complexes were probed using cyclic voltammetry and differential pulse voltammetry, which indicate an inner-sphere electron-transfer mechanism for the formation of phenanthrenequinone. The lack of O2 reactivity of [PhTttBu]Ni(3,5-DBSQ) can be rationalized by the high redox potential of the metal-ligated 3,5-DBSQ/3,5-DBQ couple.

electronic,_magnetic,_and_redox_properties_and_o2_reactivity_of_iron(ii)_and_nickel(ii)_o-semiquinon

INTRODUCTION<!>General Information.<!>[PhTttBu]NiI.<!>[PhTttBu]Ni(phenSQ).<!>[PhTttBu]Ni(3,5-DBSQ).<!>Examining the Reaction of [PhTttBu]Fe(PhenSQ) with O2 by Liquid-Injection Field Desorption Ionization Mass Spectrometry (LIFDI-MS).<!>Examining the Reaction of [PhTttBu]Fe(PhenSQ) with O2 by 1H NMR Spectroscopy and Gas Chromatography (GC).<!>Examining the Reaction of [PhTttBu]Ni(PhenSQ) with O2 by Electronic Absorption Spectroscopy, 1H NMR Spectroscopy, and GC.<!>Examining the Reaction of [PhTttBu]Ni(3,5-DBSQ) with O2 by Electronic Absorption Spectroscopy.<!>Physical Methods.<!>Computational Methods.<!>[PhTttBu]Fe(phenSQ).<!>[PhTttBu]Ni(phenSQ).<!>[PhTtBu]Ni(3,5-DBSQ).<!>Electronic and Magnetic Properties of [PhTttBu]\xe2\x88\x92 Fe(phenSQ).<!>O2 Reactivity of [PhTttBu]Fe(phenSQ).<!>Synthesis and X-ray Structures of [PhTttBu]Ni-(phenSQ) and [PhTttBu]Ni(3,5-DBSQ).<!>Electronic and Magnetic Properties of [PhTttBu]\xe2\x88\x92 Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ).<!>O2 Reactivity of [PhTttBu]Ni(phenSQ) and [PhTttBu]\xe2\x88\x92 Ni(3,5-DBSQ).<!>Redox Properties of [PhTttBu]Fe(phenSQ), [PhTttBu]Ni(phenSQ), and [PhTttBu]Ni(3,5-DBSQ).<!>CONCLUSIONS

<p>Transition-metal dioxolene complexes continue to attract much interest, in part because of their applications as redox-active ligands and their relevance to catechol dioxygenase enzymes.1–3 As a well-established redox-active ligand, dioxolene can exist in three different oxidation states: neutral (quinone), monoanionic (semiquinonate), and dianionic (catecholate) (Scheme 1). All three redox forms have been explored in transition-metal dioxolene chemistry, which led to various intriguing observations such as valence tautomerization,4–7 spin-crossover,8–14 biomimetic properties,15–18 and catalytic activities.19–24</p><p>Our interest in transition-metal dioxolene complexes stems from their biological significance. An iron(III) catecholate species was identified in the active sites of intradiol catechol dioxygenases, enzymes that catalyze the oxidative cleavage of the C1–C2 bond of catechols.16'25 The form of the enzyme that react with O2 contains a five-coordinate iron(III) site. One of the proposed mechanisms for how the iron(III) catecholate species activates O2 invokes the mixing of an iron(II) semiquinonate state into the iron(III) catecholate ground state. According to this mechanism (Scheme 2), iron(II) o-semiquinonate is the "active form" that reacts with O2 to generate an iron(III) alkylperoxo intermediate26 with a monodentate dioxolene moiety. The iron(III) alkylperoxo intermediate then undergoes a Criegee rearrangement, followed by ring opening to subsequently yield the intradiol cleavage product. This proposal has been supported by theoretical calculations27–30 and the observed correlations between the spectroscopic features and intradiol reactivity of iron(III) catecholate model compounds.31–35 Nevertheless, to the best of our knowledge, direct observation of the intradiol cleaving reactivity of a well-defined iron(II) semiquinonate species had not previously been reported.</p><p>In a recent communication, some of us reported the preparation and characterization of the first mononuclear iron(II) o-semiquinonate complex, namely, [PhTttBu]Fe-(phenSQ)36 (Figure 1). On the basis of the average C-O distance of the dioxolene ligand, 1.285(2) Å, the redox state of the dioxolene ligand was assigned as a semiquinonate. Furthermore, the existence of a five-coordinate high-spin iron(II) ion was inferred on the basis of the ligand-field (LF) transition at 935 nm (539 M−1 cm−1; Figure 1). Given the scarcity of mononuclear iron(II) o-semiquinonate species and their proposed involvement in the mechanism of O2 activation by intradiol catechol dioxygenases and their model compounds, it is highly desirable to gain insight into the electronic structure of [PhTttBu]Fe(phensQ) and to examine its reactivity with O2.</p><p>Herein, we report spectroscopic and magnetic studies in conjunction with density functional theory (DFT) calculations to further interrogate the electronic structure of [PhTttBu]Fe(phenSQ). In addition, studies of the O2 reactivity of [PhTttBu]Fe(phenSQ) led to the discovery of the first synthetic iron(II) o-semiquinonate species exhibiting intradiol cleaving reactivity. For comparison to [PhTttBu]Fe(phenSQ), two nickel o-semiquinonate analogues were prepared, namely, [PhTttBu]" Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ). The solid-state structures, electronic and magnetic properties, and redox behaviors of these new nickel complexes are presented and discussed. Finally, comparative O2 reactivity studies of the iron(II) and nickel(II) o-semiquinonate complexes highlight the fundamental effect of the metal ion on the intradiol cleaving reactivity.</p><!><p>All air- and moisture-sensitive reactions were performed under N2 using standard Schlenk techniques or carried out under an Ar or N2 atmosphere in a Vacuum Atmospheres glovebox equipped with a gas purification system. Unless otherwise noted, all reagents were purchased from commercial sources and used without further purification. Anhydrous NiI2 was purchased from Strem Chemicals. Solvents were of reagent-grade or better and were dried by passage through activated alumina and then stored over 4 Å molecular sieves prior to use. Deuterated solvents were purchased from Cambridge Isotope Laboratories and stored over 4 Å molecular sieves. [PhTttBu]Tl,37 Ti(phenSQ),38 and Tl(3,5-DBSQ)38 were prepared following published procedures.</p><!><p>Anhydrous NiI2 (1.250 g, 4.0 mmol) was ground into a fine powder and suspended in 100 mL of tetrahydrofuran (THF). After stirring for 6 h, [PhTttBu]Tl (1.204 g, 2.0 mmol) was added in small portions. After stirring for 16 h, the reaction mixture was filtered through Celite, removing TlI, and the solvent was removed in vacuo. The residue was extracted with pentane (2 × 40 mL), and the resulting red solution was filtered through Celite. Removal of the solvent under reduced pressure afforded a brown solid (919 mg, 69%). 1H NMR (C6D6): δ 15.2 (27H, br, C(CH3)3S), 11.1 (2H, s, m-(C6H5)B), 8.5 (2H, s, o-(C6H5)B), 8.3 (1H, s, p-(C6H5)B). UV–vis [toluene; λmax, nm (ε, M−1 cm−1)]: 311 (1518), 375 (sh), 413 (sh), 452 (6036), 567 (972), 841 (427), 903 (432). Anal. Calcd for C21H38BINiS3: C, 43.26; H, 6.57. Found: C, 43.50; H, 6.80.</p><!><p>A suspension of Tl(phenSQ) (83 mg, 0.20 mmol) in 30 mL of THF was added dropwise over 20–30 min to a stirring solution of [PhTttBu]NiI (117 mg, 0.20 mmol) in 10 mL of THF. A yellow precipitate gradually formed as the color of the solution turned from orange-red to purple. The mixture was stirred for 6 h and then filtered through a pad of Celite. THF was removed in vacuo, and the residue was washed with pentane (2 × 4 mL) and then extracted with pentane/diethyl ether (1:1, v/v). Filtration of the extract through a Celite pad, followed by solvent removal in vacuo, yielded a dark-purple powder (106 mg, 80%). Slow evaporation of a concentrated pentane/diethyl ether (2:1, v/v) solution of the product yielded crystals suitable for X-ray diffraction (XRD) analysis. 1H NMR (C6D6): δ 52.6 (br), 9.4 (br, C(CH3)3S), −4.1 (br). UV–vis [toluene; λmax, nm (ε, M−1 cm−1)]: 316 (9131), 398 (12877), 536 (11375), 586 (10011), 846 (710), 966 (sh). LIFDI-MS. Calcd for C35H46BNiO2S3 [(M)+, 100%]: m/z 663.2113. Found: m/z 663.2093. μeff (C6D6): 2.18(3) μB. Anal. Calcd for C35H46BNiO2S3: C, 63.27; H, 6.98. Found: C, 63.31; H, 7.18.</p><!><p>A solution of Tl(3,5-DBSQ) (178 mg, 0.42 mmol) in 50 mL of THF was added dropwise over 30 min to a stirring solution of [PhTttBu]NiI (233 mg, 0.40 mmol) in 20 mL of diethyl ether. A yellow precipitate gradually formed as the color of the solution turned from orange-red to red-purple. The mixture was stirred for 6 h and then filtered through a pad of Celite. The solvent was removed in vacuo, and the residue was extracted with pentane. Filtration of the extract through Celite followed by solvent removal in vacuo yielded a dark-brown powder (230 mg, 85%). Slow evaporation of a concentrated pentane solution of the product yielded crystals suitable for XRD analysis. 1H NMR (C6D6): δ 10.3 (sh), 9.2 (br, C(CH3)3S), −7.1 (br). UV–vis [toluene; λmax, nm (ε, M−1 cm−1)]: 300 (7328), 366 (sh), 522 (5811), 566 (sh), 842 (823), 966 (sh). LIFDI-MS. Calcd for C35H58BNiO2S3 [(M)+, 100%]: m/z 675.3052. Found: m/z 675.3093. μeff (C6D6): 2.25 μB.</p><!><p>In an Ar-filled glovebox, [PhTttBu]Fe(PhenSQ) (3.0 mg, 0.0045 mmol) was dissolved in 10 mL of toluene, and 4 mL of the solution was transferred to a 20 mL scintillation vial charged with a magnetic stir bar. The vial was then sealed with a rubber septum and removed from the glovebox. The solution was degassed by two freeze–pump–thaw cycles, and the vial was then immersed in a dry ice/acetone bath (−78 °C). With stirring, the solution was exposed to 1 atm of dry O2 at −78 °C. After 5 min, the solution was warmed to room temperature and the LIFDI-MS spectrum was taken.</p><!><p>In an Ar-filled glovebox, [PhTttBu]Fe(PhenSQ) (30 mg, 0.045 mmol) was dissolved in 10 mL of toluene in a 100 mL Schlenk flask charged with a magnetic stir bar. The solution was removed from the glovebox and degassed by two freeze–pump–thaw cycles. The flask was then immersed in a dry ice/acetone bath (−78 °C), and 1 atm of dry O2 was added through a needle. The solution was stirred at −78 °C for 30 min, then warmed to room temperature, and stirred for an additional 30 min (the pressure of O2 was released via an outlet needle as the sample warmed). The solution was concentrated to 2–3 mL in vacuo, and 0.5 M HCl (2 mL, 0.5 mmol) was added to dissociate the metal from the organic products. After the mixture was stirred for 5 min, 15 mL of H2O was added, and the sample was extracted with ethyl acetate (3 × 20 mL). The organic extracts were combined and dried over anhydrous Na2SO4. After removal of ethyl acetate in vacuo, the products were obtained and analyzed by 1H NMR spectroscopy (CDCl3) and GC.</p><!><p>[PhTttBu]Ni(PhenSQ) (2.5 mg, 0.0038 mmol) was dissolved in 100 mL of toluene in an Ar-filled glovebox, and 3 mL of the solution was transferred to a cryostat UV–vis cuvette charged with a magnetic stir bar. The cuvette was sealed with a rubber septum and removed from the glovebox. With slow stirring, 1 atm of dry O2 was bubbled gently into the solution through a needle for 5 min at room temperature. Electronic absorption spectra were collected every 15 min until no further spectral changes were observed (~27 h).</p><p>For the 1H NMR spectroscopy and GC measurements, [PhTttBu]− Ni(phenSQ) (30 mg, 0.045 mmol) was dissolved in 10 mL of toluene in a 100 mL Schlenk flask charged with a magnetic stir bar. The solution was removed from the glovebox and 1 atm of dry O2 was bubbled into the solution through a needle for 5 min. Then, a balloon filled with dry O2 was connected to the flask through a needle and a syringe. The solution was stirred for 36 h and subsequently concentrated to 2–3 mL in vacuo, and then 0.5 M HCl (2 mL, 0.5 mmol) was added. After the mixture was stirred for 5 min, 15 mL of H2O was added and the sample was extracted with ethyl acetate (3 × 20 mL). The organic extracts were combined and dried over anhydrous Na2SO4. After removal of ethyl acetate in vacuo, the products were obtained and analyzed by 1H NMR spectroscopy (CDCl3) and GC.</p><!><p>[PhTttBu]Ni(3,5-DBSQ) (10.5 mg, 0.0155 mmol) was dissolved in 100 mL of toluene in an Ar-filled glovebox. A 3 mL aliquot of the solution was transferred to a cryostat UV–vis cuvette charged with a magnetic stir bar. The cuvette was sealed with a rubber septum and removed from the glovebox. With gentle stirring, 1 atm of dry O2 was bubbled gently into the solution through a needle for 5 min at room temperature. Electronic absorption spectra were collected every 15 min over a 12 h period. No spectral changes were observed.</p><!><p>NMR spectra were recorded on a Bruker AVIII 400 spectrometer. Chemical shifts (δ) were referenced to residual protons in the deuterated solvents. Electronic absorption spectra were recorded on a Varian Cary 50 UV–vis spectrophotometer using screw-top quartz cuvettes with a 1 cm path length. Solution-state magnetic moments were determined using the Evans method.39–41 LIFDI-MS42,43 was performed on a Waters GCT Premier mass spectrometer. The static magnetic properties of [PhTttBu]Fe-(phenSQ), [PhTttBu]Ni(phenSQ), and [PhTttBu]Ni(3,5-DBSQ) were measured on samples of ground crystals using a Quantum Design MPMS-XL SQUID magnetometer operating over the temperature range 1.8–400 K at a 1000 Oe direct-current field. The data were corrected for diamagnetic contributions using Pascal constants.</p><p>Low-field (0.04 T), variable-temperature (5–200 K) Mössbauer spectra were recorded on a closed-cycle refrigerator spectrometer, model CCR4K, equipped with a 0.04 T permanent magnet, maintaining temperatures between 5 and 300 K. The samples consisted of solid powders (or crystalline material) suspended in Nujol, placed in Delrin 1.00 mL cups, and frozen in liquid nitrogen. The isomer shifts are quoted at 5 K with respect to the iron metal spectra recorded at 298 K. Mössbauer spectra were analyzed using the software WMOSS (Thomas Kent, See Co., Edina, MN).</p><p>Electrochemical experiments were performed using a CHI-620D potentiostat/galvanostat under an N2 atmosphere. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed using a standard three-electrode configuration. The working electrode was a polished glassy carbon electrode (GCE; 3.0 mm diameter, CH Instruments), the auxiliary electrode was a platinum wire, and a Ag+-coated silver wire was used as a pseudoreference electrode. Decamethylferrocene (Fc*, 1 mM) was used as the internal standard, and all potentials were referenced to Fc+/Fc through the relationship Fc+/Fc = 427 mV + Fc*+/Fc*.44</p><p>Single-crystal XRD data were obtained by mounting crystals using viscous oil onto plastic mesh and cooling them to the data collection temperature. Data were collected on a Bruker-AXS APEX II CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). Unit cell parameters were obtained from 36 data frames, 0.3° ω, from three different sections of the Ewald sphere. The data sets were treated with absorption corrections based on redundant multiscan data. The systematic absences and unit cell parameters were consistent with Cc and C2/c for [PhTttBu]Ni(3,5-DBSQ) and, uniquely, with Pbca for [PhTttBu]NiI and with P21/n for [PhTttBu]− Ni(PhenSQ). The solution in the centrosymmetric space group option, C2/c, for [PhTttBu]Ni(3,5-DBSQ) yielded chemically reasonable and computationally stable results of refinement. The structures were solved using direct methods and refined with full-matrix least-squares procedures on F2. The iodine atom in [PhTttBu]NiI was found to be disordered in two positions with a refined site occupancy ratio of 75:25. Slight disorder was observed in a tert-butyl group in [PhTttBu]Ni(3,5-DBSQ) but could not be modeled satisfactorily. The disordered groups were refined with three-dimensional Uij rigid-group restraints. All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were treated as idealized contributions. Atomic scattering factors are contained in the SHELXL 2013–2014 program libraries.45 The CIFs have been deposited with the Cambridge Crystallographic Database as CCDC 1541366–1541368.</p><!><p>Computational models were generated for each [PhTttBu]MIISQ complex and analyzed against the available experimental data. Unless otherwise stated, the initial atomic coordinates for each [PhTttBu]MIISQ model were imported from crystallographic data. DFT geometry optimizations and singlepoint calculations were performed with the ORCA, version 2.9.0 or 2.9.1, software package developed by Dr. Frank Neese.46 Both high-spin and low-spin MII models of each complex with a semiquinone radical ligand were considered, and the spin multiplicity of the complex was specified, rather than metal and ligand oxidation states. Metal atoms and the immediately ligated oxygen and sulfur atoms were described with Ahlrich's polarized triple-ζ-valence47 basis set, while the remaining atoms were modeled with the polarized split-valence basis set.48 For each spin state, computations were carried out using the spin-unrestricted formalism and either Becke's three-parameter hybrid functional for exchange along with the Lee–Yang–Parr functional for correlation (B3LYP)49,50 or Becke's functional for exchange along with Perdew's functional for correlation (BP86).51–53 Tight self-consistent-field convergence criteria were specified with an integration grid of 302 Lebedev points. For further analyses of the computed electronic structures, single-point DFT and time-dependent DFT (TD-DFT) calculations were performed on the optimized geometries. The TD-DFT results were used to simulate absorption spectra, whereby each of the 40 computed transitions was modeled as a Gaussian band with a full width at half-maximum of 2500 cm−1.</p><!><p>Starting coordinates for the initial models of [PhTttBu]Fe(phenSQ) were taken from X-ray crystallographic data. Because the magnetic susceptibility [μeff = 4.65(2) μB] and electronic absorption data indicate a high-spin metal center, the spin state of this complex was set to S = 3/2 (high-spin iron(II) coupled antiferromagnetically to an SQ− ligand radical, models Fe(phenSQ)1 [B3LYP] and Fe(phenSQ)2 [BP86]). Because model Fe(phenSQ)1 converged to an unreasonable electronic structure, a third S = 3/2 model was generated using the optimized geometry and molecular orbital (MO) descriptions from Fe(phenSQ)2 as the starting point for a geometry optimization with the B3LYP functional [model Fe(phenSQ)3]. Mössbauer parameters were derived for Fe(phenSQ)3 using the same functionals and basis sets as described above. The DFT calibration for the isomer shift (mm/s) relative to α-iron at 295 K (eq 1) was performed using experimental data for a test set of molecules, as was previously described.54 (1)δ=α(ρo−C)+β=−0.298(ρo−11580)+1.118 The computed charge density at the iron nucleus, ρ(0), for Fe(phenSQ)3 from this calculation was 11581.0971, which yields δ = 0.791 mm/s using eq 1. The same DFT calculation yielded a quadrupole splitting of ΔEQ = 2.486 mm/s.</p><!><p>The starting coordinates for the initial model of [PhTttBu]Ni(phenSQ) were taken from X-ray crystallographic data. Because the magnetic susceptibility measurement [μeff = 2.18(3) μB] indicates an S = ½ system, the spin state of the complex was modeled as S = ½ (high-spin nickel(II) coupled antiferromagnetically to an SQ− ligand radical or low-spin nickel(II) coordinated by SQ−, models Ni(phenSQ)1 [B3LYP] and Ni(phenSQ)2 [BP86]). Because model Ni(phenSQ)1 converged to an unreasonable electronic structure, a third S = ½ model was generated using the optimized geometry and MO descriptions from Ni(phenSQ)2 as the starting point for a geometry optimization with the B3LYP functional [model Ni(phenSQ)3].</p><!><p>The starting coordinates for models of [PhTttBu]Ni(3,5-DBSQ) were taken from X-ray crystallographic data. The spin state of this complex was set to S = ½ (high-spin nickel(II) coupled antiferromagnetically to an SQ− ligand radical or low-spin nickel(II) coordinated by SQ−, models Ni(3,5-DBSQ)1 [B3LYP] and Ni(3,5-DBSQ)2 [BP86]). Because model Ni(3,5-DBSQ)1 converged to an unreasonable electronic structure, a third S = ½ model was generated using the optimized geometry and MO descriptions from Ni(3,5-DBSQ)2 as the starting point for a geometry optimization with the B3LYP functional [model Ni(3,5-DBSQ)3].</p><!><p>To further interrogate the electronic structure of [PhTttBu]Fe(phenSQ), solid-state Mössbauer spectra were collected in an applied field of 0.04 T at a range of temperatures. The zero-field spectrum recorded at 100 K is displayed in Figure 2. The spectra at 6–100 K exhibit a single sharp quadrupole doublet, with an isomer shift (δ) of 0.92 mm/s and a quadrupole splitting (ΔEQ) of 2.29 mm/s, which are consistent with high-spin iron(II). Temperatures up to 200 K did not significantly affect the quadrupole splitting. A five-coordinate high-spin ferrous complex with redox-innocent ligands, {[PhTttBu]FeCl}2, which was previously characterized in our laboratories,55 exhibited Mössbauer parameters of δ = 0.96 mm/s and ΔEQ = 3.45 mm/s, similar to those of [PhTttBu]Fe(phenSQ). Recently, Fiedler, Popescu, and coworkers have explored the oxidation of iron(II) catecolate complexes with Mössbauer spectroscopy, showing that metal-based oxidation to iron(III) catecholate leads to a significantly lower isomer shift (0.5 mm/s).56 Thus, the Mössbauer data of [PhTttBu]Fe(phenSQ) strongly support its description as a high-spin iron(II) semiquinonate species.</p><p>To study the magnetic interaction between the high-spin iron(II) and the o-semiquinonate ligand, the temperature-dependent magnetic susceptibility of [PhTttBu]Fe(phenSQ) in the solid state was probed by SQUID magnetometry. As shown in Figure 3, the χT value of 2.58 cm3 K mol−1 at 300 K (μeff = 4.54 μB) comports with the solution magnetic moment at room temperature [μeff = 4.65(2) μB], confirming the same spin state in the solid state and in solution. As the temperature was lowered from 300 to 50 K, the χT value slowly decreased to 2.19 cm3 K mol−1, which is slightly higher than the spin-only value of 1.88 cm3 K mol−1 for an S = 3/2, g = 2.0 system but much lower than what is expected for the uncoupled (i.e., S = 2 and **1/2) system of 3.38 cm3 K mol−1. Thus, [PhTttBu]Fe-(phenSQ) adopts an S = 3/2 ground state derived from antiferromagnetic coupling between the high-spin iron(II) (S =2) and the o-semiquinonate radical (S = ½). The increase in χT as the temperature was raised from 50 to 300 K is likely due to the thermal population of the S = 5/2 excited state. Interestingly, Fiedler's mononuclear five-coordinate iron(II) p-semiquinonate complexes exhibit ferromagnetic coupling between the high-spin iron(II) center and the p-semiquinonateradical to yield an S = 5/2 ground state, as indicated by electron paramagnetic resonance (EPR) and DFT data.57,58 The ferromagnetic coupling observed in the iron(II) p-semi-quinonate complexes (DFT-computed J values of ~65 cm−1, H = −2JS1∙S2) was attributed to the orthogonal orientation of the magnetic orbitals.</p><p>Below 50 K, the χT values dropped sharply, which may be attributed to zero-field splitting (ZFS) and/or intermolecular interactions. The field-dependent magnetization data collected at 1.8 K (Figure S7) slowly increased at lower fields, which is indicative of intermolecular antiferromagnetic interactions. At fields above 5 T, magnetization approached saturation at 2.1 μB, well below the expected value, which is also indicative of ZFS and/or intermolecular interactions. The susceptibility data were fitted to the Hamiltonian H = −2J(S1∙S2) + DSz2 + E(Sx2 − Sy2) + gβB∙S using PHI software.59 The χT values in the high-temperature regime (300–50 K) were used to calculate a coupling constant of −127 cm−1 with g = 2.22. Fits of the low-temperature range were used to estimate the ZFS parameters of the iron(II) ion (D and E) and intermolecular interactions (zJ). Best fits were obtained for D = +14.5 cm−1, E = 0.001 cm−1, and zJ = −0.23 cm−1. No satisfactory fits were obtained without including intermolecular interactions. The field-dependent magnetization curves at different temperatures (1.8–4.5 K) are nonsuperimposable, indicating the presence of ZFS (Figure S8).</p><p>DFT calculations were performed using the broken-symmetry approach to provide a detailed description of the electronic structure of [PhTttBu]Fe(phenSQ). Starting from the molecular structure of [PhTttBu]Fe(phenSQ) determined by XRD, geometry optimizations for the S = 3/2 spin state were performed using both the BP86 and B3LYP functionals. The metric parameters and electronic structure descriptions obtained from the experimental data and DFT calculations are provided in Table 1. While model Fe(phenSQ)1 fails to predict the correct spin state of iron, the geometries and ground-state electronic structures of models Fe(phenSQ)2 and Fe(phenSQ)3 are consistent with the experimental data. However, because the TD-DFT-calculated electronic absorption spectrum for Fe(phenSQ)2 agrees poorly with the experimental data (Figure S11), only the results obtained for model Fe(phenSQ)3 will be discussed further.</p><p>The DFT-computed Mössbauer parameters for Fe-(phenSQ)3, δ = 0.79 mm/s and ΔEq = 2.49 mm/s, are in good agreement with the experimental isomer shift δ = 0.92 mm/s and quadrupole splitting ΔEQ = 2.29 mm/s (Table 2). Likewise, the calculated coupling constant, J = −79.7 cm−1, agrees reasonably well with the experimental value, J = −127 cm−1 (Table 2).</p><p>The TD-DFT-calculated electronic absorption spectrum for model Fe(phenSQ)3 is in good agreement with the experimental spectrum (Figure 4). The transitions associated with the major features in the computed spectrum were assigned on the basis of electron density difference maps (EDDMs; Figures 4 and S16). The transitions responsible for the two intense features at 384 nm (16736 M−1 cm−1) and 412 nm (14784 M−1 cm−1) have primarily phen SQ-to-FeII charge-transfer character and both phenSQ-to-FeII charge-transfer and phenSQ π → π* character, respectively. The transitions associated with the weaker features at 571 nm (3575 M−1 cm−1) and 592 nm (3693 M−1 from FeII-to-phenSQ and [PhTttBu]-to-phenSQ charge-transfer excitations. These features match with the experimental transition at 600 nm (868 M−1 cm−1). Finally, the FeII dxz/dyz to dx2−y2 transition gives rise to the weak feature at 874 nm (4M−1 cm−1) in the calculated spectrum, which corresponds to the broad band at 935 nm (539 M−1 cm−1) in the experimental spectrum.</p><p>The good agreement between the geometric structures and spectroscopic data obtained experimentally for [PhTttBu]Fe-(phenSQ) and predicted computationally for model Fe-(phenSQ)3 warrants a closer inspection of the calculated electronic structure. The qualitative MO diagram for model Fe(phenSQ)3 derived from the DFT results (Figure 5) reveals that the iron(II) ion is high-spin (S = 2), with one electron pair occupying the Fe dyz-based MO and the four unpaired spin-up electrons occupying the remaining Fe d-based MOs. The spin-up electron in the Fe dxz-based MO is coupled to the spin-down electron in the SQ π*-based MO with a modest spatial overlap of S = 0.28, giving rise to the antiferromagnetic coupling between the high-spin iron(II) and the SQ radical observed by SQUID magnetometry.</p><!><p>Numerous iron(III) catecholate complexes modeling the reactivity of intradiol catechol dioxygenases have been reported over the last 30 years.15–17,60–68 While the intradiol cleaving reactivity of the enzymes and the model complexes was often attributed to the iron(II) semiquinonate character within the iron(III) catecholate species, we are unaware of any well-characterized iron(II) semiquinonate species exhibiting intradiol cleaving reactivity. Thus, our well-characterized iron(II) o-semiquinonate complex, [PhTttBu]Fe(phenSQ), afforded a unique opportunity to test the "iron(II) semiquinonate" mechanistic hypothesis by reacting it with O2.</p><p>Dry O2 was introduced to a toluene solution of [PhTttBu]− Fe(phenSQ) at −78 °C, and the reaction mixture was allowed to warm to ambient temperature. The mass spectrum of the products (Figure 6a) was compared to the calculated mass spectra of phenanthrenequinone (Figure 6b) and diphenic anhydride (Figure 6c). The results obtained are consistent with the formation of diphenic anhydride, the intradiol cleavage product, as well as phenanthrenequinone. To further characterize the organic products, the reaction was performed on a preparative scale. The organic products were dissociated from the iron via the addition of 0.5 M HCl followed by extraction into ethyl acetate. Signals of diphenic anhydride were identifiable in the 1H NMR spectrum of the products, although they partially overlapped with the signals of other products (Figure 6d). The yields of diphenic anhydride (16%) and phenanthrenequinone (4%) were determined by GC (Figure S22). The reaction of [PhTttBu]Fe(phenSQ) with O2 is depicted in Scheme 3. Notably, the formation of diphenic anhydride through oxygenation of [PhTttBu]Fe(phenSQ) represents the first example of intradiol cleaving reactivity of a synthetic iron(II) semiquinonate complex. Furthermore, it validates our previous assertion that the observed intermediate(s) of the reaction of [PhTttBu]Fe(phenSQ) with O2 at low temperature36 is relevant to the intradiol reactivity. The reason for the low yield of the intradiol cleavage product is not clear. We propose that boronic esters derived from the phenSQ ligand may be generated because a similar product was identified in the oxygenation reaction of [PhTttBu]Co(3,5-DBSQ).36 Indeed, in addition to diphenic anhydride and phenanthrenequinone, signals consistent with 2-phenylphenanthro[9,10-d][1,3,2]dioxaborole (m/z 296.10) and 2-phenoxyphenanthro[9,10-d][1,3,2]dioxaborole (m/z 312.12) were observed in the LIFDI-MS spectrum collected on the reaction mixture of [PhTttBu]Fe(phenSQ) and O2 without treatment by acid (Figure S24).</p><!><p>Inspired by the findings of the intradiol cleaving reactivity of [PhTttBu]Fe-(phenSQ), as well as the previously reported intradiol reactivity of [PhTttBu]Co(3,5-DBSQ),36 we sought to assess the role of the metal ion in directing O2 reactivity. To this end, we prepared, characterized, and explored the O2 reactivity of two nickel(II) semiquinonate complexes, [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ).</p><p>The strategy employed for the preparation of [PhTttBu]Ni-(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) was similar to that used for the preparation of [PhTttBu]Fe(phenSQ). Metathesis of [PhTttBu]NiI with Tl(phenSQ) yielded [PhTttBu]Ni-(phenSQ) in 80% yield. Metathesis of [PhTttBu]NiI with Tl(3,5-DBSQ) yielded [PhTttBu]Ni(3,5-DBSQ) in 85% yield. Both complexes are five-coordinate, as revealed by their X-ray crystal structures, with the phenSQ and 3,5-DBSQ ligands binding to the nickel center in a bidentate fashion (Figure 7). [PhTttBu]Ni(phenSQ) adopts a square-pyramidal geometry (τ5 = 0.00), while the geometry of [PhTttBu]Ni(3,5-DBSQ) is distorted square-pyramidal (τ5 = 0.34).69 Selected bond distances are listed in Table 3. The average C–O distances for [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) are 1.283(3) and 1.285(2) Å, respectively, characteristic of semqiuinonate ligands (1.27–1.31 Å). The average Ni–O distances for [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) are 2.001(2) and 1.999(1) Å, respectively. These distances are nearly identical with the average Ni–O distance [2.002(1) Å] of a trigonal-bipyramidal high-spin nickel(II) semiquinonate complex reported by Shultz and co-workers,70 indicating that both [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni-(3,5-DBSQ) are high-spin (S = 1) nickel(II) complexes.</p><!><p>The signals in the 1H NMR spectra of both nickel semiquinonate complexes are broad and paramagnetically shifted, reflecting the paramagnetic character of the complexes. The electronic absorption spectrum of [PhTttBu]Ni(phenSQ) shows LF transitions at 846 nm (710 M−1 cm−1) and 966 nm (shoulder). Similarly, [PhTttBu]Ni(3,5-DBSQ) exhibits LF transitions at 842 nm (823 M−1 cm−1) and 966 nm (shoulder), as shown in Figure 8.</p><p>The aforementioned LF transitions are present only when the NiII ion is high-spin (S = 1). Indeed, LF transitions of previously reported five-coordinate high-spin nickel(II) complexes of tris(thioether) borate ligands are found at similar wavelengths [866 nm (54 M−1 cm−1) for [PhTttBu]Ni(NO3)71 and 845 nm (350 M−1 cm−1) for [PhTtAd]Ni(O2)72].</p><p>The temperature-dependent magnetic properties of the nickel(II) semiquinonate complexes were examined using SQUID magnetometry (Figure 9). For [PhTttBu]Ni(3,5-DBSQ), the χT value is 0.52 cm3 K mol−1 at 300 K, corresponding to μeff = 2.03 μB, which is consistent with the solution μeff of 2.25 μB at room temperature. This μeff value is slightly higher than the spin-only value of 1.73 μB expected for an S = ½ ground state but much lower than that for the uncoupled (S = 1 and ½) system (μeff = 3.32 μB), suggesting strong antiferromagnetic coupling between the high-spin NiII (S = 1) and 3,5-DBSQ radical (S = ½). The χT values are effectively constant throughout the temperature range 2–300 K, which further supports the presence of very strong antiferromagnetic coupling, leading to population of only the S = ½ state. Above 300 K, χT gradually increased because of the thermal population of the S = 3/2 excited state. The field-dependent magnetization data collected at 1.8 K show saturation behavior at fields above 5 T, consistent with an S = ½ coupled ground state (Figure S9). Fitting of the susceptibility data to the Hamiltonian H = −2J(S1∙S2) + DSz2 + E(Sx2 − Sy2) + gβB∙S using PHI software59 yielded a large J of −382 cm−1 (g = 2.24). Because the χT values remain constant even at low temperature, intermodular interactions were not considered in this fit.</p><p>Strong antiferromagnetic coupling was also observed for [PhTttBu]Ni(phenSQ). The room temperature μeff for [PhTttBu]Ni(phenSQ) in benzene-d6 is 2.18(3) μB, indicating an S = ½ ground state. The χT value is 0.505 cm3 K mol−1 (μeff = 2.0 μB; g = 2.31) at 300 K. A slightly steeper increase in χT was observed between 300 and 390 K. Additionally, below 15 K, the χT values for [PhTttBu]Ni(phenSQ) dropped gradually down to 0.31 cm3 K mol−1 at 2 K, possibly signifying intermolecular interactions via π–π-stacking pathways in the solid-state structure. The saturation behavior of the magnetization data collected at 1.8 K further supports an S = ½ coupled ground state (Figure S10). Fitting of the susceptibility data revealed a smaller [relative to [PhTttBu]Ni(3,5-DBSQ)] exchange coupling constant of J = −320 cm−1 and small intermolecular dipolar coupling, zJ = −0.57 cm−1 (g = 2.22, assuming J >> D). Notably, a five-coordinate NiII(3,5-DBSQ) complex of a triazamacrocycle ligand reported by Dei and co-workers73 exhibits strong antiferromagnetic coupling between high-spin NiII and 3,5-DBSQ ligand with μeff = 1.89 μB. The absolute value of the coupling constant was estimated to be greater than 300 cm−1 because of the lack of excited-state population. Similarly, in Shultz's TpCum,MeNi(nitronyl nitroxide semiquinone), the calculated JNi-SQ value is between −244 and −525 cm−1.70 To the best of our knowledge, our measurements represent the first accurate determination of coupling constants for five-coordinate high-spin nickel(II) complexes with antiferromagnetically coupled semiquinonate ligands.</p><p>Following the approach employed for [PhTttBu]Fe(phenSQ), broken-symmetry DFT calculations were performed for [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) to obtain detailed descriptions of the electronic structures and to correlate the electronic structures with the magnetic properties. Geometry optimizations based on the X-ray crystal structures were performed for the S = ½ spin ground state using both the BP86 and B3LYP functionals. When the B3LYP functional was used, the calculated geometric and electronic structures of [PhTttBu]Ni(phenSQ) [model Ni(phenSQ)1] and [PhTttBu]' Ni(3,5-DBSQ) [model Ni(3,5-DBSQ)1] did not agree with the experimental data (Table 3). Although the models obtained using the BP86 functional, Ni(phenSQ)2 and Ni(3,5-DBSQ)2, accurately reproduce the experimental molecular structures and are predicted to have the correct spin distribution, the TD-DFT-calculated electronic absorption spectra are inconsistent with the experimental data (Figures S12 and S13). However, when models Ni(phenSQ)2 and Ni(3,5-DBSQ)2 were subjected to further geometry optimizations using the B3LYP functional, the resulting models, Ni(phenSQ)3 and Ni(3,5-DBSQ)3, exhibited geometric and electronic structures and afforded TD-DFT-calculated absorption spectra consistent with the corresponding experimental data.</p><p>The TD-DFT-calculated absorption spectrum for the Ni(phenSQ)3 model is in excellent agreement with the experimental spectrum of [PhTttBu]Ni(phenSQ) (Figure 10). Inspection of the EDDMs provided in Figures 10 and S17 reveals that the intense feature at 397 nm (15967 M−1 cm−1) in the computed spectrum, which corresponds to the prominent feature at 397 nm (15967 M−1 cm−1) in the computed spectrum, which corresponds to the prominent feature at 398 nm (12877 M−1 cm−1) in the experimental spectrum, arises from a semiquinonate intraligand transition. Toward lower energy, the transitions associated with the computationally predicted features at 547 (3497 M−1 cm−1) and 555 nm-phenSQ corresponds to the broad band at 846 nm (710 M−1 cm−1) in the experimental spectrum, arises from a nickel(II) LF transition.</p><p>The TD-DFT calculated absorption spectrum for model Ni(3,5-DBSQ)3 and the experimental spectrum of [PhTttBu]− Ni(3,5-DBSQ) also agree quite well (Figure S14). Because these spectra and the underlying transitions are similar to those of [PhTttBu]Ni(phenSQ) described above, they will not be discussed further.</p><p>A qualitative MO diagram for model Ni(phenSQ)3 derived from our DFT results is shown in Figure 11. Consistent with our experimental data, the NiII is high-spin, with two unpaired spin-up electrons occupying the Ni(dz2)- and Ni(dx2-y2)-based MOs. The spin-up electron in the Ni(dz2)-based MO is coupled to the spin-down electron in the phenSQ π*-based MO with spatial overlap of S = 0.31, leading to the strong antiferromagnetic coupling observed by SQUID magnetometry. The calculated coupling constant, J = −273 cm−1, is in good agreement with the SQUID-derived value, J = −320 cm−1.</p><p>As expected on the basis of the experimental and TD-DFT-computed absorption spectra, the MO diagram for model Ni(3,5-DBSQ)3 (Figure S15) closely resembles that obtained for Ni(phenSQ)3 in Figure 11. Again, the spin-up electron in the Ni(dz2)-based MO is coupled to the spin-down electron in the 3,5-DBSQ π*-based MO with spatial overlap of S = 0.31, leading to strong antiferromagnetic coupling. The larger coupling constant calculated for this complex, J = −282 versus −273 cm−1 for [PhTttBu]Ni(phenSQ), agrees with our SQUID data, which show that [PhTttBu]Ni(3,5-DBSQ) features stronger antiferromagnetic coupling between the NiII ion and the semiquinonate ligand. Similarly strong antiferromegnetic coupling between high-spin nickel(II) and a ligand derived radical, superoxide, has been proposed to best describe the electronic structures of nickel–dioxygen adducts, including those prepared directly from nickel(I) and O2.74,75</p><!><p>With a thorough understanding of the electronic structures of [PhTttBu]Ni(phenSQ) and [PhTttBu]" Ni(3,5-DBSQ) in hand, their O2 reactivity was investigated. Stirring a toluene solution of [PhTttBu]Ni(phenSQ) under O2 at room temperature resulted in the slow decomposition of the complex, as indicated by the gradual loss of electronic absorption bands at 536, 586, and 846 nm (Figure 12). To identify the organic products, the reaction of [PhTttBu]Ni-(phenSQ) with O2 was conducted on a preparative scale and monitored by electronic absorption spectroscopy. After complete decomposition of [PhTttBu]Ni(phenSQ), 0.5 M HCl was added to liberate the ligand-derived products. The 1H NMR spectrum of the organic products lacks resonances due to diphenic anhydride. Indeed, GC indicated that phenanthrenequinone was produced in 14% yield along with a negligible amount of diphenic anhydride (ca. 0.5% yield; Figure S23).</p><p>The lack of intradiol cleaving reactivity of [PhTttBu]Ni-(phenSQ) contrasts with the reactivity displayed by [PhTttBu]− Fe(phenSQ), highlighting the crucial role of the metal ion in effecting the intradiol cleavage. The general intradiol cleavage mechanism invokes the formation of a metal(III) alkylperoxo intermediate.26,76,77 Because the redox potential for the nickel(III)/nickel(II) couple is much higher than that for the iron(III)/iron(II) couple, a nickel(III) alkylperoxo intermediate would not form as readily as an iron(III) alkylperoxo intermediate. Therefore, in the case of [PhTttBu]Ni(phenSQ), the rate of electron transfer from the phenSQ ligand to O2 outcompetes the rate of formation of the nickel(III) alkylperoxo intermediate. As a result, phenanthrenequinone was formed rather than diphenic anhydride.</p><p>In contrast to its phenSQ counterpart, [PhTttBu]Ni(3,5-DBSQ) is inert toward O2. No spectral change was observed after stirring of a toluene solution of [PhTttBu]Ni(3,5-DBSQ) under an O2 atmosphere for 12 h (Figure S19). The results of the O2 reactivity studies on [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) is less stable than the one-electron-oxidation product of [PhTttBu]Ni(phenSQ).[PhTttBu]Ni(3,5-DBSQ) are summarized in Scheme 4. The difference in the O2 reactivity between [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ) implies that the dioxolene ligand also plays an important role in affecting the O2 reactivity, which was further interrogated through electrochemical studies (vide infra).</p><!><p>The redox properties of [PhTttBu]Fe(phenSQ), [PhTttBu]Ni-(phenSQ), and [PhTttBu]Ni(3,5-DBSQ) were probed using CV and DPV. The cyclic voltammograms of the three complexes are shown in Figure 13. The CV of [PhTttBu]Fe(phenSQ) shows irreversible oxidation and reduction events. Interestingly, while the oxidation process is irreversible at scan rates up to 1 V/s, detection of the anodic current associated with the reduction couple is only observed at faster scan rates, which suggests strongly that the one-electron reduction product of [PhTttBu]Fe(phenSQ) is also relatively unstable (Figure S25). By contrast, the cyclic voltammogram of [PhTttBu]Ni-(phenSQ) exhibits quasi-reversible oxidation and reversible reduction processes, suggesting that the kinetic stability of the oxidation and reduction products of [PhTttBu]Ni(phenSQ) is much greater than that of [PhTttBu]Fe(phenSQ). [phTttBu]Ni-(3,5-DBSQ) exhibits a reversible reduction and an irreversible oxidation wave, indicating that on the time scale of the voltammetry experiments the one-electron-oxidation product of [PhTttBu]Ni(3,5-DBSQ) is less stable than the one-electronoxidation product of [PhTttBu]Ni(phenSQ).</p><p>To garner information regarding the redox potentials at thermodynamic equilibrium for the irreversible redox events, differential pulse voltammograms were collected (Figure 14). Oxidation of [PhTttBu]Fe(phenSQ) and [PhTttBu]Ni(phenSQ) was observed at −0.10 and 0.05 V (vs Fc+/Fc0), respectively. These redox events are attributed to ligand-based phenSQ/phenQ redox cycling, which occur at similar potentials for other MII(phenSQ) complexes.78,79 The higher potential of the phenSQ/phenQ couple for [PhTttBu]Ni(phenSQ), compared to that for [PhTttBu]Fe(phenSQ), can be attributed to (a) a greater LF stabilization energy (LFSE) for high-spin NiII d8 versus high-spin FeII d6 and (b) a stronger antiferromagnetic exchange in [PhTttBu]Ni(phenSQ). A similar trend was observed by Pierpont et al. in their studies of the [TpCum,Me]− M(3,5-DBSQ) series (M = ZnII, CuII, and CoII).80 In their case, the zinc(II) analogue, which has no LFSE and no exchange interaction between the diamagnetic ZnII ion and the semiquinonate radical, has an oxidation potential that is 0.15 and 0.17 V lower than those of the copper(II) and cobalt(II) analogues, respectively. The oxidation of [PhTttBu]Ni(3,5-DBSQ) to [PhTttBu]Ni(3,5-DBQ)+ occurs at 0.28 V, which is 0.23 V higher than the potential at which [PhTttBu]Ni-(phenSQ) is oxidized to [PhTttBu]Ni(phenQ)+. These data indicate that [PhTttBu]Ni(3,5-DBSQ) is more resistant toward oxidation of the semiquinonate ligand than [PhTttBu]Ni-(phenSQ), which provides an explanation for why [PhTttBu]− Ni(3,5-DBSQ) does not react with O2, whereas [PhTttBu]Ni-(phenSQ) does.</p><p>The oxidation potentials of [PhTttBu]Fe(phenSQ) and [PhTttBu]Ni(phenSQ) also provide clues as to whether the oxidation product phenQ is generated through an outer-sphere or inner-sphere electron-transfer mechanism. The redox potential of O2/O2− is around −1.0 V versus SCE (ca. – 1.5 V vs Fc+/Fc0),81 which is much lower than the redox potential of phenSQ/phenQ. Thus, direct electron transfer from phenSQ to O2 is unlikely. Instead, phenQ may be generated through an inner-sphere electron-transfer mechanism, which involves binding of O2 to the metal, followed by electron transfer from the phenSQ ligand to the M(O2) moiety. Under this mechanism, the low O2 affinity of nickel(II) disfavors the O2 binding and, therefore, makes the formation of phenQ sluggish.</p><p>On the reduction side of the differential pulse voltammograms, [PhTttBu]Fe(phenSQ) and [PhTttBu]Ni(phenSQ) are reduced at −0.92 and −1.00 V, respectively. These events are assigned as the phenSQ/phenCat redox couple. The reduction of [PhTttBu]Ni(3,5-DBSQ) at −0.94 V is assigned to a 3,5-DBSQ/3,5-DBCat redox couple. A comparison of the reduction potentials of [PhTttBu]Ni(3,5-DBSQ) and [PhTttBu]− Ni(phenSQ) reveals that it is easier to reduce 3,5-DBSQ to its catecholate form, which explains the inability to isolate an iron(II) semiquinonate complex with 3,5-DBSQ as the ligand.</p><!><p>The geometric and electronic structures of a previously reported iron(II) o-semiquinonate complex, [PhTttBu]Fe-(phenSQ),36 and two new nickel(II) o-semiquinonate complexes, [PhTttBu]Ni(phenSQ) and [PhTttBu]Ni(3,5-DBSQ), were elucidated by X-ray crystallography, optical and NMR spectroscopies, SQUID magnetometry, and DFT calculations. All three complexes contain high-spin MII ions antiferromagnetically coupled to the semiquinonate radicals, with [PhTttBu]− Ni(3,5-DBSQ) having the largest exchange interaction (J = −382 cm−1), followed by [PhTttBu]Ni(phenSQ) (J = −320 cm−1) and then [PhTttBu]Fe(phenSQ) (J = −127 cm−1). The trend of the magnitude of exchange interactions between the MII ions and the semiquinonate ligands is well reproduced by the DFT calculations.</p><p>The fact that [PhTttBu]Fe(phenSQ) exhibits intradiol cleaving reactivity provides new evidence supporting the theory that the iron(II) semiquinonate character within the iron(III) catecholate species could be responsible for the O2 reactivity of intradiol catechol dioxygenases. The O2 reactivity studies further reveal the importance of the metal ion on the intradiol cleaving reactivity because changing the metal ion from FeII to NiII abolishes the intradiol reactivity and makes the oxidation of phenSQ to phenQ more pronounced. The metal-ion effect is caused by the difference in the redox potentials of the metal(III)/metal(II) couples and, likely, differences in the O2 binding affinity. When the metal(III)/metal(II) potential is high, as in the case of [PhTttBu]Ni(phenSQ), the NiII ion acts as a mediator for the inner-sphere electron transfer from phenSQ to O2, generating phenQ. On the other hand, when the midpoint potential of the metal(III)/metal(II) couple is sufficiently low, the formation of the metal(III) alkylperoxo intermediate outcompetes the inner-sphere electron transfer and, thus, the reaction produces primarily the intradiol cleavage product. Last, the lack of any O2 reactivity of [PhTttBu]Ni(3,5-DBSQ) indicates that the midpoint potential of the semi-quinonate/quinone couple also affects the outcome of the O2 reactions. This assertion is further supported by the results of the electrochemical studies, which show that the midpoint potential for the 3,5-DBSQ/DBQ couple is higher than that of the phenSQ/phenQ couple.</p>

PubMed Author Manuscript

The Influence of Metal-Doped Graphitic Carbon Nitride on Photocatalytic Conversion of Acetic Acid to Carbon Dioxide

Metal-doped graphitic carbon nitride (MCN) materials have shown great promise as effective photocatalysts for the conversion of acetic acid to carbon dioxide under UV–visible irradiation and are superior to pristine carbon nitride (g-C3N4 , CN). In this study, the effects of metal dopants on the physicochemical properties of metal-doped CN samples (Fe-, Cu-, Zn-, FeCu-, FeZn-, and CuZn-doped CN) and their catalytic activity in the photooxidation of acetic acid were investigated and discussed for their correlation, especially on their surface and bulk structures. The materials in the order of highest to lowest photocatalytic activity are FeZn_CN, FeCu_CN, Fe_CN, and Cu_CN (rates of CO2 evolution higher than for CN), followed by Zn_CN, CuZn_CN, and CN (rates of CO2 evolution lower than CN). Although Fe doping resulted in the extension of the light absorption range, incorporation of metals did not significantly alter the crystalline phase, morphology, and specific surface area of the CN materials. However, the extension of light absorption into the visible region on Fe doping did not provide a suitable explanation for the increase in photocatalytic efficiency. To further understand this issue, the materials were analyzed using two complementary techniques, reversed double-beam photoacoustic spectroscopy (RDB-PAS) and electron spin resonance spectroscopy (ESR). The FeZn_CN, with the highest electron trap density between 2.95 and 3.00 eV, afforded the highest rate of CO2 evolution from acetic acid photodecomposition. All Fe-incorporated CN materials and Cu-CN reported herein can be categorized as high activity catalysts according to the rates of CO2 evolution obtained, higher than 0.15 μmol/min−1, or >1.5 times higher than that of pristine CN. Results from this research are suggestive of a correlation between the rate of CO2 evolution via photocatalytic oxidation of acetic acid with the threshold number of free unpaired electrons in CN-based materials and high electron trap density (between 2.95 and 3.00 eV).

the_influence_of_metal-doped_graphitic_carbon_nitride_on_photocatalytic_conversion_of_acetic_acid_to

Introduction<!>Materials and Reagents<!>Preparation of CN and Metal-Doped CN<!>Material Characterization<!>Chemical Composition<!><!>Bulk Structure<!><!>Bulk Structure<!><!>Surface Structure<!><!>Surface Structure<!><!>Surface Structure<!>Photocatalytic Oxidation of Acetic Acid<!>Property Correlations<!><!>Conclusion

<p>Photocatalysis has attracted great interest as an energy-efficient, low-cost, and relatively safe method for chemical conversions related to pollution abatement, and in the production of platform chemicals and other valuable substances (Hagfeldt & Graetzel, 1995; Hoffmann et al., 1995; Fujishima et al., 2000; Fujishima et al., 2008). Exploiting the advantages of photocatalysis requires the development of semiconducting materials having desirable characteristics such as high surface area, low electron-hole pair recombination rate, suitable band-gap energy based on light source, and the appropriate type and amount of bulk and surface defects. Several reports have focused on explaining the correlation between energy band gap and photocatalytic performance (Srikhaow & Smith, 2013; Dante et al., 2019), while other studies have probed the defect characteristics of semiconductor materials (Anantachaisilp et al., 2014; 2015). Recently, graphitic carbon nitride (g-C3N4, CN) has gained attention in the photocatalysis research field, with potential applications in the decomposition of organic pollutants, in water splitting, and for carbon dioxide reduction (Dinesh & Chakma, 2019; Li et al., 2019; Volokh et al., 2019; Wang et al., 2020; H-g.; Zhang et al., 2018). It is easy to synthesize, exhibits high thermal and chemical stability, and exhibits a moderate band gap (2.7 eV) and an absorption edge at 450 nm. Despite this, some enhanced visible light activity CN materials show a high recombination rate of photogenerated electron-hole pairs, which results in low photocatalytic performance (Dong et al., 2013). From literature, the dopants may contribute toward the suppression of recombination of charge carriers and enhancing the photocatalytic activity of carbon nitride (Barrio et al., 2021). Metal doping is one strategy for suppressing the recombination of photogenerated electron holes in semiconductors. This method is considered an effective way to extend the light absorption range, modify the electronic structure, and enhance surface properties, all of which can improve the photocatalytic activity of g-C3N4. Doping of copper into mesoporous C3N4 (mpg-C3N4) doubled the photocatalytic activity of the material relative to pure mpg-C3N4 for the degradation of methyl orange. This was due to a higher separation rate and greater mobility of the photogenerated carriers (Le et al., 2016). In addition, Fe-doping into g-C3N4 resulted in enhanced light absorption and photocatalytic activity, and the resulting material could be reused five times without any change in activity (Tonda et al., 2014). Bi-metallic doping can result in even higher photocatalytic activities, with Fe and P co-doped g-C3N4 materials being active photocatalysts for Rhodamine B photodegradation and hydrogen production (Hu et al., 2014). Higher activities for dye degradation were observed for co-doped Fe and P g-C3N4 relative to singly doped (with Fe or P) CN or undoped CN, probably due to the narrower band gap, larger specific surface area, and lower degree of polymeric condensation. Optoelectronic properties often are linked with the activity of CN photocatalysts, with less discussion on contributions from the surface and the bulk structure.</p><p>Reversed double-beam photoacoustic spectroscopy (RDB-PAS) is a newly utilized technique for investigating the surface properties of metal oxides and O/S doped g-C3N4 (Chuaicham, et al., 2020). Fingerprint energy-resolved distribution of electron traps (ERDTs) combined with conduction band bottom position (CBB) patterns have highlighted differences in surface structure among heteroatom-doped g-C3N4 samples, which could not be observed by other standard methods such as powder X-ray diffraction (PXRD) and infrared spectroscopy. Unpaired electrons in the g-C3N4 structure, as detected by electron spin resonance (ESR), were reported to be related to the materials' photocatalytic activity (Li et al., 2018; Xia et al., 2019). These findings not only provide important surface structural information but also describe the relationship between structural features and photocatalytic activity (Chuaicham, et al., 2020; Nitta et al., 2018; Nitta et al., 2019). In this work, the bulk and surface structural properties of single- and bi-metal-doped g-C3N4 powders, synthesized by a facile method, were elucidated using complementary techniques with the aim to explore relationships between these and the photoactivity of these materials for the degradation of acetic acid. Acetic acid emissions contribute negatively to global warming (Budsberg et al., 2020), and this chemical is a typical degradation product from wastewater remediation (Badawi et al., 1992). While this acid is of low toxicity, it is quite stable, being oxidation-resistant under ambient conditions (Li et al., 1991). This work chose acetic acid as a model compound of a persistent organic compound in the photocatalytic conversion of acetic acid to carbon dioxide in order to represent a highly effective photocatalyst in oxidation processes.</p><!><p>All chemicals used in this work were of analytical grade. Crystalline urea (Kemaus, Australia) was used for the preparation of g-C3N4 (CN). Copper (II) acetate monohydrate [Cu(CH3COO)2‧H2O] (Chem-supply, Gillman, Australia), zinc acetate dihydrate [Zn(CH3COO)2‧2H2O] (Univar, IL, United States), and anhydrous iron (II) acetate [Fe(CH3COO)2] (Aldrich, Auckland, New Zealand) were used without any purification as metal sources. Acetic acid was purchased from Wako Pure Chemical Industries (Japan). In addition, 65% nitric acid (HNO3) was purchased from RCI Labscan (Thailand) with 70% perchloric acid (HClO4) being obtained from Qrec, New Zealand.</p><!><p>All samples were prepared as in a previous report (Dante et al., 2021), which utilized metal-doped carbon nitride as a sensing material for glucose. In the first step, pristine CN was prepared by the pyrolysis of urea. In a typical preparation, 25 g of urea was placed in an alumina crucible with a cover and was calcined at 873 K in an air atmosphere for 4 h (controlled heating/cooling rates of 25 K min−1). After cooling, the product (CN) was ground to obtain a fine powder. Subsequently, metal-doped CN was synthesized by ultrasonic impregnation, applying ultrasonic irradiation to a suspension of CN (416 mg) in aqueous metal acetate solution (33.2 mg of the appropriate metal acetate in 10 ml of deionized water). Each suspension was sonicated for 2 h and then filtered and washed with water several times. The resulting samples were dried in an oven at 338 K for 24 h to remove water, ground to a powder, and then stored in a desiccator. The obtained metal-doped CN samples were denoted as Fe_CN, Cu_CN, and Zn_CN, depending on metal dopant. The preparation of co-metal-doped CN (FeCu_CN, FeZn_CN, and CuZn_CN) followed the same procedure, dispersing CN into the aqueous solution containing 16.6 mg of each metal acetate.</p><!><p>X-ray diffraction patterns were recorded on an X-ray diffractometer (XRD, SmartLab, Rigaku) with Cu Kα radiation over a 2θ scan range between 10° and 80°. Solid stage electron spin resonance (ESR) (Bruker ELEXSYS, ER083CS) measurements were obtained at room temperature. For this, a 20-mg portion of each sample was transferred to an ESR tube to run in X-band, at a microwave power of 20 mW, a microwave frequency of 9.850 GHz, a modulation amplitude of 1 G, and a modulation frequency of 100 kHz. ICP-MS measurements involved adding 5 mg of metal dopant CN sample in 10 ml of 1:1 v/v mixed acid solution (HNO 3 /HClO 4 ) and standing overnight to ensure complete dissolution prior to analysis (ICP-MS, PerkinElmer NexION® 2000 instrument). Additionally, the elemental composition of materials was analyzed using an XPS spectrophotometer (Kratos, Axis Ultra DLD) and EDX equipped with a scanning electron microscope (FE-SEM, Hitachi, SU-8010). FE-SEM imaging utilized the following instrument settings: 5.0-kV electron-acceleration voltage, 10.0-μA current, and 3.0-nm working distance. Each sample was coated for 10 s with gold using an ion sputter coater (JFC-1600, JEOL). Specific surface area (SSA) and pore size distributions of metal-doped CN samples were determined based on nitrogen (N2) adsorption-desorption isotherms at 77 K (Autosorb-6, Quantachrome Instrument). Prior to analysis, surface moisture was evaporated by pre-heat treatment (353 K, 2 h). The surface morphologies of the samples were also conducted on a field-emission scanning electron microscope (FE-SEM, JSM-7400F, JEOL) in the secondary-electron image mode.</p><p>ERDT/CBB patterns were measured by RDB-PAS analysis following a previous report (Chuaicham, et al., 2020). Samples were placed into a PAS cell, and prior to measurement, N2 saturated with methanol vapor was flowed through the cell for 30 min. The sample cell was then moved to an acrylic box under N2 flow and irradiated using a light beam from a xenon lamp (Bunkokeiki, Tokyo, Japan, BK1) equipped with a grating monochromator from 650 to 350 nm. The obtained PAS signal, generated by simultaneously irradiated 35-Hz modulated 625-nm LED light, was detected using a digital lock-in amplifier. Photoacoustic spectra were then recorded with reference to the photoacoustic spectrum of graphite.</p><p>The optical properties of CN-based materials were investigated using a UV–vis diffuse reflectance spectrometer (UV–vis DRS, V670, Jasco), with barium sulfate as a reference. Photoluminescence analysis (PL) utilized a photoluminescence spectrofluorometer (Horiba: FluoroMax4), with an excitation wavelength of 320 nm and an excitation slit/emission slit ratio of 2:1. Photocatalytic activity measurements.</p><p>A 30-mg portion of photocatalyst was suspended in 5 ml of 5% v/v acetic acid solution in a Pyrex tube sealed with a rubber septum. Photoirradiation was performed using a mercury arc lamp (Eiko-sha 400) at a wavelength > 290 nm. The estimated UV light (365 nm) flux was ca. 1 μmol s−1 for a 5-ml suspension. Prior to UV light irradiation, the suspension was stirred in the dark for 60 min to reach an adsorption–desorption equilibrium and then sonicated for 30 s to prevent agglomeration. While maintaining an anaerobic atmosphere, the reaction mixture was then irradiated while regulating the temperature of the vessel at 298 K with a water circulation system. The amount of generated CO2 was determined at 30-min time intervals using gas chromatography (GC, GC-8A, Shimadzu).</p><!><p>The metal compositions of CN-based samples, obtained using three techniques (EDX, XPS, and ICP-OES), are reported in Table 1. SEM-EDX mapping showing the localization of elements in CN-based materials is highlighted in Figures 1, 2. The images indicate the presence of very low levels of metal incorporation on the surface of all samples, while XPS spectra (Supplementary Figure S1) indicate that the surface concentrations of metals could be lower than XPS detection limits. Similar with previous studies (Praus et al., 2020; Lin et al., 2021) that reported the presence of hydroxyl groups on the CN sheets, oxygen was found in all samples (EDX mapping images). Oxygen-containing CN materials have shown high activity in photocatalytic hydrogen evolution (Huang et al., 2020) and photocatalytic dye degradation (Praus et al., 2020). On the other hand, the bulk concentrations of metal (s) in the samples were quantified by the ICP-OES technique and are reported in Table 1. The detectable % Fe (0.076 ± 0.021) and Zn (0.021 ± 0.016) in the pristine CN could be due to trace metals in the urea precursor. Also, trace metals in the iron (II) acetate, copper (II) acetate, and Zn (II) acetate reagents result in the added-up metal amount being detected in metal-doped CN materials, being relative to those in the pristine CN. No copper is detected in CuZn_CN probably due to the lower copper affinity in the CN structure.</p><!><p>Comparative elemental composition of CN-based materials by SEM-EDX, XPS and ICP-MS analyses.</p><p>EDX mapping for single metallic doping systems (A) Fe_CN, (B) Cu_CN, and (C) Zn_CN.</p><p>EDX mapping for bi-metallic doping systems (A) FeCu_CN, (B) FeZn_CN, and (C) CuZn_CN.</p><!><p>A pristine CN sample was synthesized by urea pyrolysis at 873 K in air by modification of an established procedure (Xu et al., 2013; Maeda et al., 2018). Powder X-ray diffraction (PXRD) patterns of all CN-based samples, shown in Figure 3, exhibit a major peak at 27.7° corresponding to the (002) planes with an interlayer distance of 0.322 nm, which arises from typical interlayer stacking of hexagonal CN, and a minor peak at 13° from the (100) planes with an interplanar distance of 0.680 nm arising from in-plane structural packing motifs (Hu et al., 2014). Apart from those of pristine CN and metal-doped CN, no impurity peaks were detected even though the pyrolysis time was shorter than that used in the literature. The high intensities of the (100) and (002) peaks in the metal-doped CN samples (Fe_, FeCu_, FeZn_, CuZn_, Zn_ CN, and Cu_CN) indicated their improved crystallinity relative to undoped CN. No PXRD peaks corresponding to metals, or metal oxides, were detected, as expected due to the low metal loadings (shown in Table 1). Nevertheless, a slight shifting of the (002) peak to a lower 2-theta angle was observed for metal-doped CN samples, which suggested some modification of the graphitic stacking of CN as a consequence of metal doping, resulting in an increased interlayer distance (Zhang et al., 2018). However, varying the metal dopant does not result in any significant differences in the diffraction patterns.</p><!><p>XRD patterns of undoped CN and metal-doped CN, and an enlarged view of the (002) peak.</p><!><p>Solid-state ESR spectra, shown in Figure 4A, contain a symmetric ESR signal at g = 2.0036 with respect to delocalized free electrons on the aromatic ring of the CN materials (Zhang et al., 2013; Dante et al., 2021). The higher intensity of such ESR signals in metal-doped CN samples, in comparison to those seen in pristine CN, is consistent with previous work (Liu et al., 2017; Zheng et al., 2018). This indicates that the electron density or charge mobility in metal-doped CN samples is greater than in undoped CN (Chen et al., 2019). An additional ESR signal at g = 2.07, corresponding to Cu2+, can be referred to the Spin-Hamiltonian parameter along the x and y axes (g xx = g yy = g ⊥) (Arizaga et al., 2008). The Spin-Hamiltonian parameter in the z-direction (g zz = g ||) around g = 2.3 (Arizaga et al., 2008), and the hyperfine coupling constant (A||), could not be detected for Cu_CN. Signals from Fe2+ and Zn2+ were also undetectable possibly due to the amount of these metal dopants being lower than the ESR detection limit. The relative signal intensity ratios for metal-doped CN/pure CN at g = 2.0036 are plotted in Figure 4B with the result that doping with Fe appears to afford CN-based materials containing more free unpaired electrons than is the case with other metal dopants or CN alone. The Fe-containing CN materials, FeZn_CN, FeCu_CN, Fe_CN, are ranked first, second, and third in terms of the number of free unpaired electrons in the CN-based materials.</p><!><p>(A) ESR spectra of undoped CN and metal-doped CN at room temperature and (B) increase in relative intensity at g = 2.0036.</p><!><p>Since photocatalytic reactions occur on the material surface, an understanding of the surface properties is of paramount importance. UV–Vis diffused reflectance spectra of all samples are shown in Figure 5A, and these reflectance spectra can be transformed to the corresponding absorption spectra by applying the Kubelka–Munk function (Jones et al., 2019). The obtained absorption spectra, given in Supplementary Figure S2, indicate that pristine CN and metal-doped CN samples absorb light in both the UV and visible regions. Pristine CN semiconductors show an absorption range from 200 to 450 nm, which originates from charge transfer from the valence band populated by the N 2p orbital to the conduction band formed by the C 2p orbital (Wang et al., 2020). The band-gap energies of CN and CuZn_CN materials were found to be slightly higher than 3.0 eV, which is in line with the results obtained by Dante et al. (2021) and Katsumata et al. (2019) for metal-doped CN. The extended absorption spectra of metal-doped CN materials shifted into the visible range (400–50 nm), as seen in other reports (Dante et al., 2021; Liu et al., 2017; Nguyen Van et al., 2020). On the other hand, the absorption characteristics of CuZn_CN were found to be similar to pristine CN. The FeZn_CN sample showed the most optical response absorbance in the visible region, while the pristine CN showed the lowest response. The band-gap energies (Eg) of metal-doped CN materials (Table 2), as estimated from Tauc plots (Figure 5B,C), were slightly smaller than that of CN. The CN-based materials containing Fe show enhanced light absorption and narrowing of the energy band gap (FeZn_CN; E g = 2.84 eV), whereas those with Cu loadings exhibit a relatively high visible light response, in comparison to other metal-doped CN systems (Supplementary Figure S2). Photoluminescence (PL) results, as reported in Supplementary Figure S2, were obtained using an excitation wavelength of 320 nm. The PL spectrum of CN (Supplementary Figure S2) displays an emission peak around 457 nm (2.7 eV) caused by the transition of lone pairs to the π* conduction band (Guo et al., 2016). Results indicate that PL spectral intensities for metal(s)-doped CN spectra are significantly lower than that of pristine CN. A possible reason for this is trapping of photogenerated electrons by metal-doping sites, which leads to slower electron-hole recombination rates (Nguyen Van et al., 2020; Cui et al., 2021). Nevertheless, XPS spectra (Supplementary Figure S1) suggested the very small amount of metal incorporated on the CN surface, insufficient to be detected by the surface characterization technique. While these results agree well with those from SEM-EDX investigations (Table 1), these limitations prevent the elucidation of any interactions between metal dopants and CN sheets.</p><!><p>(A) UV–Vis diffuse reflectance spectra and estimation of band-gap energies of undoped CN, (B) single-metal-doped CN, and (C) co-metal-doped CN samples.</p><p>Physical properties and photocatalytic activities of CN and metal-doped CN. In this work, two groups of materials were classified based on their high (green) and low CO2 evolution (black).</p><p>The total density of electron traps (dET) in the unit of μmol g−1.</p><p>The dET in the range of 2.95–3.00 eV in the unit of μmol g−1.</p><!><p>Nitrogen adsorption–desorption isotherms were utilized to analyze the surface of CN materials. As shown in Supplementary Figure S3 and Table 2, the specific surface area (SSA) and porosity of metal-doped CN materials vary with the dopant system. CuZN_CN has the highest SSA (97.72 m2 g−1), with all other metal-doped CN exhibiting lower SSA than pristine CN. The adsorption isotherms of pristine CN and metal-doped CN materials are given in Supplementary Figure S3, and all can be classified as type IV isotherms, which indicates the presence of mesopores. Average pore sizes and pore volumes, as calculated by the Barrett–Joyner–Halenda method, are summarized in Table 2, with FeZn_CN having an average pore size of 11.3 nm, about threefold larger than other CN materials. Figure 6 highlights the morphologies of undoped and metal-doped CN particles. All samples are composed of randomly packed thin sheet structures, constructed from the aggregation of micron-size plate-like particles, which agreed well with the observed H3 hysteresis nitrogen adsorption–desorption isotherms. The introduction of metal dopants does not appear to impact sample morphology, as indicated by the similarities in particle images. However, as stated earlier, metal doping results in slight shifting of peaks in PXRD patterns (Figure 3) along with variations in ESR signal intensities (Figure 4) and optical properties (Figures 5, 6) in CN materials, which underlines the effect of low-level metal dopants on the material structure.</p><!><p>ERDT patterns with CBB positions of CN and metal-doped CN samples. The number in < > denotes the total density of ETs, with the units of μmol g−1. VBT, valence band top.</p><!><p>Reversed double-beam photoacoustic spectroscopy (RDB-PAS) can provide information about electron accumulation in electron traps (ETs) near the conduction band bottom (CBB) on the surface of CN-based materials. Figure 6 highlights a plot of the distribution of electron traps (ERDT) versus CBB patterns, with ERDT and CBB reflecting the surface and bulk structure, respectively, of the CN-based materials. With the similar CBB, it can be concluded that the introduction of metal dopant (s) gives no effect to the bulk structure, detecting similar CBB for CN, Zn_CN, Fe_CN, and CuZn_CN materials. On the other hand, the modified bulk structures of Cu_CN, FeCu_CN, and FeZn_CN were obtained after the metal-doping process. The positions of ERDTs of Zn_CN are similar to those of pristine CN indicating neither bulk nor surface structure disruption from the introduction of Zn into the CN structure. Note that extended ERDT positions, at around 2.0 eV, were observed in the case of Cu_CN, CuZn_CN, and FeZn_CN, reflecting the modified surface structure due to metal doping. With the figures in⟨ ⟩ denoting the total density of ETs in the unit of μmol g−1, there is no correlation between the specific surface area (SSA) and the density of ETs found in CN-based materials, inconsistent with Nitta's work (Nitta et al., 2019) that reported the high density of ETs for the high SSA samples. The high-density state of ETs found in FeZn_CN is the same as that of the pristine CN material. The high-density state reflecting the high number of accumulated electrons in ETs is around 2.8 eV for the pristine CN, being localized above CBB for most samples except Cu_CN. The Cu_CN material has the high-density state below CCB (0.3 eV higher energy than that of the pristine CN). Therefore, the presence of accumulated electrons in ETs between the energy band gap can suppress the electron-hole pair recombination in photocatalysts. Nevertheless, electrons may be preferably exited from the high-density state that localized below CBB.</p><!><p>The photocatalytic activity of CN-based materials toward the oxidation of acetic acid to carbon dioxide (CO2) was examined (Mozia et al., 2010; Nosaka et al., 1996; Ohtani et al., 2008). Carbon dioxide evolution rates for processes employing different CN materials are summarized in Table 2, and these indicate that Fe doping significantly enhances the activity of CN-based photocatalysts. Acetic acid decomposition over Fe_CN, FeCu_CN, and FeZn_CN generated CO2 at a rate 1.6–1.8 times higher than that of pristine CN. The highest CO2 evolution rate (13.37 μmol h−1), obtained from FeZn_CN, mirrors results obtained by Yuan et al. (2002) who found that Fe- and Zn-doped titania is far more effective as a photocatalyst for phenol degradation than single metal-doped titania alone. However, from Table 2, Cu_CN and Zn_CN give higher CO2 evolution rates than CuZn_CN, so it cannot always be concluded that the addition of two metal dopants gives superior performance. Although doping of Fe into CN affords a high activity CN-based photocatalyst system, there is no clear connection between the rates of CO2 evolution obtained from such photocatalysts and the other properties listed in Table 2.</p><!><p>Correlations between the activity of photocatalysts and their properties are not easy to establish with certainty. For example, a CN material having high crystallinity was reported as a promising photocatalyst due to its low resistant carrier transfer, extended light absorption range, and suppressed electron-hole pair recombination (Yang et al., 2021). However, metal-doped CN materials with relatively low crystallinity have also shown enhanced photocatalytic activity relative to pristine CN (Wang et al., 2009; Hu et al., 2014). Other properties (optical and electronic) of semiconducting materials may also significantly influence the activity of photocatalysts (Hu et al., 2014; Tonda et al., 2014; Li et al., 2016).</p><p>Results herein indicate that the highest CO2 evolution rate was obtained from photocatalysis using FeZn_CN, even though Cu_CN shows the highest level of crystallinity. There is no clear correlation between all parameters related to CN materials: crystallinity, optical properties (PL, DRS, band-gap energies), specific surface area, and photocatalytic activity. Furthermore, a plot of the CO2 evolution rate from acetic acid oxidation under UV irradiation and electron-trap density at 2.95–3.00 eV is given in Figure 7A. From this, CN-based materials can be categorized into two groups. The first group, giving high CO2 evolution rates, includes Cu_CN, and Fe-containing CN materials and the rest giving low CO2 evolution rates are included in the second group. Figure 7B shows that the bulk concentration of Fe in samples correlates well with the relative intensity of the ESR signal at g = 2.0036. This correlation can thus be used to classify materials as high activity photocatalysts for conversion of acetic to carbon dioxide (as shown in the green oval). Similar correlations with respect to Cu or Zn bulk concentrations can be observed, as summarized in Supplementary Figure S4. All Fe-containing CN samples show high densities of accumulated electrons at about 2.95–3.00 eV. Previously reported work (Hu et al., 2014) has suggested that photogenerated electrons are trapped by the Fe-doping sites, promoting efficient separation of photogenerated electrons and holes, resulting in high photocatalytic activity. Accordingly, it may be concluded that the increase in electron-trap density at 2.95–3.00 eV (with regard to that of the pristine CN) is related to the high photoactivity of Fe-doped CN systems (Fe_CN, FeCu_CN, and FeZn_CN) toward acetic acid oxidation. As has been previously reported (Nitta et al., 2018) for patterns of energy-resolved distribution of electron traps (ERDT), the energies might be overestimated as the DOS and density of states at the top of the valence band are negligible. As actual photoexcitation occurs from the high DOS position (energy) part of VB, but not VBT, for titanium(IV) oxide, this energy discrepancy is estimated to be about 0.1–0.2 eV. The higher-energy ERDT might be shallow electron traps located just below CBB. The shallow trap state can capture photoexcited electrons, migrate to the surface, and take part in photocatalysis. Hence, trapped shallow electrons considerably improve photocatalytic activity. In the case of the deep trapped state, these electrons are easily recombined, which leads to poor photocatalytic activity (Choudhury et al., 2014; Ruan et al., 2020; Wang et al., 2021). However, the photocatalytic behavior of Cu_CN cannot be explained by this hypothesis. It might be possible that the higher light absorption intensity in the visible light region extended up to 800 nm (Supplementary Figure S2) makes Cu_CN to be highly light responsive, resulting in the enhanced photocatalytic activity in acetic acid oxidation. As discussed earlier (shown in Figure 4B), the relative intensity of the ESR signal at g = 2.0036 corresponds to the number of free unpaired electrons or defects at the localized π-conjugated carbon structure, and that there is a correlation between the number of delocalized unpaired electrons in CN-based materials and the rates of CO2 evolution. Such correlations are applicable for classifying these materials as high-performance photocatalysts in terms of CO2 evolution rate, i.e., Fe-doped CN (Fe_CN, FeCu_CN, and FeZn_CN) and Cu_CN materials, corresponding to the relatively ESR intensity (g = 2.003) of 1.3 and above. The presence of delocalized π-electrons in CN materials described herein, and their relationship with photocatalytic activity for chemical conversion, is in excellent agreement with previous work (Guan and Zhang, 2019; Lin et al., 2021).</p><!><p>(A) Plot of CO2 evolution rate vs electron-trap density at 2.95–3.00 eV, and (B) the correlation between ESR signal relative intensity at g = 2.0036 and the bulk concentrations of Fe in carbon nitride-based materials. The materials in the green oval gave high CO2 evolution rates, as defined in Table 2.</p><!><p>The effects of the introduction of metal dopants on the bulk and surface structure of carbon nitride were studied in relation to their photocatalytic activity in acetic acid oxidation. Powder XRD and ESR techniques confirmed the graphitic stacking and crystallinity in CN-based materials and the presence of free unpaired electrons on the aromatic CN sheets. The enhanced photooxidation rate of acetic acid for Fe-containing CN systems could be the result of the high ET density at the trap state between 2.95 and 3.00 eV and the high number of unpaired electrons available in the CN structure. Relationships between the bulk and surface structure of metal-doped CN materials, as studied by complementary techniques, could be used to classify CN materials as being of high or low photoactivity. Further investigations to probe the interactions between metals and carbon nitride, such as synchrotron measurements at various metal loadings, may allow for high-resolution spectroscopic data to be obtained, which will allow the elucidation of the oxidation state and chemical environment of the metal dopants. Determination of overall degradation byproducts from photocatalytic oxidation of acetic acid by using the metal-doped carbon nitride materials should be carried out to confirm the selectivity of such oxidation.</p>

PubMed Open Access

Round Robin Evaluation of MET Protein Expression in Lung Adenocarcinomas Improves\nInterobserver Concordance

Introduction: Overexpression of the mesenchymal-epithelial transition (MET) receptor, a receptor tyrosine kinase, can propel the growth of cancer cells, and portends poor prognoses for patients with lung cancer. Evaluation of MET by immunohistochemistry is challenging, with MET protein overexpression varying from 20% to 80% between lung cancer cohorts. Clinical trials using MET protein expression to select patients have also reported a wide range of positivity rates and outcomes. Materials and Methods: To overcome this variability, the Lung Cancer Mutation Consortium (LCMC) Pathologist Panel endeavored to standardize evaluation of MET protein expression with \xe2\x80\x9cRound Robin\xe2\x80\x9d conferences. This panel used randomly selected Aperio-scanned formalin-fixed paraffin-embedded lung cancer specimens stained by MET immunohistochemistry for the LCMC 2.0 study (N = 838). Seven pathologists in separate laboratories scored images of 5 initial cases and 2 subsequent rounds of 39 cases. The pathologists\xe2\x80\x99 scores were compared for consistency using the intraclass correlation coefficient. Issues affecting reproducibility were discussed in Round Robin conferences between rounds, and steps were taken to improve scoring consistency, such as sharing reference materials and example images. Results: The overall group intraclass correlation coefficient comparing consistency of scoring improved from 0.50 (95% confidence interval, 0.37-0.64) for the first scoring round to 0.74 (0.64-0.83) for the second round. Discussion: We found that the consistency of MET immunohistochemistry scoring is improved by continuous training and communication between pathologists.

round_robin_evaluation_of_met_protein_expression_in_lung_adenocarcinomas_improves\ninterobserver_con

Introduction<!>Population<!>MET Immunohistochemistry and digital image scanning<!>Scoring<!>Statistical analyses<!>Exploratory Round<!><!>Round Robin II<!>Discussion

<p>The mesenchymal-epithelial transition (MET) proto-oncogene on chromosome 7q21-31 encodes MET, a transmembrane tyrosine kinase receptor. After MET is activated by its ligand, hepatocyte growth factor (HGF), it initiates signaling through several downstream pathways to promote cellular proliferation, differentiation, angiogenesis, and survival. In lung cancers, increased MET or HGF expression can drive tumorigenesis and are associated with poor prognoses.(1-4) Cigarette smoke is a factor that promotes c-MET addiction in lung cancer.(5)</p><p>MET genomic alterations, such as MET amplification and/or MET gene exon 14 (METex14) splice-site mutations, can increase MET protein expression.(6-9) Investigational efforts continue to provide additional insights regarding diagnostic, prognostic and therapeutic implications for MET genomic alterations. These studies have refined our understanding that MET genomic alterations are enriched in pulmonary sarcomatoid carcinomas;(10). MET ex14 splice-site mutations are also reported in other lung neoplasms, brain gliomas and tumors of unknown primary origin.(11)</p><p>MET protein expression may also be upregulated as a resistance mechanism in response to tyrosine kinase inhibitor therapy for epidermal growth factor receptor- (EGFR-) mutated lung adenocarcinomas.(12, 13) ERBB3 signaling resultant from MET amplification is described as an underlying mechanism attributed to gefitinib resistance.(14) Dual blockade of EGFR and MET may be a therapeutic strategy for tumors that harbor both an EGFR mutation and MET gene amplification, mutation, and/or increased expression or signaling.(13-15)</p><p>Several MET inhibitors, including monoclonal antibodies and small molecule inhibitors, are under investigation in lung cancer clinical trials. Individuals whose tumors harbor a high MET gene copy number and/or amplification and/or METex14 skipping mutations have demonstrated therapeutic response to tyrosine kinase inhibitors such as crizotinib (6, 7, 9, 16, 17) and cabozantinib.(6, 18) Onartuzumab (MetMab), a humanized monovalent monoclonal antibody that blocks HGF binding to MET, was associated with improved survival in patients with advanced lung cancers and high MET expression by immunohistochemistry (IHC) in a Phase II trial, but subsequently failed to show benefit in a Phase III study.(19-21) Tivantinib (ARQ 197), a small molecule inhibitor of MET, was studied in a Phase III clinical trial, but the overall trial failed to show benefit.(22) However, subgroup analysis demonstrated that tivantinib improved survival in patients with MET protein overexpression.(22)</p><p>Thus, despite some setbacks, MET inhibition has produced benefit in molecularly defined subsets of individuals with lung cancer, such as those with MET protein overexpression (tivantinib trial subgroup analysis), high MET gene amplification, and METex14 skipping (case report collection and promising clinical trial data). MET inhibition continues to be an area of investigation in lung cancers of all types.(23-25) Crizotinib, cabozantinib, capmatinib, tepotinib, glesatinib, merestinib, and savolitinib are all in phase II trials as MET inhibitors.(26) Rilotumumab and ficlatuzumab are under investigation as monoclonal antibodies that target the MET ligand, HGF.(25, 26)</p><p>These studies have highlighted the complexities with MET as a potential biomarker. Analysis of only MET gene amplification by fluorescence in-situ hybridization misses some patients with METex14 splice site mutations and/or protein over-expression who might respond to therapy and vice versa.(27) To add to the complexity, increased HGF ligand can over-activate MET signaling and may decrease MET receptor protein due to feedback inhibition; yet, these patients may still respond to monoclonal antibodies that target HGF.(28)</p><p>MET protein demonstrates variable expression in normal lung with heterogeneous expression in most lung tumors.(29) The staining, scoring, and positivity cut-offs for MET are not standardized and there has been a wide range of reported positivity rates for MET expression from 20% to 80% in different studies.(1, 2, 30) Given the intense investigation of MET as a potential biomarker with predictive utility for improved outcomes in the context of the use of MET-targeted therapies, it is important that pathologists refine the evaluation of cancer specimens for MET expression.</p><p>This need for better standardization became apparent for our Lung Cancer Mutation Consortium 2.0 (LCMC 2.0) pathologist group when, at a monthly teleconference early in the LCMC study (February 2014), the MET protein expression positivity rates for the first 150 LCMC specimens at 4 sites ranged from 27% to 83%. This wide range of positivity findings between sites propelled the initiation of a quality assurance effort with a series of "Round Robin" tests, a series of tests that are performed independently by separate laboratories several times with analyses of variance. In the LCMC 2.0 pathologist Round Robin effort, 7 pathologists performed a series of 3 rounds of evaluation of 83 total cases, with analysis and discussion between rounds. This endeavor improved the consistency of MET scores between pathologists.</p><!><p>LCMC 2.0 was a collective effort by 16 cancer centers across the United States to study genetic and protein biomarkers in patients with stage IV or recurrent lung adenocarcinomas (31). Eligibility requirements for this study were Eastern Cooperative Oncology Group performance status of 0, 1, or 2, expected survival of more than 6 months, no prior treatment with targeted therapy, diagnosis of metastatic disease after May 1, 2012, and adequate tissue for molecular analyses. Of 1367 enrolled patients, 1009 patients were confirmed to have both a diagnosis of adenocarcinoma and adequate pathologic material for continuation in the biomarker study. Of 904 patients for whom at least 1 biomarker was assessed, 838 were successfully stained by MET IHC. All sites obtained local Institutional Review Board approval and patient consent for participation in this study. The overall findings from LCMC 2.0 are published separately.(32)</p><!><p>Formalin-fixed paraffin-embedded (FFPE) tumor blocks were collected from patients enrolled in the LCMC 2.0 biomarker study. Lung adenocarcinoma diagnosis and specimen adequacy were confirmed for each specimen by evaluation of hematoxylin and eosin (H&E) stained slides by 2 pathologists. Unstained slides from FFPE specimens were stained for MET protein expression with the optimized prediluted CONFIRM anti-Total c-MET (SP44) rabbit monoclonal primary antibody by Ventana Medical Systems (Tucson AZ, Catalog number 790-4430), as described in the Ventana product library (productlibrary.ventana.com, search "790-4430"). In short, FFPE tumor tissue blocks were cut at 4 μm thickness and placed onto positively charged slides, deparaffinized, placed on a Ventana Benchmark XT autostainer, hybridized with the primary SP44 antibody, analyzed with the Ventana ultraview universal DAB detection kit, stained with hematoxylin and bluing reagent, and cover-slipped. The conditions for this general protocol were optimized at each participating LCMC 2.0 site.</p><p>Slides were scanned at 40X power with an Aperio AT2 scanner at the University of Colorado Denver Biorepository. The Aperio AT2 utilizes an LED light source with a cold white temperature (CCT of 5700K), for a natural white illumination of the specimen. The AT2 also utilizes an ICC color correction profile to maintain digital slide image color as close as possible to what can be viewed under the microscope. The standard image format of the Aperio AT2 is SVS, which is a standard pyramid tiles TIFF with JPEG2000 image compression. Compression quality was set to produce a 30:1 JPEG2000 image compression. The pathologists accessed images on the Spectrum™ Web Service, a shared biorepository imaging website hosted by the University of Colorado. Pathologists used ImageScope software downloaded on their on-site computers to view the images.</p><!><p>Pathologists at multiple sites received specialized training and written guidelines for MET scoring prior to scoring. MET protein expression was evaluated for the percentage of tumor cells with no, faint, moderate, or strong staining. A cut point of at least 50% of tumor cells with moderate-to-strong MET staining for a final diagnosis of positive or negative was used as defined by the "original" MetMab clinical trial diagnostic criteria (20, 27). Scores were also calculated with the histology score (H-score), a semi-quantitative calculation in which tumor cells with stronger staining are given more weight in the assessment of the overall protein expression of the tumor as follows:</p><p>H-score = 1 × (% of faintly stained tumor cells) + 2 × (% of moderately stained tumor cells) + 3 × (% of strongly stained tumor cells).(27) Thus, the H score ranged from 0 to 300.</p><!><p>A nested random-effect model was used to evaluate the reproducibility of the MET protein expression technique. This statistic is based on analysis of variance models and describes how strongly quantitative measurements between multiple observers resemble each other.(33) The intraclass correlation coefficient (ICC) is calculated by the formula ICC=σinter2∕(σinter2+σintra2), where σinter2is the variance between specimens and σintra2 is the pooled within-specimen variance. For each round of protein expression scoring, overall group and individual pathologists' scores were compared with ICC analysis. The ICC has a range from 0 (no correlation) to 1 (perfect correlation). The interobserver agreement was interpreted as follows: < 0.2, "poor"; 0.2 to 0.39, "fair"; 0.40 to 0.59, "moderate"; 0.60 to 0.79, "good"; and 0.80 to 1.00, "very good."(33)</p><!><p>An exploratory round of investigation was prompted by the discovery of a wide range of MET protein positivity rates, 27% to 83%, between LCMC 2.0 study sites. During a preliminary internal investigation, cases scored as "positive" (n = 66) at the University of Colorado were reviewed to identify "borderline positive" cases arbitrarily defined as those called "positive" (>50%), but with ≤ 70% tumor cells with moderate-to-strong staining and/or an H score ≤ 200. Thirty-nine percent (26/66) of positive cases fit into this "borderline positive" category. Out of these 26 "borderline positive" cases, 5 cases were randomly selected for whole slide digital scanning and rescoring of the cases by 6 of the multi-institutional LCMC pathologists (Figure 1, A-E).</p><p>The percentages of tumor cells with moderate-to-strong staining for the 5 cases as scored by the 7 pathologists were compared (Figure 2A). Only one case was concordantly determined to be positive by all 7 pathologists. Two cases were scored as "positive" with moderate-to-strong staining in ≥ 50% of tumor cells by 6 of the 7 pathologists; 1 case was scored as positive by 3 pathologists and negative by 4; and one case with an original score of 55% was scored as negative by the 6 additional pathologists. An H-score system with a cut-off of ≥ 150 for positivity showed a similar pattern of concordance for the same cases (Figure 2B).</p><p>The LCMC pathologists discussed the results by teleconference and concluded that although it was still possible that differences in preanalytical factors, such as specimen processing and staining, could contribute to the variable positivity rates between sites, there was enough interobserver variability from pathologist scoring of the same images during this exploratory round to warrant further efforts to standardize scoring. It was also noted that MET heterogeneity inherently complicated scoring with most cases exhibiting a heterogeneous staining pattern of MET, that is, highly variable tumor-staining intensities in different tumor areas. All 7 pathologists agreed to score and discuss more cases as a "Round Robin" quality assurance project.</p><!><p>Provide matched H&E slide images for the next round of MET image scoring to facilitate tumor cell confirmation.</p><p>Keep the MET-scoring training document open for reference during scoring.</p><p>Provide example MET IHC images of Round 1 cases with concordant results between pathologists for "faint", "moderate", and "strong" staining (Figure 4, A-I).</p><p>Perform final scoring at low power (10X objective).</p><p>Self-adjust scoring higher or lower for consistency with the group positivity rate from Round Robin 1.</p><!><p>For Round Robin 2, 40 cases not included in Round Robin 1 were randomly selected for scoring by the 7 LCMC pathologists; 1 of these cases was excluded from analysis due to poor image quality. The average, lowest, and highest scores for each case are depicted in Figure 5. Statistical analysis revealed "good" agreement with an ICC of 0.74 (95% confidence interval, 0.64-0.84). The average MET positivity rate was 66% (range, 46%-92%) and the average H-score was 173 (range, 43-280).</p><p>The overall average MET H-score for all 78 cases from Round Robins I and II was 165 (H-score range, 43-280). The average H-score was < 125 for 14 specimens, 125 to 175 for 35 specimens, and > 175 for 29 specimens. A comparison of the individual pathologists' ICCs demonstrated improved individual scoring consistency for all 7 pathologists between rounds, with improvement from "good" consistency with an average ICC of 0.64 (range, 0.43-0.76) for the first round to "very good" consistency with an average of 0.82 (range, 0.75-0.93) for the second round. During the pathologist teleconference to discuss the results, the ability to improve scoring consistency with communication was appreciated, but with the caveat that the heterogeneous staining pattern of MET remained a cause of inherent difficulty for scoring standardization.</p><!><p>Pathologists have the training and expertise to develop and refine the criteria to improve interobserver reproducibility of MET IHC evaluation. Scoring of protein expression on MET IHC-stained slides involves both the "art" of observing what is seen on the slide and the "science" of recording what is observed in a semi-quantitative manner. There have been other efforts for standardization of IHC evaluation of other proteins with initiatives lead by pathologists to collectively determine reliable criteria and practical metrics for interpretative concordance amongst pathologists (34); however, MET IHC evaluation has been notoriously challenging with an exceptionally high variation of positivity rates between studies.(1, 2, 30)</p><p>The collective experience of the LCMC 2.0 pathologists in the MET-scoring exercise has highlighted the importance of direct and continuous communication between pathologists when studies involve slide evaluation by multiple participants. With a Round Robin effort and good communication between the LCMC 2.0 pathologists, the overall group intraclass correlation coefficient improved from "moderate" (0.50) agreement for the first scoring round to "good" (0.74) agreement for the second round. Although the efforts to improve interobserver concordance in scoring were successful, standardized scoring of MET may continue to be inherently challenging due to its heterogeneous expression which complicates setting an accurate cut-off for positivity.(35)</p><p>This pathologist-driven effort to standardize scoring for MET has corroborated the conclusions of other studies that have emphasized the critical importance of communication among pathologists to achieve standardized scoring. Efforts to standardize scoring of other lung cancer receptor proteins, such as EGFR (34) and ALK (36), have also benefited from efforts to enhance direct communication between pathologists. Programmed death ligand-1 (PD-L1) is another heterogeneous lung cancer protein with clinical relevance for which harmonization of scoring is in progress, such as with the PD-L1 Blueprint endeavor.(37) The pathologist-initiated quality assurance project described here to standardize scoring of MET serves as a model for the use of "Round Robin" conferences to improve the evaluation and development of any potential biomarker.</p><p>In this study, scores from glass slides were not compared to scores from digital images due to the complexities associated with multiple pathologists performing both scores and the introduction of other variables between the scoring of the glass slides and digital images, such as training to improve the standardization of scoring. These complexities could complicate any analyses of differences between scores. However, a future study could be performed with a side-by-side comparison of the matched glass slides and digital images, with a defined wash out period between scoring rounds. Furthermore, an approach that incorporates machine learning to explore its potential as an adjunct tool for the standardization of MET scoring could be performed with this same set of cases. Previous studies have corroborated the effectiveness of machine learning–based scoring of IHC. (38-42).</p><p>With clinical trials in progress, evidence is growing for MET genomic biomarkers, such as MET gene amplification (43) and METex14 splice-site mutations (9), to predict response to MET inhibitor therapy. Evaluation of MET protein expression and assessment of downstream signaling may play a role in determining which patients with MET genomic aberrations will most likely benefit from MET-targeted therapy.(22) The practical implementation of standardized MET IHC evaluation for protein expression by pathologists sets the foundation for advancement of MET as a biomarker.</p><p>To date, only limited studies are available in the literature that investigate the correlation between MET IHC expression and MET genomic sequencing data.(44, 45) The utility of MET IHC will likely ultimately be dependent upon the extent to which protein expression is able to reliably complement NGS findings. Future studies to refine the standardization of MET IHC scoring should be performed with an appropriate volume of cases, with routine peer monitoring to ensure quality control. Moreover, it is important to correlate MET IHC scoring with findings from genomic sequencing in the context of clinical outcomes with MET-targeted therapy.</p>

PubMed Author Manuscript

DNA 5-Methylcytosine-Specific Amplification and Sequencing

DNA 5-methylcytosine (5mC)-specific mapping has been hampered by severe DNA degradation and the presence of 5-hydroxymethylcytosine (5hmC) using the conventional bisulfite sequencing approach. Here, we present a 5mC-specific whole-genome amplification method (5mC-WGA), with which we achieved 5mC retention during DNA amplification from limited input down to 10 pg scale with limited interference from 5hmC signals, providing DNA 5mC methylome with high reproducibility and accuracy.

dna_5-methylcytosine-specific_amplification_and_sequencing

<p>DNA 5mC methylation, as the main postreplication epigenetic mark in mammals, plays crucial roles in various biological pathways including gene expression,1 developmental regulation,2 and tumorigenesis.3 Insights into the functionality of DNA methylation mainly rely on the gold standard bisulfite sequencing (BS-seq), which allows single-base resolution mapping of 5mC with quantification of the methylation level at each site.4,5 However, the bisulfite treatment is harsh and degrades most DNA.6 Bisulfite-free methods have been developed in recent years to avoid severe DNA degradation.7 Alternatively, a method that can faithfully amplify the 5mC pattern during DNA amplification would be highly desirable for subsequent sequencing such as BS-seq or loci-specific detection approaches. In addition, the conventional BS-seq cannot discriminate 5-hydroxymethylcytosine (5hmC), the oxidative product of 5mC, from the sole 5mC methylation, which restrains studies of DNA methylation dynamics.</p><p>Oxidative bisulfite sequencing (oxBS-seq) has been developed for specific 5mC methylome mapping,8 but its application has been hampered by severe DNA degradation under the oxidative conditions and cannot be applied for limited input samples. Genome-wide mapping of 5hmC, when combined with bisulfite sequencing for analysis, also makes the 5mC-specific detection possible.9,10 However, 5hmC exists in lower abundance and can be highly dynamic. Simple subtraction of 5hmC signals from the whole-genome bisulfite sequencing may introduce additional variations into the DNA 5mC detection. Because all these methods still rely on bisulfite treatment, which degrades most DNA, 5mC-specific sequencing or detection from limited input is still a challenge.</p><p>DNA methylation is maintained by three canonical DNA methyltransferases, namely, DNMT1, DNMT3A, and DNMT3B. While DNMT3A and DNMT3B are responsible for the de novo methylation, DNMT1 installs methyl groups donated by S-adenosylmethionine (SAM) on cytosines that are located on hemimethylated CpG sites during DNA replication.11 Inspired by the conservative methylation regulation during DNA replication, we developed a whole-genome amplification method with specific retention of 5mC starting from a limited input amount, a conceptually "simple" but potentially highly useful approach by preamplifying the limited DNA sample to a higher abundance with retention of 5mC, which we named 5mC-retained whole-genome amplification (5mC-WGA). Amplified products are compatible with most downstream detection methods, with 5mC modification replicated faithfully during the amplification. Followed with the standard bisulfite sequencing, our method can identify 5mC sites with limited interference from 5hmC, as DNMT1 cannot replicate 5hmC during our isothermal amplification.</p><p>Our design started with the incorporation of the strand displacement amplification with the DNMT1 methylation (Figure S1a,b). Isothermal DNA amplification allows DNMT1 to work under its optimal temperature.12 We started our tests with human brain genomic DNA with well-defined CpG methylation information already available. With an optimized combination of DNA polymerase phi29 and DNMT1, we successfully amplified input DNA by at least 100-fold (Figure S2), ranging from 10 ng down to 10 pg with 5mC marks retained faithfully (Table S1). We picked both a known 5mC hypermethylated region (a locus in NFATC1) and a nonmethylated region (a locus in MAPK8IP2) for low-throughput validation13 (Figure S3a). The amplified products were subjected to bisulfite treatment, PCR amplification with predesigned primers, and Sanger sequencing. The results showed that 5mC-WGA amplified the hypermethylated sites with high accuracy but did not produce methylation within the nonmethylated region (Figure 1a). The amplified products were further subjected to MeDIP-seq (methylated DNA immunoprecipitation and sequencing) along with bulk control. The results again demonstrated faithful retention of the DNA methylation during amplification (Figures 1b and S3b).</p><p>We then proceeded to the high-throughput sequencing for more accurate validation of our method with amplified products starting from 10 pg, 100 pg, and 1 ng genomic DNA, respectively. We still detected expected retention of methylation when we amplified targeted loci. However, we did observe relatively low genome coverage with lower input down to 10 pg, which is also commonly observed with single-cell bisulfite sequencing. A careful analysis on the high-throughput data revealed that the decrease in genome coverage is mainly due to the biased priming with random hexamer during the strand displacement amplification. With primer-derived artifacts and sequence-dependent hybridization kinetics, amplification bias would be introduced when the input amount is limited.14 Therefore, to minimize priming bias, we incorporated a unique DNA primase into our amplification system. While most primases use NTPs as substrates to produce RNA primers, TthPrimPol uses dNTPs to synthesize DNA primers during polymerization. A recent study applied TthPrimPol in DNA amplification together with the highly processive strand displacement polymerase phi29 to initiate a true random priming process.15 Such combination accomplished near-complete whole-genome amplification with high reproducibility and superb genome coverage, which could partially solve the priming bias issue for 5mC-WGA.</p><p>We therefore performed preliminary tests on the primer-free system. Early trials showed that TthPrimPol, phi29, and DNMT1 work compatibly in our reaction buffer. With a welltuned amplification system and an optimal methylation condition (Figure 2), we can acquire similar 5mC-retained amplification verified by our low-throughput analysis on specific loci. We then proceeded to test the three-enzyme whole-genome amplification system.</p><p>We amplified 10 pg of genomic DNA purified from mES cells, and the products were subjected to bisulfite treatment followed by library construction, which altogether we named as 5mC-WGA-BS. We analyzed our sequencing results together with two control samples: one as 10 pg of gDNA without any treatment and the other amplified without DNMT1 under the same condition. When aligned to the bulk positive control, our amplified samples all showed methylation retention with a little lower methylation level compared to the direct bisulfite bulk control sample, while all CHG and CHH levels remained low (Figure 3a). Our negative control without DNMT1 yielded a quite low methylation level, which confirmed that DNMT1 is responsible for methylation maintenance during amplification. The GC content bias analysis did not show any significant bias toward certain GC contents, suggesting a universal amplification of 5mC-containing regions. Compared with the standard commercial library construction kits, our method did not incorporate bias during amplification (Figure S4). Hierarchical clustering based on CpG methylation levels revealed high reproducibility among our replicate libraries generated from low inputs (Figure 3b). The high reproducibility was also verified by the high correlation among three different samples compared with one another (Figure 3c). The well-overlaid methylation metagene plots showed a typical methylation pattern where TSS regions show low methylation while gene body regions enrich DNA methylation (Figure 3d). In contrast to the random-hexamer-assisted amplification we used at the beginning, the primer-free system achieved higher specificity and reliability in 5mC retention (Figure S5).</p><p>To examine class heterogeneity, we analyzed all unmethylated, methylated, and hydroxymethylated C sites derived from 5mC-WGA-BS, oxBS-seq, and TAB-seq (a direct 5hmC readout method). The oxBS-seq data were generated from our same input, while TAB-seq data were acquired from a published data set.16 All methods were analyzed along with bisulfite sequencing from bulk input to simultaneously estimate methylation levels and hydroxymethylation levels by extracting information from both BS-seq and 5mC-WGA-BS, or oxBS-seq, or TAB-seq based on maximum likelihood methylation levels (MLML).17 While our method showed a similar pattern to that of oxBS-seq, TAB-seq demonstrated a different trend as expected (Figure 4a). Our method demonstrated a higher correlation with oxBS-seq in 5mC detection than TAB-seq as expected. In addition, subtraction of whole-genome bisulfite sequencing with 5mC-WGA-BS also detects most 5hmC sites (Figure S6). 5mC-WGA-BS showed a high correlation with oxBS-seq in 5mC spotting (Figure 4b). 5hmC tends to exist at lower abundance than 5mC and marks more dynamic 5mC sites. All the replicates showed a high correlation to detected 5hmC sites (Figure S7a), confirming 5hmC as a derivative of 5mC. In addition, because of the known enrichment of 5hmC on gene bodies, we did notice a decrease in the 5mC methylation level detected using 5mC-WGA-BS compared to that of the conventional bisulfite sequencing (Figure 4c). The 5hmC sites revealed from 5mC-WGA-BS (subtraction from conventional BS-seq, see Supporting Information for details) showed high correlation with results from 5hmC-Seal that captures 5hmC-containing DNA fragments (Figure S7b). Examples are plotted to show results from different approaches, confirming that 5mC-WGA-BS faithfully uncovers 5mC sites and can help extract 5hmC information (Figures 4d and S8).</p><p>This 5mC-WGA-BS method works well with 10 pg of isolated genomic DNA. Preliminary tests using five cells also showed the retention of 5mC as expected, with CHG and CHH levels remaining low (Figure S9a). The metagene plots demonstrated a similar pattern when the input was amplified in the presence of DNMT1 but no methylation pattern when amplified without DNMT1 (Figure S9b). The ternary plot and the correlation analysis with oxBS-seq both corresponded to results from the bulk genomic DNA libraries (Figure S9c,d). Our trials at the single-cell level suggested a requirement of a further optimized lysis condition to eliminate potential genomic DNA degradation but still ensure full denaturation of the chromatin to be ready for amplification. Perhaps a microfluidics device or other procedures could help in the future. Because phi29 effectively amplifies only long DNAs, the method currently could be employed only to genomic DNA rather than synthetic probes or short DNA fragments.</p><p>With our high-throughput results, we also investigated the methylation activity of DNMT1 in the presence of opposite CpG or 5hmCpG. DNMT1 was reported to exhibit an enzymatic methylation activity opposite to CpGs, which is about 1/10 of that opposite to 5mCpGs and around 1/3 opposite to 5hmCpGs compared with that opposite to 5mCpGs.18 These results are consistent with the percentages of the potential reactive sites for methylation based on our analyses. However, the interference on quantitative 5mCpGs detection is quite limited, as we only observed low methylation levels that stemmed from these activities (Figure S10a,b). While potential de novo sites account for about 10% of the detected methylated sites, they tend to be low in their methylation levels. Thus, the interference from de novo methylation is limited to lower than 5% (Figure S10a). Our method showed little activity with CpH methylation, especially when compared with CpG methylation (Figure S10c). All these analyses suggested that our approach is a reliable 5mCpG-specific detection method for limited input materials. Future studies may further elucidate potential activities of DNMT1 at unmethylated CpGs, 5hmCpGs, and CpHs.</p><p>In conclusion, we present a 5mC-specific whole-genome amplification system for simultaneous DNA amplification and methylation in a one-pot, primer-free reaction. The amplified products could be subjected to bisulfite sequencing or other detection platforms for faithful methylome mapping or detection using limited input materials. This methylation-retained amplification approach could enable facile detection of 5mC in clinical samples such as DNA from biopsy or cell-free DNA in plasma in the future.</p>

PubMed Author Manuscript

A QSTR-Based Expert System to Predict Sweetness of Molecules

This work describes a novel approach based on advanced molecular similarity to predict the sweetness of chemicals. The proposed Quantitative Structure-Taste Relationship (QSTR) model is an expert system developed keeping in mind the five principles defined by the Organization for Economic Co-operation and Development (OECD) for the validation of (Q)SARs. The 649 sweet and non-sweet molecules were described by both conformation-independent extended-connectivity fingerprints (ECFPs) and molecular descriptors. In particular, the molecular similarity in the ECFPs space showed a clear association with molecular taste and it was exploited for model development. Molecules laying in the subspaces where the taste assignation was more difficult were modeled trough a consensus between linear and local approaches (Partial Least Squares-Discriminant Analysis and N-nearest-neighbor classifier). The expert system, which was thoroughly validated through a Monte Carlo procedure and an external set, gave satisfactory results in comparison with the state-of-the-art models. Moreover, the QSTR model can be leveraged into a greater understanding of the relationship between molecular structure and sweetness, and into the design of novel sweeteners.

a_qstr-based_expert_system_to_predict_sweetness_of_molecules

Introduction<!><!>Introduction<!>Experimental dataset and data curation<!><!>Experimental dataset and data curation<!>Molecule representation<!>Multidimensional scaling<!>Classification models<!>Partial least squares discriminant analysis (PLSDA)<!>N-nearest neighbors (N3)<!>Reduction and selection of molecular descriptors<!>Model validation<!>Software<!>Clustering sweet and non-sweet chemicals<!><!>Clustering sweet and non-sweet chemicals<!><!>Clustering sweet and non-sweet chemicals<!>QSTR models based on molecular descriptors<!><!>QSTR models based on molecular descriptors<!><!>QSTR models based on molecular descriptors<!>Assessment of the QSTR-based expert system<!><!>Assessment of the QSTR-based expert system<!><!>Assessment of the QSTR-based expert system<!><!>Applicability domain assessment<!><!>Applicability domain assessment<!>Comparison and final discussion of the classification performance<!>Author contributions<!>Conflict of interest statement

<p>Taste chemistry has become an important field of research for many disciplines, especially food chemistry. In fact, there exists a keen interest in research related to taste perception, since developments in molecular biology and biochemistry have provided the background for sweet-taste chemistry. Taste evocation is the result of soluble chemicals with different osmotic, endothermic and exothermic properties that interact with biological membranes on the taste buds on the tongue in different ways. Thus, the different tastes could be separated on the basis of the nature of such reactions; however, the mechanisms of how these interactions occur are not completely elucidated. Five accepted basic tastes exist: sweetness, bitterness, saltiness, sourness, and umami (also known as savory; Damodaran et al., 2008).</p><p>Li et al. (2002) described for the first time the sweet taste chemoreceptor, which is a G protein-coupled receptor (GPCR) constituted by the T1R2 and T1R3 subunits. This sweet chemoreceptor is able to recognize sweet stimuli produced by distinct sweeteners, such as carbohydrates, artificial sweeteners, amino acids, peptides, and proteins. Subsequently, Morini et al. (2011) proposed the use of the term "receptor-mediated taste" instead of "basic taste" due to the fact that the tastes are sensed by means of specific receptors and other mechanisms not necessarily mediated by the receptors. Thus, the human perception of these tastes varies from person to person, and it may be related to slight differences in psychology, anatomy, receptor function, concentration of the taste, or interaction with other substances.</p><p>Among the receptor-mediated tastes, sweetness is considered as the most important in a wide variety of foods, since it produces a pleasant sensation. Sucrose, the most common sweetener, is used as the international standard for measuring the sweetness of chemical compounds. Sucrose imprints a clean-sweet sensation without other aftertastes even at high concentrations, and it is obtained from economic renewable sources (sugar cane and sugar beets). Unlike the sweet taste, bitterness is usually perceived as an unpleasant receptor-mediated taste, although in some products such as tea, cocoa, coffee, beer, tonic water, or olives, the bitter taste is considered desirable. In this case, the quinine alkaloid is used as the standard for measuring relative bitterness. It is frequently used as a food additive. Finally, tastelessness could be defined as the lack of taste (insipid) or the loss of a perceived taste (e.g., sweet, bitter, sour, salty; Damodaran et al., 2008). Since both diabetic/dietetic medicines and foods should contain low-calorie sweeteners, preferably with a clean-taste, the pharmaceutical and food industries deal with the rational design and synthesis of potential compounds to be used as alternative sweeteners (Damodaran et al., 2008; Morini et al., 2011).</p><p>During the synthesis of new sweeteners, some variations in the chemical structure of a scaffold may change sweet molecules to non-sweet chemicals (tasteless, bitter, sour, and salty; Damodaran et al., 2008). In order to deal with this problem, scientist have been using approaches based on the Quantitative Structure-Activity/Property Relationships (QSAR/QSPR) to predict the sweetness of compounds to be synthesized. The QSAR/QSPR theory is an effective tool to build mathematical relationships between activities/properties of substances and their chemical structures, which is encoded by means of molecular descriptors (Todeschini and Consonni, 2009). Several Quantitative Structure-Taste Relationships (QSTRs) for predicting the sweetness of chemicals were proposed in the past years and are summarized in Table 1. The earlier work included compounds such as perillartine and aniline derivatives (Iwamura, 1980; van der Wel et al., 1987), sweet and bitter aldoxime derivatives (Kier, 1980), perillartine derivatives, aspartyl dipeptides, and carbosulfamates (Takahashi et al., 1982, 1984; Miyashita et al., 1986a,b; Okuyama et al., 1988), as well as sulfamate derivatives (Spillane and McGlinchey, 1981; Spillane et al., 1983, 1993, 2000, 2002, 2003, 2006, 2009; Spillane and Sheahan, 1989, 1991; Drew et al., 1998; Kelly et al., 2005). Moreover, two QSTR models to discriminate sweet, tasteless and bitter compounds have been proposed (Rojas et al., 2016a). Recently, Chéron et al. (2017) performed a predictive model for the discrimination of sweet and bitter molecules and the subsequent use of sweet compounds for predicting their relative sweetness (RS) property. In addition, some other recent studies remark the importance of the conformation-independent QSPR models for predicting the RS of sweet molecules (Rojas et al., 2016b; Ojha and Roy, 2017). Additionally, several recent scientific reviews regarding the applications of QSTRs are also available (Walters, 2006; Spillane and Malaubier, 2014; Rojas et al., 2016c).</p><!><p>Summary of the performances of the QSTR classification models reported in the literature for predicting sweet taste of molecules.</p><p>CART, classification and regression tree; d, number of descriptors; DA, discriminant analysis; kNN, k-nearest neighbors; LDA, linear discriminant analysis; LLA, linear learning machine; Ntrain, number of molecules in the training set; Ntest, number of molecules in the test set; SIMCA, soft independent modeling by class analogy; QDA, quadratic discriminant analysis; RF, Random Forest; SLR, simple linear regression.</p><p>Not available.</p><p>Calculated as the ratio of correctly classified molecules to the total number of molecules (Accuracy).</p><p>Number of components for SIMCA analysis.</p><p>Number of components considering for the DA analysis.</p><!><p>The purpose of the work presented here was to build a QSTR-based expert system for the prediction of sweetness using a dataset of 649 molecules (435 sweet, 133 tasteless, and 81 bitter chemicals). To the best of our knowledge, this is the largest database of sweet chemicals ever used for predicting the sweetness of substances. The proposed expert system combines a structural similarity analysis and two QSTR models. Similarity structural analysis is based on extended-connectivity fingerprints (ECFPs), while the QSTR models are based on molecular descriptors (MDs) and N3 (N-nearest neighbors) and PLSDA (partial least squares discriminant analysis) classifiers. The proposed QSTR-based expert system was developed keeping in mind the five principles defined by the Organization for Economic Co-operation and Development (OECD) to make it applicable (OECD, 2007). The predictive ability of the model was properly evaluated by means of appropriate internal and external validation procedures. In addition, the chemical information of the molecular descriptors included in the QSTR models was interpreted and the model applicability domain properly defined.</p><!><p>Each chemical compound can be experimentally associated with a predominant taste such as sweet, bitter, sour, and salty standards by trained panelists using a sip and spit method (Spillane et al., 1993, 2009). The initial experimental database, which is named TastesDB, was comprised of 727 chemicals retrieved from several scientific publications (refer to Table S1 for details of these publications). Each substance was associated with an experimental taste class (sweet, tasteless, or bitter). In this study, the tasteless and bitter categories were merged into a general non-sweet class, because the major scientific interest was in the identification of sweet compounds rather than bitter or tasteless chemicals. In fact, several studies on sweetness and taste have been conducted to discover and describe natural and synthetic sweeteners, sweetness potentiators and bitter blockers, to propose methods for characterizing different aspects of consumers' perception of sweetness. These perceptions are crucial aspects to be considered in order to improve the flavor, sweetness, texture, appearance, and physical properties in the development of food products (Damodaran et al., 2008).</p><p>The dataset was curated to remove molecules associated with wrong or problematic molecular structures, according to the following steps:</p><!><p>Pentadin, thaumatin, monellin, curculin, miraculin, brazzein, and mabinlin sweet proteins, were removed;</p><p>Disconnected molecular structures (salts), such as tripotassium glycyrrhizinate or aspartame-acesulfame salts, were retained;</p><p>For each molecule, the canonical Simplified Molecular Input Line Entry System (SMILES) strings were obtained from the designed molecular structure;</p><p>Tasteless and bitter classes were merged into a non-sweet class, as we wanted to focus on the prediction of sweetness vs. non-sweetness;</p><p>Stereoisomers belonging to different taste classes (ambiguous molecules) were excluded (e.g., D-Arginine and L-Arginine, which are experimentally sweet and bitter compounds, respectively).</p><p>Amongst sweet molecules with the same SMILES strings, only one was retained (e.g., maltose and lactose).</p><!><p>The curated TastesDB dataset consisted of 649 molecules divided into two subsets of 435 sweet and 214 non-sweet (133 tasteless and 81 bitter) compounds, respectively (Table S1). QSTR studies regarding the prediction of the sweetness receptor-mediated taste were conducted by considering only homogeneous families of sweeteners (Iwamura, 1980; Kier, 1980; Spillane and McGlinchey, 1981; Takahashi et al., 1982, 1984; Spillane et al., 1983, 1993, 2000, 2002, 2003, 2006, 2009; Miyashita et al., 1986a,b; van der Wel et al., 1987; Okuyama et al., 1988; Spillane and Sheahan, 1989, 1991; Drew et al., 1998; Kelly et al., 2005), limiting their ability to predict the sweetness of other kinds of sweeteners. In order to generalize the predictiveness of the QSTR-based expert system, we used a dataset that covered a large chemical space of both sweet and non-sweet molecules. For example, derivatives of sucrose, abruside, acesulfame, isovanillic, mogroside, periandrin, saccharin, rebaudioside, cyclamate, suosan, aspartame, aspartyl dipeptides, glycyrrhizin, as well as several other heterogeneous compounds were included.</p><!><p>Structural characteristics of molecules were represented by means of both binary fingerprints and molecular descriptors. Binary fingerprints provide a holistic view of the molecular structure in terms of the presence/absence of identified molecular fragments. In particular, ECFPs (Rogers and Hahn, 2010) were used to represent molecular structures taking into account the information of the circular atom neighborhoods. ECFPs can be rapidly calculated and capture the common structural features of molecules by representing the presence/absence of particular substructures in a binary manner. For each molecule, a binary vector with 2,048 bits was calculated by using 2 bits per structural pattern and a maximum pattern length of 2.</p><p>In addition, classical molecular descriptors (MDs) were calculated, which are numbers that encode specific chemical/structural information of molecules (Todeschini and Consonni, 2009). The calculation of molecular descriptors on disconnected structures has been widely studied during the last years (Mauri et al., 2016). In the study presented here, the Dragon 7 approach (Kode srl, 2016) has been chosen, which consists of the application of the original definition and algorithm of the considered descriptors. If the original algorithm cannot be directly applied on disconnected structures, the Dragon approach provides a modification of the descriptor's original definition to allow the calculation since such modification is consistent with the theoretical sense of the descriptor.</p><p>In both cases, a two-dimensional molecular representation was selected instead of a geometrical representation to avoid irreproducible 3D structure optimizations. 3D descriptors could add valuable chemical information; however, since they require the geometrical optimization of molecules, the descriptor values can be affected by differences between 3D conformers with similar energies (Pearlman, 1998). In addition, the search of the minimum in the conformational energy hypersurface of molecules by means of an adequate optimization method involves high computational costs and long time. For this reason, the use of a conformation-independent molecular representation emerges as an alternative when dealing with the prediction of the sweetness and the relative sweetness (Rojas et al., 2016a,b; Chéron et al., 2017; Ojha and Roy, 2017).</p><!><p>Multidimensional scaling (MDS; Kruskal, 1964) is a well-known multivariate method for unsupervised data exploration. MDS reconstructs similarities/dissimilarities between pairs of molecules by projecting data in a reduced hyperspace. In this way, data interpretation is facilitated. After the selection of a suitable number of dimensions to consider, a scatter plot of molecules provides a visual representation of the projected distances and can be used to analyze the relationships between chemicals as well as to identify clusters.</p><!><p>Since sweetness is a qualitative discrete response, classification approaches were used to establish mathematical relationships between the chemical/structural features of molecules and the modeled classes (sweet and non-sweet).</p><!><p>PLSDA (Wold et al., 2001) is a well-known classifier that combines the properties of partial least squares regression (PLS2-based method) with the linear discrimination capability of a classification technique. In brief, this analysis finds relationships between the matrix of molecular descriptors and the class vector by calculating latent variables (LVs), which are orthogonal linear combinations of the original variables (descriptors). When dealing with PLSDA, molecular descriptors were autoscaled.</p><!><p>The recently proposed N3 classifier (Todeschini et al., 2015) is based on local molecular similarities. Thus, a molecule is classified by taking into account the class to which the most similar molecules (i.e., neighbors) belong. The neighbor contribution is weighted by the molecule similarity rank, whose role is modulated by an alpha parameter to be optimized. Range scaling and the average Euclidean metric were used when dealing with the N3 classifier.</p><p>The optimal number of latent variables (PLSDA) and the alpha parameter (N3) were optimized according to the lowest classification error in cross-validation.</p><!><p>The V-WSP unsupervised variable reduction method (Ballabio et al., 2014) was used to reduce the presence of multicollinearity, redundancy, and noise in the initial pool of molecular descriptors. This method is a modification of the algorithm proposed by Wootton, Sergent, and Phan-Tan-Luu (WSP) for the selection of a subset of well-distributed points for design of experiments (DOE). In brief, V-WSP selects a subset of descriptors from the pool of candidates, in such a way as to have a minimal correlation from each descriptor in the defined multidimensional space. In addition, one of the fundamental steps of QSAR studies is the supervised selection of descriptors in order to build a parsimonious and predictive model based on a subset of informative descriptors. To this end, the Genetic Algorithms-Variable Subset Selection (GA-VSS) technique (Leardi and González, 1998) was coupled with both PLSDA and N3 classification methodologies in order to find the optimal subset of molecular descriptors. The essence of the GA-VSS is to start from an initial random population of chromosomes (i.e., models), which are binary vectors indicating the presence or absence of a given descriptor within the model. Then, an evolutionary process is performed and new chromosomes are generated by combination of chromosomes of the initial population (crossover) and/or random inclusion/exclusion of variables (mutation). If the new models have a reduced classification error, they are included in the population of chromosomes at the expenses of the worst ones, which are discarded.</p><!><p>Models were validated by means of an external test set constituted by 30% of the total number of molecules. Since the initial dataset was populated by a significant number of sweet substances, test molecules were randomly selected by maintaining the class proportion. Thus, the training set included 488 molecules (327 sweet chemicals and 161 non-sweet chemicals) and the test set was comprised of the remaining 161 molecules (108 sweet chemicals and 53 non-sweet chemicals). This partition guaranteed similar representation of the modeled classes. Training molecules were used for the supervised selection of molecular descriptors and the calibration of the QSTR-based expert system, while test molecules were used only to evaluate its prediction ability. A cross-validation protocol based on five cancelation groups divided in venetian blinds was used during the GA-VSS procedure (Ballabio and Consonni, 2013). The QSTR-based expert system was further validated by Monte Carlo (leave-many-out) random sub-sampling validation (Krakowska et al., 2016). The Monte Carlo approach defines many subsets by drawing samples in a random way from the available classes, based on a chosen number of iterations. Therefore, in each iteration, molecules were randomly divided into training (80%) and evaluation (20%) sets. The QSTR-based expert system was calibrated each time on the training molecules and then used to predict the class of evaluation molecules. The performance of the Monte Carlo validation was finally assessed by comparing the cumulative predictions vs. the experimental classes of test molecules.</p><p>Quality of the classification models was evaluated by means of sensitivity (Sn) and specificity (Sp) of classes (Ballabio and Consonni, 2013). Sensitivity of the sweet class was calculated as the ratio of the number of sweet compounds correctly classified to the total number of sweet compounds, while the specificity of the sweet class was calculated as the ratio of the number of non-sweet compounds correctly classified to the total number of non-sweet compounds. Since it is a two-class problem, the sensitivity of the sweet class corresponds to the specificity of the non-sweet class and vice versa. In addition, the non-error rate (NER) was calculated as the average of sensitivity values of sweet and non-sweet classes (Ballabio and Consonni, 2013). NER was used instead of Accuracy (which is the ratio of correctly classified molecules to the total number of molecules) to better estimate classification performance in the presence of unbalanced classes; non-sweet molecules are in fact less represented and constitute the 33% of the total number of molecules only.</p><!><p>HyperChem software (Hypercube Inc.)1 was used for representing the molecular structure, and the SMILES strings were obtained by using Babel software (O'Boyle et al., 2011). Molecular descriptors and extended connectivity fingerprints were calculated by means of DRAGON version 7 (Kode srl, 2016), while data curation and filtering of the dataset were carried out by means of a KNIME workflow written by the authors (Berthold et al., 2008). Data analysis and model calculations were performed in a MATLAB environment (MathWorks)2. The V-WSP variable reduction toolbox (Ballabio et al., 2014) was used to perform descriptors reduction, the classification toolbox for MATLAB (Ballabio and Consonni, 2013) was used for model calibration and the PCA toolbox for MATLAB (Ballabio, 2015) was used for both multidimensional scaling and molecular descriptors analysis. Genetic Algorithms variable subset selection was performed in MATLAB by means of code written by the authors. Classification toolbox and PCA toolbox are available at the Milano Chemometrics and QSAR Research Group website (http://michem.disat.unimib.it/chm/download/softwares.htm).</p><!><p>The 488 training molecules were initially used to perform a structural similarity exploratory analysis based on their extended connectivity fingerprints. To this end, molecular similarities were quantified by means of the Jaccard-Tanimoto similarity coefficient (Jaccard, 1912) and used to produce a multidimensional scaling (MDS) of the dataset. Figure 1 presents the MDS scores of the first two coordinates.</p><!><p>MDS plot of the two first coordinates (explained variance equal to 69.85%) for the training set molecules. Sweet molecules are marked with blue circles, and non-sweet molecules are market with cyan circles.</p><!><p>Three clusters (S1, S2, and C3) were identified in the MDS space, corresponding to three groups of molecules with specific structural similarities. Cluster S1 was comprised of 143 sweet molecules (Table S2), which have a common scaffold, as represented in Figure 2. The main characteristic of this molecular scaffold is the presence of the aspartic amino acid. However, other sweet chemicals with the same scaffold, but also containing benzene rings, are located in cluster C3, such as aspartame and N-(L-aspartyl)-1,1-diaminoalkane 5, along with some special cases of aspartyl derivatives (e.g., super aspartame, cyanoarylurea aspartame, aspartic acid fenchyl ester, and aspartame-acesulfame salt). The 107 molecules grouped in Cluster S2 (Table S3) included 100 sweet compounds (e.g., sucrose) and just 7 non-sweet compounds, such as the 6-Chloro-6-deoxy-D-galactose (tasteless), as well as a limited number of molecules exhibiting bitter taste (e.g., picrocrocin, methyl-α-D-2,6-dideoxy-gluco-pyranoside, methyl-α-D-3,6-dideoxy-gluco-pyranoside, methyl-α-D-4,6-dideoxy-gluco-pyranoside, and solanine). Finally, the remaining 399 chemicals and, in particular, the majority of non-sweet compounds are grouped in cluster C3 (Table S4).</p><!><p>Common chemical scaffold of sweeteners grouped in cluster S1.</p><!><p>Since the structural similarity analysis provided a satisfactory grouping of chemicals in terms of their taste, a QSTR-based expert system was considered as a suitable strategy to optimize the discrimination of sweet and non-sweet molecules. This system was structured as follows: the first step consisted of the identification of the cluster associated with a target molecule, using the ECFP-based structural similarity analysis; for example, if the molecule was assigned to cluster S1 or S2, it was likely to be predicted as sweet molecule. The second step consisted of the application of the QSTR models based on specific molecular descriptors which were calibrated using molecules included in cluster C3 to enhance the class discrimination in this chemical space.</p><!><p>The 297 training molecules belonging to cluster C3 were used to calibrate two different QSTR models based on the N3 and PLSDA approaches. The molecules were described by 3,763 conformation-independent Dragon descriptors. Descriptors with constant and near-constant values or those descriptors affected by missing values were excluded from the analysis. Moreover, to reduce the potential overfitting of the models due to highly correlated variables, the V-WSP unsupervised variable reduction approach was applied to further exclude another 1,255 descriptors at a correlation threshold of 0.95. The remaining 875 molecular descriptors were submitted to the subsequent supervised selection. This was carried out in two sequential steps: (1) GA-VSS (coupled with both N3 and PLSDA classifiers) was initially performed separately on each of the 18 blocks of molecular descriptors, and (2) the descriptors selected from each block were merged and a subsequent GA-VSS was carried out. The selection of the final sets of descriptors was performed by taking into account the NER classification parameter, as well as a balanced ratio between specificity and sensitivity of the sweet class. Two final models, each one based on six conformation-independent descriptors, were obtained with an optimal alpha of 1.5 for N3 and one latent variable (LV) for PLSDA.</p><p>The classification performance of the N3 model in fitting (NER = 0.748, Snsweet = 0.764, Spsweet = 0.732) and cross-validation (NER = 0.738, Snsweet = 0.750, Spsweet = 0.726), and the performance of the PLSDA classifier in fitting (NER = 0.722, Snsweet = 0.636, Spsweet = 0.809) and cross-validation (NER = 0.711, Snsweet = 0.607, Spsweet = 0.815) suggest a suitable capability of these models for predicting sweet taste inside cluster C3. The comparable performance in fitting and validation of the models indicate that these classifiers exhibit an overall balanced discrimination between the sweet and non-sweet classes with absence of potential overfitting. Descriptor details of the N3 and PLSDA models are shown in Table 2.</p><!><p>Details of the conformation-independent Dragon molecular descriptors included in the N3 and PLSDA models in cluster C3.</p><!><p>A graphical interpretation of the mechanistic effect of each descriptor in predicting the sweetness in the N3 models is not feasible because it is a local non-linear classifier; however, we attempted to explain the role of descriptors according to their chemical meaning. CATS2D_04_AL, CATS2D_05_AL (Renner et al., 2006) represent the frequency of hydrogen-bond acceptors and lipophilic atoms at a topological distance of 4 and 5 bonds, respectively. They indicate that sweetness of molecules may be attributed to the molecular hydrophobicity or the hydrophilic-lipophilic balance (HLB; Birch et al., 1994; Rojas et al., 2016a). Thus, the hydrophilic group works as an anchor allowing the fitting of the hydrophobic zone of the sweetener into hydrophobic binding sites in the sweet taste receptor (Yuasa et al., 1994). In fact, the presence of lipophilic atom pairs at a distance of 5 bonds (CATS2D_05_LL) already proved relevant in describing molecular relative sweetness (Rojas et al., 2016b). In addition, sweetness may also be influenced by the number of nitrogen and oxygen atom pairs (Carhart et al., 1985) at a topological distance of 3 bonds in the molecule (F03[N-O]) (Rojas et al., 2016a). Finally, the nCconj descriptor [number of non-aromatic conjugated carbon (sp2)], Balaban U index (Balaban and Balaban, 1991; which relates to the degree of branching of the molecule) and the number of aromatic carbons bonded to two aromatic carbon and one electronegative atom (O, N, S, P, Se, or halogens) (C-026) (Ghose et al., 1998) are also important for predicting the sweetness in the local non-linear N3 classifier.</p><p>Considering the PLSDA classifier, analysis of the model coefficients for the sweet class suggests that sweetness can be described by the CATS2D_04_AP, CATS2D_02_DN, and F03[C-S] descriptors. Figure 3 shows the coefficients of descriptors describing the sweet molecules. The selected CATS2D descriptors encode the presence of (1) pairs of hydrogen-bond donors (D) and negatively charged atoms (N) at a topological distance of 2 (CATS2D_02_DN) and (2) pairs of bond acceptors (A) (i.e., all N or O with at least one available lone pair electron) and positively charged atoms (P) separated by 4 bonds (CATS2D_04_AP). In fact, the presence of the positive-negative pharmacophores in the scaffold at a topological distance of 2 bonds was introduced for predicting the relative sweetness of molecules (Rojas et al., 2016b). F03[C-S] suggests that the sweetness is also related to the frequency of carbon-sulfur atom pairs in the skeleton at a distance of 3 bonds.</p><!><p>Coefficients for training descriptors in the PLSDA model for the sweet class.</p><!><p>Coefficients for the non-sweet class of molecules have the same value but an opposite sign with respect to those of the sweet class. Thus, the descriptors associated with the non-sweet class correspond to the Moran autocorrelation of lag 1 weighted by I-state (MATS1s), the aromatic ratio (ARR) and the distance/detour ring index of order 7 (D/Dtr07). Moran autocorrelation of lag 1 weighted by I-state (MATS1s) is a descriptor calculated by applying the Moran coefficient (Moran, 1950) to the molecular graph by using the intrinsic state(s) as the atomic property. Positive values of the Moran coefficient produce positive spatial autocorrelations, whereas negative values of the coefficient are related to negative spatial autocorrelations. The distance/detour ring index of order 7 (D/Dtr07) (Randić, 1997) is a topological descriptor reflecting the ratio between the lengths of the shortest to the lengths of the largest through-bond paths between any pair of vertices belonging to 7-membered rings. The distance/detour ring in combination with other ring descriptors, such as the aromatic ratio (ARR) (i.e., ratio of the number of aromatic bonds to the total number of non-H bonds), indicates that non-sweetness is related to the presence of aromatic rings.</p><p>Since N3 and PLSDA models are based on different descriptors/modeling methods, a consensus analysis (Baurin et al., 2004) was applied in order to join information and predictions from these two sources. Individual models contain varying extents of noise (especially when dealing with large and heterogeneous datasets and noisy endpoints), which can be reduced by averaging the predictions of the models. The main assumption of consensus modeling is that the strengths of one model should compensate for the weaknesses of others models and vice versa. Therefore, each molecule was predicted only if the two QSTR models classified it in the same class; otherwise, it was not classified. The classification performance of the consensus approach in calibration (NER = 0.852, Snsweet = 0.792, Spsweet = 0.913, not assigned = 33%) and cross-validation (NER = 0.831, Snsweet = 0.772, Spsweet = 0.890, not assigned = 32%) confirms the main assumption of the consensus strategy by improving the overall prediction performance. On the other hand, the number of non-assigned molecules increased considerably. However, since the molecules of concern are those of cluster C3, the drawback of having non-assigned chemicals can be accepted in favor of increased classification performance.</p><!><p>Once the models were calibrated using the molecules of the C3 cluster, the QSTR-based expert system was assembled for the prediction of sweetness of the entire dataset. Figure 4 shows the structure of the proposed QSTR-based expert system. In particular, for any new target molecule, the sweetness prediction can be carried out on the basis of the following procedure:</p><!><p>1. Calculate ECFP vector for the target molecule and then its Jaccard-Tanimoto average distance to the molecules included in Clusters S1 (ds1) and S2 (ds2), respectively;</p><p>2a. If ds1 and ds2 are lower than defined thresholds (0.6 and 0.8, respectively), then the target molecule is classified as sweet, because of its high structural similarity to sweet molecules of clusters S1 or S2;</p><p>2b. Alternatively, if ds1 and ds2 are higher than the thresholds, then the target molecule is predicted by means of the consensus model based on the QSTR N3 and PLSDA classifiers.</p><p>Workflow of the basic steps of the QSTR-based expert system for predicting the sweetness of chemicals.</p><!><p>The thresholds described in step 2a. were rationally chosen by analyzing the distribution of average similarities of each training molecule with respect to molecules of the three clusters. These distributions define a threshold value equal to 0.6 (Figure 5A) and a threshold value of 0.8 (Figure 5B) for cluster S1 and cluster S2, respectively.</p><!><p>Histogram plot of the Jaccard-Tanimoto average similarity of the training molecules from molecules grouped in cluster S1 (A) and cluster S2 (B).</p><!><p>Performance in classification of the QSTR-based expert system is listed in Table 3. Performance of the Monte Carlo validation based on 1,000 iterations (NER = 0.887, Snsweet = 0.927, Spsweet = 0.848, non-assigned = 20.5%) confirms the predictive power of the model. Finally, the 161 test molecules were used to assess the external predictive ability of the QSTR-based expert system. The results confirmed the predictive ability of the model (NER = 0.848, Snsweet = 0.880, Spsweet = 0.816, non-assigned = 19.3%). Model stability in fitting, validation and prediction, indicates that the proposed model does not exhibit potential overfitting, although the percentage of non-assigned molecules is c.a. 20%. Thus, the expert system presented in this paper could be useful to chemists who are dealing with the prediction of sweetness of both synthesized (virtual screening) and un-synthesized chemicals.</p><!><p>Performance of the QSTR-based expert system based on the "strict" consensus.</p><!><p>Every QSTR prediction should be associated with a specific estimation of the applicability domain (OECD, 2007), in order to get an assessment of the prediction reliability. The applicability domain (AD) assessment of the QSTR-based expert system can be implemented on the basis of the following procedure:</p><!><p>1. Calculate ECFP vector for the target molecule and then its Jaccard-Tanimoto average distance to the molecules included in Clusters S1 (ds1) and S2 (ds2), respectively;</p><p>2a. If ds1 and ds2 are lower than defined thresholds (0.6 and 0.8, respectively), then the target molecule is inside the AD of the QSTR-expert model, because it can be assumed to be grouped together with molecules included in clusters S1 and S2;</p><p>2b. Alternatively, if ds1 and ds2 are higher than thresholds, the applicability domain assessment is carried out by comparing the leverage of the target molecule with respect to the leverage threshold for the PLSDA classifier; while an analysis of the distribution of average similarities is used for the N3 classifier.</p><!><p>Thus, any target molecule should satisfy one of these conditions to be inside the AD of the QSTR-based expert system, otherwise its sweetness prediction could be an extrapolation.</p><!><p>The classification performance of both models included in the proposed QSTR-based expert system is considered appropriate, as well as the simplicity of the workflow of the expert system and the small number of molecular descriptors included in N3 and PLSDA models. The models presented in Table 1 from the existing literature were mainly calibrated by using small datasets and homogeneous sets of molecules, thus hampering the model generalization ability toward different types of chemicals (i.e., limited applicability domain). In addition, the majority of the studies did not perform validation of the QSTR models (Iwamura, 1980; Takahashi et al., 1982; Spillane et al., 1983; Miyashita et al., 1986b; Spillane and Sheahan, 1989, 1991). Thus, our QSTR-based expert system can be considered as a more general model for accurate prediction of sweetness of both un-evaluated and un-synthesized potential sweeteners exhibiting diverse scaffolds (i.e., a more general applicability domain). Additionally, this study provides the first QSTR model for sweetness prediction based on an expert system that (i) considers the use of both extended connectivity fingerprints and molecular descriptors and (ii) integrates the results from a structural similarity analysis along with consensus QSTR model predictions.</p><p>Several factors may affect the calibration of QSTR models for sweetness prediction such as the presence of unclear tastes of some sweeteners (i.e., multisapophoric or potential multisapophoric molecules). For instance, acesulfame potassium, sodium saccharin, hernandulcin, stevioside, and isocoumarin derivatives along with some sugar derivatives deliver bitterness in addition to sweetness. Their taste depends on the concentration of such molecules in solution (Birch et al., 1994). For molecules having more than two tastes, the taste perception may be complex (Shamil et al., 1987). For these reasons, humans are unlikely to discriminate these differences when dealing with multisapophoric molecules and this limitation may be due to the receptor saturation on the taste buds of the tongue or the polarization of the taste receptors (Birch et al., 1994).</p><p>On the other hand, sweeteners could exist in several equilibrium conformations that minimize their energy (Morini et al., 2011) and have more than one AH-B sites (Spillane and Sheahan, 1989; Damodaran et al., 2008); therefore, it is complex and difficult to define the active conformation and how such AH-B sites interact with the sweet-taste receptor to evoke the human sensation of sweetness. Moreover, the real interaction receptor-sweetener is not completely known. For instance, some compounds bind to the sweet receptor but they are not recognized as sweet (false positives), and other substances do not bind to the sweet receptor but are perceived as sweet (false negatives; Bassoli et al., 2008).</p><p>The simplicity and the satisfactory predictive ability of the QSTR-based expert system presented in this paper makes it a valid tool for scientists attempting to propose sweet molecular candidates either by synthesis or by virtual screening of very large available libraries. Thus, this model constitutes a starting point to understand the structure-taste relationships of molecules in which further evaluations could be addressed: (i) the conformational states of sweeteners, (ii) the mechanism of interactions between receptors and sweeteners (molecular docking and calculation of energies of binding), (iii) the measurement of the relative sweetness, and (iv) the identification of possible safety issues before using molecules as potential low-calorie sweeteners.</p><!><p>CR and DB conceived the workflow, CR and FG curated the dataset, CR performed the calculations, and wrote the manuscript. All the authors contributed equally to the scientific planning, discussion and to the manuscript revision</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

Me₃SiSiMe₂(O^<i>n</i>Bu): a disilane reagent for the synthesis of diverse silacycles <i>via</i> Brook- and retro-Brook-type rearrangement

Herein, a readily available disilane Me 3 SiSiMe 2 (O n Bu) has been developed for the synthesis of diverse silacycles via Brook-and retro-Brook-type rearrangement. This protocol enables the incorporation of a silylene into different starting materials, including acrylamides, alkene-tethered 2-(2-iodophenyl)-1Hindoles, and 2-iodobiaryls, via the cleavage of Si-Si, Si-C, and Si-O bonds, leading to the formation of spirobenzosiloles, fused benzosiloles, and p-conjugated dibenzosiloles in moderate to good yields. Preliminary mechanistic studies indicate that this transformation is realized by successive palladiumcatalyzed bis-silylation and Brook-and retro-Brook-type rearrangement of silane-tethered silanols.

me₃sisime₂(o^<i>n</i>bu):_a_disilane_reagent_for_the_synthesis_of_di

Introduction<!>Results and discussion<!>Conclusions

<p>Silacycles have attracted increasing attention because they have shown unique physical, optoelectronic, and physiological properties in medicinal chemistry and materials science. 1 In this context, considerable efforts have been devoted to the development of synthetic methods for silacycles, which is a prerequisite for fully discovering their application potentials. 2,3 Among them, of particular interest to synthetic chemists is the synthesis of silacycles via the cleavage of C-Si bonds. 3 Typical strategies include direct annulation of silicon-based frameworks 3a-n and C-Si/C-C bond exchange reaction of small ring systems based on the existence of a high ring strain. 3o-r However, these methods generally require transition-metal catalysts to assist the activation of C-Si bonds. Transitionmetal-free catalyzed annulation to assemble silacycles by cleaving C-Si bonds is still elusive so far.</p><p>The Brook rearrangement enables an intramolecular migration of a silyl group from carbon to oxygen atoms via a hypervalent silicon species, 4 which was initially introduced by Brook 5 and was demonstrated to be a reversible process. 6 Its reverse process, namely retro-Brook rearrangement, can, in turn, be achieved by the transfer of a silyl group from oxygen to carbon atoms (Scheme 1A). 7 Obviously, the Brook and retro- Brook rearrangements allow the cleavage of a C-Si bond and the formation of a C-Si bond under transition-metal-free catalysis. 8 On the other hand, the existing studies are limited to the translocation of a single silyl group from the starting materials. The migration of two different silyl groups in one event has not been reported so far. In this context, we want to explore the synthesis of silacycles via the migration of two different silyl groups in the Brook and retro-Brook rearrangement. The design and synthesis of silane-tethered silanols is undoubtedly the primary task and challenge to achieve this hypothesis.</p><p>Disilanes have been among the most versatile silylation reagents in organic synthesis. Over the past few decades, a myriad of methods for accessing organosilanes from disilanes have focused on the activation of Si-Si bonds for the development of mono-silylation involving aryl halides or cyanides, alkenes, and C-H bonds and bis-silylation of alkynes, alkenes, carbenes, and palladacycles (Scheme 1B). [9][10][11][12][13] Particularly, the pioneering work that realizes the bis-silylation of in situ generated palladacycles with hexamethyldisilane via the cleavage of Si-Si bonds has recently been reported by Zhang,Cheng,and us. 12,13 These advances have inspired us to modify hexamethyldisilane, namely the replacement of the methyl group with an oxygen-containing group, for the synthesis of silane-tethered silanols, which were further converted into silacycles via Brook and retro-Brook-type rearrangement. Herein, we disclose a Brook and retro-Brook-type rearrangement strategy for the synthesis of diverse silacycles, including spirobenzosiloles, fused benzosiloles, and p-conjugated dibenzosiloles, by employing a readily available disilane reagent Me 3 SiSiMe 2 (-O n Bu) that could be prepared by a simple treatment of pentamethylchlorodisilane with n-butanol in the presence of NEt 3 at room temperature (Scheme 1C). Notably, the cleavage of Si-Si, Si-C, and Si-O bonds is involved in the transformation.</p><!><p>We initiated the studies by investigating the reaction of acrylamide 1a with 1-butoxy-1,1,2,2,2-pentamethyldisilane 2a. To our delight, the anticipated spirobenzosilole 3a was indeed afforded in 66% yield by using a simple catalytic system composed of Pd(OAc) 2 and K 2 CO 3 in DMF at 90 C. Encouraged by these initial results, various parameters were screened, and the optimized reaction conditions are as follows: 1a (0.2 mmol), 2a (0.3 mmol), Pd(OAc) 2 (10 mol%), PPh 3 (20 mol%), and K 2 CO 3 (0.6 mmol) in DMA (2 mL) at 90 C under N 2 for 6 h (see the ESI † for details). Next, several disilanes 2b-h were tested. As shown in Scheme 2, when the n-butyl group of disilane 2a was replaced by other functional groups such as n-hexyl, benzyl, cyclohexyl, and 2-oxopropyl, all of them could afford the desired product 3a, albeit in a lower yield. Unexpectedly, disilane 2f was unreactive. Finally, disiloxane 2g was found to produce product 3a in 58% yield.</p><p>With the optimal reaction conditions and disilane reagent conrmed, the scope of acrylamides 1 was subsequently examined. Gratifyingly, this protocol was applicable to a large variety of acrylamides 1 to afford spiro[benzo [b]silole-3,3 0indolin]-2 0 -ones 3a-u in moderate to good yields (Scheme 3). Note that the replacement of iodine atoms with bromine atoms on the acrylamide showed good reactivity, delivering the product 3a in 66% yield. Moreover, the three substructures of acrylamides 1 were systematically investigated. Regarding different substituents on the nitrogen atom, methyl or ethyl group substituted acrylamides 1b and 1c were competent substrates, while acrylamide 1d with a Ts group could not give the target product 3d under the standard conditions. For the 2iodoaniline fragment, a broad range of functional groups on the benzene ring, including electron-donating groups (Me and OMe), modiable halogen groups (F and Cl), and even strong electron-withdrawing groups (CF 3 , CO 2 Me, and NO 2 ), were well tolerated (3e-n). Meanwhile, the structure of 3g was unambiguously conrmed by X-ray crystallography. Their electronic properties seem to affect the reactivity, since substrates 1l-n with strong electron-withdrawing groups, especially CO 2 Me and NO 2 groups, resulted in a diminished yield. Finally, the compatibility was further demonstrated by testing the key 2phenylalkene moiety. Both the benzene ring containing ortho or para substituents and the naphthalene ring could survive, affording spirobenzosilole 3o-u in moderate to good yields. Nevertheless, a slight modication of the reaction conditions was required when substrates bearing F groups were used (3q and 3t). When the reaction was scaled up to 1 mmol, 70% spirocyclic product 3a could also be obtained.</p><p>To highlight the generality of this domino Heck/ silacyclization, we envision that fused benzosiloles can be synthesized by a domino Heck/ortho C-H functionalization of aryl iodides. Therefore, 2-(2-iodophenyl)-1-(2-methylallyl)-1Hindole was employed to react with disilane 2a under the above reaction conditions. To our delight, indolo[2,1-a]silolo [4,3,2-de] isoquinolines 5a could smoothly be produced in 58% yield by the cleavage of Si-Si and Si-O bonds. Encouraged by these results, the scope of 2-(2-halophenyl)-1-(2-methylallyl)-1Hindoles was then explored (Scheme 4). Bromine atoms instead of iodine atoms on the substrate 4a 0 were subjected to the standard conditions, which could give product 5a, albeit in a lower yield. Satisfactorily, substrates 4b-g being diversely substituted (Me, F, and Cl) on the indole ring were able to undergo this domino Heck/silacyclization with disilane 2a to provide the desired products 5b-g in moderate yields. Differently, using substrate 4h required relatively mild conditions.</p><p>To emphasize the versatility of the disilane reagent Me 3 -SiSiMe 2 (O n Bu), we next attempted to synthesize p-conjugated dibenzo [b,d]siloles by performing the reaction of 2-iodo-1,1 0biphenyl with Me 3 SiSiMe 2 (O n Bu) 2a. However, no anticipated product was observed under the above standard conditions. Subsequently, PPh 3 was found to suppress the reaction, since dibenzo [b,d]siloles 7a could be afforded in 52% yield by the removal of PPh 3 . Encouraged by these results, the optimal reaction conditions that could furnish 64% of 7a were established by the screening of various parameters (see the ESI † and Scheme 5). Aerward, a series of substituted 2-iodobiphenyls were examined. Delightfully, the electron-donating group (Me and OMe) on the benzene ring could be tolerated for the silacyclization reaction with disilane 2a, thus delivering the desired products 7b-k in moderate yields. Unfortunately, this protocol was not applicable to substrates 6l-o bearing an electronwithdrawing group. The possible reason is that the protonation of palladacycles formed by substrates 6l-o is easier than bis-silylation (see the ESI †).</p><p>To gain insight into the reaction mechanism, a range of control experiments were performed (Scheme 6). The reaction of acrylamides 1a with disiloxane 2h or 2i could afford product 3a in 27% and 50% yields, respectively, under the optimal reaction conditions (eqn (1) †). Moreover, hexamethyldisiloxane (TMSOTMS) was detected by gas chromatography in the model reaction of 1a with 2a (eqn (2), see the ESI †). These results indicated that the silicon source of product 3a came from the dimethylsilyl group generated by disilane 2a via the cleavage of Si-Si and Si-O bonds. Interestingly, two disilylated products 8a and 8a 0 as well as product 3a were isolated in 43%, 27%, and 16% yields when 1a and 2a were reacted under the standard conditions for 20 minutes (eqn (3) †). Therefore, we speculated that two disilylated products 8a and 8a 0 were the reaction intermediates (the structure of 8a was absolutely conrmed by X-ray crystallography). Finally, a spiropalladacycle 14 that could Scheme 5 Variations of the 2-iodobiphenyls (6). a Reaction conditions: 6 (0.2 mmol), 2a (0.24 mmol), Pd(OAc) 2 (10 mol%), K 2 CO 3 (3 equiv.), and DMF (2 mL) at 90 C under a N 2 atmosphere for 12 h. b 2-Bromobiphenyl was used. c 2-Iodo-4 0 -methyl-1,1 0 -biphenyl was used. d 2-Iodo-3 0 -methyl-1,1 0 -biphenyl was used.</p><p>be prepared from acrylamides and stoichiometric Pd(PPh 3 ) 4 was employed for the reaction with disilane 2a (eqn (4) †). Unexpectedly, no product 3b was observed in the absence of Pd(OAc) 2 and PPh 3 . Surprisingly, extra addition of Pd(OAc) 2 and PPh 3 could give product 3b in 60% yield. These results suggested that the spiropalladacycle as a reaction intermediate underwent transmetalation with intermediate G, rather than direct oxidative addition with disilane 2a, to furnish disilylated products 8a and 8a 0 , which were then converted into the desired product 3a.</p><p>To verify our hypothesis and propose the possible formation process of 3a from 8a or 8a 0 , we conducted several control experiments (Scheme 7). The silacyclization of disilylated products 8a and 8a 0 , respectively, was conducted in the absence of Pd(OAc) 2 and PPh 3 , and the product 3a was obtained in 66% and 60% yields as expected (eqn (1) †). Besides, TMSOTMS was also detected (see the ESI †). These results demonstrated that the transformation of 8a and 8a 0 into 3a involved the cleavage of the Me 3 Si-C bond, which did not require the assistance of palladium catalysts. On the basis of these results from eqn (1) † and previous work reported by Smith and Takeda,15 we speculated that 8a and 8a 0 undergo a Brook-and retro-Brook-type rearrangement to afford 3a (path c and path d). To capture carbanion species F and F 0 , two common electrophilic reagents, such as iodomethane and benzyl bromide, were added for the silacyclization of 8a and 8a 0 (eqn ( 2) and (3) †). However, the corresponding products F-1 and F 0 -2 were not observed. Therefore, these results are more favorable to this pathway involving synergetic Brook/retro-Brook-type rearrangement (path d).</p><p>Based on the results of mechanistic experiments as well as reported work, 12,13,15 a plausible mechanism for the synthesis of silacycles was proposed (Scheme 8). Initially, oxidative addition followed by intramolecular Heck-cyclization of acrylamides 1a to Pd(0) species forms intermediate A, which then undergoes a C-H activation to afford spiropalladacycle B. Next, spiropalladacycle B produces disilylated products 8a/8a 0 and regenerates Pd(0) by sequential transmetalation with intermediate G generated by disilane 2a, reductive elimination and further hydrolysis (path a). 8a and 8a 0 then undergo a synergetic Brook/retro-Brook-type rearrangement to afford 3a and a trimethylsiloxy anion, which could be converted into TMSOTMS (path d). 16 Notably, another possible pathway that synthesizes disilylated products 8a/8a 0 by direct oxidative addition of spiropalladacycle B with disilane 2a is ruled out by the results of eqn ( 4) † (path b).</p><!><p>In conclusion, we have disclosed the rst example of divergent synthesis of silacycles via a Brook-and retro-Brook-type rearrangement strategy by employing a readily accessible disilane reagent Me 3 SiSiMe 2 (O n Bu). In this novel transformation, divergent silacycles, such as spirobenzosiloles, fused benzosiloles, and p-conjugated dibenzosiloles, can be produced in moderate to good yields by an unprecedented complex process composed of a bis-silylation of a palladacycle and a Brook-and retro-Brook-type rearrangement. Notably, mechanistic studies reveal that bis-silylation of the palladacycle is completed by a transmetalation process. Further applications of the disilane reagent Me 3 SiSiMe 2 (O n Bu) and the rearrangement are still in progress in our laboratory.</p>

Royal Society of Chemistry (RSC)

Carbohydrate-auxiliary assisted preparation of enantiopure 1,2-oxazine derivatives and aminopolyols

An approach to enantiopure hydroxylated 2H-1,2-oxazine derivatives is presented utilizing the [3 + 3] cyclisation of lithiated alkoxyallenes and an L-erythrose-derived N-glycosyl nitrone as precursors. This key step proceeded only with moderate diastereoselectivity, but allowed entry into both enantiomeric series of the resulting 3,6-dihydro-2H-1,2-oxazines. Their enol ether double bond was then subjected to a hydroboration followed by an oxidative work-up, and finally the auxiliary was removed. The described three-step procedure enabled the synthesis of enantiopure hydroxylated 1,2-oxazines. Typical examples were treated with samarium diiodide leading to enantiopure acyclic aminopolyols. We also report on our attempts to convert these compounds into enantiopure hydroxylated pyrrolidine derivatives.

carbohydrate-auxiliary_assisted_preparation_of_enantiopure_1,2-oxazine_derivatives_and_aminopolyols

<!>Introduction<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>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<!>Experimental<!>Typical procedure for the preparation of 1,2-oxazine derivatives by addition of a lithiated alkoxyallene to nitrone 1a (Procedure 1)<!>Typical procedure for hydroborations of 1,2-oxazines (Procedure 2)<!>Typical protocol for glycosyl bond cleavage (Procedure 3)<!>Typical procedure for the reactions with samarium diiodide (Procedure 4)<!>

<p>This article is part of the Thematic Series "Synthesis in the glycosciences II".</p><!><p>During the last few decades carbohydrate-derived nitrones have turned out to be particularly attractive tools for the synthesis of structurally complex compounds [1–4]. Employed mainly as 1,3-dipoles in cycloadditions [5–6] or as imine analogues in nucleophilic additions [7–8], these nitrones very often furnish the corresponding products in a highly selective manner. In this context, reactions of lithiated alkoxyallenes with enantiopure nitrones are particularly of interest since they lead by a [3 + 3] cyclisation process to 1,2-oxazine derivatives with excellent diastereoselectivity [9]. We previously reported on the unusually diverse synthetic potential of carbohydrate-derived 1,2-oxazines allowing the smooth and flexible preparation of various highly functionalised compounds, including de novo syntheses of carbohydrates and their mimetics, as well as N-heterocycles [10–12]. Although the reactions of lithiated alkoxyallenes, with nitrones bearing substituents with stereogenic centres at the carbon atom, were studied in our group in great detail [13], N-glycosyl-substituted nitrones have so far not been used as electrophiles for this purpose. This type of nitrone has been introduced and broadly studied by Vasella and co-workers [14–20] and has also been used by other groups [21–25]. They observed moderate to high diastereoselectivities for 1,3-dipolar cycloadditions and for nucleophilic additions. Successful applications of these easily removable auxiliaries in the syntheses of biologically active agents were also reported [26–31]. Apart from the obvious reactivity of N-glycosyl nitrones of type 1 leading to five-membered heterocycles A or to N,N-disubstituted hydroxylamine derivatives B, a twofold nucleophilic addition of an excess of organometallic reagents furnishing compounds of type C (Nu1 = Nu2) was described and discussed by Goti et al. (Scheme 1) [32]. In selected examples, the synthesis of differently substituted products (Nu1 ≠ Nu2) was possible by consecutive additions of the appropriate Grignard reagents [33]. Here we report on the application of a nitrone with an L-erythronolactone-derived auxiliary for the synthesis of 3,6-dihydro-2H-1,2-oxazine derivatives of type D. Their selected transformations, including hydroboration of the enol ether moiety, oxidative work-up, glycosyl cleavage, and samarium diiodide-induced reactions, are presented as well.</p><!><p>Reactivity of N-glycosyl nitrones 1 towards dipolarophiles and nucleophiles leading to products of type A, B, C and D.</p><!><p>In continuation of our recent exploration of L-erythrose-derived nitrones for the synthesis of 3,6-dihydro-2H-1,2-oxazine derivatives [34], we turned our attention to benzaldehyde-derived nitrone 1a, which is readily available in a three-step procedure starting from L-arabinose. L-Erythronolactone was first prepared [35] and was subsequently treated with N-benzylhydroxylamine [36], and the resulting product was oxidised with activated MnO2 [37] to furnish the desired compound 1a in 51% overall yield. The initial experiment with 1a was carried out under typical conditions with 2.4 equiv of lithiated methoxyallene at −78 °C in THF. Similarly to previous results for more rigid cyclic nitrones [38], the formation of the intermediate N-hydroxylamines 2 (Scheme 2) was clearly observed. These primarily formed compounds were not isolated, but (in the presence of a drying agent) they underwent slow cyclisation in Et2O solution at room temperature to furnish the desired 1,2-oxazine derivative as a mixture of separable diastereomers (3S)-3a and (3R)-3a in 25% and 8% yield, respectively (Table 1, entry 3). The 1,2-oxazines were accompanied by a complex mixture of by-products, from which only two compounds 4 (1%) and 5 (3%) could be isolated in pure form (Figure 1). After tedious optimisation with respect to stoichiometry, temperature, time, concentration, etc. (selected results are presented in Table 1), we found that running the reaction from −130 to −80 °C, followed by standing overnight at room temperature, allowed the synthesis of 3a with an overall yield of 75% and a ratio of diastereomers of ca. 2:1 (49% and 22% after separation of the isomers, Table 1, entry 7). When the reaction was scaled up to 3.50 g of 1a the expected diastereomers of 1,2-oxazines 3a were obtained with no decrease in yield (78%). As illustrated in Scheme 2, lithiated (2-trimethylsilyl)ethoxyallene and benzyloxyallene were also examined under the optimised reaction conditions and furnished the expected diastereomers of 1,2-oxazine derivatives 3b and 3c in 51% and 65% yield, respectively.</p><!><p>Additions of lithiated alkoxyallenes to L-erythrose-derived nitrone 1a leading to 3,6-dihydro-2H-1,2-oxazine derivatives 3 via the respective N-hydroxylamines 2.</p><p>Selected reaction conditions of nitrone 1a with lithiated methoxyallene.</p><p>aReactions performed in 1.1 mmol scale with respect to 1a. bCrude product. cCombined yield of isolated (3S)-3a and (3R)-3a. dOnly the major diastereomer (3S)-3a was detected.</p><p>By-products 4 and 5 isolated from the reaction of nitrone 1a with lithiated methoxyallene.</p><!><p>The mixtures of diastereomers of crude 1,2-oxazines 3a–c were easily separated by standard column chromatography and characterised by spectroscopic methods. However, in certain cases additional purification was necessary to obtain analytically pure samples. The absolute configuration of the newly generated stereogenic centre could not be determined based on NMR techniques. For instance, in the 1H NMR spectra of the diastereomeric products (3S)-3a and (3R)-3a, the signals of the benzylic protons assigned to C-3 of the 1,2-oxazine ring appear as singlets at 4.85 and 4.48 ppm, respectively. Due to the unhindered rotation of the auxiliary moiety similar correlation peaks in NOE experiments were observed for both isomers. Fortunately, the minor product (3R)-3a isolated as an amorphous solid could be recrystallised from ethyl acetate solution to give crystals suitable for an X-ray crystallographic analysis (Figure 2). The X-ray analysis of (3R)-3a shows a well-defined half-chair conformation of the 1,2-oxazine ring, with four carbon atoms in plane and with ONCC and OCCC torsion angles of −60° and 13°, respectively. The bulky N-substituent occupies a pseudo-equatorial position and the phenyl group is in a pseudo-axial position. Since characteristic shift patterns in the 1H NMR spectra for both diastereomeric series are observed, the configuration at C-3 of the TMSE derivatives (b) and benzyloxy analogues (c) could be assigned as well with high certainty.</p><!><p>Single-crystal X-ray analysis of (3R)-3a (ellipsoids are drawn at a 50% probability level).</p><!><p>The stereochemical outcome observed for the reactions studied also deserves some comment. Whereas lithiated methoxy- and TMS-ethoxyallene yielded the diastereomers in ca. 2:1 ratio, in the case of lithiated benzyloxyallene a significant switch of the selectivity to an approximate 1:2 ratio was observed. According to the model proposed in the literature [7,18,20] for the addition of nucleophiles, the stereochemical course is governed by a competition of steric and electronic effects. As presented in Figure 3, the bulky benzyloxy substituent favours the anti-addition and hence yields the (3R)-configured product as the major compound. In contrast, the less hindered lithiated methoxyallene enables a syn-attack supported by a "kinetic anomeric effect", stabilizing the respective transition state [20] and furnishing the (3S)-configured 1,2-oxazine. In all tested examples the level of diastereoselectivity was only low to moderate, and the interpretation should therefore not be exaggerated. Alternative conformations explaining the results are certainly possible.</p><!><p>Model proposed for the addition of lithiated allenes to nitrone 1a.</p><!><p>The fragmentation of the primarily formed allenyl N-hydroxylamines of type 2 leading to 1,3-dienes such as 4 (Figure 1) by retro-nitroso-ene reaction was discussed in earlier work [13], but the formation of pyrrole derivative 5 is unprecedented for the reactions of nitrones and lithiated alkoxyallenes. The 1H NMR spectrum of 5 shows four singlets (3H each) assigned to three methyl groups and one methoxy substituent. Additionally, a broad singlet at δ = 8.98 ppm attributed to the NH functionality, an OH group at 2.11 ppm (dd, J ≈ 4.0, 8.1 Hz) coupling with the adjacent methylene group, and a characteristic set of multiplets of the [1,3]dioxolane and the phenyl moieties could be found. The signals of four quaternary carbon atoms in the 13C NMR spectrum evidenced the presence of the pyrrole structure. Finally, the HMBC experiment proved the proposed substitution pattern at the pyrrole ring. HRMS and elemental analysis allowed identification of 5 as a 2,3,4,5-tetrasubstituted pyrrole derivative.</p><p>As shown in Table 1, entries 1–3, a higher excess of lithiated methoxyallene resulted in a significant decrease in the yield of 1,2-oxazine derivatives. For example, in the case of 10 equiv of lithiated methoxyallene (Table 1, entry 1) only a trace amount of (3S)-3a (<5%) and numerous side products were found in the crude product, including compound 5. On the other hand, no pyrrole 5 could be detected when only a slight excess of methoxyallene was used (Table 1, entry 5) or when the reaction was performed at lower temperatures (Table 1, entries 6 and 7). These observations prompted us to postulate that the surprising formation of 5 is the result of a double addition of lithiated methoxyallene to nitrone 1a as illustrated in Scheme 3, the crucial step being the (reversible) opening of the tetrahydrofuran ring of the primary addition product E. The resulting new nitrone F can then react with the second equiv of lithiated methoxyallene to give the double adduct G. After aqueous work-up, hydroxylamine derivative H underwent a ring-closure to 1,2-oxazetidine derivative I. It is known that this class of compounds can suffer a thermally induced [2 + 2] cycloreversion involving N–O bond cleavage [39–40], which, in our case, led to the formation of methyl acrylate and imine J. This imine underwent subsequent cyclisation to zwitterion K and two proton shifts, probably via 3-exomethylene compound L, finally led to pyrrole 5. This mechanism is certainly speculative but offers a possibility to explain the formation of the tetrasubstituted pyrrole 5. The structure of the intermediate double-addition product H suggests that other by-products may be formed, e.g., two different N-allenylmethyl-substituted 3,6-dihydro-2H-1,2-oxazines or an isomeric pyrrole derivative. The numerous sets of signals in the 1H NMR spectrum as well as additional spots seen on TLC of the crude reaction mixtures support this assumption, but none of these possible by-products could be isolated.</p><!><p>Speculative mechanistic suggestion for the formation of tetrasubstituted pyrrole derivative 5.</p><!><p>With respect to the enormous importance of polyhydroxylated N-heterocycles as carbohydrate-mimicking glycosidase inhibitors [41–45], the introduction of an additional hydroxyl moiety into 1,2-oxazine derivatives was an essential goal in several studies by our group [34,46–48]. A series of 5-hydroxy-1,2-oxazine derivatives was successfully prepared by the well known hydroboration/oxidation protocol, with yields and selectivities strongly depending on the relative configuration of the employed 1,2-oxazine derivative and on the presence of additives [34,48]. In general, syn-configured 1,2-oxazines (with respect to the relative configuration at C-3 and the neighbouring stereogenic centre at the carbohydrate-derived C-3-substituent) were found to be excellent substrates, leading to the desired alcohols exclusively with very high degrees of stereoselectivity. In an extension of these studies, selected compounds of type 3 were hydroxylated following the general methodology. As shown in Scheme 4, each of the (3S)-configured 1,2-oxazines 3a and 3b furnished a pair of hydroxylated products 6, 7 and 8, 9, respectively, in high combined yields (83% and 93%) and almost the same ratio (approximately 3:2) of cis-trans/trans-trans isomers. The stereoselectivity for this series is apparently only low. On the other hand, for (3R)-3a a ca. 3:1 ratio of hydroxylated products was observed based on the 1H NMR spectrum of the crude mixture; however, the minor product 10 was isolated in 13% yield only, whereas the major product was obtained in a satisfying 66% yield. We assume that the observed low facial selectivity results predominantly from the moderate hindrance exhibited by the neighbouring phenyl substituent, which in the case of the (3S)-series probably occupies a pseudo-equatorial position in the half-chair conformation of the 3,6-dihydro-2H-1,2-oxazine derivatives 3. The higher stereoselectivity of the hydroboration of (3R)-3a is probably caused by the pseudo-axial location of the phenyl group (as evidenced by the X-ray analysis, Figure 2), shielding one side more efficiently. The carbohydrate-derived N-substituent is relatively far away from the two reacting carbon atoms and very likely has no strong, direct influence on the observed diastereoselectivities.</p><!><p>Introduction of a 5-hydroxy group into 1,2-oxazine derivatives 3 by a hydroboration/oxidation protocol; (a) BH3·THF (4.0 equiv), THF, −30 °C to rt, 3 h; (b) NaOH, H2O2 (30%), −10 °C to rt, overnight.</p><!><p>All products 6–11 obtained by the hydroboration/oxidation protocol were easily purified and separated by column chromatography and finally deprotected by treatment with acid. This afforded a series of the desired, highly functionalised tetrahydro-2H-1,2-oxazine derivatives 12–15, including ent-12 and ent-13 (Table 2). Reaction of the trans-trans-configured 4-methoxy-1,2-oxazines 6 and 10 with a methanolic solution of HCl (1 M) at elevated temperatures enabled the smooth cleavage of the glycosyl bond to give the N-unsubstituted derivatives 12 and ent-12 in high yields (Table 2, entries 1 and 5). The cis-trans-configured compound pair 7 and 11 provided similar results, yielding the expected enantiomers 13 and ent-13 (Table 2, entries 2 and 6). As expected, the enantiomers show nicely matching optical rotations with opposite sign. In the case of the TMSE-protected derivative 9, selective removal of the N-protective group could be achieved under the applied conditions. After prolonged reaction times (16 h) there was no significant change in the tested sample. A complete deprotection of 9 leading to dihydroxylated compound 15 was possible in high overall yield (75%) by using ion-exchange resin DOWEX-50 at 50 °C (Table 2, entry 4). As shown for compound 8, simultaneous cleavage was also possible, and the analytically pure compound 14 was isolated in comparable yield (Table 2, entry 3). Alternatively, demethylation of 12 by treatment with boron tribromide at low temperatures [49] also provided the expected compound 14 (Table 2, entry 7); however, the analytically pure sample of this compound could only be isolated in 18% yield. Therefore, the protocol applying a TMS-ethyl substituent as a more easily removable O-protective group turned out to be much more effective. All enantiopure 1,2-oxazines 12–15 were isolated as colourless crystals, which were prone to sublimation.</p><!><p>Acid-induced cleavages of N- and O-protective groups of 5-hydroxy-1,2-oxazine derivatives 6–11; conditions: (a) HCl (1 M) in MeOH, 40 °C, 3.5 h; (b) DOWEX-50, EtOH, 50 °C, 4 d; (c) BBr3 (3 equiv), CH2Cl2, −78 °C (1 h) then rt, overnight.</p><p>aReaction time prolonged to 10 days; boverall yield for two steps; cmelting point and spectroscopic data correspond with the sample of compound 14 obtained from 8 (Table 2, entry 3).</p><!><p>The 1H NMR spectrum of trans–trans configured 14 also deserves a short comment. Similarly to the previously described 2,4- and 2,5-dimethyltetrahydro-1,2-oxazine derivatives [50], significant long-range couplings could be observed in the 1H NMR spectrum. The low-field shifted multiplet (4.12–4.19 ppm) assigned to the equatorial 6-H showed additional couplings of <2.5 Hz. However, due to 4-H/5-H overlapping, selective decoupling of this complex spin system was not possible. An indirect proof for the observed phenomenon was found in the 1H NMR spectrum (Supporting Information File 1) of 14 prior to purification, i.e., still containing BBr3, which acts here as a shift reagent. The influence of the coordinated boron species resulted not only in a strong low-field shift but it also simplified the spectrum, and thus, only geminal and vicinal couplings (J = 12.2 Hz and J = 5.4 Hz) for the equatorial 6-H could be found. On the other hand, a possible nitrogen and/or ring inversion usually measurable at lower temperatures should be taken into account [51]. As expected, no significant changes in the shift pattern were observed in a series of 1H NMR spectra measured at elevated temperatures, both in methanol-d4 (up to 50 °C) and DMSO-d6 (up to 80 °C). Moreover, in the 13C NMR spectrum only one set of sharp signals was observed.</p><p>Due to their similarity to carbohydrate derivatives, hydroxylated 1,2-oxazines such as 12–15 may already have interesting biological activity, but their functional groups also open several options for subsequent transformations into other relevant compound classes. By reductive ring opening the corresponding amino polyols should be accessible. Since compounds of type 12 contain a benzylamine substructure, standard methods that may possibly attack this moiety, such as catalytic hydrogenation, should be avoided. As an alternative, samarium diiodide is an attractive reagent for this purpose. Apart from its extraordinary potential for the formation of new carbon–carbon bonds [52–54], the cleavage of N–O bonds in a chemoselective fashion is also well documented [55–57]. The application of samarium diiodide for 1,2-oxazine ring opening allowed efficient syntheses of numerous polyhydroxylated heterocycles, such as pyrrolidine [46], azetidine [47], furan [58], and pyran derivatives [59]. Gratifyingly, the treatment of tetrahydro-2H-1,2-oxazine derivatives 12 and 13 with an excess of SmI2 in tetrahydrofuran smoothly provided the expected amino alcohols 16 and 17 in excellent yields (Scheme 5).</p><!><p>Samarium diiodide-induced ring opening of tetrahydro-2H-1,2-oxazine derivatives 12 and 13.</p><!><p>In order to compare the behaviour of a compound still bearing the N-auxiliary, we converted tetrahydro-2H-1,2-oxazine 7 into the O-benzylated derivative 18 under standard conditions (Scheme 6). Treatment of this protected compound with samarium diiodide furnished a complex mixture of products from which only the two amino alcohols 19 and 20 were isolated, in low yield. The formation of 20 could be explained by a subsequent SmI2-mediated reduction of the C=N bond formed by ring opening of 19, which contains a hemiaminal moiety. This suggestion is supported by the 1H NMR spectrum of 19 in which a second set of signals could be easily detected. Thus, the direct use of 1,2-oxazine derivatives still containing the carbohydrate-derived auxiliary at the nitrogen is apparently not sufficiently selective during samarium diiodide-promoted reactions.</p><!><p>Reaction of tetrahydro-2H-1,2-oxazine 18 with samarium diiodide. (a) NaH (1.4 equiv), BnBr (1.2 equiv), DMF, 0 °C to rt, overnight.</p><!><p>The successful transformation of N-benzyl-substituted tetrahydro-2H-1,2-oxazine derivatives into polyhydroxylated pyrrolidine derivatives [46] prompted us to select compound 13 as a precursor and to examine the described methods with this substrate. First, the free hydroxy group was protected as a trimethylsilyl ether and, after SmI2-induced ring opening, the expected product 22 was clearly identified based on TLC monitoring. However, the attempted isolation and purification of this compound by column chromatography provided amino alcohol 17 as the only product in high yield (92%). The limited stability of the TMS protective group is evident from the results presented in Scheme 7. Treatment of freshly prepared unpurified 22 with an excess of mesyl chloride and triethylamine yielded a complex product mixture. The isolated compounds 23–26 clearly indicate that the migration of the TMS group not only takes place in an intramolecular fashion to the terminal hydroxy function to furnish 24, but it also occurs intermolecularly leading to the disilylated mesylamide 23. The desired pyrrolidine derivative 25 was obtained only as a minor product (5%). The major isolated component, N,O-dimesylated pyrrolidine 26 (35%) derives from 25 by TMS-cleavage and subsequent mesylation of the OH group.</p><!><p>Attempted synthesis of pyrrolidine derivatives from precursor 13. (a) TMSCl (1.5 equiv), imidazole, DMAP, CH2Cl2, rt, overnight; (b) SmI2, THF, 1.5 h, rt; (c) CHCl3, rt, overnight; (d) MsCl (4 equiv), Et3N, CH2Cl2, rt, overnight.</p><!><p>To overcome these apparent difficulties, tert-butyldimethylsilyl-protected compound 27 was prepared. Samarium diiodide-mediated ring opening under standard conditions furnished the expected amino alcohol 28 in excellent yield (Scheme 8). An attempted cyclisation of 28 using tosyl chloride in the presence of triethylamine was not successful but led to N-tosylated compound 31 in 24% yield. A partial epimerisation at the benzylic position and slow decomposition of precursor 28 could also be observed under the reaction conditions applied, and none of the desired pyrrolidine derivatives could be found in the crude product. Purification on a silica gel column yielded two fractions containing a mixture of the C-4 epimeric N,O-di-tosylated compounds (14%, ca. 1:1 ratio) and a mixture of the respective tosylamides (41%, ca. 4:1 ratio). Additional chromatography of the latter fraction enabled isolation of compound 31 in the pure state (24%). Isolation of other by-products was not possible.</p><!><p>Synthesis of TBS-protected tetrahydro-2H-1,2-oxazine 27 and its transformation into pyrrolidine derivatives 29, 30 and 32. (a) TBSCl (2.0 equiv), imidazole, DMAP, CH2Cl2, rt, 5 d; (b) SmI2, THF, 1.5 h, rt; (c) MsCl (2.0 equiv), Et3N, CH2Cl2, rt, overnight; (d) LDA (5.4 equiv), rt, 16 h; (e) pTsCl (2.2 equiv), Et3N, CH2Cl2, rt, overnight; (f) CBr4 (1.2 equiv), PPh3 (1.2 equiv), Et3N (1.1 equiv), CH2Cl2, rt, overnight; (g) HCl (1 M) in MeOH, rt, 3 d.</p><!><p>Fortunately, the use of mesyl chloride was more efficient to achieve cyclisation of 28. Application of this reagent afforded pyrrolidine derivative 29 in acceptable overall yield. The different reaction outcome observed for the transformations of 28 with the two sulfonyl chlorides is probably a consequence of the bulkiness of the TBS group. The small sulfene intermediate, generated from mesyl chloride, smoothly reacts with the terminal OH group to give the respective mesylate, which subsequently cyclises to afford pyrrolidine 29. On the other hand, the more bulky tosyl chloride competitively attacks the amino group. As illustrated in Scheme 8, the attempted conversion of 29 into the free secondary amine 30 by treatment with LDA [60] was not very efficient. The target compound was accompanied by a mixture of dihydropyrrole derivatives, which were very likely formed by deprotonation at the benzylic position and subsequent elimination.</p><p>Finally, freshly prepared unpurified 28 was subjected to the conditions of an Appel reaction [61] providing, after 16 hours at room temperature, pyrrolidine derivative 30 in 33% yield. Again, the relatively low efficacy could be explained by the destructive role of the base required for the subsequent cyclisation step. Cleavage of the TBS-moiety under acidic conditions furnished the desired hydroxylated pyrrolidine 32 in good yield. An attempted direct conversion of the unprotected amino diol 17 into 32 by treatment with tetrabromomethane in the presence of triphenylphosphine gave no satisfactory results, possibly due to the formation of the corresponding oxirane and its diverse, subsequent reactions.</p><!><p>We achieved the efficient synthesis of enantiopure hydroxylated tetrahydro-2H-1,2-oxazine derivatives using, in the key step, lithiated alkoxyallenes and a phenyl-substituted nitrone 1a bearing an L-erythronolactone-derived auxiliary as starting materials. Moderate levels of diastereoselectivity were observed for the formation of the 1,2-oxazine ring and for the subsequent hydroboration step. However, due to the easy separation of the formed products by standard column chromatography, the presented protocol opens up access to enantiopure products with both absolute configurations in different relative configurations, in a relatively short time. The described procedure supplements known protocols employing terpene units [62] and carbohydrate-derived auxiliaries [63–64] for the asymmetric synthesis of the 1,2-oxazine derivatives. More recently, the use of (–)-menthol as a chiral auxiliary was presented for the separation of diastereomeric 6H-1,2-oxazines [65–66]. Subsequent transformations of the newly prepared tetrahydro-2H-1,2-oxazines, utilizing samarium diiodide as the key reagent for the chemoselective ring opening, enable a smooth access to novel phenyl-substituted aminopolyols. Their transformation into hydroxylated pyrrolidine derivatives so far proceeds only with moderate efficacy, but this may certainly be optimised in the future.</p><!><p>General methods. Reactions were generally performed under an inert atmosphere (argon) in flame-dried flasks. Solvents and reagents were added by syringe. Solvents were purified with a MB SPS-800-dry solvent system. Triethylamine was distilled from CaH2 and stored over KOH under an atmosphere of argon. Other reagents were purchased and used as received without further purification unless stated otherwise. Products were purified by flash chromatography on silica gel (230–400 mesh, Merck or Fluka). Unless stated otherwise, yields refer to analytically pure samples. NMR spectra were recorded with Bruker (AC 250, AC 500, AVIII 700) and JOEL (ECX 400, Eclipse 500) instruments. Chemical shifts are reported relative to TMS or solvent residual peaks (1H: δ = 0.00 ppm [TMS], δ = 3.31 ppm [CD3OD], δ = 7.26 ppm [CDCl3]; 13C: δ = 49.0 ppm [CD3OD], δ = 77.0 ppm [CDCl3]). Integrals are in accordance with assignments and coupling constants are given in Hertz. All 13C NMR spectra are proton-decoupled. For detailed peak assignments, 2D spectra were measured (COSY, HMQC, HMBC). IR spectra were measured with a Nexus FT-IR spectrometer fitted with a Nicolet Smart DuraSample IR ATR. MS and HRMS analyses were performed with a Varian Ionspec QFT-7 (ESI–FT ICRMS) instrument. Elemental analyses were obtained with a Vario EL or a Vario EL III (Elementar Analysensysteme GmbH) instrument. Melting points were measured with a Reichert apparatus (Thermovar) and are uncorrected. Optical rotations ([α]D) were determined with a Perkin–Elmer 241 polarimeter at the temperatures given. Single crystal X-ray data were collected with a Bruker SMART CCD diffractometer (Mo Kα radiation, λ = 0.71073 Å, graphite monochromator); the structure solution and refinement was performed by using SHELXS-97 [67] and SHELXL-97 [67] in the WINGX system [68]. CCDC-864241 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.</p><!><p>Lithiated methoxyallene was generated under an atmosphere of dry argon by treating a solution of methoxyallene (357 mg, 0.42 mL, 5.06 mmol) in dry THF (20 mL) with n-BuLi (2.5 M in hexanes; 2.0 mL, 5.0 mmol) at −40 °C. After 5 min, the resulting mixture was cooled to −130 °C (n-pentane/liq. N2 bath), and a solution of nitrone 1a (606 mg, 2.30 mmol) in dry THF (15 mL) was added under vigorous stirring. The partially solidified mixture was allowed to reach −80 °C within 1.5 h and was quenched with water. Then, warming to room temperature was followed by extraction with Et2O (3 × 25 mL), and the combined organic layers were stirred overnight with the drying agent (MgSO4). When cyclisation of the primarily formed allene adducts was complete (TLC monitoring, hexane/ethyl acetate 4:1, p-anisaldehyde stain) the solvents were removed in vacuo to yield a light orange oil (763 mg). The crude material was filtered through a short silica gel pad (hexane/ethyl acetate 3:1) to yield a mixture of diastereomers (574 mg, 75%, 2:1 ratio), which were separated by column chromatography (silica gel, hexane/ethyl acetate 7:1, gradient to 5:1) to give (3S)-3a (380 mg, 49%, first eluted) as a pale yellow oil and (3R)-3a (170 mg, 22%) as a colourless solid. An analytically pure sample of (3R)-3a was obtained by recrystallisation from ethyl acetate.</p><p>(3S,3a'S,4'S,6a'S)-2-(2',2'-Dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4'-yl)-4-methoxy-3-phenyl-3,6-dihydro-2H-[1,2]oxazine ((3S)-3a): +133.4 (c 1.12, CHCl3); 1H NMR (CDCl3, 700 MHz) δ 1.32, 1.41 (2 s, 3H each, 2 Me), 3.47 (s, 3H, OMe), 4.03 (d, J = 9.5 Hz, 1H, 6'-H), 4.23–4.27 (ddbr, J ≈ 3.5, 9.5 Hz, 1H, 6'-H), 4.30 (dd, J = 4.3, 13.7 Hz, 1H, 6-H), 4.40 (s, 1H, 4'-H), 4.62 (dtbr, J ≈ 2.0, 13.7 Hz, 1H, 6-H), 4.85 (sbr, 1H, 3-H), 4.87 (dtbr, J ≈ 1.3, 4.3 Hz, 1H, 5-H), 4.88–4.90 (m, 2H, 3a'-H, 6a'-H), 7.26–7.33, 7.34–7.38 (2 m, 5H, Ph) ppm; 13C NMR (CDCl3, 126 MHz) δ 24.5, 26.3 (2 q, 2 Me), 54.8 (q, OMe), 63.5 (d, C-3), 67.3 (t, C-6), 76.6 (t, C-6'), 81.2, 84.2 (2 d, C-3a', C-6a'), 92.1 (d, C-5), 94.7 (d, C-4'), 111.6 (s, C-2'), 128.0, 128.3, 129.7, 136.2 (3 d, s, Ph), 154.9 (s, C-4) ppm; IR (ATR) : 3085–2840 (=C-H, C-H), 1670 (C=C), 1225, 1075, 1055 (C-O) cm−1; ESI–TOF (m/z): [M + Na]+ calcd for C18H23NNaO5, 356.1474; found, 356.1479; Anal. calcd for C18H23NO5 (333.4): C, 64.85; H, 6.95; N, 4.20; found: C, 64.81; H, 6.98; N, 4.15.</p><p>(3R,3a'S,4'S,6a'S)-2-(2',2'-Dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4'-yl)-4-methoxy-3-phenyl-3,6-dihydro-2H-[1,2]oxazine ((3R)-3a): mp 110–113 °C; crystals suitable for X-ray analysis were obtained from AcOEt solution by cooling (fridge); Crystal data: C18H23NO5, M = 333.37, orthorhombic, a = 5.6042(12) Å, b = 16.756(4) Å, c = 17.839(4) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1675.2(6) Å3, T = 133(2) K, space group P2(1)2(1)2(1), Z = 4, Mo Kα, 23651 reflections measured, 4186 independent reflections (Rint = 0.0178). R1 = 0.0307 (I > 2σ(I)); wR(F2) = 0.0782 (all data); GOOF(F2) = 1.048. −87.0 (c 1.36, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.34, 1.44 (2 s, 3H each, 2 Me), 3.49 (s, 3H, OMe), 3.89 (d, J = 9.9 Hz, 1H, 6'-H), 4.03 (dd, J = 4.0, 9.9 Hz, 1H, 6'-H), 4.41 (ddd, J = 1.7, 3.2, 14.3 Hz, 1H, 6-H), 4.48 (sbr, 1H, 3-H), 4.54 (ddd, J = 1.6, 2.4, 14.3 Hz, 1H, 6-H), 4.72 (s, 1H, 4'-H), 4.80 (dd, J = 4.0, 6.1 Hz, 1H, 6a'-H), 4.90 (tbr, J ≈ 3.0 Hz, 1H, 5-H), 5.03 (d, J = 6.1 Hz, 1H, 3a'-H), 7.27–7.34, 7.36–7.40 (2 m, 5H, Ph) ppm; 13C NMR (CDCl3, 126 MHz) δ 24.7, 26.3 (2 q, 2 Me), 54.6 (q, OMe), 63.6 (d, C-3), 65.2 (t, C-6), 74.5 (t, C-6'), 81.1 (d, C-6a'), 81.5 (d, C-3a'), 91.7 (d, C-5), 96.4 (d, C-4'), 112.0 (s, C-2'), 127.8, 128.3, 129.0, 138.1 (3 d, s, Ph), 153.1 (s, C-4) ppm; IR (ATR) : 3060–2840 (=C-H, C-H), 1675 (C=C), 1220, 1100, 1050 (C-O) cm−1; ESI–TOF (m/z): [M + Na]+ calcd for C18H23NNaO5, 356.1474; found, 356.1470; Anal. calcd for C18H23NO5 (333.4): C, 64.85; H, 6.95; N, 4.20; found: C, 64.85; H, 6.83; N, 4.11.</p><!><p>To a solution of 1,2-oxazine (3S)-3a (268 mg, 0.80 mmol) in dry THF (20 mL), a solution of BH3·THF (1 M in THF, 3.2 mL, 3.2 mmol) was added at −30 °C. The solution was warmed to room temperature and stirred for 3 h, then cooled to −10 °C and an aq NaOH solution (2 M, 4.8 mL) followed by H2O2 (30%, 1.6 mL) were added. Stirring at room temperature was continued overnight. After addition of a sat. aq Na2S2O3 solution, the layers were separated, the water layer was extracted with Et2O (3 × 15 mL), the combined organic layers were dried with MgSO4 and filtered, and the solvents were removed under reduced pressure. The crude products (321 mg, 3:2 ratio) were separated by chromatography column (silica gel, hexane/ethyl acetate 1:1) to give 5-hydroxy-1,2-oxazines 6 (92 mg, 33%, first eluted) and 7 (141 mg, 50%) as hygroscopic, colourless semisolids.</p><p>(3S,4S,5S,3'aS,4'S,6'aS)-2-(2',2'-Dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4'-yl)-4-methoxy-3-phenyl-[1,2]oxazinan-5-ol (6): +131.2 (c 1.02, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.28, 1.35 (2 s, 3H each, 2 Me), 2.60 (d, J = 1.9 Hz, 1H, OH), 2.90 (s, 3H, OMe), 3.41 (ddd, J = 1.4, 6.7, 9.4 Hz, 1H, 4-H), 3.66–3.72 (m, 2H, 5-H, 6-H), 3.93 (d, J = 9.4 Hz, 1H, 3-H), 3.93 (d, J = 9.5 Hz, 1H, 6'-H), 4.07 (dd, J ≈ 11, 16 Hz, 1H, 6-H), 4.19 (dd, J = 4.4, 9.5 Hz, 1H, 6'-H), 4.41 (s, 1H, 4'-H), 4.81 (dd, J = 4.4, 6.1 Hz, 1H, 6a'-H), 4.86 (d, J = 6.1 Hz, 1H, 3a'-H), 7.28–7.42 (m, 5H, Ph) ppm; 13C NMR (CDCl3, 126 MHz) δ 24.5, 26.2 (2 q, 2 Me), 60.5 (q, OMe), 67.6 (d, C-3), 70.6 (d, C-5), 71.4 (t, C-6), 77.4 (t, C-6'), 81.3 (d, C-6a'), 84.4 (d, C-3a'), 87.6 (d, C-4), 94.8 (d, C-4'), 111.7 (s, C-2'), 128.3, 128.8*, 136.8 (2 d, s, Ph) ppm; *higher intensity; IR (ATR) : 3440 (O-H), 3090–2830 (=C-H, C-H), 1205, 1055 (C-O) cm−1; ESI–TOF (m/z): [M + Na]+ calcd for C18H25NNaO6, 374.1580; found, 374.1581; Anal. calcd for C18H25NO6 (351.4): C, 61.52; H, 7.17; N, 3.99; found: C, 61.43; H, 7.15; N, 3.85.</p><p>(3S,4R,5R,3a'S,4'S,6a'S)-2-(2',2'-Dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4'-yl)-4-methoxy-3-phenyl-[1,2]oxazinan-5-ol (7): +138.2 (c 1.41, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.28, 1.34 (2 s, 3H each, 2 Me), 2.44 (d, J = 7.7 Hz, 1H, OH), 3.10 (s, 3H, OMe), 3.21 (mc, 1H, 4-H), 3.75 (sbr, 1H, 5-H), 3.82 (d, J = 12.2 Hz, 1H, 6-H), 3.94 (d, J = 9.4 Hz, 1H, 6'-H), 4.21 (dd, J = 4.6, 9.4 Hz, 1H, 6'-H), 4.36 (dd, J = 1.4, 12.2 Hz, 1H, 6-H), 4.45 (d, J = 2.3 Hz, 1H, 3-H), 4.60 (s, 1H, 4'-H), 4.81 (tbr, J ≈ 5.2 Hz, 1H, 6a'-H), 4.94 (d, J = 6.1 Hz, 1H, 3a'-H), 7.24–7.31, 7.42–7.45 (2 m, 5H, Ph) ppm; 13C NMR (CDCl3, 126 MHz) δ 24.5, 26.3 (2 q, 2 Me), 59.3 (q, OMe), 62.7 (d, C-3), 65.3 (d, C-5), 71.0 (t, C-6), 77.5 (t, C-6'), 80.4 (d, C-4), 81.1 (d, C-6a'), 84.5 (d, C-3a'), 95.6 (d, C-4'), 111.6 (s, C-2'), 127.8, 128.3, 129.5, 136.4 (3 d, s, Ph) ppm; IR (ATR) : 3455 (O-H), 3090–2830 (=C-H, C-H), 1215, 1085, 1050 (C-O) cm−1; ESI–TOF (m/z): [M + Na]+ calcd for C18H25NNaO6, 374.1580; found, 374.1579; Anal. calcd for C18H25NO6 (351.4): C, 61.52; H, 7.17; N, 3.99; found: C, 61.43; H, 7.17; N, 3.87.</p><!><p>1,2-Oxazine 6 (425 mg, 1.21 mmol) was dissolved in 1 N HCl in MeOH (14 mL) and heated at 40 °C for 3.5 h (TLC monitoring, hexane/AcOEt 1:2, potassium permanganate stain). Then the mixture was allowed to reach room temperature, quenched with sat. aq NaHCO3 solution and extracted with Et2O (3 × 30 mL). The combined organic layers were dried with MgSO4 and filtered, and the solvents were removed. The purification by column chromatography (silica gel, dichloromethane/methanol 40:1) yielded 12 (201 mg, 79%) as a colourless solid.</p><p>(3S,4S,5S)-4-Methoxy-3-phenyl-[1,2]oxazinan-5-ol (12): mp 110–112 °C; +60.1 (c 1.05, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 3.02 (s, 3H, OMe), 3.37 (tbr, J ≈ 8.5 Hz, 1H, 4-H), 3.71–3.81 (m, 2H, 5-H, 6-H), 3.91 (d, J = 9.2 Hz, 1H, 3-H), 4.14 (dd, J = 4.1, 9.7 Hz, 1H, 6-H), 2.60, 5.48 (2 sbr, 2H, NH, OH), 7.31–7.38, 7.40–7.43 (2 m, 5H, Ph) ppm; 13C NMR (CDCl3, 126 MHz) δ 60.4 (q, OMe), 67.0 (t, C-3), 70.9 (d, C-5), 72.3 (t, C-6), 86.9 (d, C-4), 128.4, 128.6, 128.7, 136.3 (3 d, s, Ph) ppm; IR (ATR) : 3405–3260 (O-H, N-H), 3065–2830 (=C-H, C-H), 1105, 1055 (C-O) cm−1; ESI–TOF (m/z): [M + H]+ calcd for C11H16NO3, 210.1130; found, 210.1127; Anal. calcd for C11H15NO3 (209.2): C, 63.14; H, 7.23; N, 6.69; found: C, 63.14; H, 7.23; N, 6.66.</p><!><p>To a solution of SmI2 (ca. 0.1 M in THF, 15 mL, ~1.5 mmol) at room temperature was added dropwise a solution of 5-hydroxy-1,2-oxazine 12 (102 mg, 0.49 mmol) in degassed THF (10 mL). After the mixture was stirred for 3 h it was quenched with sat. aq sodium potassium tartrate solution and extracted with Et2O (20 mL), and then with CH2Cl2 (3 × 15 mL). The combined organic layers were dried with MgSO4, filtered and the solvents were removed under reduced pressure to give the spectroscopically pure product as a yellow oil in almost quantitative yield. Filtration through a short silica gel pad (dichloromethane/methanol 15:1) yielded 16 (97 mg, 94%) as a colourless oil.</p><p>(2S,3S,4S)-4-Amino-3-methoxy-4-phenylbutane-1,2-diol (16): +12.2 (c 1.48, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 3.31 (dd, J = 2.1, 5.3 Hz, 1H, 3-H), 3.39 (s, 3H, OMe), 3.57 (dd, J = 4.6, 11.1 Hz, 1H, 1-H), 3.72 (dd, J = 6.1, 11.1 Hz, 1H, 1-H), 3.77–3.80 (m, 1H, 2-H), 4.58 (d, J = 5.3 Hz, 1H, 4-H), 7.28–7.33, 7.36–7.43 (2 m, 5H, Ph) ppm; 13C NMR (CDCl3, 126 MHz) δ 55.9 (d, C-4), 59.3 (q, OMe), 63.4 (t, C-1), 70.7 (d, C-2), 83.0 (d, C-3), 127.1, 128.1, 128.8, 138.6 (3 d, s, Ph) ppm; IR (ATR) : 3490–3230 (O-H, N-H), 3065–2810 (=C-H, C-H), 1075 (C-O) cm−1; ESI–TOF (m/z): [M + H]+ calcd for C11H18NO3, 212.1292; found, 212.1282.</p><!><p>Experimental procedures and characterisation data.</p><p>1H NMR and 13C NMR spectra of synthesised compounds.</p>

PubMed Open Access

Cardiac Myosin Binding Protein C and Its Phosphorylation Regulate Multiple Steps in the Cross-Bridge Cycle of Muscle Contraction

Cardiac myosin binding protein C (c-MyBPC) is a thick filament protein that is expressed in cardiac sarcomeres and is known to interact with myosin and actin. While both structural and regulatory roles have been proposed for c-MyBPC, its true function is unclear; however, phosphorylation has been shown to be important. In this study, we investigate the effect of c-MyBPC and its phosphorylation on two key steps of the cross-bridge cycle using fast reaction kinetics. We show that unphosphorylated c-MyBPC complexed with myosin in 1:1 and 3:1 myosin:c-MyBPC stoichiometries regulates the binding of myosin to actin (KD) cooperatively (Hill coefficient, h) (KD = 16.44 \xc2\xb1 0.33 \xce\xbcM, and h = 9.24 \xc2\xb1 1.34; KD = 11.48 \xc2\xb1 0.75 \xce\xbcM, and h = 3.54 \xc2\xb1 0.67) and significantly decelerates the ATP-induced dissociation of myosin from actin (K1k+2 values of 0.12 \xc2\xb1 0.01 and 0.22 \xc2\xb1 0.01 M\xe2\x88\x921 s\xe2\x88\x921, respectively, compared with a value of 0.42 \xc2\xb1 0.01 M\xe2\x88\x921 s\xe2\x88\x921 for myosin alone). Phosphorylation of c-MyBPC abolished the regulation of the association phase (K1k+2 values of 0.32 \xc2\xb1 0.02 and 0.33 \xc2\xb1 0.01 M\xe2\x88\x921 s\xe2\x88\x921 at 1:1 and 3:1 myosin:c-MyBPC ratios, respectively) and also accelerated the dissociation of myosin from actin (K1k+2 values of 0.23 \xc2\xb1 0.01 and 0.29 \xc2\xb1 0.01 M\xe2\x88\x921 s\xe2\x88\x921 at a 1:1 and 3:1 myosin:c-MyBPC ratios, respectively) relative to the dissociation of myosin from actin in the presence of unphosphorylated c-MyBPC. These results indicate a direct effect of c-MyBPC on cross-bridge kinetics that is independent of the thin filament that together with its phosphorylation provides a mechanism for fine-tuning cross-bridge behavior to match the contractile requirements of the heart.

cardiac_myosin_binding_protein_c_and_its_phosphorylation_regulate_multiple_steps_in_the_cross-bridge

<!>Preparation of Contractile Proteins<!>PKA Phosphorylation of c-MyBPC<!>Immunological Techniques<!>Transient Kinetic Measurements<!>ATPase Assays<!>Statistical Analysis<!>Protein Preparation<!>Myosin_c-MyBPC Binding to Actin<!>ATP-Induced Actomyosin_c-MyBPC Dissociation<!>ATPase Activity<!>DISCUSSION<!>Effects of c-MyBPC on the Kinetics of Binding of Myosin to Actin<!>Effects of c-MyBPC on the ATP-Induced Dissociation of Myosin from Actin<!>Effects of c-MyBPC on Actomyosin ATPase Rates<!>Functional Effects of c-MyBPC Phosphorylation on Cross-Bridge Kinetics

<p>Cardiac myosin binding protein C (c-MyBPC) is a thick filament-associated, sarcomeric protein located in discrete transverse bands in the C zone of the A band, which consists of eight immunoglobulin-like domains and three fibronectin type 3 domains that are designated C0–C10. These domains have been shown to specifically bind to the myosin tail (C10),1 titin (C8–C10),2 myosin subfragment 2 (S2) (C1–C2), and actin (C0–C1).3,4 The cardiac isoform includes a cardiac specific domain motif or m domain between C1 and C2 that undergoes phosphorylation by cAMP-dependent protein kinase A (PKA) in response to β-adrenergic agonists5 to affect cardiac output, and a proline/alanine rich linker connecting C0 and C1 that has been proposed to contain a possible actin binding region.4</p><p>c-MyBPC is being increasingly shown to have an important role in the regulation of muscle contraction; however, the precise mechanism is still unclear. Most data show the effects of c-MyBPC regulation in the presence of the thin filament. Some reports suggest that there is a direct interaction with tropomyosin (Tm),6 and others suggest that c-MyBPC is important for regulating force generation in fibers;7,8 however, whether the regulatory effects of c-MyBPC are due to a change in the inherent properties of cross-bridge formation and whether c-MyBPC acts in a manner that is independent of the thin filament regulatory proteins are unknown. Mutations in c-MyBPC are a leading cause of familial hypertrophic cardiomyopathy (FHC), which further underlines its necessity for normal cardiac function.9 c-MyBPC knockout (KO) mice display an overall sarcomere structure that is unaffected but exhibit symptoms of cardiac hypertrophy,10 suggesting that c-MyBPC is not required for sarcomere assembly but for correct sarcomere function. This is further reinforced by studies with c-MyBPC KO mice that show that the lack of c-MyBPC accelerates cross-bridge cycling and rates of force development and increases shortening velocity and rates of force redevelopment and relaxation in permeabilized muscle fibers.7,11–13</p><p>As a potential mechanism to account for the effects of c-MyBPC in limiting myofilament contractile properties, Hoffman and colleagues proposed a model in which c-MyBPC acts as an internal load within the thick filament that opposes shortening in such a way that c-MyBPC is tethered to myosin S2, thereby limiting myosin head position and/or mobility,14 and this idea was further examined in the work of Calaghan and colleagues.15 The tethering model may have an important part to play in c-MyBPC's regulation of contractile function; however, it is unclear that it accounts for the entire mechanism. Studies containing only the C1–C2 fragment with the associated phosphorylation sites of c-MyBPC showed that this fragment can affect contractile function alone in myocytes, and the ability of heavy meromyosin to move actin in in vitro motility assays without the remaining c-MyBPC domains and most significantly without the light meromyosin (LMM) binding domains, which is central to the idea of a tethering mechanism.16–18 Other studies have shown that the C0–C1 N-terminal region of c-MyBPC can bind the S1–S2 hinge region of myosin in a manner that is independent of a tether mechanism; however, although necessary, the regulatory role of the C0–C1 domain of c-MyBPC is unclear.19 Several investigations using in vitro biochemistry methods17–21 and structural methods6,22–27 have also provided evidence that the C0–C1 and C1–C2 regions of c-MyBPC may bind F actin in addition to binding the S2 region of myosin, and a further structural study has suggested that most of the N-terminal domains bind and are ordered on actin in such a way that they can modulate Tm function as well as block binding of myosin to actin.6 PKA-mediated phosphorylation of c-MyBPC is believed to accelerate cross-bridge kinetics,5,16,28 as many studies involving skinned fibers have shown increases in rates of force development and stretch activation in response to PKA stimulation;28–31 however, other studies do not support this view.32,33 The exact mechanism therefore is still unknown, and it has not been demonstrated whether c-MyBPC affects discrete steps of the cross-bridge cycle. Fiber and in vitro motility studies do not record discrete measurements of individual steps in the cross-bridge cycle of muscle contraction. It is therefore very difficult to apportion observations seen in the experiments when c-MyBPC is present to specific kinetic interactions. The stopped-flow assay has a long track record in measuring cross-bridge kinetics. It is able to accurately measure the fast reaction rates, but more importantly, it allows the measurement of kinetics of specific steps during muscle contraction. It is therefore possible to measure the effects of c-MyBPC and its phosphorylation at each stage of the cross-bridge cycle of muscle contraction.</p><p>The binding of S1 to actin and the hydrolysis of ATP with the subsequent dissociation of the two binding partners are two of the principal steps in the cross-bridge cycle of muscle contraction and can be described by Schemes 1 and 2. Both steps are very rapid events; e.g., in the absence of ATP, myosin binds actin in solution with a very high affinity (Kass ≈ 107 M−1) to form the actomyosin complex, but the presence of ATP reduces the association constant to <104 M−1. The cross-bridge cycle has been extensively studied, but only in the absence of thick filament regulatory proteins, and defining the regulation of c-MyBPC on specific steps in the cross-bridge cycle is a prerequisite for improving our understanding of muscle contraction. This is very important as c-MyBPC is indicated in almost 40% of all hypertrophic cardiomyopathies (HCM);9 therefore, improving our understanding of how c-MyBPC works will give us a better understanding of the pathophysiology of this disease. This is important for devising sarcomere treatment-based therapies for the treatment of HCM and heart failure such as that featured in the study of Malik.34 The purpose of this study, therefore, is to define how c-MyBPC directly affects the kinetics of key steps in the cross-bridge cycle. The mechanism of c-MyBPC regulation as described above is still unclear, and there is currently no information regarding its effects on the kinetics of the key steps in the cross-bridge cycle of muscle contraction or whether cross-bridge regulation occurs in a manner that is independent of the thin filament regulatory proteins. It is possible that like another thick filament protein, regulatory light chain (RLC), which has been shown to modulate cross-bridge cycling in skeletal muscle35 and cardiac muscle36 in a phosphorylation-dependent manner, c-MyBPC may be a critical thick filament regulator of muscle contraction.</p><p>The current widely accepted model for the cross-bridge cycle of muscle contraction was presented by Geeves et al. in 1984.37 This model consists of an ATPase-driven cycle of cross-bridge attachment, cross-bridge "rotation", and ligand dissociation and binding where strain is an important factor.38 The binding of myosin to actin and thus the cross-bridge cycle are regulated by thin filament proteins in a Ca2+-dependent manner in both skeletal and cardiac muscle,39,40 whereby Tm sterically blocks the myosin binding sites on actin in the absence of Ca2+.41,42</p><p>In this study, we have prepared full-length c-MyBPC purified from porcine ventricular tissue because this system closely resembles the human heart in terms of the expression profile of contractile proteins as well as in vivo function. Stopped-flow measurements using full-length myosin and actin were conducted in the absence and presence of c-MyBPC, and the ATPase activity of the myosin motor was also investigated using the NADH-coupled system. In this way, we were able to quantify the effects of c-MyBPC and its phosphorylation on both transient and steady-state kinetics of critical steps in the cross-bridge cycle that govern the regulation of cardiac muscle contraction.</p><!><p>c-MyBPC protein was purified from porcine left ventricular tissue using the method of Hartzell and Windfield.43 Briefly, 150 g of porcine left ventricular tissue was homogenized in 750 mL of buffer A [50 mM KCl, 2 mM EDTA, 20 mM Tris-HCl (pH 7.9), and 15 mM 2-mercaptoethanol] for approximately 2 min in a Waring blender. The homogenate was centrifuged at 3000g for 15 min. The pellet was washed three times with buffer A, twice more with buffer A containing 1% Triton, and a further three times with buffer A. The final pellets were resuspended with a polytron homogenizer in 400 mL of EDTA-PO4 buffer (pH 5.9) (10 mM EDTA-Na2, 124 mM NaH2PO4, and 31 mM Na2HPO4). The homogenate was centrifuged at 10000g for 20 min, and the resulting pellet was extracted a second time. The two supernatants were pooled and concentrated by ammonium sulfate precipitation (55% saturation). The precipitated protein was dissolved in 300 mM NaCl, 5.2 mM K2HPO4, 4.8 mM NaH2PO4, 2 mM NaN3, 0.1 mM EDTA, and 3 mM 2-mercaptoethanol and dialyzed overnight. The protein extract was then chromatographed according to the method of Starr and Offer.44 The purity of the c-MyBPC protein was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Cardiac myosin from porcine left ventricular tissue was prepared according to the method of Taylor and Weeds,45 and porcine skeletal actin was prepared according to the method of Spudich and Watt.46</p><!><p>The exogenous catalytic subunit of PKA from bovine heart (Sigma) was used to phosphorylate c-MyBPC according to the method of Tong et al.29 The PKA catalytic subunit was resuspended in a 60 mM KCl, 10 mM MgCl2, 2 mM ATP buffer such that the concentration was 100 units/μL, and this was added to the c-MyBPC protein solution to give a final PKA concentration of 1 unit/μL, where 1 unit is defined as the quantity of enzyme that will transfer 1.0 pmol of phosphate from [γ-32P]ATP to hydrolyzed and partially dephosphorylated casein per minute at pH 6.5 and 30 °C. The c-MyBPC/PKA solution was incubated at 30 °C for 1 h, and the resulting sample was checked for the presence of phosphorylated c-MyBPC by Pro-Q Diamond staining (Molecular Probes) according to the manufacturer's protocol.</p><!><p>The phosphorylation states of the three known c-MyBPC residues that are targets for PKA phosphorylation (i.e., serines 275, 284, and 304, of the human c-MyBPC sequence) were probed using phospho-specific antibodies raised against phosphate-conjugated serine 275, 284, and 304 peptides in SPF rabbits (21st Century Biochemicals, Marlboro, MA) and were subsequently affinity purified. Western blots were used to determine the relative level of basal and PKA-induced phosphorylation of residues 275, 284, and 304. To allow the rabbit purified anti-serine 275, 284, and 304 were used at a dilution of 1:500, and an anti-rabbit horseradish peroxidase secondary antibody was used at a dilution of 1:5000 for detection and qualitative comparison. Total c-MyBPC levels were also probed with a c-MyBPC specific antibody (Santa Cruz) at a dilution of 1:500. Blots were scanned and quantified using a FluorChem E imaging system (proteinsimple, Santa Clara, CA).</p><!><p>All rapid kinetic measurements were performed with a standard Applied Photophysics stopped-flow system. Pyrene fluorescence was excited at 365 nm and monitored through a KV389 filter. The reactant concentrations stated in the text and figures are those after mixing in the stopped flow, unless stated otherwise. All experiments were conducted at 25 °C in buffer containing 20 mM MOPS (pH 7.0), 500 mM KCl, and 5 mM MgCl2.</p><!><p>The steady-state ATPase activity of myosin in the absence and presence of c-MyBPC was measured using the NADH-coupled assay47,48 in a TECAN Infinite M1000 pro fluorescent plate reader. Increasing concentrations of myosin and c-MyBPC were mixed with 3.5 μM actin in a solution of 20 mM MOPS (pH 7.0), 500 mM KCl, and 10 mM MgCl2. The reaction was started by mixing the protein solution with an equal volume of 2× reaction mix (2 mM phosphoenolpyruvate, 1.2 μM NADH, 10 mM ATP, and 0.04 unit/μL pyruvate kinase/lactate dehydrogenase), and the absorbance of NADH at 340 nm was monitored. Readings were taken every minute for 1 h or until the reaction went to completion. The absorbance at 340 nm was converted to ADP concentration and plotted versus time. ATPase rates were calculated from the slope of the graph and plotted as a function of myosin concentration.</p><p>All stopped-flow and ATPase assays were conducted with myosin and c-MyBPC present at a 1:1 ratio or at physiological myosin:c-MyBPC ratios of 3:1 or 7:1 as described by Craig et al.49</p><!><p>Steady-state and transient cross-bridge kinetics in the absence and presence of c-MyBPC were compared by one-way analysis of variance (ANOVA) or a Student's t test as appropriate. Measurements performed prior to and following c-MyBPC phosphorylation were compared by a Student's t test. Data were averaged from at least three separate experiments, and P values of <0.05 were considered statistically significant.</p><!><p>c-MyBPC was extracted and purified from porcine hearts to produce ~50 mg of protein from ~150 g of starting tissue. The purified protein seen in Figure 1A produced a single band on a 10% SDS–PAGE gel of ~140 kDa, which corresponded to values from previously published studies involving endogenous c-MyBPC. Figure 1B shows the effects of PKA treatment on purified endogenous c-MyBPC. Purified endogenous c-MyBPC was virtually unphosphorylated, like a protein treated with alkaline phosphatase; however, treatment with PKA using the method of Tong et al.29 produced a robust band concomitant with the c-MyBPC protein. To determine the relative levels of PKA-induced phosphorylation at individual c-MyBPC phosphorylation residues prior to and following PKA treatment, phosphospecific antibodies for serines 275, 284, and 304 were used and Western blot analysis was conducted (Figure 1S of the Supporting Information). Similar to results from Pro-Q Diamond phosphorylation staining, basal phosphorylation levels prior to PKA treatment were low in residues 275 and 284 and not detected in residue 304; however, PKA treatment produced a significant increase in the magnitude of the phosphorylation signal in all three residues. Further analysis of the PKA signal size was conducted by quantifying the relative signal obtained at residues 275, 284, and 304 prior to and following PKA treatment. The analysis showed that the magnitude of the signal produced by the phosphorylation of c-MyBPC was ~4- and ~5-fold higher at residues 275 and 284, respectively, than the magnitude of the unphosphorylated signal. The relative change in the phosphorylation signal due to PKA treatment at residue 304 could not be accurately determined because of the absence of a basal phosphorylation signal. It is important to note that although our data confirm that PKA does phosphorylate all three residues, our analysis does not provide direct information about the actual stoichiometry of the phosphorylation at each residue.</p><!><p>The binding of the myosin_c-MyBPC complex to actin can be followed by monitoring the decrease in fluorescence of pyrene-labeled actin (pyr.actin). Figure 2A shows representative fluorescence transients observed at 25 °C when 1 and 27.5 μM myosin_c-MyBPC complex is mixed with 1 μM pyr.actin in the stopped-flow fluorimeter. The observed transients can be described well by a single-exponential equation with a Kobs of 0.38 s−1 for 1 μM myosin_c-MyBPC complex and 14.7 s−1 for 27.5 μM. Kobs values showed a linear dependence on increasing myosin concentration as shown in Figure 2B; however, in the presence of unphosphorylated c-MyBPC, there is clearly a shift to a sigmoidal dependence, which suggests a regulatory and cooperativity effect of c-MyBPC on binding of myosin to actin. Figure 2B also shows the effect of increasing the ratio of c-MyBPC in the myosin_c-MyBPC complex. When c-MyBPC and myosin are present in a complex in a 1:7 or 1:3 c-MyBPC:myosin ratio, the lag phase is shorter than when c-MyBPC and myosin are present in a complex in a 1:1 ratio. This suggests that the regulatory effect is dependent on c-MyBPC concentration. Figure 2C shows the effect of adding PKA-treated c-MyBPC to the complex. Kobs values show a linear dependence on increasing myosin_c-MyBPC.PKA concentration for all three complex ratios in a manner similar to that of the binding of myosin alone, thus suggesting that phosphorylation of c-MyBPC abolishes the regulatory effect. The second-order rate constants for binding of myosin to actin alone and binding of myosin_c-MyBPC.PKA to actin (K1k+2) are listed in Table 1 and were estimated from a linear fit to the Kobs versus myosin or myosin_c-MyBPC plot to be 0.35 ± 0.02 M−1 s−1 for myosin alone or 0.32 ± 0.02, 0.33 ± 0.01, and 0.33 ± 0.03 M−1 s−1 for myosin_c-MyBPC.PKA in 1:1, 3:1, and 7:1 myosin:c-MyBPC ratios, respectively. The Kobs of binding of myosin_c-MyBPC to actin with unphosphorylated c-MyBPC exhibited a sigmoidal dependence on protein concentration rather than a linear one; therefore, binding departs from Scheme 1, and a second-order rate constant (K1k+2) could not be calculated from the plot. The fraction of myosin_c-MyBPC bound to actin (KD) is also presented in Table 1 and was calculated via the Hill equation to be 6.79 ± 0.09 μM with a cooperativity h of 5.89 ± 0.22 for a 7:1 myosin:c-MyBPC ratio, 11.48 ± 0.75 μM with an h of 3.54 ± 0.67 for a 3:1 myosin:c-MyBPC ratio, and 16.44 ± 0.33 μM with an h of 9.24 ± 1.34 for a 1:1 myosin:c-MyBPC ratio. Binding of c-MyBPC to actin alone (Figure 2D) shows a modest linear increase in the Kobs values with an increase in c-MyBPC concentration, and a much slower second-order rate constant compared to that of binding of myosin and actin alone (Table 1). Thus, c-MyBPC directly binds to actin, but its relative contribution to the measured actin binding signal in the presence of myosin is significantly smaller than that of myosin.</p><!><p>The ATP-induced dissociation of the actomyosin complex can be followed by monitoring the increase in the fluorescence of pyr.actin after the addition of ATP. Figure 3A shows representative fluorescence transients observed at 25 °C when 2 μM actomyosin_c-MyBPC is mixed with 50 and 500 μM ATP in the stopped-flow fluorimeter. The observed transient for myosin_c-MyBPC was described well by a single-exponential equation at both 50 and 500 μM ATP with observed rate constants (Kobs) of 22 s−1 at 50 μM ATP and 190 s−1 at 500 μM ATP. The second-order rate constants for ATP-induced dissociation of actomyosin, actomyosin_c-MyBPC, and actomyosin_c-MyBPC.PKA (K1k+2) as described in Scheme 2 are listed in Table 1 and were estimated from the linear fit to the plot of Kobs versus ATP concentration.</p><p>The second-order rate constant for ATP-induced dissociation from actin in the absence of c-MyBPC (K1k+2) was measured to be 0.42 ± 0.01 M−1 s−1. In the presence of unphosphorylated c-MyBPC where myosin and c-MyBPC are in a 1:1 ratio, the rate of dissociation decreases ~3.5-fold compared to that for myosin alone with a second-order rate constant of 0.12 ± 0.01 M−1 s−1, suggesting that the presence of c-MyBPC slows the dissociation of myosin from actin in the presence of ATP. This regulatory effect of c-MyBPC does not appear to be cooperative unlike the case for the binding of myosin to actin. In the presence of unphosphorylated c-MyBPC where myosin and c-MyBPC are in a 7:1 ratio, the rate of dissociation is similar to that for myosin alone with a second-order rate constant of 0.39 ± 0.02 M−1 s−1; however, at a 3:1 ratio, the rate of dissociation is reduced ~2-fold relative to that for myosin alone with a second-order rate constant of 0.22 ± 0.01 M−1 s−1. The ATP-induced dissociation of actin from myosin in the presence of phosphorylated c-MyBPC where myosin and c-MyBPC are in a 1:1 ratio also decreases ~1.5-fold relative to that for actomyosin alone with a second-order rate constant of 0.23 ± 0.01 M−1 s−1, but it is increased ~2-fold compared with that for ATP-induced actomyosin dissociation in the presence of unphosphorylated c-MyBPC. This regulatory effect also does not appear to be cooperative, and the phosphorylation of c-MyBPC appears to partially weaken the effects of c-MyBPC regulation on ATP-induced dissociation of actin from myosin. The rate of ATP-induced dissociation of actin from myosin in the presence of phosphorylated c-MyBPC where myosin and c-MyBPC are in a 7:1 ratio did not significantly deviate from the rate of dissociation of myosin from actin alone; however, at a 3:1 ratio, the rate of ATP-induced dissociation of actin from myosin decreases ~1.5-fold compared to that for myosin alone with a second-order rate constant of 0.29 ± 0.01 M−1 s−1 and is significantly increased ~0.5-fold compared to the rate of unphosphorylated c-MyBPC at a 3:1 ratio with myosin (Table 1).</p><!><p>The steady-state ATPase activity of myosin in the absence and presence of c-MyBPC and c-MyBPC.PKA where myosin and c-MyBPC are at a 1:1 or 3:1 myosin:c-MyBPC ratio was measured using the NADH-coupled assay.47,48 The release of Pi and ADP from the myosin motor can be conveniently followed by the change in absorbance of NADH at 340 nm after the addition of ATP. The change in absorbance over time in all cases was linear with a negative slope, and from these data, a plot of ADP production versus time was generated. The slope of this plot yielded the steady-state ATPase rate, and this was normalized by plotting the steady-state ATPase rate versus myosin_c-MyBPC concentration. Plots of the steady-state ATPase rate versus myosin and myosin_c-MyBPC concentration are shown in Figure 4. The steady-state ATPase rate of myosin alone is described as a linear function of its concentration as 0.015 ± 0.002 s−1. The steady-state rate of ATPase with myosin_c-MyBPC at a 1:1 or 3:1 myosin:c-MyBPC ratio is described as a sigmoidal function of its concentration, and the rate could not be accurately measured; however, the presence of c-MyBPC appears to significantly slow ATPase rates at low myosin concentrations (Figure 4). Kcat is the maximal activated ATPase rate of myosin_c-MyBPC, and KATPase is the concentration of myosin_c-MyBPC needed to reach half-maximal activation of myosin ATPase activity. The values of these two quantities are 0.24 ± 0.01 s−1 and 8.66 ± 0.15 μM for myosin and c-MyBPC in a 1:1 ratio and 0.23 ± 0.02 s−1 and 5.96 ± 0.20 μM for myosin and c-MyBPC in a 3:1 ratio, respectively.</p><p>The steady-state ATPase rate of myosin in the presence of phosphorylated c-MyBPC at 1:1 and 3:1 ratios with myosin was also determined. The ATPase rate for myosin in the presence of phosphorylated c-MyBPC at 1:1 and 3:1 ratios with myosin was described as a linear function of myosin_c-MyBPC.PKA concentration and was calculated to be 0.006 ± 0.001 and 0.005 ± 0.001 s−1, respectively. The rates in the presence of phosphorylated c-MyBPC were reduced compared to the rate for myosin alone; however, at least in the case of myosin_c-MyBPC at a 1:1 ratio, c-MyBPC phosphorylation appeared to relieve the inhibitory effect of c-MyBPC on ATPase rates at low myosin concentrations (Figure 4).</p><!><p>The purpose of this study was to elucidate the functional effects of c-MyBPC on the kinetics of key steps in the cross-bridge cycle of cardiac muscle contraction. Previous studies using skinned cardiac fibers or in vitro motility assays have demonstrated a regulatory role for c-MyBPC in cardiac muscle contraction;5,7,11–18 however, the role of c-MyBPC in the regulation of specific steps within the cross-bridge cycle has yet to be clearly established. In this study, we demonstrate that c-MyBPC regulates at least two key steps in the cross-bridge cycle that modulate cross-bridge association and dissociation from actin, and that some of the effects of c-MyBPC are mediated by affecting the cooperative behavior of cross-bridges, even in the absence of thin filament regulation. Finally, we show that PKA-induced phosphorylation of c-MyBPC regulates rates of actomyosin interactions, thereby providing the heart with a molecular mechanism that modulates the rates of cross-bridge cycling to match circulatory demands.</p><!><p>The kinetic pathway that describes the binding ofo myosin to actin is described in Scheme 1, and as expected, binding of myosin to actin in the absence of c-MyBPC exhibited Kobs values that were dependent upon myosin concentration, and the second-order rate constants could be easily determined from the gradient of the plot of Kobs versus myosin concentration (Figure 2). Our data show that unphosphorylated c-MyBPC regulates the binding of myosin to actin and that the mechanism of regulation does not adhere to the kinetic pathway represented in Scheme 1. The mechanism of regulation is cooperative as indicated by the sigmoidal relationship of Kobs when both unphosphorylated c-MyBPC and myosin are present, compared to the linear relationship of Kobs when only myosin is present (Figure 2). This cooperative binding by the porcine myosin as a consequence of the presence of unphosphorylated c-MyBPC was exhibited also when in the presence of a recombinant mouse full-length c-MyBPC (data not shown), suggesting that these effects are not species specific but rather a characteristic of c-MyBPC. At low myosin concentrations, the presence of c-MyBPC appears to inhibit the binding of myosin to actin as a lag is observed in the increase in the rate of binding of myosin to actin (Figure 2B), consistent with an inhibitory effect of c-MyBPC on myosin binding. This inhibition also appears to be dependent on c-MyBPC concentration, as at a 7:1 myosin:c-MyBPC ratio, the lag is smaller and the inhibition is more easily overcome at relatively low myosin concentrations. However, at 1:3 and 1:1 c-MyBPC:myosin ratios, the lag becomes progressively longer and a much higher myosin concentration is needed to overcome the inhibition of c-MyBPC on myosin binding to actin. Because the physiological ratio of myosin to c-MyBPC in the sarcomere is thought to be as high as 3:1 in the C-zone,20,49 it is reasonable to suggest that c-MyBPC would impart significant inhibition to the binding of myosin to actin in vivo. At higher myosin concentrations, there appears to be c-MyBPC-induced activation, whereby binding of myosin to actin is accelerated when compared with binding of myosin to actin alone (Figure 2B).</p><p>Previous studies suggested that c-MyBPC binds actin via its N-terminal6,8,18,20–27,50,51 or C-terminal52 domains; however, the stage of the cross-bridge cycle at which c-MyBPC interacts with actin has not been established. The cooperativity observed when myosin binds actin in the presence of c-MyBPC even at a 1:7 c-MyBPC:myosin ratio may suggest that c-MyBPC can bind actin together with myosin. Our data do not distinguish whether c-MyBPC is detached from myosin when myosin binds actin, or when the c-MyBPC domain binds to actin. We observed, however, that c-MyBPC does bind actin alone (Figure 2D), but the rate is slower compared to the rate of binding of myosin to actin; thus, it is unlikely that direct binding of c-MyBPC to actin significantly contributes to the observed acceleration of actomyosin association with an increasing myosin concentration. The observed lag phase in the binding of myosin to actin, which is dependent on c-MyBPC concentration, can be explained by a mechanism in which c-MyBPC inhibits binding of myosin to actin, perhaps by tethering the S2 region of myosin.14,15 Alternatively, it may be due to direct competition by c-MyBPC and myosin for actin binding sites, such that at low myosin concentrations c-MyBPC is able to bind actin more efficiently. Although binding of c-MyBPC to actin had only modest effects on Kobs, this binding could be significant in vivo as recent structural studies have suggested that the N-terminal domains of c-MyBPC bind actin in the same region as Tm at low Ca2+ concentrations, which may lead to the destabilization of tropomyosin from the blocked position, thereby favoring thin filament activation.6,24,27 Therefore, even weak binding of c-MyBPC to actin could significantly activate the thin filament to accelerate rates of force development.</p><p>The activation of binding of myosin to actin in the presence of c-MyBPC at higher myosin concentrations suggests that c-MyBPC facilitates a degree of synergy between adjacent S1 heads and adjacent myosin molecules that enhances cooperative binding to actin, such that initial binding of S1 heads recruits binding of additional S1 heads. In the absence of c-MyBPC, myosin will bind actin with a 1:1 stoichiometry, resulting in a linear relationship (Figure 2B); however, the presence of unphosphorylated c-MyBPC gives rise to more efficient S1 binding, which increases the number of S1 heads that interact with actin, perhaps because of changes in S1 alignment and/or stabilization of S1 heads that favors actin binding. Alternatively, c-MyBPC may give rise to a reordering of the S1 heads at low myosin concentrations toward actin binding sites,6,27 thereby accelerating cross-bridge binding and transitions to strongly bound states. Although our data do not support a clear mechanism by which c-MyBPC interacts with adjacent myosins, there is some evidence that the C0 domain and the proline-alanine (PA) domain may play a role in this mechanism as there has been some suggestion that these domains interact with the S1–S2 myosin hinge and the RLC regions of myosin.19,53 It is conceivable, therefore, that c-MyBPC could interact with adjacent RLC molecules to better stabilize the S1 heads of more than one myosin molecule. Taken together, these data suggest that c-MyBPC enhances force generation by enhancing cross-bridge binding and cooperative recruitment by direct effects on cross-bridge binding and/or orientation, and indirectly via actin binding, which would enhance thin filament activation.</p><!><p>Dissociation of myosin from actin in the absence of c-MyBPC exhibited Kobs values that increased with an increasing ATP concentration,54–56 and the second-order rate constants as described in Scheme 2 could be easily determined from the gradient of the plot of Kobs versus myosin concentration (Figure 3B,C). In the presence of unphosphorylated c-MyBPC, the second-order rate constant for dissociation was decreased ~3.5- and ~2-fold when c-MyBPC was in 1:1 and 1:3 ratios with myosin, respectively, compared with that for myosin alone; however, at a low c-MyBPC concentration (i.e., at a 1:7 ratio with myosin), c-MyBPC had only small effects on dissociation rates (Figure 3B). The reduction in dissociation rates in the presence of c-MyBPC compared to that with myosin alone suggests that c-MyBPC slows cross-bridge detachment and agrees with in vitro motility data that show that actin filament sliding velocity is reduced in the presence of unphosphorylated c-MyBPC.18,20 These studies suggest that c-MyBPC directly interacts with actin at high ATP concentrations because of the lower affinity of myosin S1 for actin under those conditions, and this interaction could lead to the reduction in the rate of dissociation that we observe in our studies. It has been suggested that a population of slowly cycling cross-bridges could give rise to a viscous drag force that limits muscle shortening velocity,57 and it is conceivable that slowly cycling cross-bridges could significantly impact cross-bridge behavior in regions of the sarcomere where c-MyBPC is abundant (i.e., present at a 1:3 ratio with myosin). Furthermore, because c-MyBPC may interact with more than one actin molecule,27 even weak interactions between c-MyBPC and actin could have significant effects on muscle contraction in vivo. Our in vitro studies also agree well with previous studies utilizing skinned fiber preparations that showed that biochemical extraction, or the genetic ablation of c-MyBPC, accelerates shortening velocity,13,14,58 presumably because of a weakened inhibition of c-MyBPC on cross-bridge detachment.</p><p>Although there is strong evidence that supports binding of c-MyBPC to actin, interactions of c-MyBPC with S2 at this stage of the cross-bridge cycle cannot be ruled out, as it would be possible that a direct interaction between c-MyBPC and S2 could delay one or even both of the S1 heads from detaching, thus creating the viscous drag without directly binding to actin. Regardless of the mechanism, our data show, in agreement with others,18,20 that c-MyBPC slows cross-bridge detachment rates and therefore acts as a "brake" on the contractile apparatus.</p><!><p>The ATPase activity in the absence and presence of c-MyBPC was determined using the NADH-coupled assay to assess the functional effects of c-MyBPC on the overall rate of ATPase turnover. The data show that the ATPase rate of myosin alone was directly proportional to myosin concentration, which was exhibited as a linear curve, consistent with the 1:1 stoichiometry of actomyosin binding (Figure 4). In the presence of c-MyBPC, the curve exhibited a sigmoidal relationship (Figure 4) similar to that observed with the actomyosin association transient kinetic data. The lag phase exhibited by the ATPase curve confirms in the steady state that at high c-MyBPC concentrations (i.e., at a 1:1 or 1:3 c-MyBPC:myosin ratio), c-MyBPC directly inhibits binding of myosin to actin. Slowed ATPase rates at low myosin concentrations and high c-MyBPC concentrations could be due to both slowed cross-bridge association (Figure 2D) and dissociation (Figure 3B). The inhibition of ATPase rates at high c-MyBPC concentrations is overcome only at a certain myosin concentration threshold, which restores myosin binding; however, the maximal ATPase rate in this instance in the absence of c-MyBPC is not significantly different from that for myosin alone. Our results show that the ATPase activity of the myosin motor is inhibited at low myosin concentrations, consistent with previous studies that showed that c-MyBPC inhibits actomyosin ATPase activity18,59,60 in the presence of ATP, while in the absence of ATP myosin binding is restored.59</p><p>Enhanced cross-bridge binding due to c-MyBPC with increasing myosin concentrations and slower cross-bridge detachment rates enhance force generation by augmenting the number of force-generating cross-bridges at a given time and their duty ratio, which is consistent with the leftward shift in the force–pCa relationship observed in skinned fibers incubated with N-terminal c-MyBPC peptides.8,17,18 A slowing of the ATPase rates in the presence of c-MyBPC could be beneficial in vivo via prevention of premature cross-bridge detachment and, thereby, a truncation in the duration of the systolic ejection phase.28,58,61</p><!><p>Some studies in skinned myocardium have shown that phosphorylation of c-MyBPC accelerates cross-bridge kinetics,28–30 while other studies show no effect of PKA-induced phosphorylation of c-MyBPC on cross-bridge kinetics.32,33 It has been shown that phosphorylation of c-MyBPC increases the proximity of myosin heads to actin, thereby relieving the tethering constraint imposed by c-MyBPC on myosin heads and increasing the probability of actomyosin interaction.62 Other studies involving direct binding of c-MyBPC to actin showed that phosphorylation of c-MyBPC reduces the binding affinity of N-terminal domains of c-MyBPC for actin,25 suggesting that c-MyBPC phosphorylation may act to reduce drag on cross-bridge cycling and accelerate cross-bridge detachment. Our studies showed that during cross-bridge association, phosphorylation of c-MyBPC abolishes the lag phase of binding of the cross-bridge to actin, resulting in a linear Kobs versus myosin concentration relationship, which is directly proportional to myosin concentration (Figure 2C), consistent with the kinetic mechanism described in Scheme 1. The second-order rate constant is not significantly different from that for myosin binding alone, and this along with the elimination of the lag phase and sigmoidal curve exhibited by the binding of myosin in the presence of unphosphorylated c-MyBPC strongly suggests that phosphorylation of c-MyBPC abolishes the inhibitory effects of c-MyBPC on actomyosin interactions at low myosin concentrations. Phosphorylation of c-MyBPC, therefore, accelerates the attachment step of the cross-bridge cycle relative to binding in the presence of unphosphorylated c-MyBPC, and the second-order rate constant of this step is closely comparable to that observed for myosin alone (Table 1).</p><p>With respect to the ATP-induced dissociation of myosin from actin, phosphorylation of c-MyBPC when present at a 1:1 or 1:3 ratio with myosin significantly increased the rate of dissociation relative to the rate of dissociation in the presence of unphosphorylated c-MyBPC (Table 1). In the presence of both dephosphorylated and phosphorylated c-MyBPC, the Kobs versus ATP concentration curves were linear, suggesting that the regulation of c-MyBPC on ATP-induced dissociation is not cooperative (Figure 3B,C) and adheres to the kinetic mechanism described in Scheme 2. This result possibly suggests that c-MyBPC could be bound to actin at this stage of the cross-bridge cycle, in accordance with the idea that c-MyBPC binds actin directly and acts as a brake on cross-bridge kinetics,6,8,18,20,24,25,27 which is removed when c-MyBPC is phosphorylated. Because Kobs values for rates of dissociation of porcine cardiac myosin alone are ~20% faster than the rate of association, we can speculate that PKA-induced acceleration of cross-bridge dissociation results in accelerated ATPase rates and a shortened cross-bridge duty ratio. In contrast, when c-MyBPC is unphosphorylated, the reduction in kinetics due to cooperative activation of cross-bridge association slows the overall rate of ATPase turnover.</p><p>In addition to c-MyBPC, there are several targets of β-agonist stimulation in the myocyte that are critical regulators of in vivo cardiac function. Increased sympathetic drive is known to accelerate Ca2+ cycling in the myocyte and enhances the rate and amount of Ca2+ that is released and sequestered by the sarcoplasmic reticulum (SR) (reviewed in 63 and 64). Phosphorylation of phospholamban relieves the inhibition on the Serca2a Ca2+-ATPase pump, allowing more entry of Ca2+ into the SR, and phosphorylates ryanodine receptors in the SR, allowing greater Ca2+ release, thereby enhancing the amplitude of the Ca2+ transient and reducing its duration and contributing to accelerated systolic and diastolic function. At the level of the myofilaments, β-adrenergic stimulation principally phosphorylates the thin filament protein troponin I, resulting in altered interactions with troponin C to weaken its Ca2+ binding affinity, thereby allowing accelerated rates of force relaxation (reviewed in refs 65 and 66). Data from this study support a model (depicted in Figure 2S of the Supporting Information) in which c-MyBPC appears to have a dual role in regulating cross-bridge kinetics, which directly affects at least two steps of the cross-bridge cycle in a phosphorylation-dependent manner. c-MyBPC inhibits cross-bridge binding at low myosin concentrations, either by binding to myosin directly or by competing with myosin for actin binding sites, and slows cross-bridge dissociation rates to prolong cross-bridge attachment time, with the net result being enhanced force generation that is sustained for a longer duration. Phosphorylation of c-MyBPC relieves the inhibitory delay on cross-bridge binding and speeds cross-bridge dissociation such that the overall rate of cross-bridge cycling is accelerated. Therefore, in conjunction with accelerated Ca2+ handling properties, c-MyBPC and troponin I phosphorylations at the myofilament level fine-tune cross-bridge behavior and motor function to match the augmented contractile requirements of the heart under conditions of increased workload.</p>

PubMed Author Manuscript

RNF38 Encodes a Nuclear Ubiquitin Protein Ligase that Modifies p53

The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

rnf38_encodes_a_nuclear_ubiquitin_protein_ligase_that_modifies_p53

INTRODUCTION<!>Cell Culture and transfections<!>Constructs<!>Expression and purification of RNF38 from Baculovirus<!>Antibodies<!>Binding studies<!>Ubiquitination assay \xe2\x80\x93 in vitro<!>Ubiquitination assay \xe2\x80\x93 in vivo<!>Immunofluorescence<!>RNF38 is a nuclear protein<!>Identification of nuclear localization signals<!>RNF38 is an E3 ubiquitin ligase<!>The RING domain limits RNF38 overexpression<!>RNF38 is a p53 binding protein<!>RNF38 ubiquitinates p53 in vitro and in vivo<!>Overexpression of RNF38 alters p53 localization<!>DISCUSSION

<p>The p53 tumor suppressor gene (TP53) has been termed "the guardian of the genome" [1] and it is one of the most frequently mutated genes in human cancers [2, 3, 4, 5]. Mutations in the regulators of p53, such as Arf, Mdm2 and Pirh2, are also commonly seen in cancer cells [6, 7, 8]. Considering the extensive involvement of p53 in tumorigenesis, understanding p53 regulation is important to the continued development of anti-cancer strategies.</p><p>In a search for new p53 interactors, Lunardi et al. employed an affinity purification approach with Drosophila p53 in which they identified many novel p53 binding proteins, including the little studied protein, RING finger protein 38 (RNF38) [9]. Although human RNF38 has been previously cloned [10], its function is currently unknown.</p><p>RNF38 mRNA is widely expressed in a variety of human tissues [10], and evolutionary conservation suggests an important cellular function. Interestingly, RNF38 is located on an area of chromosome 9 (9p13) that is frequently deleted in multiple cancers such as ganglioglioma [11], lung cancer [12, 13], hepatocellular carcinoma [14], and in the 9p13-24 deletion seen in the lymphoid blast transformation of chronic myeloid leukemia [15].</p><p>Many proteins containing a RING finger motif act as ubiquitin protein ligases [16, 17]. We wanted to explore the possibility that RNF38 might have E3 activity and whether or not p53 might be a substrate. p53 is known to be regulated by several ubiquitin protein ligases, the best studied of which is Mdm2/Hdm2. [18, 19, 20]. Other E3 ligases that can modify p53 include WWP1, Pirh2 and Cop1 [21, 22, 23]. Here we find that RNF38 does possess E3 ubiquitin ligase activity and can recognize p53 as a substrate for ubiquitination. In addition, overexpression of RNF38 alters the nuclear localization of p53.</p><!><p>All cell lines were obtained from the Tissue Culture Shared Resource at the University of Colorado Cancer Center. Human embryonic kidney 293 (HEK293) and HEK293T cells were maintained in DMEM (Hyclone) supplemented with 10% fetal bovine serum and antibiotics. H1299 cells were grown in RPMI (Hyclone) supplemented with 10% fetal bovine serum and antibiotics. Transfections were performed with Lipofectamine (Invitrogen).</p><!><p>cDNA for RNF38 isoform 1 was obtained by RT-PCR from HEK293 mRNA and cloned into pcDNA 3.1(+) (Invitrogen). The RNF38 cDNA insert and all other constructs derived by PCR were fully sequenced. GFP fusions were made in pEGFP-C1 (Clontech). PCR primers and cloning details are provided in the supplementary materials. The mammalian expression vector pEBG-SrfI was obtained from Yusen Liu. Vector pMT 123 expressing HA-ubiquitin was obtained from Mathias Treier.</p><!><p>A six histidine tag was added to the amino terminus of RNF38 by PCR and cloned into the baculovirus recombination plasmid pVL1393. The Baculovirus Core Facility at the University of Colorado Cancer Center performed the recombination into baculovirus and growth in SF9 cells. RNF38 was purified from extracted nuclei using immobilized Co++ ("Talon", Clontech) affinity chromatography. Full details are provided in the supplementary data.</p><!><p>Mouse monoclonal anti-p53 (1C12) was purchased from Cell Signaling. Rabbit anti-p53 (sc-6243), mouse monoclonal anti-p53 (sc-126), rabbit anti-RNF38 (sc-102096), rabbit anti-GFP (sc-8334) and mouse monoclonal anti-PML (sc-966) were purchased from Santa Cruz Biotechnology. A second rabbit anti-RNF38 antibody (AP12816a) was acquired from Abgent. Mouse anti-NR1 (#05-432) was obtained from Upstate Biotechnology. Horseradish peroxidase (HRP) conjugated rat anti-HA antibody (3F10) was from Roche Applied Science.</p><!><p>GST and GST-p53 fusion proteins were purified from E. coli BL21 transformed with pGEX-4T-2-(p53). Either 2 μg of purified RNF38 in GST binding buffer (50 mM NaCl, 20 mM Tris pH 7.5, 1 mM MgCl2, 1 mM DTT, 0.1% Thesit and 1.0 mg/mL BSA) or 850 μg of 293T whole cell extract in lysis buffer (50 mM NaCl, 20 mM Tris pH 7.5, 1 mM MgCl2, 1.0% Trition X-100, 1 mM Na3VO4, 1 mM DTT and 1 mM PMSF plus protease inhibitors) were incubated at 4° C for 30 minutes with equal amounts (2 μg) of GST or GST-p53. Preblocked (5% BSA) glutathione-Sepharose beads (G.E. Healthcare Lifesciences) were then added to the mixtures and incubated for 2 hours at 4° C with mixing. The beads were then washed with GST wash buffer (50 mM NaCl, 20 mM Tris pH7.5, 1 mM MgCl2, 1 mM DTT and 0.1% Thesit) and subjected to western blot analysis.</p><!><p>Purified RNF38 was mixed with 50 μg/ml E1, E2 and 0.5 mg/ml HA-ubiquitin in ubiquitin buffer (50 mM KCl, 20 mM HEPES pH 7.4, 5 mM MgCl2, 1 mM DTT and 1 mM ATP) and incubated for 30 minutes at 30° C with mixing at 600 rpm. The reaction products were analyzed by western blot with anti-RNF38 (sc-102096) and anti-HA antibodies.</p><p>For in vitro p53 ubiquitination reactions, p53 was immunoprecipitated from HEK293T cells lysed in RIPA+ buffer (150 mM NaCl, 20 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 1.0% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF plus protease inhibitors). Mouse anti-p53 (1C12) or mouse anti-NR1 control (0.75 μg) was added to 500 μg of precleared whole cell lysate and incubated overnight. Antibody complexes were then bound to preblocked (5% BSA) protein G-agarose (Sigma-Aldrich P-4691) for two hours followed by extensive washing: twice with RIPA+, twice with SNNTE (0.5 M NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 5% sucrose, 1% NP-40), twice with 200 mM glycine pH 2.5, and twice with ubiquitin buffer. The washed beads were then used in in vitro ubiquitination reactions with or without purified RNF38 as described above. Following the ubiquitination reactions, the beads were washed twice with RIPA+ and then analyzed by western blot with anti-p53 and anti-HA antibodies.</p><!><p>HEK293T cells were transfected with pMT 123 (HA-ubiquitin) plus pEBG or pEBG-RNF38. 24 hours post-transfection cells were passaged and allowed to grow for 48 hours. At 72 hours post-transfection the cells were incubated for 4 hours in 25 μM MG132 (Sigma-Aldrich) then collected in lysis buffer (100 mM NaCl, 20 mM Tris pH 7.5, 5 mM EDTA, 10% glycerol, 1% NP-40, 1 mM DTT, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 10 mM NEM plus protease inhibitors). Immunoprecipitation of p53 was performed using 1 μg of mouse anti-p53 (sc-126) antibody to 1.5 mg of precleared whole cell extract followed by binding to preblocked protein G-agarose. Bound complexes were washed with lysis buffer then analyzed by western blot with rat anti-HA antibody.</p><!><p>Cultured cells were seeded on glass coverslips, transfected and 24 hours later fixed with 4% para-formaldehyde in PBS. Cells were permeabilized with cold methanol, rinsed with PBS, and blocked with 2% nonfat milk, 0.1% Tween-20 in PBS. Primary antibody rabbit anti-p53 (sc-6243) was used at 2 μg/ml in 1% BSA, 0.5% Triton X-100 in PBS and incubated for 1 hour at room temperature. Primary antibody rabbit anti-RNF38 (AP12816a) was used at 5 μg/ml and anti-PML (sc-966) was used at 4 μg/ml incubated overnight at 4° C. Cells were washed with 0.05% Tween-20 in PBS and incubated with the appropriate fluorescent secondary (Alexa Fluor 568 goat anti-rabbit or Alexa Fluor 488 goat anti-mouse, Invitrogen) at 2 μg/ml for 1 hour. Nuclei were counterstained with 0.2 μg/ml Hoechst 33258 (Kodak). Coverslips were mounted in gelvatol and examined with a Nikon Eclipse Ti-S microscope fitted with the appropriate fluorescence filters, DS-Qi1 camera and NIS-Elements imaging software.</p><!><p>In their study identifying p53-interacting proteins in Drosophila, Lunardi et al. also examined the intracellular localization of 41 mammalian orthologs, including RNF38 [9]. Immunofluorescence of epitope-tagged RNF38 overexpressed in U2OS cells revealed diffuse nuclear staining. However, the data presented was a supplementary figure containing an image of a single cell with no mention of which RNF38 isoform was used or if any other staining patterns were observed. As a first step toward understanding RNF38 function, we sought to confirm and expand this result.</p><p>We first examined RNF38 in HEK293T cells. Endogenous RNF38 was detectable by immunofluorescence with only one of two commercially available anti-RNF38 antibodies (AP12816a) under the conditions employed. Similar to the results of Lunardi et al., we find endogenous RNF38 is also localized to the nucleus in HEK293T cells (Fig. 1A). RNF38 is predicted to have three isoforms. Isoform 1 is the largest, containing 515 amino acids, whereas isoform 2 lacks amino acids 5-54, and isoform 3 lacks amino acids 1-84. According to the manufacturer, the AP12816a antibody was raised against amino acids 47-77 of RNF38, a region present in isoform 1 but partially or entirely lacking in the other isoforms. We chose to focus on isoform 1, henceforth referred to as simply "RNF38".</p><p>The endogenous RNF38 signal was fairly weak, so to further investigate RNF38 nuclear localization, we constructed a GFP-tagged RNF38 expression vector. As shown in Figure 1, transient expression (Fig. 1D, second row) resulted in nuclear localization of GFP-RNF38. Note also that the stronger signal from overexpressed GFP-RNF38 allows more detail to be observed, and the protein appears to be excluded from nucleoli.</p><!><p>To identify the motif(s) responsible for RNF38 nuclear localization we analyzed its amino acid sequence with several nuclear localization signal (NLS) prediction programs. cNLS Mapper [24] identified amino acids 57-71 as a possible NLS, and amino acids 115-131 were identified as a possible NLS by NLStradamus [25]. We then created a series of GFP fusions to examine the role these sequences might play in RNF38 localization (Fig. 1C).</p><p>Transient expression of GFP in HEK293T cells results in a uniform staining pattern throughout the nucleus and cytoplasm (Fig. 1D). In contrast, the GFP-RNF38 fusion protein appears restricted to the nucleus. GFP fusion to either amino acids 57-71 or 115-131 is insufficient to confer complete nuclear localization, though a larger GFP fusion to amino acids 57-131 is completely localized to the nucleus . This region contains both of the basic residue-rich sequences identified as putative NLSs and thus appears to function as a bipartite NLS. Deletion of this region results in loss of exclusive nuclear localization with the GFP-RNF38 57-131 fusion protein localized in both cytoplasm and nucleus.</p><!><p>In addition to ubiquitination of their target proteins, many E3 ubiquitin ligases undergo autoubiquitination in vitro [16, 17]. Figure 2 shows in vitro ubiquitination assays using His-tagged RNF38 purified from baculovirus infected SF9 cells. RNF38 clearly undergoes robust autoubiquitination and shows specificity in interacting with E2s. Of the three E2 proteins tested (UbcH2, UbcH5b and UbcH7), only UbcH5b supported RNF38 autoubiquitination. These findings demonstrate RNF38 is an active ubiquitin protein ligase (E3).</p><!><p>We found RNF38 to be resistant to overexpression by transfection despite using a variety of expression plasmids and cell lines (including MDA-MB-231, H1299 and HEK293, data not shown). Since an active RING domain might limit expression through autoubiquitination or down-regulation of some other cellular component, we removed the C-terminal RING domain from RNF38 (RNF38 ΔRING) to explore the issue.</p><p>Truncation of the RING domain greatly increased RNF38 expression (Fig. S1A). In two cell types (HEK293T and H1299), transfection with GFP alone or GFP-RNF38ΔRING yielded many GFP-positive cells, whereas with full-length GFP-RNF38, only rare GFP-positive cells were observed. Western blot analysis of these cells confirmed the results (Fig. S1B).</p><p>Successful overexpression of full-length RNF38 constructs was detectable on western blots using derivatives of the vector pEBG in HEK293T cells (Fig. S1C). The pEBG plasmid contains an SV40 origin of replication, allowing amplification of the plasmid in HEK293T cells which express SV40 large T antigen. Treatment of cells with the proteasome inhibitor MG132 did not increase RNF38 expression, nor was a prominent ladder of ubiquitinated RNF38 species observed, suggesting that autoubiquitination may not be the mechanism limiting overexpression of full-length RNF38 (Fig. S1C). Additional experiments will be required to identify the mechanism(s) regulating RNF38 expression.</p><!><p>We have examined the p53-RNF38 binding interaction first reported by Lunardi et al. using a different affinity purification technique employing a fusion protein between human p53 and glutathione S-transferase (GST). Purified RNF38 binds specifically to GST-p53 but not GST alone (Fig. 3A, upper panel). Similarly, endogenous RNF38 in lysates of HEK293T cells specifically binds GST-p53 but not GST alone (Fig. 3B).</p><!><p>To test whether p53 is a substrate for ubiquitination by RNF38, endogenous p53 was immunoprecipitated from HEK293T lysate and added to in vitro ubiquitination reactions. Control immunoprecipitates using an unrelated antibody (anti-NR1) were treated similarly. We found that p53 was ubiquitinated in an RNF38-dependent manner under these conditions (Fig. 3C).</p><p>Next, we asked whether we could detect RNF38-dependent ubiquitination of p53 in cells. HEK293T cells were transfected with an expression vector for HA-tagged ubiquitin and either GST or GST-RNF38. p53 was recovered by immunoprecipitation and analyzed by immunoblotting for HA-ubiquitin (Fig. 3D). GST-RNF38 clearly increased p53 ubiquitination in vivo, as evidenced by the ladder of higher molecular weight species reacting with HA (ubiquitin) antibody. We note that the p53 complexes isolated from cells appear more extensively modified than those produced in vitro, suggesting there may be additional factors or conditions involved in p53 ubiquitination in cells.</p><p>Investigation of possible effects of RNF38 on p53 turnover has been hindered by the resistance of RNF38 to overexpression. As mentioned earlier, detection of biochemical levels of exogenous RNF38 has only been achieved in HEK293T cells, in which SV40 large T antigen prevents normal p53 turnover [26]. Experiments in other systems, including H1299 cells (which lack p53 expression), have been inconclusive due to insufficient/undetectable RNF38 expression.</p><!><p>Since biochemical analysis of possible RNF38 effects on p53 turnover has been uninformative due to the technical issues described above, we turned to fluorescence microscopy to allow visualization of any effects on intracellular localization of p53. HEK293T cells were transfected with GFP-RNF38 or GFP alone, then fixed and stained for p53. In untransfected and GFP-transfected HEK293T cells, p53 is distributed diffusely throughout the nucleus with a small minority of cells (13 %) showing punctate staining (Figure 4). In cells transfected with GFPRNF38, p53 redistributed to large punctate structures in the nucleus in the majority of GFP-RNF38 positive cells (69 %).</p><p>Since p53 has been previously identified in PML (promyelocytic leukemia-associated protein) nuclear bodies [27, 28], we analyzed the localization of PML and p53 in RNF38 transfected cells. A typical cell with a punctate p53 phenotype is shown in Figure 4C. In these cells many, though not all, p53 foci associate directly adjacent to PML nuclear bodies. The number of p53 and PML foci vary from cell to cell, and while many p53 foci colocalize with PML foci, a few of each type do not. These data suggest that overexpression of RNF38 can change the distribution of p53 within the nucleus from diffuse to punctate, and many of these foci are associated with PML nuclear bodies.</p><!><p>This study has begun the functional characterization of the evolutionarily conserved protein, RNF38. We have shown that RNF38 (isoform 1) is localized to the nucleus in several cell types and have identified a bipartite NLS capable of mediating this localization. We have expressed and purified RNF38 from baculovirus-infected SF9 cells and found that purified RNF38 protein is an active ubiquitin protein ligase that exhibits specificity with the E2 protein with which it interacts. We have confirmed Lunardi et al.'s findings that RNF38 is a p53-binding protein, shown that RNF38 can ubiquitinate p53 in vitro, and demonstrated that overexpression of RNF38 increases p53 ubiquitination in vivo.</p><p>Although our efforts to elucidate the dynamics of the p53-RNF38 interaction biochemically were hindered by limited RNF38 overexpression, fluorescence microscopy revealed that overexpression of RNF38 in HEK293T cells alters the intracellular distribution of p53 from a diffuse nuclear pattern to punctate foci, many of which are associated with PML nuclear bodies. Association of p53 with PML nuclear bodies has been previously described in a number of instances [27-30], and it has been suggested that PML nuclear bodies may be sites of functional regulation of p53 [31]. In this regard, our data indicating altered p53 localization upon RNF38 overexpression are intriguing. While insufficient to illuminate the exact functional interplay between these two proteins, these results do provide additional evidence for a regulatory relationship between RNF38 and p53. Taken together with our other findings, a case can be made that RNF38 may be a biologically significant regulator of p53. It remains to future studies to elaborate the full functional details of such regulation.</p>

PubMed Author Manuscript

A Comprehensive Approach to the Profiling of the Cooked Meat Carcinogens 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and their Metabolites in Human Urine

A targeted liquid chromatography/tandem mass spectrometry-based metabolomics-type approach, employing a triple stage quadrupole mass spectrometer in the product ion scan and selected reaction monitoring modes, was established to profile 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and their principal metabolites in urine of omnivores. A mixed-mode reverse phase cation exchange resin enrichment procedure was employed to isolate MeIQx, and its oxidized metabolites, 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-IQx) and 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), which are produced by cytochrome P450 1A2 (P450 1A2). The phase II conjugates N2-(\xc3\x9f-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and N2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)-sulfamic acid were measured indirectly, following acid hydrolysis to form MeIQx. The enrichment procedure permitted the simultaneous analysis of PhIP; N2-(\xc3\x9f-1-glucosidurony1)-2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine; N3-(\xc3\x9f-1-glucosidurony1)-2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine; 2-amino-1-methyl-6-(4\xe2\x80\xb2-hydroxy)-phenylimidazo[4,5-b]pyridine (4\xe2\x80\xb2-HOPhIP); and the isomeric N2- and N3-glucuronide conjugates of the carcinogenic metabolite, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which is formed by P450 1A2. The limit of quantification (LOQ) for MeIQx, PhIP, and 4\xe2\x80\xb2-HO-PhIP was ~5 pg/mL; the LOQ values for 8-CH2OH-IQx and IQx-8-COOH were, respectively, <15 pg/mL and <25 pg/mL; and the LOQ values for the glucuronide conjugates of PhIP and HONH-PhIP were 50 pg/mL. The metabolism was extensive: Less than 9% of the dose was eliminated in urine as unaltered MeIQx and <1% was eliminated as unaltered PhIP. Phase II conjugates of the parent amines accounted for up to 12% of the dose of MeIQx, and up to 2% of the dose of PhIP. 8-CH2OH-IQx and IQx-8-COOH accounted for up to 76% of the dose of MeIQx, and the isomeric glucuronide conjugates of HONH-PhIP accounted for up to 33% of the dose of PhIP that were eliminated in urine within 10 hours of meat consumption. P450 1A2 significantly contributes to the metabolism of both HAAs, but with marked differences in substrate specificity. P450 1A2 primarily catalyzes the detoxification of MeIQx by oxidation of the 8-methyl group, whereas it catalyzes the bioactivation of PhIP by oxidation of the exocyclic amine group.

a_comprehensive_approach_to_the_profiling_of_the_cooked_meat_carcinogens_2-amino-3,8-dimethylimidazo

INTRODUCTION<!>EXPERIMENTAL PROCEDURES<!>Materials and Methods<!>General Methods<!>2-Amino-3-methylimidazo[4,5-f]quinoxaline-8-carbaldehyde, 8-CH2OH-IQx, IQx-8-COOH, and MeIQx-N2-SO3H<!>Biosynthesis of NOH-MeIQx-N2-Gl and MeIQx-N2-Gl<!>Biosynthesis of 4\xe2\x80\xb2-HO-PhIP, N2-(\xc3\x9f-1-glucosidurony1)-2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (PhIP-N2-Gl), N3-(\xc3\x9f-1-glucosidurony1)-2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (PhIP-N3-Gl), N2-(\xc3\x9f-1-glucosidurony1)-N-hydroxy-2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N2-Gl) and N3-(\xc3\x9f-1-glucosidurony1)-N-hydroxy-2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N3-Gl) Conjugates<!>Human Subjects and Meat Consumption<!>Solid-phase Extraction (SPE) of MeIQx and PhIP and their Metabolites from Urine<!>LC-ESI/MS/MS Analyses<!>Calibration Curves<!>Enzyme and Acid Hydrolysis Assays<!>Statistical Analysis<!>Rapid SPE and LC-ESI/MS/MS Analysis of MeIQx, 8-CH2OH-MeIQx, IQx-8-COOH, PhIP, PhIP-N2-Gl, 4\xe2\x80\xb2-HO-PhIP, HON-PhIP-N2-Gl, and HON-PhIP-N3-Gl in Urine of Omnivores<!>Performance of the Analytical Method<!>Estimation of MeIQx and PhIP Urinary Metabolites, and Correlations Among Urinary Metabolic Ratios (MRs)<!>Indirect Measurement of Phase II Detoxification Products of MeIQx and PhIP<!>Discussion

<p>Urine is a useful biological matrix for the assessment of recent exposures to carcinogens, since large quantities can be obtained noninvasively. Moreover, the characterization of the urinary metabolic profiles of the genotoxicants can provide an estimate of the relative extent of bioactivation, as opposed to detoxification, undergone by the chemicals in vivo (1). These measurements can also reveal inter-individual differences in metabolism due to genetic polymorphisms that code for enzymes involved xenobiotic metabolism; such differences can affect the genotoxic potency of many procarcinogens. A number of biomarkers of carcinogens present in tobacco smoke (1), as well as biomarkers of the hepatocarcinogen aflatoxin B1 (2,3), have been measured in human urine.</p><p>The measurement of urinary biomarkers of carcinogens can also be used to assess the efficacy of chemoprotective agents in modulating the activities of phase I and II enzymes involved in carcinogen metabolism (4,5). This is an area of research that we are currently investigating and involves heterocyclic aromatic amines (HAAs), a class of carcinogens formed in high-temperature-cooked meats (6).</p><p>2-Amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are two of the most mass-abundant carcinogenic heterocyclic aromatic amines (HAAs) formed in cooked meats: Concentrations can range from less than 1 part per billion (ppb) to greater than 15 ppb in meats prepared under common household cooking conditions (7). Both compounds induce tumors in multiple organs of rodents during long-term feeding studies (6). Putative DNA adducts of MeIQx (8) and PhIP (9-12) have been detected in human tissues. Thus, the chronic consumption of foods containing these HAAs constitutes a potential human health hazard: the Report on Carcinogens, Eleventh Edition, of the National Toxicology Program, concluded that several prevalent HAAs, including MeIQx and PhIP, are "reasonably anticipated" to be human carcinogens (13).</p><p>The metabolism of MeIQx and PhIP has been extensively studied in vitro with tissue fractions, purified and recombinant enzymes (14-17), and hepatocytes (18,19), as well as in vivo in experimental laboratory animals (18,20-22), and in humans (5,23-28). The major pathways of metabolism of MeIQx and PhIP are depicted in Schemes 1 and 2. Metabolic activation occurs by cytochrome P450 mediated N-oxidation of the exocyclic amine group, to produce 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) and 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONHPhIP). This oxidation step is catalyzed primarily by P450 1A2 in liver and by P450s 1A1 and 1B1 in extrahepatic tissues (16,17). The HONH-HAAs undergo further metabolism by N-acetyltransferases (NATs) or by sulfotransferases (SULTs), to produce highly reactive esters that bind to DNA (29).</p><p>Competing pathways of metabolism serve as mechanisms of detoxification for both HAAs. In the case of MeIQx, P450 1A2 also catalyzes oxidation of the 8-CH3 group to form 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-IQx), which undergoes further oxidation by P450 1A2, to form 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH) (30). This latter metabolite is the major detoxification product of MeIQx in human hepatocytes (31), and in humans (23). It is noteworthy that IQx-8-COOH is not formed in rodents or nonhuman primates (21,31). Human P450s catalyze oxidation at the 4′ position of the phenyl ring of PhIP, to form 2-amino-1-methyl-6-(4′-hydroxy)-phenylimidazo[4,5-b]pyridine (4′-HO-PhIP); however, the rates of formation of 4′-HO-PhIP are significantly lower than the rates of HONH-PhIP formation (16,22,32). Although 4′-HO-PhIP is a minor metabolic product of human P450 1A2, it is a principal detoxification product of PhIP in rodents and non-human primates (18,21).</p><p>MeIQx and PhIP also undergo detoxification by phase II conjugation reactions. MeIQx undergoes sulfamation, to form N2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)-sulfamic acid (MeIQx-N2-SO3H), the process is catalyzed by sulfotransferase 1A1 (SULT1A1) (33). A second phase II metabolite of MeIQx is the glucuronide conjugate, N2-(ß-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx-N2-Gl), which is formed by uridine diphosphate glucuronosyltransferases (UGTs), apparently UGT1A isoforms (34-36). An N2-glucuronide conjugate of HONH-MeIQx has been characterized as N2-(ß-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (HON-MeIQx-N2-Gl) (37). Both PhIP and HNOH-PhIP undergo conjugation by UGT1A isoforms to produce N2- and N3-glucuronide conjugates (34-36). The glucuronide conjugates of HNOH-MeIQx and HONH-PhIP are viewed as detoxification products (27).</p><p>The analysis of unaltered MeIQx and PhIP, and their metabolites, in human urine is an analytical challenge, because usually only ~1 to several μg of each compound is ingested per day, in individuals eating well-done meat (38). Thus, the concentrations of these HAAs and their metabolites are often well below the ppb level in urine. The polar and ionic nature of the metabolites also presents difficulties for their isolation along with their parent HAAs, from thousands of other components in the urine matrix. Various analytical approaches have been devised to isolate MeIQx or PhIP from human urine: such techniques have included solvent extraction (39,40), solid-phase extraction (SPE) (41), use of molecularly imprinted polymers (28), and immunoaffinity methods (24), followed by quantification by gas chromatography and negative ion chemical ionization mass spectrometry (GC-NICI-MS) (39,40,42), or LC-ESI/MS/MS (28,41), or alternatively followed by fluorescence detection (25). [14C]-MeIQx, [14C]-PhIP, and [14C]-radiolabeled metabolites have been identified in human urine by accelerator mass spectrometry (AMS) (23,27,43). Urinary metabolites have also been detected by liquid chromatography-electrospray ionization/mass spectrometry/tandem mass spectrometry (LCESI/MS/MS) (5,26), or indirectly, after chemical reduction or acid hydrolysis of HONH-PhIP conjugates, with detection LC-ESI/MS/MS or GC-NICI-MS (44,45).</p><p>To our knowledge, no report in the literature has described the simultaneous analysis of MeIQx and PhIP, and their principal metabolites, in human urine. The concurrent analysis of these biomarkers is important since the urinary excretion levels of either MeIQx or PhIP can only serve as an approximate measure for one another, in assessment of exposures in humans consuming unrestricted diets (46). Moreover, assessment of the efficacies of chemoprotective agents requires a multipurpose analytical method that can be employed to quantitate both HAAs and their metabolites in urine. We recently reported a facile solid phase extraction (SPE) method to isolate PhIP and several of its major metabolites in human urine; its use is followed by quantitative measurements by LC/MS (47). In this article, we describe a refinement of the SPE procedure to create a rapid, one-step method to isolate MeIQx and PhIP, and several of their P450 1A2-derived metabolites in urine, followed by quantitative LC-ESI/MS/MS analysis. With this validated method, we have examined the interrelationship between the oxidative metabolism of MeIQx and that of PhIP, in urine samples from 10 volunteers.</p><!><p>Caution: MeIQx, PhIP, and several of their derivatives are potential human carcinogens and should be handled with caution in a well-ventilated fume hood with the appropriate protective clothing.</p><!><p>MeIQx, PhIP, and 2-amino-3-trideutromethyl-8-methylimidazo[4,5-f]quinoxaline ([2H3C]-MeIQx) and 2-amino-1-trideutromethyl-6-phenylimidazo[4,5-b]pyridine ([2H3C]-PhIP) (both at 99% isotopic purity) were purchased from Toronto Research Chemicals (Toronto, ON, Canada). NADPH, NADH, glucose-6-phosphate, uridine-5′-diphosphoglucuronic acid (UDPGA), glucose-6-phosphate dehydrogenase, alamethicin, ß-glucuronidase Type IX-A from E. coli, and sulfatase from abalone entrails Type VIII, were all purchased from Sigma (St. Louis, MO). Pooled male rabbit liver microsomes (New Zealand White) were purchased from BD Biosciences (Woburn, MA). Male SD rat liver microsomes of animals pretreated with polychlorinated biphenyls (PCBs, Aroclor-1254) were obtained from Moltox (Boone, NC). Human liver microsomal samples were from Tennessee Donor Services, Nashville, TN, and were a gift kindly provided by Dr. F. P. Guengerich, Vanderbilt University. The P450 1A2 protein expression and metabolic activity were previously characterized (16). All solvents used were high-purity B & J Brand® from Honeywell Burdick and Jackson (Muskegon, MI). ACS reagent grade HCO2H (88%) was purchased from J.T. Baker (Phillipsburg, NJ), Retain CX resins (30 mg) were purchased from ThermoFisher Scientific (Palm Beach, FL), and Baker C18 solid-phase extraction (SPE) resins (500 mg) were purchased through Krackeler Scientific Inc. (Albany, NY). All other chemical reagents were ACS grade, and purchased from Sigma Aldrich.</p><!><p>Mass spectra of synthetic and biosynthetic derivatives were obtained on a Finnigan Quantum Ultra triple stage quadrupole mass spectrometer (Thermo Electron, San Jose, CA). Typical instrument tuning parameters were: capillary temperature 275 °C, source spray voltage 3.5 kV, sheath gas setting 35, tube lens offset 95, capillary offset 35, and source fragmentation 15 V. Argon, set at 1.5 mTorr, was used as the collision gas. Analyses were conducted in positive ionization mode. NMR-resonance experiments assignment experiments were carried out at 25°C and 45°C for the rabbit and human liver PhIP-N-glucuronides, respectively, on a Bruker Avance DRX 600 MHz spectrometer equipped with a triple resonance cryoprobe (Bruker BioSpin Corporation, Billerica, MA). The 1H chemical shifts were referenced directly from the DMSO-d6 multiplet at 2.50 ppm. A standard DQF-COSY experiment was employed to collect 1024 t1 increments over a 7507 Hz spectral window. Selected NMR spectra are provided in the Supporting Information. HPLC separations of biomarkers were done with an Agilent (Palo Alto, CA) Model 1100 HPLC system equipped with a photodiode array detector, equipped with a Rheodyne 7725i (Rhonert Park, CA) manual injector.</p><!><p>MeIQx (5 mg, 2.3 μmol) in dioxane (20 mL), was oxidized with selenium dioxide (5 mg, 45 μmol), by heating the solution at reflux for 5 h, as previously described (31). The two major products, the 8-carbaldehyde derivative of MeIQx and IQx-8-COOH, were formed in approximately 30 and 60% yields, respectively. Treatment of the reaction mixture with NaCNBH3 (5 mg, 78 μmol, 1 h at 37 °C) resulted in complete reduction of the aldehyde to 8-CH2OH-IQx (31). MeIQx-N2-SO3H was prepared by treatment of MeIQx with a 1.2 mol excess of chlorosulfonic acid in anhydrous pyridine, as previously described (48).</p><!><p>These metabolites were prepared by incubating MeIQx or HONH-MeIQx (0.5 mM) (or [2H3C]-MeIQx, [2H3C]-HONH-MeIQx) with 20 mg of liver microsomal protein from rats pretreated with PCBs in 10 mL of 100 mM Tris-HCl buffer (pH 7.5), containing 10 mM MgC12, and UDPGA (5 mM) for 6 h at 37 °C (49). The microsomal mixture was preincubated with alamethicin (50 μg/mg protein) on ice for 15 min, prior to addition of the MeIQx compounds. The glucuronide metabolites were isolated as previously described (47). HONH-MeIQx was synthesized as previously described (50).</p><!><p>The biosynthesis of PhIP-N2-Gl was carried out with rabbit liver microsomal protein (1 mg/mL), and the biosynthesis of PhIP-N3-Gl was done with human liver microsomal protein (2 mg/mL) in 100 mM Tris-HCl buffer (pH 7.5), containing 10 mM MgC12, PhIP (0.5 mM), and UDPGA (5 mM). The mixture was preincubated with alamethicin (50 μg/mg protein) on ice for 15 min, followed by incubation at 37 °C for 6 h. The products were isolated as previously described (47). The isomeric HON-PhIP-N2-Gl and HON-PhIP-N3-Gl metabolites were prepared from human or rat liver microsomes as previously described (47). 4′-HOPhIP was produced from the oxidation of PhIP with rat liver microsomes pretreated with PCBs (47).</p><!><p>The analyses of MeIQx and PhIP metabolites were conducted on urine samples from male volunteers who participated in a previous investigation; and full details were reported previously (5,51). In brief, each subject consumed 275 g of cooked minced beef patties that had been fried without added oil or fat for 6 min on each side, using a hot metal griddle at 300 °C, until the meat was well-browned. The average amount of MeIQx ingested was 920 ng, and the average amount of PhIP ingested was 4,950 ng (51). A urine collection was then commenced over 10 h, and urine samples were stored at -80 °C. A subset of samples were sent blind coded on dry ice to the Wadsworth Center for further analyses. This study was approved by the Institutional Review Board at the Wadsworth Center.</p><!><p>The enrichment procedure was previously reported (47). In this present work-up, the organic precipitation and vacuum concentration steps were omitted prior to SPE. The urine samples (1.0 mL) were acidified with HCO2H (88% v/v, 20 μL) and centrifuged at 15,000 g for 2 min, to remove particulates. The supernatants were applied to ThermoFisher HyperSep Retain CX (30 mg resin) cartridges that had been prewashed with CH3OH containing 5% NH4OH (1 mL), followed by 2% HCO2H in H2O (1 mL). The resins were attached to a vacuum manifold, under slight positive pressure (~5 inches of Hg), to achieve a flow rate of the eluent of approximately 1 mL/min. After application of the samples, the cartridges were washed with 2% HCO2H in H2O (1 mL), followed by 2% HCO2H in CH3OH (1 mL), H2O (1 mL) and 5% NH4OH (1 mL). The resin was allowed to run to dryness. Next, the biomarkers were eluted from the resin with CH3OH containing 1% NH4OH (1.5 mL). This solvent was allowed to absorb into the resin for 3 min, prior to gentle manual elution of the solvent with a disposable 1 mL syringe. The extract was collected into Eppendorf tubes (2.0 mL), and placed in a ventilated hood for 15 min to allow the NH3 to evaporate. The extracts were concentrated to approximately 0.1 mL, by vaccum centrifugation. Then, the samples were transferred into silylated glass conical vials (0.35 mL volume) from MicroLiter Analytical Supplies, Inc. (Suwanee, GA) and evaporated to dryness by vacuum centriguation. The samples were resuspended in 1:1 H2O:DMSO (20 μL).</p><!><p>Chromatography was performed with an Agilent 1100 series capillary LC system (Agilent Technologies, Palo Alto, CA) equipped with an Agilent Zorbax-SB-C18 column (0.3 × 250 mm; 5 μm particle size). Analytes were separated by a gradient. The A solvent contained 0.01% HCO2H in H2O, and the B solvent contained 0.01% HCO2H and 5% H2O in CH3CN. The flow rate was set at 6 μL/min, starting at 100% A and holding for 1 min, followed by a linear gradient to 60% B at 35 min, and then to 100% B at 36 min, and holding for 4 min. The gradient was reversed to the starting conditions over 1 min, and a post-run time of 14 min was required for re-equilibration. The mass-spectral data were acquired on a Finnigan™ Quantum Ultra Triple Stage Quadrupole MS; data manipulations were carried out with Xcalibur version 2.07 software. Analyses were conducted in the positive ionization mode and employed an Advance nanospray source from Michrom Bioresources Inc. (Auburn, CA). The spray voltage was set at 2100 V; the in-source fragmentation was -10 V; and the capillary temperature was 220 °C. No sheath or auxiliary gas was used. The peak widths (in Q1 and Q3) were set at 0.7 Da, and the scan width was 0.002 Da.</p><p>The following transitions and collision energies were used for the quantification of MeIQx, PhIP, and their metabolites: MeIQx and [2H3C]-MeIQx: 214.1 → 199.1 and 217.1 → 199.1, at 30 eV; 8-CH2OH-IQx and [2H3C]-8-CH2OH-IQx: 230.1 → 212.1, 197.1 and 233.1 → 215.1, 197.1, at 20 or 30 eV; 8-COOH-IQx and [2H3C]-8-COOH-IQx: 244.1 → 198.1, 183.1 and 247.1 → 201.1, 183.1, at 20 or 30 eV; PhIP and [2H3C]-PhIP: 225.1 → 210.1 and 228.1 → 210.1 at 33 eV; 4′-HO-PhIP: 241.1 → 226.1 at 35 eV; isomeric PhIP-N-Gl and [2H3C]-PhIP-N-Gl: 401.1 → 210.1 and 404.1 → 210.1 at 55 eV; isomeric HON-PhIP-N-Gl and [2H3C]-HON-PhIP-N-Gl: 417.1 → 225.1 and 223.1 and 420.1 → 228.1 and 225.1 @ 34 eV. The dwell time for each transition was 10 ms. Argon was used as the collision gas and was set at 1.5 mTorr. Product ion spectra were acquired on the protonated molecules [M+H]+, scanning from m/z 50 to 500 at a scan speed of 250 amu/s using the same acquisition parameters as above.</p><!><p>Calibration curves were generated in triplicate by the addition of a fixed amount of [2H3C]-PhIP and [2H3C]-MeIQx (100 pg), and [2H3C]-8-CH2OH-IQx (300 pg) and 0, 6, 12, 16, 20, 40, 60 or 100 pg of the unlabeled standards per 1.0 mL urine from a volunteer who had not consumed cooked meat for at least 48 h. [2H3C]-IQx-8-COOH was added at a level of 300 pg/mL of urine, and the unlabeled standard was added at 0, 30, 60, 80, 100, 200, 300 or 500 pg per mL of urine. The calibration curves of PhIP metabolites were also constructed in triplicate with [2H3C]-PhIP-N3-Gl, [2H3C]-HON-PhIP-N2-Gl, and [2H3C]-HON-PhIP-N3-Gl added at a fixed concentration of 1000 pg per mL of urine, and the unlabeled analytes were each added at concentrations of 0, 60, 120, 160, 200, 400, 600, or 1000 pg/mL of urine. The calibration data were fitted to a straight line using the ordinary least-squares method with equal weightings. Each urine sample was subjected to the SPE processing conditions described above. The within-day and between-day precisions for MeIQx and PhIP and their metabolites were calculated in triplicate or quadruplicate as described (52), with use of urine samples from three different subjects collected during 10 hr, after consumption of cooked meat (5). The measurements were done on three different days over a time period of 1 month. The response of the signal of 4′-HO-PhIP was assumed to be comparable to PhIP and [2H3C]-PhIP was employed as an internal standard to estimate the levels of 4′-HO-PhIP.</p><!><p>Urine samples from meat-eaters (0.5 mL) were spiked with the isotopically labeled internal standards of MeIQx and PhIP, or their metabolites, and then diluted with 0.5 mL of 100 mM sodium acetate buffer (pH 5.5) or 0.5 mL of 100 mM sodium phosphate buffer (pH 6.5). Enzyme hydrolysis was conducted with ß-glucuronidase (14 U/mL) and sulfatase (2 U/mL) at 37 °C for 4 h. The samples were then diluted with glacial acetic acid (1 mL) and processed by SPE. Urine samples (0.5 mL) were also subjected to acid hydrolysis in 50% (v/v) glacial acetic acid or in 1 N HCl, by heating at 80 °C for 8 h. Upon cooling, the samples were processed by SPE. Under these acid hydrolysis conditions, MeIQx-N2-SO3H, MeIQx-N2-Gl, and PhIP-N2-Gl were quantitatively hydrolyzed to the parent amines, whereas the PhIP-N3-Gl was stable and <5% of the conjugate underwent hydrolysis.</p><!><p>Spearman's rank correlation coefficient (rs) determinations for the urinary metabolites and unmetabolized HAAs were done with GraphPad Prism® Version 4 software (San Diego, CA).</p><!><p>A facile SPE procedure previously developed to purify PhIP and its metabolites from urine (47) was employed for the concurrent isolation of MeIQx, 8-CH2OH-MeIQx and IQx-8-COOH. We determined that the treatment of urine with an organic solvent, to precipitate salt and protein, and the ensuing vaccum concentration step, to remove the organic solvent, are not required prior to SPE. The recovery of the analytes, the purity of the extracts, and the limit of quantification (LOQ) of MeIQx, PhIP, and their metabolites were not affected by the omission of these pre-SPE processing steps. The exclusion of these procedures has resulted in a rapid, one-step, high-throughput method for isolation of these HAA urinary biomarkers.</p><p>The analyses of MeIQx, 8-CH2OH-MeIQx, and IQx-8-COOH in urine from an omnivore who had refrained from eating cooked meat for 48 h (pre-exposure) and in urine collected over a 10 h time point, after consumption of grilled meat (post-exposure), is shown in Figure 1. The compounds were monitored by LC-ESI/MS/MS. The transition employed to monitor MeIQx ([M+H]+ → [M+H-15]+• at 34 eV) produces a radical cation. The product ion arises by homolytic cleavage of the 3-N-CH3 bond (53). The transition used to monitor 8-CH2OH-IQx ([M+H]+ → [M+H-18]+ at 20 eV) is due to the loss of H2O from the alcohol group (30). The transition employed to monitor IQx-8-COOH ([M+H]+ → [M+H-46]+ at 20 eV) is attributed to loss of H2O and CO (30). These collision energy values provided the maximum sensitivity in the selective reaction monitoring (SRM) mode. MeIQx was not detected in the pre-exposure urine sample, but it and its linear, tricyclic ring isomer, 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) (54) were observed in urine collected after meat consumption by the subject (Figure 1). The internal standard [2H3C]-8-CH2OH-IQx was discerned in both pre- and post-exposure urine extracts; however, the background signal of the transition employed to monitor 8-CH2OH-IQx was elevated in the pre-exposure urine, and numerous isobaric interfering peaks were observed in the urine extract after meat consumption. The transitions employed to measure IQx-8-COOH and [2H3C]-IQx-8-COOH were even less selective. The internal standard was barely resolved from interfering peaks in the urine extract analyzed after meat consumption.</p><p>The Finnigan™ Quantum Ultra triple stage quadrupole MS has the capability of scanning at enhanced resolution, thereby increasing the mass resolving power from ~500 (m/Δm at full width at half maximum (FWHM) peak height), when it is scanning at 0.7 Da in either Q1 or Q3 (55), to a resolution of ~2000 for MeIQx and its oxidized metabolites, when the scan width is set at 0.1 Da at FWHM resolution. However, the use of the enhanced resolution scan mode did not significantly improve the signal to noise for the transitions of 8-CH2OH-IQx, 8-IQx-8-COOH or their internal standards (data not shown).</p><p>The collision energy used to fragment these oxidized metabolites of MeIQx was then increased from 20 to 30 eV. Under the higher collision energy conditions, both metabolites undergo a second fragmentation at the 3-N-CH3 bond and lose a CH3 radical (30). Although the response of the signals was decreased at 30 eV, there was a dramatic diminution in the background signals at all transitions ([M+H]+ → ([M+H-33]+ and [M+H]+ → ([M+H-36]+ for 8-CH2OH-IQx and [2H3C]-8-CH2OH-IQx, respectively; and [M+H]+ → ([M+H-61]+ and [M+H]+ → ([M+H-64]+ for IQx-8-COOH and [2H3C]-IQx-8-COOH, respectively): at 30 eV, the target compounds and internal standards of both metabolites appear as distinct peaks (Figure 2). 8-CH2OH-IQx or 8-IQx-8-COOH were not detected in the pre-dose urine extract, but both metabolites were readily discernible in the urine sample after meat consumption. Remarkably, the high sensitivity provided by the Quantum Ultra triple quadrupole MS with the Michrome Advance nanospray source has enabled us to acquire good-quality product ion spectra of the urinary 8-CH2OH-IQx and IQx-8-COOH metabolites. The spectra are in good agreement with the spectra of the reference compounds (Figure 3) (30). The product ion spectra of MeIQx and MeIgQx were also successfully acquired on urine samples that underwent acid-hydrolysis, resulting in an increase in the concentrations of these HAAs by up to 5-fold (vide infra) (Figure 4).</p><p>The analyses of PhIP, 4′-HO-PhIP, PhIP-N2-Gl, PhIP-N3-Gl, HON-PhIP-N2-Gl, and HON-PhIP-N3-Gl in urine of the subject, pre- and post-meat consumption, are shown in Figure 5. None of these biomarkers were present in urine at detectable levels, when the subject had refrained from eating meat. However, PhIP and all of its metabolites, except for PhIP-N3-Gl, were detected in the urine of the subject after consumption of meat. The product ion spectra of HON-PhIP-N2-Gl and HON-PhIP-N3-Gl are in excellent agreement to the spectra of the reference compounds previously published (data not shown) (47). Product ion spectra of PhIP and 4′-HO-PhIP were successfully acquired on urine samples that underwent acid hydrolysis: acid treatment increased the concentrations of these compounds by up to several-fold (vide infra) (Figure 6).</p><p>The relative amounts of isomeric glucuronide conjugates of PhIP formed by human liver microsomes in vitro, and their formation in vivo, based on urinary elimination, appear at variance. Human liver microsomes fortified with UDPGA produced 10-fold greater amounts of PhIP-N3-Gl than PhIP-N2-Gl (47,56), yet PhIP-N2-Gl is the main isomer known to be present in urine of meat eaters (Figure 5, tR 27.1 min) (27,43). To confirm the sites of conjugation and structural assignments of these isomeric glucuronide conjugates, we re-examined, by 1H NMR spectroscopy, the human and rabbit liver microsomal PhIP-N-Gl metabolites: the glucuronide conjugate formed by rabbit liver was previously characterized as PhIP-N2-Gl, whereas the conjugate produced by human liver was assigned as the PhIP-N3-Gl (56). Our 1H NMR spectral data (Supporting Information, Figures S-1A – S-1C, and Table S-1), are consistent with the structures previously assigned: PhIP-N3-Gl is the major human liver microsomal metabolite, and PhIP-N2-Gl is the principal glucuronide conjugate produced by rabbit liver microsomes. Despite the strong preference, by human liver UGTs, to glucuronidate PhIP at the N3 imidazole atom, only the PhIP-N2-Gl isomer is eliminated in urine at levels above the limit of detection. However, the amounts of PhIP-N2-Gl eliminated in urine are low, and the direct glucuronidation of PhIP appears to be a minor route of detoxication of PhIP in these 10 subjects of our pilot study.</p><!><p>The recoveries of each internal standard [2H3C]-MeIQx and [2H3C]-PhIP (100 pg/mL); [2H3C]-8-CH2OH-IQx and [2H3C]-IQx-8-COOH (each at 300 pg/mL); PhIP-N2-Gl, HON-PhIP-N2-Gl and HON-PhIP-N3-Gl (each at 1000 pg/mL), added to urine prior to sample processing, were consistently between 40 and 80%, based on the response of the signals to those of pure standards measured by LCESI/MS/MS. The response of the signals of the processed internal standards is a function of the recoveries of the compounds and the potential ion suppression effects of the urine matrix (41). The calibration curves for MeIQx, 8-CH2OH-IQx and IQx-8-COOH (generated from three independent replicates per calibrant level), constructed from urine samples from a subject who had refrained from eating cooked meat for 48 h, displayed good linearity (R2 >0.997) Supporting Information (Figure S-2). The calibration curves for PhIP, PhIP-N3-Gl, HON-PhIP-N2-Gl, and HON-PhIP-N3-Gl have previously been reported (47): the new sets of calibration curves of these biomarkers obtained in the current study are presented in the Supporting Information (Figure S-3). The LOQ values were derived based on a threshold of 10σ SD units above the background signal levels (57) in the urine samples from three volunteers, during the pre-exposure phase of the study. The LOQ values were ~5 pg/mL for MeIQx, PhIP, and 4′-HO-PhIP, whereas the LOQ for 8-CH2OH-IQx was 10 pg/mL, and the LOQ for IQx-8-COOH was 35 pg/mL. The LOQ values for the glucuronide conjugates of PhIP and HONH-PhIP were estimated at 50 pg/mL.</p><p>The precision CV (%) of the estimates in the calibration curve at the lowest calibrant levels of MeIQx and 8-CH2OH-IQx (6 pg/mL) was ≤11%, and the CV (%) for IQx-8-COOH at the lowest calibrant levels (30 pg/mL) was ≤10%; the precision values improved at the higher calibrant levels. The performance of the method was assessed by the within-day and between-day estimates and precision of measurements of MeIQx, 8-CH2OH-IQx, IQx-8-COOH, PhIP-N2-Gl, HON-PhIP-N2-Gl, and HON-PhIP-N3-Gl, in urine samples from three randomly selected volunteers determined over 3 separate days (n = 3 or 4 independent measurements per day), within a time period of 1 month. The results are summarized in Table 1. The within-day and between-day CV (%) in estimates of MeIQx, 8-CH2OH-IQx, and IQx-8-COOH were well below 10.0%. The performance of the method was previously reported for PhIP and its N2- and N3-glucuronides of HONH-PhIP (47); a similar degree of precision is observed in these newly assayed urine samples (Table 1), and in the within-day and between-day CV (%) values, which are also below 10%, Supporting Information (Table S-2). Thus, the analytical method is precise, and intra-day and inter-day estimates for the quantification of MeIQx and PhIP, and their metabolites, are highly reproducible.</p><!><p>The estimates of MeIQx and PhIP, and their metabolites in urine samples from 10 subjects are summarized in Table 2. Both HAAs underwent extensive metabolism: the proportion of unaltered MeIQx ranged from 2.2 to 8.7% of the ingested dose, whereas the proportion of unmetabolized PhIP ranged from 0.2 to 0.8% of the ingested dose. The major pathway of metabolism of MeIQx occurred through oxidation of the C8-methyl group to form IQx-8-COOH, but the major pathway of PhIP metabolism occurred through oxidation of the exocyclic amine group, to form HONH-PhIP. The latter metabolite underwent glucuronidation to form the isomeric N-glucuronide conjugates. 4′-HO-PhIP was a minor component, but it was detected in the urine of all subjects, after meat consumption. 4′-HO-PhIP can form at the low ppb concentration in beef cooked well-done (58,59). Hence, the 4′-HO-PhIP present in urine can either be a minor metabolite of PhIP or it may be derived from unaltered 4′-HOPhIP ingested in the cooked beef. The amounts of 4′-HO-PhIP excreted in urine at the 10 h time point of these subjects are minor, and the levels ranged from an equivalent of 0.2 to 1.0% of the ingested dose of PhIP.</p><p>The urinary metabolic ratio (MR) is often used as an indirect method of assessing drug metabolizing enzyme activity in vivo (60). We observed that the extent of MeIQx and PhIP metabolism and the MR (% dose of urinary metabolite/% dose of unmetabolized urinary HAA) for several oxidative urinary metabolites of MeIQx and PhIP were correlated for a given subject. The Spearman's rank correlation coefficient for the percentage of the dose eliminated in urine as unmetabolized MeIQx and PhIP was rs = 0.86. The correlation coefficient relating the MR of the two major oxidative metabolites of MeIQx and PhIP, IQx-8-COOH/MeIQx and HON-PhIP-N2-Gl/ PhIP, was rs = 0.92; the correlation for IQx-8-COOH/MeIQx and HON-PhIP-N3-Gl/PhIP was rs = 0.78; the correlation for IQx-8-COOH/MeIQx and 8-CH2OH-IQx/MeIQx was rs = 0.60; and the correlation for HON-PhIP-N3-Gl/PhIP and HON-PhIP-N2-Gl/PhIP was rs = 0.90. These correlations were all significant (p value two-tailed α < 0.05), except for the correlation of the MR relating IQx-8-COOH/MeIQx and 8-CH2OHIQx/MeIQx (rs 0.64, P value α = 0.054) (Figure 7). The inter-relationship between these latter two metabolites could be obscured because P450 1A2 catalyzes the further oxidation of IQx-8-CH2OH to form IQx-8-COOH (30).</p><!><p>MeIQx-N2-SO3H, MeIQx-N2-Gl, and HON-MeIQx-N2-Gl did not bind to the mixed-mode reversed phase-cation exchange SPE resin. The poor binding of the sulfamate was not surprising, given the compound's strong polarity and negative charge. MeIQx-N2-Gl and HON-MeIQx-N2-Gl did bind to the SPE resin, when applied as pure standards in 1.8% HCO2H. However, the binding of the metabolites to the SPE resin was poor, when applied in acidified urine, regardless of pre-processing of the urine samples by organic precipitation, to remove salt and protein. The poor binding of these two MeIQx glucuronide conjugates was unexpected, given that the pKa values for MeIQx (pKa 5.94) and PhIP (pKa 5.65) are similar (61), and given that the binding of the urinary glucuronide metabolites of PhIP to the SPE resin was satisfactory.</p><p>Acid hydrolysis of MeIQx-N2-SO3H and MeIQx-N2-Gl quantitatively transforms these conjugates to MeIQx (62). The increase in the amount of MeIQx, after acid hydrolysis of urine, was used as a means to estimate the contribution of N2-sulfamation and N2-glucuronidation to the metabolism of MeIQx (24,62). We concurrently measured, following acid treatment, the amounts of PhIP and the 4′-HO-PhIP; this latter metabolite can undergo further metabolism to form 4′-sulfate or 4′-glucuronide conjugates (18,27). The LC-ESI/MS/MS traces of MeIQx, PhIP, and 4′-HO-PhIP in urine from a subject who ate meat, before and after acid hydrolysis of urine, are shown in Supporting Information (Figure S-4). The SPE procedure is highly effective in purifying these HAAs from acidified urine: the acid hydrolysis treatment increased the amounts of all of three biomarkers. The contribution of phase II conjugation, to the metabolism of MeIQx and PhIP in urine samples from four subjects, is summarized in Figure 8. The amounts of MeIQx, PhIP and 4′-HO-PhIP increased by 3 to 5-fold, following acid hydrolysis. Approximately 8 – 13% of the ingested dose of MeIQx was present as phase II conjugates, but <3% of the ingested dose was recovered as PhIP or 4′-HO-PhIP, following acid hydrolysis.</p><p>The pretreatment of urine with a mixture of ß-glucuronidase and arylsulfatase did not increase the urinary concentration of MeIQ because both MeIQx-N2-SO3H and MeIQx-N2-Gl are resistant to these hydrolytic enzymes (37). The isomeric PhIP-N-Gl conjugates are poor substrates of ß-glucuronidase, whereas ß-glucuronidase treatment of urine resulted in complete hydrolysis of HONPhIP-N3-Gl, but HON-PhIP-N2-Gl remained intact (data not shown). The relative susceptibilities of these glucuronide conjugates of PhIP to ß-glucuronidase are consistent with a previous observation (63). We did observe a modest ~2.5-fold increase in the urinary concentrations of PhIP and 4′-HO-PhIP, following enyzme hydrolysis with a combination of arylsulfatase and ß-glucuronidase (data not shown). The amount of the isomeric PhIP-N2-Gl conjugate present in urine samples was just at the LOQ (~50 pg/mL), whereas the level of PhIP-N3-Gl was below the LOQ (Figure 5). These findings demonstrate that direct phase II conjugation is more important for the detoxification of MeIQx than for the detoxification of PhIP, in agreement with the findings of a previous study (24).</p><!><p>A facile, one-step SPE enrichment procedure was devised to isolate MeIQx and PhIP, and several of their principal metabolites, in urine of omnivores. With this clean-up procedure, twenty urine samples can be processed in one day. Both HAAs undergo extensive metabolism. In the case of MeIQx, the major urinary metabolite is IQx-8-COOH, a detoxification product (31). The primary metabolite of PhIP is HON-PhIP-N2-Gl, a conjugate of HONH-PhIP, which is a genotoxic metabolite that covalently adducts to DNA (64). The formation of both IQx-8-COOH (30,31) and HONH-PhIP (14,16) is catalyzed by P450 1A2. Thus, P450 1A2 serves a dual role in the metabolism of HAAs: it catalyzes both the bioactivation and detoxification of MeIQx, but it only catalyzes the bioactivation of PhIP.</p><p>Comprehensive analyses of urinary metabolites of MeIQx and PhIP in humans are limited to studies that have employed 14C-labeled compounds (23,27,43). In a pilot study, five volunteers who were about to undergo colorectal cancer surgery, were given the dietary equivalent of 14C-labeled MeIQx in a capsule (23). Between 20 and 59% of the ingested dose of MeIQx was excreted in urine within 26 h. Unmetabolized MeIQx and the five principal urinary metabolites were estimated by HPLC with liquid scintillation counting. The estimates of MeIQx and the formed metabolites were (noted as the range in percentages of the ingested dose): MeIQx (0.7- 2.8%); MeIQx-N2-SO3H (0.6 – 3.1%); 8-CH2OH-IQx (1.0 – 4.4%); MeIQx-N2-Gl (1.6 – 6.3%), HON-MeIQx-N2-Gl (1.4 – 10.0%); and IQx-8-COOH (8 - 28%).</p><p>In this current study, the rates of metabolism and elimination of MeIQx in healthy subjects ingesting MeIQx in well-done cooked beef were greater than the rates determined in the subjects who underwent colorectal surgery; this discrepancy may be reflective of the poorer health status of those subjects (23). We estimated, by LC-ESI/MS/MS, that from 60 to 85% of the ingested dose of MeIQx was eliminated in urine of our healthy subjects, as a combination of unaltered MeIQx, P450 1A2 derived metabolites, and phase II conjugates, within 10 h of consumption of cooked beef (Table 2). IQx-8-COOH and 8-CH2OH-IQx (30,31) combined accounted for 34.8 – 76.2% of the ingested dose (Table 2), underscoring the strong contribution of P450 1A2 to the metabolism of MeIQx.</p><p>The estimates of unaltered PhIP (0.2 – 0.8% of the dose), as well as the contribution of HONPhIP-N2-Gl (14.1 – 29.7% of the dose) and HON-PhIP-N3-Gl (1.0 – 3.5% of the dose) to the metabolism of PhIP, are in good agreement to values previously estimated in the 10-h urine samples of these subjects (5), and the estimates of these PhIP metabolites formed are comparable to the data obtained by liquid scintillation counting of radioactivity (27), or by LC-ESI/MS/MS in other feeding studies (26).</p><p>The smaller percentage of the dose of PhIP that is eliminated in urine compared to MeIQx, at the 10-h time point of our study, is due to the slower rate of elimination of PhIP: As much as 30 to 50% of the ingested dose of PhIP is excreted in urine between 8 – 48 h post-meat consumption (5,27,43). In human metabolic studies conducted with 14C-PhIP, the isomeric HON-PhIP-N-glucuronide conjugates were reported to account for approximately 50% of the dose eliminated in urine within 24 h, and combined with 4′-HO-PhIP conjugates, PhIP-N-Gl, and unaltered PhIP, accounted for 60 – 82% of the dose of PhIP eliminated in urine within 24 h (27). We have also detected, by LC/MS, significant quantities of HON-PhIP-N-glucuronide conjugates in urine of our subjects at the 10 to 24-h time point (unpublished observations) (5). Thus, our LC/MS method covers a large proportion of the PhIP dose that is eliminated in urine.</p><p>A m e t h o d was recently developed to measure 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-PhIP) in urine of omnivores (28). 5-HO-PhIP is a solvolysis product of N-acetoxy-PhIP, a penultimate metabolite that reacts with DNA (Scheme 2) (28,40). The analysis of 5-HO-PhIP required heat treatment of urine with hydrazine, under acidic pH conditions, to hydrolyze potential glucuronide conjugates of 5-HO-PhIP, prior to LC/MS (28). The authors estimated high levels of 5-HO-PhIP in human urine, following consumption of meat (28). In a previous study, we did not detect 5-HO-PhIP in urine of omnivores (47), but we did identify several urinary analytes at m/z 417.1, an m/z that is consistent with the molecular weight of protonated glucuronide conjugates of hydroxylated-PhIP (47). The full scan product ion spectra of these analytes were acquired under elevated collision-induced dissociation conditions to fragment the aglycone ions [M+H – 176]+; however, the product ion spectra of these analytes differed from the spectra of either 4′-HO-PhIP or 5-HO-PhIP (47). These urinary components appeared to be isobaric interferents. The major pathways of biotransformation of 14C-PhIP have been characterized in human urine (27,43) and in human hepatocytes (19): Neither 5-HO-PhIP nor its glucuronide or sulfate conjugates were identified as prominent metabolites. Quantitative LC/MS methods with a stable, isotopically labeled internal standard will be required to determine the amounts of 5-HO-PhIP in urine of omnivores (28).</p><p>The prominent role of P450 1A2 in the metabolism of MeIQx and PhIP in humans was previously inferred from a pharmacokinetics study on subjects given furafylline (39), a selective and mechanism-based inhibitor of P450 1A2 (65), prior to consumption of cooked beef. In that study, P450 1A2 was estimated to account for 90% of the elimination of MeIQx and 70% of the elimination of PhIP (39). Consistent with that finding, we previously showed that the pretreatment of human liver microsomes with various amounts of furafylline led to a concentration-dependent inhibition of 8-CH2OH-IQx, IQx-8-COOH, HONH-MeIQx, and HONH-PhIP formation by up to 95% (16,30). The formation of 8-CH2OH-IQx and IQx-8-COOH, and the glucuronide conjugates of HONH-MeIQx and HONH-PhIP, was also inhibited to a similar degree in human hepatocytes pretreated with furafylline (19,31).</p><p>We examined the extent of MeIQx and PhIP metabolism and compared the urinary MR values for several of their P450 1A2-catalyzed oxidation products. The use of MR values and their correlations must be interpreted with caution. A high urine flow rate can limit the utility of MR in assessment of enzyme metabolizing activity in vivo, if the elimination of the parent compound or the metabolite is dependent upon the flow rate of urine (60). In a previous study, the renal clearance of MeIQx and PhIP was reported not to be urine flow-dependent (39). In the present pilot study of 10 subjects, we observed that the renal clearances of MeIQx, 8-CH2OH-IQx, IQx-8-COOH, HON-PhIP-N2-Gl, HON-PhIP-N2-Gl, and 4′-HO-PhIP were independent of urine flow rate (Spearman rs values < 0.52, two-tailed α, p > 0.14, urine volume vs percent dose of biomarkers collected in urine over 10 h), but the elimination of PhIP did appear to be correlated with urine flow output (rs = 0.74, two-tailed α, p = 0.02). The urinary MR value relating the two major oxidation metabolites of MeIQx and PhIP, IQx-8-COOH/MeIQx and HON-PhIP-N2-Gl/PhIP were strongly correlated among the 10 subjects of the study (Figure 7). These correlations support the notion that P450 1A2 is an important enzyme in the metabolism of both procarcinogens in vivo. A study on a larger number of subjects will be required before we can firmly establish the MR values and the inter-relationship between P450 1A2-mediated metabolism of MeIQx and that of PhIP.</p><p>The large contribution of IQx-8-COOH to the metabolism of MeIQx is unexpected, when the steady-state kinetic parameters of P450 1A2 for the formation of IQx-8-CH2OH, the precursor to IQx-8-COOH, are considered. Based on kinetic studies with human liver microsomes, the catalytic efficiency of P450 1A2 in the first oxidation step of the 8-methyl group of MeIQx to form IQx-8-CH2OH (kcat/Km 0.03, Vmax 0.1 nmol/min/mg protein, Km 3.1 μM) is ~10-fold lower than the catalytic efficiency of P450 1A2 in the production of HONH-MeIQx (kcat/Km 0.26, Vmax 3.9 nmol/ min/mg of protein, Km 15 μM) (30). Thus, we postulate that a portion of the HONH-MeIQx metabolite formed in vivo undergoes enzymatic reduction back to MeIQx (66), which ultimately undergoes oxidation at the 8-methyl group to form IQx-8-CH2OH and IQx-8-COOH (Scheme 1).</p><p>HON-MeIQx-N2-Gl was not measured, due to its poor binding to the SPE resin. The acid treatment of HON-MeIQx-N2-Gl produces the deaminated derivative 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline with high yield. This biomarker was employed as an indirect measure of urinary HON-MeIQx-N2-Gl, by GC/MS (67). HON-MeIQx-N2-Gl was deduced to account for 9.4 ± 3.0% (mean ± SD) of the dose, and varied in range from 2.2 to 17.1% (mean ± SD) of the dose in 66 subjects. We observed that 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline also bound poorly to the SPE resin, thus precluding its measurement by our method. Glucuronidation seems to be a less important pathway for the metabolism of HONH-MeIQx than for the metabolism of HONH-PhIP in either humans (23), or in human hepatocytes (31). Further work will be required to develop and optimize the isolation HON-MeIQx-N2-Gl from human urine, for its quantification by LC-ESI/MS/MS.</p><p>A previous study reported the metabolism of PhIP in wild-type, P450 1A2-null, and P450 1A2-humanized mice in detail, using a metabolomic approach, in which metabolites were characterized by ultraperformance liquid chromatography-time-of-flight mass spectrometry analysis (68). In that study, the dose of PhIP given to mice was 10 mg/kg, a dose that is about 150,000-fold higher than the dose consumed by humans in our current study. It would be of great interest to determine whether high resolution mass spectrometer instrumentation can be employed to examine the urinary metabolome of HAAs in humans, following consumption of cooked meat.</p><p>In summary, we have established a robust SPE method to measure MeIQx and PhIP and several of their major urinary metabolites that are produced by P450 1A2. With this validated analytical method, we plan to explore the influence of genetic polymorphisms that encode enzymes involved in xenobiotic metabolism, and assess the efficacy of chemoprotective dietary constituents to modulate the metabolism of these two potential human carcinogens.</p>

PubMed Author Manuscript

Lipid class composition of membrane and raft fractions from brains of individuals with Alzheimer's disease

Perturbation of the homeostasis of brain membrane lipids has been implicated in the pathomechanism of Alzheimer's disease (AD). The ε4 allele of the apolipoprotein E gene (APOE) confers an increased risk, in a dosage-dependent manner, for brain amyloid-β accumulation and the development of sporadic AD. An effect of the APOE genotype on brain lipid homeostasis may underlie the AD risk associated with the ε4 allele. In this research, we examined an effect of APOE ε4 on the lipid class composition of crude membranes and raft-enriched fractions of brains. We applied enzymatic reaction-based methods for the quantification of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and sphingomyelin. Our results indicate that brain lipid class composition was neither significantly altered in AD subjects nor affected by the presence of the APOE ε4 allele.

lipid_class_composition_of_membrane_and_raft_fractions_from_brains_of_individuals_with_alzheimer's_d

<!>Introduction<!>Enzymes and reagents<!>Human brain tissues<!>Fractionation of membrane lipids and lipid rafts<!>Measurement of phospholipid class<!>Statistics<!><!>Discussion<!>Declaration of competing interest<!>Transparency document

<p>No change was found in the composition of lipid classes of brains with Alzheimer's disease.</p><p>The APOE ε4 allele did not affect lipid class composition of the brain membrane or rafts.</p><p>The enzymatic measurement of phospholipids is applicable to brain tissues.</p><!><p>Alzheimer's disease (AD) is a polygenic neurodegenerative disease characterized clinically by progressive memory loss and, eventually, dementia. The genetic heritability of the sporadic form has been estimated to be 60%–80% [1,2]. The brains of AD patients exhibit loss of synapses and neurons, as well as the presence of neuropathological hallmarks such as senile plaques and neurofibrillary changes. The cores of the senile plaques are composed of aggregated amyloid-β (Aβ) peptide, which is generated from a neuronal transmembrane protein called amyloid precursor protein (APP), and trigger AD pathogenesis.</p><p>Altered metabolism of brain membrane lipids has been implicated in the pathogenesis of AD. This hypothesis is based upon multiple lines of evidence. The apolipoprotein E (apoE)-encoding gene (APOE) is the strongest genetic risk factor for a sporadic form of AD. In humans, three polymorphic APOE alleles (ε2, ε3 and ε4) encode three isoforms carrying amino acid substitutions at residues 112 and 158: apoE2 [Cys112, Cys158], apoE3 [Cys112, Arg158], and apoE4 [Arg112, Arg158], respectively. AD risk is two–four-fold higher for subjects heterozygous for the ε4 allele and eight- to 12-fold for homozygous individuals [3]. Genetic loci close to the ABCA7, TREM2, and SORL1 genes, which may be implicated in lipid metabolism, have also been shown to be associated with sporadic AD [4].</p><p>The pathogenic potency of Aβ species depends on the length of the C-terminus and the amount of the protein accumulated in the brain. Aβ42 and Aβ43, which have longer C-termini, are more prone to form aggregates, and have more potent pathogenicity. γ-Secretase, an intramembrane aspartic protease, catalyzes the final step in the generation of Aβ, and determines pathogenicity by creating the C-terminus. The local membrane lipid microenvironment has a potent effect on γ-secretase activity and cleavage sites. Increased cholesterol in membrane lipids augments Aβ production and shows a positive correlation with AD development [5]. In vitro assays for the γ-secretase cleavage of APP revealed that subtle changes in phospholipid composition greatly modify the activity of γ-secretase. Phosphatidylserine was shown to decrease γ-secretase activity, but increased the relative production of shorter Aβ species, whereas phosphatidylinositol competitively inhibited the γ-secretase cleavage of APP [[6], [7], [8]]. Previous reports have claimed that the specificity of γ-secretase cleavage sites was modified to alter relative production of longer Aβ species in sporadic AD and aged brains [[9], [10], [11], [12]]. In addition to their effect upon Aβ biogenesis, membrane lipids affect Aβ degradation and aggregation. Interactions with cholesterol, gangliosides and phospholipids influence the aggregation of amphiphilic Aβ on the cell membrane [13,14].</p><p>The molecular mechanisms underlying the increase in AD susceptibility incurred by the presence of the APOE ε4 allele and the modification of γ-secretase activity in the AD brain remain unclear. However, these effects could be closely associated, because apoE plays a pivotal role in the regulation of brain lipid homeostasis. Several previous studies have evaluated brain lipid composition using conventional methods, in which lipids were separated with thin-layer chromatography and quantified by phosphorus analysis of spots. However, these methods have technical difficulties, which have hampered accurate quantification [15]. To examine the lipid class composition of AD brains with or without the ε4 allele, we used enzymatic reaction-based measurements of phospholipids, which were developed by a coauthor of this paper [[16], [17], [18], [19], [20]].</p><!><p>Choline oxidase and lipoprotein lipase were obtained from Wako Pure Chemical Industries (Osaka, Japan). Phospholipase D and glycerophospholipid-specific phospholipase D were purchased from Biomol International (Plymouth meeting, PA). Amine oxidase was provided by Asahi Kasei Pharma (Tokyo, Japan). l-Amino acid oxidase, l-glycerol-3-phosphate oxidase, and sphingomyelinase were obtained from Worthington (Lakewood, NJ), Roche Diagnostics (Mannheim, Germany), and Sigma-Aldrich (St. Louis, MO), respectively. Calf intestine alkaline phosphatase and horseradish peroxidase were obtained from Oriental Yeast (Osaka, Japan). Purified phosphatidylcholine (PC), phosphatidic acid (PA), L-α-palmitoyl-oleoyl phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) were purchased from Avanti Polar lipids (Alabaster, AL). The fluorescent probes Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine) and Stop Reagent were purchased from Thermo Fisher Scientific (Waltham, MA).</p><!><p>Frozen brain tissues from the temporal cortices of 20 AD patients and 10 age-matched control subjects without neurological disease were obtained from the Brain Bank for Aging Research, Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan). All AD patients fulfilled the criteria of the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Associations for probable AD. Formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin, Klüver–Barrera's method and Gallyas–Braak's silver impregnation. We also performed immunohistochemistry using antibodies against phosphorylated tau (monoclonal, AT8, Innogenetics, Themes, Belgium), Aβ peptides (monoclonal, 12B2, Immuno-Biological Laboratories, Gunma, Japan), and ubiquitin (polyclonal, Sigma-Aldrich) as previously described [21]. We examined 10 cases heterozygous for the APOE ε4 allele and 20 cases negative for the APOE ε4 allele. All of the study subjects or their next of kin gave written informed consent for the brain donation, and the Shiga University of Medical Science Review Board approved the study protocol.</p><!><p>Frozen brain tissues were homogenized using a motor driven Teflon/glass homogenizer (15 strokes) in four volumes of homogenization buffer (Tris at pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The homogenates were centrifuged at 1500 g to remove nuclei and cellular debris. The supernatants were then ultracentrifuged at 100,000 g for 20 min on a TLA 100.4 rotor (Beckman, Palo Alto, CA, USA). The resulting membrane fraction pellet was resuspended in homogenization buffer.</p><p>Fractionation of lipid rafts was performed using buoyant discontinuous sucrose density gradient ultracentrifugation [11] with some modifications. Briefly, 70% by weight of each membrane fraction prepared from 200 mg of brain tissue was suspended in 40% sucrose in MES-buffered saline (25 mM MES at pH 6.5, 150 mM NaCl) containing 1% CHAPSO. Resuspended membrane fractions were placed at the bottom of discontinuous sucrose density gradients of 35% and 5% sucrose and centrifuged at 260,000 g for 4 h. An interface of the 5%/35% sucrose layers was carefully collected and re-centrifuged. The resultant pellet, the lipid raft faction, was washed twice and resuspended in HEPES buffer (25 mM HEPES at pH 7.0, 150 mM NaCl, 5 mM CaCl2, 5 mM MgCl2).</p><!><p>Lipids in the membrane and lipid raft fractions were extracted using the method of Folch et al. [22], and subsequently dissolved in 1% Triton X-100 solution. The concentrations of PA, PC, PE, PS, and SM were measured as previously described [[16], [17], [18], [19], [20]]. Briefly, PA, PC, PE, and PS were hydrolyzed using phospholipase D to release glycerol-3-phosphate, choline, ethanolamine, and serine, respectively, which were then oxidized with l-glycerol-3-phosphate oxidase, choline oxidase, amine oxidase, and l-amino acid oxidase to generate equimolar H2O2. SM was hydrolyzed with sphingomyelinase to release phosphorylcholine, which was then dephosphorylated using alkaline phosphatase. Choline was oxidized with choline oxidase to betaine and two H2O2 molecules. Finally, the production of H2O2 was assessed by Amplex Red assays using a microplate reader (Infinite M200; Tecan, Männedorf, Switzerland). Standard curves were obtained using purified PA, PC, PS, PE, and SM as described above.</p><!><p>Statistical significance was determined using the non-parametric Mann-Whitney U test in all experiments.</p><!><p>Demographic and neuropathological findings of subjects.</p><p>Lipid classes in membrane fractions from brain tissues.</p><p>Upper table shows concentrations (nmol/g brain tissue, means ± S. E. M.) and lower table shows the ratios to PC (means ± S. E. M., p versus the control by Mann-Whitney U test).</p><p>Lipid classes in lipid raft fractions from brain tissues.</p><p>Upper table shows concentrations (nmol/g brain tissue, means ± S. E. M.) and lower table shows the ratios to PC (means ± S. E. M., p versus the control by Mann-Whitney U test).</p><!><p>In this study, we applied enzymatic reaction-based methods to the quantification of PC, PE, PS, PA, and SM in lipids extracted from human brains. These methods used hydrolyzation and then the oxidization of each phospholipid head group to generate hydrogen peroxide. Each phospholipid has linear stoichiometry with hydrogen peroxide within the biological range. The benefits of these methods include high-sensitivity, low-cost, and simplicity, and can achieve accurate and comprehensive measurements.</p><p>We found no significant differences in lipid class composition between the brains of AD patients and non-demented controls. Previous studies have indicated that AD brains have a significant decrease in PE [4,[26], [27], [28], [29]], PC [29], and PI [4,27] compared with control brains. Increases in phospholipid degradation intermediates such as glycerophosphorylethanolamine, glycerophosphorylcholine, and phosphodiesters were detected [[29], [30], [31]], suggesting enhanced catabolism of membrane phospholipids in the AD brains. However, these findings are not universal in the literature; inconsistent results, such as no differences in PC [[26], [27], [28]] and increases in PS [26], have been also reported. All of these studies used conventional methods with thin-layer chromatography and phosphate quantification.</p><p>The mechanisms that underlie the link between apoE isoforms and AD are not yet well understood, although both Aβ-dependent and Aβ-independent mechanisms have been suggested. ApoE-containing lipoproteins play a role in lipid delivery, but their role in brain lipid homeostasis remains undefined. A previous study using 31P nuclear magnetic resonance revealed that an AD-associated decrease in PC and PE in the brain was more marked in subjects with the ε4/ε3 genotype than in those with ε3/ε3 [32]. The level of phosphoinositol biphosphate (PIP2) was also reduced in the brains of ε4 carriers, possibly because of increased expression of a PIP2-degrading enzyme named synaptojanin 1 compared with the ε3 counterparts [33]. The brain lipid abnormalities in apoE-null mice shared some similarities with those of AD patients [34]. Pettergrew et al. [35] reported that there were no significant differences in phospholipid composition between ε4-negative (ε3/ε3) and ε4-positive (ε3/ε4 and ε4/ε4) brains with AD. The results of our study supported this result, and suggested that apoE4 does not affect the lipid class composition of brain membranes.</p><p>We did not find an effect of the APOE ε4 allele on the lipid class composition of raft-enriched fractions from the brains we studied. A previous study using lipid rafts isolated from human frontal cortex in non-demented subjects aged from 24 to 85 years revealed that PE increased with age in women but not in men; SM decreased in men, but not in women; and total polar lipids exhibited significant increases in both sexes [36]. Martín et al. [37], however, reported that the lipid class composition of the lipid rafts from the frontal cortex of brains from individuals with AD was not significantly different from that of healthy subjects, although the lipid rafts from AD-affected brains displayed altered acyl chain saturation. Our result was consistent with their report [37].</p><p>The potential limitations of this study include deviations in sexes, and differences in the mean age among subject groups. When selecting brain samples, we prioritized the APOE genotype and AD pathology over on sex and age. Additionally, alternative methods of isolation of specialized membrane structures or domains such as synaptosomes may result in different conclusions. We also focused on lipid class on polar head groups in this study, but fatty acyl chain length, saturation, and double-bond isomerization are also important in membrane lipid structure.</p><!><p>The authors declare no conflict of interest.</p><!><p>ApplicartionApplicartion</p>

PubMed Open Access

Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other proteins, including those from viruses. The MLK3 SH3 domain contains a six-amino-acid insert corresponding to the n-Src insert, suggesting that MLK3 may bind additional peptides. Here, affinity selection of a phage-displayed combinatorial peptide library for MLK3's SH3 domain yielded a 13-mer peptide, designated “MLK3 SH3–interacting peptide” (MIP). Unlike most SH3 domain peptide ligands, MIP contained a single proline. The 1.2-Å crystal structure of the MIP-bound SH3 domain revealed that the peptide adopts a β-hairpin shape, and comparison with a 1.5-Å apo SH3 domain structure disclosed that the n-Src loop in SH3 undergoes an MIP-induced conformational change. A 1.5-Å structure of the MLK3 SH3 domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MIP bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding. Moreover, SH3 domains of MLK1 and MLK4, but not MLK2, also bound MIP, suggesting that the MLK1–4 family may be differentially regulated through their SH3 domains. In summary, we have identified two distinct peptide-binding sites in the SH3 domain of MLK3, providing critical insights into mechanisms of ligand binding by the MLK family of kinases.

identification_of_two_distinct_peptide-binding_pockets_in_the_sh3_domain_of_human_mixed-lineage_kina

Introduction<!>MIP isolated from a phage-displayed combinatorial peptide library<!><!>Alanine scanning of phage-displayed MIP identifies residues critical for binding and structure of the peptide<!><!>Properties of synthetic MIP<!><!>Architecture of the SH3 domain of MLK3<!><!>The structure of MLK3 SH3–MIP complex reveals a novel binding site on the SH3 domain<!>Trp-10 and Arg-12 in MIP are critical for the intermolecular interaction with the MLK3 SH3 domain<!>Comparison of the apo and MIP-bound MLK3 SH3 structures reveals closed and open conformations of the n-Src loop modulated by the ligand<!><!>MIP binds to a noncanonical binding site on the SH3 domain of MLK3 in proline-independent manner<!><!>MIP binds to a noncanonical binding site on the SH3 domain of MLK3 in proline-independent manner<!>MIP and NS5A peptide bind the SH3 domain of MLK3 in competitive fashion<!><!>Binding of MIP is conserved across most of the SH3 domains of MLKs<!><!>Discussion<!>Protein expression and purification for affinity selections and ELISAs<!>Isolation of peptide ligand to the SH3 domain of MLK3<!>Sequence analysis of phage-displayed MIP<!>Alanine scanning of phage-displayed MLK3 MIP<!>Peptide synthesis<!>IC50 determination for MIP binding to the MLK3 SH3 domain<!>Gene synthesis, cloning, protein expression, and purification for structural analysis<!>Crystallization<!>Data collection and structure determination of apo, MIP-, and NS5A-bound MLK3 SH3<!>Analytical size-exclusion chromatography<!>Competitive ELISA to determine competition between MIP and NS5A<!>Specificity ELISA to determine MIP and NS5A peptide binding to other members of MLK subfamily<!>Data resources<!>Author contributions<!>

<p>The human interactome has been estimated to contain ∼650,000 interactions (1) with many protein–protein interactions facilitated by modular domains (2–4). Among different techniques used to map the interaction of domains, phage display has proven successful at mapping their peptide binding preferences (5–8). This approach is based on the observation that peptides isolated through affinity selection of combinatorial peptide libraries usually bind to biologically relevant sites on the domain. Thus, it is possible to predict the candidate interacting partners of the domain in a particular target protein through a database search with the primary structure of the selected peptide ligands (9–12). Furthermore, the isolated peptides have the potential to serve as inhibitors or activators of specific cellular interactions in proof-of-principle experiments for drug development (13–15).</p><p>One of the most widely studied protein interaction modules is the Src homology 3 (SH3)3 domain (16–18). There are ∼300 SH3 domains in the human genome, present in over 200 proteins (19). Typically, SH3 domains bind proline-rich (PXXP) motifs (7, 20–22), and their specificity can often be readily revealed through examination of peptides affinity-selected from phage-displayed combinatorial peptide libraries (6, 7). However, several SH3 domains bind noncanonical motifs; for example, the Gads SH3 domain binds SLP-76 via an RXXK motif (24), the Eps8 SH3 binds e3b1/Abi-1 (25, 26) and RN-tre (27, 28) via a PXXDY motif (29), and the Fyn SH3 domain binds SKAP55 via an RKXXYXXY motif (30). Interestingly, structural studies show that most of the noncanonical ligand sequences are recognized by the same molecular surface of the SH3 domains (16, 17) that is used for binding proline-rich motifs.</p><p>A small number of SH3 domains, such as the Pex13p (31), p67phox (32), and Vav (33) SH3 domains, bind noncanonical peptides via a different surface on the domain. More specifically, the SH3 domain of Pex13p (31) can independently bind two substrates with Pex14p interacting with the canonical site via PXXP motif and Pex5p interacting with a second noncanonical ligand-binding site in a proline-independent fashion. In the case of the p67phox SH3–p47phox interaction, both canonical and noncanonical binding surfaces facilitate interactions with respective PXXP and nonproline motifs present within the same p47phox-derived peptide sequence; this bivalent interaction results in a high-affinity and specific protein–protein interaction (32). Finally, the N-terminal SH3 domain of Vav cannot bind Pro-rich ligands because its RT loop (located between β1 and β2 strands of SH3 domain) contains a tetraproline insertion, which blocks accessibility of the canonical PXXP binding pocket; instead, it uses a different surface to bind the C-terminal SH3 domain of Grb2 (33).</p><p>Mixed-lineage protein kinase 3 (MLK3) is widely expressed in a variety of normal and cancerous tissues, such as brain, lung, liver, heart, breast, kidney, pancreas, and skeletal muscle (34–36). MLK3, also known as MAP3K11, is a serine/threonine protein kinase involved in regulating the JNK, p38, and ERK pathways (37–42). It is composed of several modules: an SH3 domain, a kinase domain, tandem leucine zippers, a Cdc42/Rac interactive binding (CRIB) motif, several linker regions, and a proline-rich C-terminal tail (39, 43). According to the BioGRID interaction database (44), MLK3 is involved in 36 unique interactions with several being mediated through the SH3 domain of MLK3. The SH3 domain has been implicated in the autoinhibition of the kinase domain through an intramolecular interaction via a nonconsensus single proline motif located between the leucine zipper and CRIB motif in the full-length MLK3 protein. Disruption of that intramolecular interaction results in increased activity of MLK3 (43). The SH3 domain of MLK3 also plays role in several other protein–protein interactions. The hepatitis C virus nonstructural 5A (HCV NS5A) protein was found to interact with the SH3 domain of MLK3 via a PXXPXR motif. Replacement of the two key prolines to alanine significantly decreases the interaction and NS5A's ability to block MLK3-induced apoptosis (45). The SH3 domain of MLK3 was also shown to interact with proline-rich regions in a hematopoietic progenitor kinase 1 (HPK1) (46, 47) and a germline center kinase (GCK) (42). Both kinases regulate the JNK pathway in an MLK3-dependent manner (42, 46).</p><p>Recently, MLK3 has been implicated in intracellular vesicle transport and motor neuron disease (48, 49), and given that the MLK3 SH3 domain contains a six-amino-acid insert, corresponding in position to the n-Src loop (50), we wondered whether the MLK3 SH3 domain might have novel binding properties. Here, we report the discovery of a novel noncanonical binding site on the SH3 domain of MLK3 that is topographically different from previous publications on noncanonical recognition by SH3 domains (31–33). The newly identified binding surface is composed of an extended n-Src loop (located between β2 and β3 strands) of the MLK3 SH3 domain, which opens up to bind a peptide ligand, named "MLK3 SH3–interacting peptide" (MIP), which lacks the canonical PXXP motif. Interestingly, when MIP binds to this site, it competes with binding of the HCV NS5A–derived peptide (PXXPXR) to the canonical binding pocket on the SH3 domain of MKL3. As the extended n-Src loop is conserved among the four members of the MLK1–4 protein family, we hypothesize that cellular interacting proteins exist that bind the SH3 domains of the MLK family of proteins in a unique MIP-like manner.</p><!><p>We utilized a GSH S-transferase (GST) fusion to the SH3 domain (amino acids 43–104) of human MLK3 (Fig. S1) for affinity selection of a phage-displayed combinatorial 12-mer peptide library (51). Following three rounds of affinity selection, we isolated a single clone displaying a 12-mer peptide, NH2-IRINPNGTWSRQ-COOH. As confirmed by phage ELISA, the clone bound to the wildtype (WT) form of the MLK3 SH3 domain (Fig. 1). Early experiments suggested that residues beyond the C terminus of the 12-mer peptide might contribute to binding, so we decided to characterize the binding properties of a 19-mer peptide, NH2-AIRINPNGTWSRQAETVES-COOH, named "MIP." Truncation of MIP revealed that the entire 19-mer sequence is necessary for binding to GST-MLK3 SH3 (Fig. S2); for instance, deletion of Ala-1 (Fig. S2A) or deletion of several residues from the C terminus (Fig. S2B) resulted in lack of target binding.</p><!><p>Isolation of MIP from phage-displayed combinatorial peptide library. Following three rounds of affinity selection with a phage-displayed combinatorial peptide library, ANL7 (51), a single peptide ligand, MIP (NH2-AIRINPNGTWSRQAETVES-COOH; insert (underlined) and flanking region), was identified to bind a GST-MLK3 SH3 domain fusion protein. ELISA shows the phage-displayed MIP to bind to the WT form of the MLK3 SH3 domain (SH3 WT) but not the GST fusion partner. The presence of virions in the supernatant was determined by coating the wells of the microtiter plate with an anti-M13 mAb (anti-M13 Ab). Virions, which were retained in the microtiter plate wells, were detected with anti-M13 mAb conjugated to HRP. Experiments were performed in duplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. See also Fig. S1.</p><!><p>To determine which residues in MIP(1–19) are critical for binding, we performed systematic alanine replacement of the sequence and evaluated the variants in phage ELISA (Fig. 2). Alanine replacements at Ile-2, Arg-3, Ile-4, Asn-5, Asn-7, Gly-8, Thr-9, Trp-10, and Arg-12 were detrimental for the intermolecular interaction with GST-MLK3 SH3(43–104) (Fig. 2). Surprisingly, mutation of Pro-6 to Ala had no effect on binding (Fig. 2), indicating a noncanonical form of peptide binding to the MLK3 SH3 domain. Alanine replacements of Gln-13 through Ser-19 also had no effect on SH3 binding (Fig. 2); however, deletion of any of the C-terminal residues compromised binding to the MLK3 SH3 domain in the phage-displayed format (Fig. S2B) but not as a fusion protein (see Figs. 4 and S4). We interpret this as an indication that the C-terminal peptide region (amino acids 14–19) of MIP serves as a linker necessary to facilitate binding in the phage-displayed format.</p><!><p>Identification of residues critical for binding to MLK3 SH3 via alanine scanning of phage-displayed MIP. The MIP sequence, NH2-AIRINPNGTWSRQAETVES-COOH, was fused to the N terminus of the pIII capsid protein of bacteriophage M13 (51) to facilitate type 3 pentavalent display. Systematic alanine replacements in the peptide were generated by Kunkel mutagenesis (11); Ala-1 and Ala-14 were replaced with Val. A truncated version of MIP (ΔA1) served as a negative control as deletion of Ala-1 resulted in loss of binding to MLK3 SH3. All phage-displayed variants were evaluated for their binding to GST-MLK3 SH3 via phage ELISA. The signal for binding of MIP (WT) to GST-MLK3 SH3 is set as 100%, and other values are normalized to it. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. See also Fig. S2.</p><!><p>Using competition ELISA, we determined the half-maximal inhibitory concentration (IC50) value of the MIP(1–19). In this assay, the GST-MLK3 SH3(43–104) protein was first incubated with increasing concentrations of unlabeled MIP (AIRINPNGTWSRQAETVES) as competitor and then introduced to a 96-well ELISA plate coated with biotinylated MIP (AIRINPNGTWSRQAETVES-K-biotin) captured via NeutrAvidin. The GST-MLK3 SH3(43–104) protein showed no cross-reactivity with NeutrAvidin or blocking agent; as anticipated, the GST tag showed no interactions with the MIP(1–19). The IC50 value for MIP binding the SH3 domain of MLK3 was observed to be 15.8 ± 0.4 μm (Fig. 3), which is in the range of binding affinities (i.e. single- to triple-digit μm) reported for multiple peptide–SH3 domain interactions (29, 52, 53).</p><!><p>Binding properties of synthetic MIP. To determine the IC50 value of MIP, a GST-MLK3 SH3(43–104) domain fusion protein was preincubated with increasing concentration of unlabeled MIP (AIRINPNGTWSRQAETVES) as competitor and then allowed to interact with biotinylated MIP (AIRINPNGTWSRQAETVES-K-biotin) immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP, and the levels are presented as percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. The curve fitting was performed with GraphPad Prism 6.0 software.</p><!><p>The sequence alignment of the SH3 domain of MLK3 and five other human SH3 domains (Src, Fyn, Eps8, p67phox, and Grb2) revealed that the n-Src loop sequence of MLK3 SH3 contains an elongated insert of five residues (Fig. 4A). We solved the X-ray structure of the apo MLK3 SH3(44–103) domain at 1.5-Å resolution and refined it to an Rwork/Rfree of 18.6%/21.7% (Table 1). The final model has all residues in the allowed region of the Ramachandran plot. The overall structure reveals a five-stranded antiparallel β-barrel, a characteristic of all known SH3 domains, and three loops (RT, n-Src, and 310 helix), commonly involved in the recognition of both proline-rich and nonproline ligands (16–18) (Fig. 4B). The residues that extend the MLK3 n-Src loop form a 310 helix, which is not commonly present in SH3 domains (Fig. 4B).</p><!><p>The crystal structure of MLK3 SH3 illustrates the mode of binding of the phage display-generated MIP. A, sequence alignment of the SH3 domains of human MLK3, Src, Fyn, Eps8, p67phox, and Grb2 was generated using Clustal Omega (23). Note the extended n-Src loop of MLK3 SH3. An asterisk indicates positions that have a single, fully conserved residue. A colon indicates conservation between groups of strongly similar properties, scoring > 0.5 in the Gonnet PAM 250 matrix. A period indicates conservation between groups of weakly similar properties, scoring ≤ 0.5 in the Gonnet PAM 250 matrix. B, pictured are two views of the ribbon-form structure of apo MLK3 SH3(44–103). The structure reveals a five-stranded antiparallel β-barrel flanked by the RT loop, n-Src loop, 310 helix loop, and distal loop. A second 310 helical structure is found in the extended n-Src loop. C, schematic of the crystal packing of MLK3 SH3 bound to MIP. Because the C terminus of MIP (13′–19′) had no clear electron density, it was not modeled. Although MLK3 SH3(41–105)-MIP(1–19) was expressed as a fusion protein, there was no intramolecular binding of MIP by SH3. Instead, the protein crystallized as a nonfunctional dimer with two chains in each unit cell (gray box). Chain A of the MLK3 SH3 dimer (cyan) binds the MIP from an adjacent Chain A (shown in blue; see zoom), whereas Chain B of the dimer (pink) binds the MIP (red) of an adjacent Chain B from a different symmetry mate. D, the MIP fits in a pocket formed by the n-Src loop of MLK3 SH3. Shown is the transparent surface rendering of MLK3 SH3 with a bound MIP. E, several residues are involved in the binding of MIP (blue) to the MLK3 SH3 domain (cyan). Hydrogen bonds formed by the interaction of MIP with MLK3 SH3 are denoted by dashed lines with distances in angstroms (Å). In particular, Trp-10′ and Arg-12′ are integral in forming several key hydrogen bonds. See also Figs. S3 and S4.</p><p>Statistics for SH3–MIP complex and apo SH3</p><p>a Values in parentheses represent the highest resolution shell.</p><p>b CC 1/2 is a value for determining the data quality. Its value here is in %.</p><p>c From PROCHECK (63).</p><!><p>To determine the structure of the MLK3 SH3–MIP complex, we constructed a fusion protein MLK3 SH3(41–105)-MIP(1′–19′) where the prime denotes residues of the peptide. This construct was expressed in bacteria and purified as described under "Experimental procedures." Because the fusion protein elutes as a higher order oligomer from a gel-filtration column (Fig. S3), we expected the interaction to occur between two separate molecules of SH3-peptide fusion rather than an intramolecular interaction. The structure of MLK3 SH3(41–105)-MIP(1′–19′) was solved to 1.2-Å resolution and refined to an Rwork/Rfree of 13.6%/15.8% (Table 1). The final model has all residues in the allowed region of the Ramachandran plot. As shown in Fig. 4C, the crystallographic asymmetric unit (gray box) contains two protomers that are labeled Chain A (cyan, domain; blue, peptide) and Chain B (pink, domain; red, peptide). The interaction of the SH3 domain of MLK3 with MIP occurs between two protomers that are related by crystallographic symmetry. The first residue in the SH3 sequence (Tyr-41) and the C terminus of MIP (13′–19′) have no clear electron density and, therefore, were not modeled.</p><p>The MIP binds in a pocket formed between the n-Src loop and β-strands 2–4 (Fig. 4D). As shown in Fig. 4E, multiple residues are involved in the binding of MIP to the SH3 domain of MLK3. Most notably, several hydrogen bonds contact Trp-10′ in the MIP sequence. The backbone amide group of Trp-10′ interacts with Asp-74, and the carbonyl group interacts with the backbone amide of Ala-76. Notably, both Asp-74 and Ala-76 are found within the MLK3-unique insert in n-Src loop of the SH3 domain. The Trp-10′ indole nitrogen atom hydrogen bonds with the carbonyl group of Trp-83 located on the edge of β3 strand. Gly-8′ and Arg-12′ also contribute to hydrogen bonding; the carbonyl group of Gly-8′ interacts with the hydroxyl group of Ser-72 in the SH3 domain, and the guanidine moiety of Arg-12′ forms a salt bridge with the side-chain carboxylate of Asp-80.</p><!><p>To evaluate the importance of MIP residues Trp-10′ and Arg-12′ for binding to the MLK3 SH3 domain, we generated three fusion proteins composed of the SH3 domain linked via its C terminus to MIP(1–13)W10A, MIP(1–13)R12A, and the truncated version of the WT ligand, MIP(1–13). These purified proteins were analyzed by size-exclusion chromatography to determine their oligomeric state. Although the WT construct MLK3 SH3(41–105)-MIP(1–13) eluted as a higher order oligomer, both mutant constructs elute at a volume corresponding to the size of a monomeric fusion protein (Fig. S4). Mutation of either Trp-10′ or Arg-12′ to Ala resulted in a loss of binding to MLK3 SH3 (Fig. S4), consistent with alanine scanning results of phage-displayed MIP (Fig. 2).</p><!><p>Comparison of the unbound and ligand-bound structures of MLK3 SH3 domain revealed a closed and open conformation of the n-Src loop, respectively (Fig. 5A). As modeled in Fig. 5B, the closed conformation of the loop precludes the peptide from the groove. To accommodate MIP as described previously, the n-Src loop undergoes a dramatic conformational change. In particular, residues Ile-77 and Ser-78, which participate in the 310-helical structure, as well as the following Gly-79 undergo dramatic spatial movements required to facilitate binding of MIP to the SH3 domain of MLK3 as demonstrated by the change of positions of the α-carbons in these residues (Fig. 5C). The rest of the SH3 structure does not undergo any significant conformational changes upon binding with an r.m.s.d. of 0.41 over 40 residues (Fig. 5A).</p><!><p>Conformational changes in the n-Src loop induced by MIP binding. A, crystal structure of apo MLK3 SH3(44–103) (tan) overlaid with MIP-bound MLK3 SH3(41–105) domain (cyan) shows a dramatic shift in the n-Src loop region, whereas the structure of the remainder of the domain is relatively unchanged. B, the n-Src loop of the unbound form of MLK3 SH3 must open up to allow MIP to bind the SH3 domain. Without this conformational change, MIP (blue) would not fit into the domain as evidenced by the clash indicated by the red arrow. C, a close-up of residues Ile-77, Ser-78, and Gly-79 in the n-Src loop in the bound (cyan) versus unbound (tan) structures. Measurements (Å) of the change of positions of the α-carbons in these residues demonstrate the spatial movement required to facilitate the interaction with MIP.</p><!><p>Based on our structure, MIP fits into a novel binding site facilitated by a single n-Src loop and the edge of the β3 strand (Fig. 4D). In contrast, interactions with the canonical binding site commonly require involvement of three loops: RT loop, n-Src loop, and 310 helix loop (20, 24, 31). The SH3 domain of MLK3 was previously reported to interact with the HCV NS5A protein (UniProt entry W8GG88) (45). The authors mapped the interaction to a PXXPXR motif, KAPTPPPRRRR (aa 2320–2331), present at the C terminus of the NS5A protein. To learn whether this peptide binds a site distinct from MIP, we fused the NS5A motif, APPIPPPR from a different HCV genotype (UniProt entry A0A076MER2; aa 2322–2333) previously crystallized with the SH3 domain of c-Src (Protein Data Bank (PDB) code 4QT7), to the SH3 domain of MLK3 and prepared crystals for X-ray diffraction. The crystals diffracted to 1.5 Å with an Rwork/Rfree of 18.8%/22.8%. Fig. 6A shows the overlay of the NS5A-bound structure compared with the MIP-bound structure. The NS5A sequence crystallized here is from a different genotype of HCV but carries the same PXXPXR motif that is conserved across all HCV genotypes (54, 55). Alignment of both complexes (Fig. 6A) shows that the two binding sites are on the opposite faces of the SH3 domain.</p><!><p>Novel noncanonical binding pocket on the SH3 domain of MLK3. A, an overlay of the SH3 domain from MLK3 (cyan) bound to MIP (blue) versus the SH3 domain from MLK3 (light green) bound to a canonical proline-rich peptide (green) derived from HCV NS5A. B, the NS5A peptide (APPIPPPR) interacts with several residues of the SH3 domain via hydrogen bonds, including Asp-58, Glu-59, Asp-80, Trp-83, Asn-98, and Tyr-99. The NS5A peptide makes contacts with the canonical binding pocket composed of the n-Src loop, RT loop, and 310 helix loop. C, the alignment of the sequences of MLK3 SH3 with the key interacting residues highlighted. Residues in black appear in the structure; key residues for MIP binding are shown in blue underlined text, whereas those crucial for NS5A binding are shown in green underlined text. Note that although Asp-80 and Trp-83 are involved in binding for both peptides only the Asp-80 side chain is shared between both structures. D, shown is the transparent surface model of MLK3 bound to NS5A. E, shown is the transparent surface model of SH3 domain of MLK3 bound to MIP in the same orientation as in D. MIP interacts with a noncanonical pocket formed by the extended n-Src loop, located on the opposite face of the SH3 molecule as compared with the canonical pocket. Note the change in orientation of the n-Src loop (top of domain).</p><!><p>The NS5A peptide interacts with the canonical polyproline-binding site (Fig. 6D) by forming contacts with residues Asp-58 and Glu-59 of the RT loop, residues Asn-98 and Tyr-99 of the 310 helix, residues Asp-80 of the n-Src loop, and residue Trp-83 of the β3 strand from MLK3 SH3 (Fig. 6, B and C). Conversely, MIP fits into a novel binding site defined by the n-Src loop and the edge of the β3 strand (Figs. 6E and 4D).</p><p>Interestingly, Asp-80 and Trp-83 of the MLK3 SH3 domain participate in binding both peptides; however, in the case of Trp-83, the side chain is implicated in interactions with the NS5A peptide (Fig. 6B), whereas its backbone carbonyl interacts with MIP (Fig. 4E). The Asp-80 side chain is the only one involved in both interactions (Figs. 6, A and B, and 4E); it is oriented differently depending on which peptide is bound and interacts with arginine residues present in both peptides (Fig. 6A).</p><!><p>Because Asp-80 in the SH3 domain of MLK3 participates in binding both peptide ligands, competition experiments were performed to determine whether MIP and NS5A peptide can bind the SH3 domain in an exclusive or simultaneous manner. The GST-MLK3 SH3(43–104) protein was first incubated with increasing concentrations of soluble MIP peptide (AIRINPNGTWSRQAETVES) or NS5A peptide (KKAPTPPPRRRR-GGG-K) and then transferred to a 96-well microtiter plate coated with biotinylated MIP peptide (Fig. 7A) or biotinylated NS5A peptide (Fig. 7B), which was captured on the microtiter plate well surface via immobilized NeutrAvidin. It was seen that the free NS5A peptide competes with immobilized MIP for binding to GST-MLK3 SH3(43–104) with an IC50 value of 12.9 ± 0.6 μm and that the free MIP peptide competes with immobilized NS5A for binding GST-MLK3 SH3 with an IC50 value of 6.6 ± 1.6 μm. This supports the crystallographic data in which Arg-12′ of MIP and Arg-8′ of NS5A would suffer a steric clash if bound at the same time as they induce a different side chain orientation in Asp-80 of the MLK3 SH3 domain (Fig. 6A). Additionally, previously described experiments noted the importance of Arg-12′ in the MIP peptide for overall binding (Fig. S4).</p><!><p>Competition ELISA between MIP and NS5A peptide. To determine whether the MIP and NS5A peptides competitively bind the SH3 domain of MLK3, a GST-MLK3 SH3(43–104) fusion protein was preincubated with increasing concentrations of unlabeled MIP peptide or unlabeled NS5A peptide as competitor and then allowed to interact with biotinylated MIP (A) or biotinylated NS5A (B) peptide probes immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of the SH3 domain was detected with an anti-GST antibody conjugated to HRP, and the signal levels are presented as a percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. Curve fitting was performed with OriginPro 2017.</p><!><p>MLK3 is one of the four members of the MLK subfamily (MLK1–4) (39, 56). To predict the likelihood of MIP cross-reacting with the rest of the subfamily, we generated protein homology models for SH3 domains of MLK1, MLK2, and MLK4 using the solved structure of MLK3 SH3 domain bound to MIP as a template (Fig. S6A). Although the primary sequences of the SH3 domains of MLK1, -2, and -4 are ∼60% identical to that of MLK3 (MLK1, 60.0%; MLK2, 66.15%; MLK4, 60.0%) (Fig. S6B), structurally the domains are predicted to be very similar based on Swiss-Prot modeling (Fig. S6A). Notably, four key residues involved in binding of MIP (Ser-72, Asp-74, Asp-80, and Trp-83) are conserved (Fig. S6B), and the overall predicted 3D structure of the n-Src loop and the rest of the molecule is highly preserved (Fig. S6A). To evaluate whether MIP and NS5A peptides can interact with SH3 domains of MLK1, MLK2, and MLK4, we constructed and purified GST fusions to each of these SH3 domains and examined their binding by ELISA. As can be seen in Fig. 8, the NS5A peptide could bind the GST-SH3 domain fusions of all (MLK1–4) purified proteins, whereas MIP bound the SH3 domains of MLK1, MLK3, and MLK4. Interestingly, the MIP peptide did not bind the SH3 domain of MLK2; comparison of the primary structures of MLK2 SH3 with the rest of the SH3 domains of the MLK subfamily (Fig. S6B) revealed a cysteine instead of a serine (MLK1) or alanine (MLK3 and MLK4) in the n-Src loop.</p><!><p>Binding of MIP and NS5A peptides to SH3 domain of other members of MLK subfamily (MLK1–4). To determine whether MIP and NS5A can interact with SH3 domains of MLK1–4 proteins, the purified GST-MLK1–4 SH3 domains were incubated with biotinylated peptides, NS5A peptide (KKAPTPPPRRRR-GGG-K-biotin), MIP (AIRINPNGTWSRQAETVES-K-biotin), and MIP-R12A (AIRINPNGTWSAQAETVES-K-biotin), immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. See also Fig. S6.</p><!><p>To our knowledge, this is the first report presenting insights into mechanisms of ligand binding by the SH3 domain of MLK3. Previously, a crystal structure of another member of the MLK subfamily, the SH3 domain of MLK2 (PDB code 2RF0), was determined. However, no structure of an SH3–ligand complex has been available until now. Our studies revealed a noncanonical binding site on the surface of MLK3 SH3 that is localized at the opposite face of the molecule compared with the site that accepts proline-rich motifs (Fig. 6). An examination of the crystal structures of several SH3 domains bound to noncanonical peptide ligands revealed that all bind the same side of the SH3 domain as do canonical proline-rich peptides (18), suggesting that the MLK SH3 domain has a novel binding site primarily defined by the extended n-Src loop (Fig. 4). In addition, as the key residues involved in the interaction of the SH3 domain with the MIP ligand are conserved across MLK1–4 (Fig. S6B), we suspected this noncanonical mode of SH3–peptide interaction to be potentially conserved across the other members of the MLK subfamily. Indeed, we found that MIP binds the SH3 domains of MLK1, MLK3, and MLK4 but not of MLK2 (Fig. 8).</p><p>In this study, we also mapped the location of the canonical binding site on MLK3 SH3 by solving a structure of the SH3 domain of MLK3 bound to the NS5A-derived peptide (APPIPPPR). Superposition of these two structures (Fig. 6) showed that the canonical PXXP and MIP-binding site are located at opposite faces of the molecule. However, based on the same overlay in Fig. 6, we also hypothesize that certain residues on the SH3 domain of MLK3 could be shared between those two different binding sites. MIP interaction with MLK3 SH3 requires involvement of Ser-72, Asp-74, Asp-80, and Trp-83 (Fig. 6C and 4E). Conversely, interaction of NS5A peptide with MLK3 SH3 seems to be dependent on Asp-58, Glu-59, Asp-80, Trp-83, Asn-98, and Tyr-99 (Fig. 6, B and C). Although the Trp-83 residue interactions are different for each peptide, the Asp-80 side chain is involved in interactions with arginines on both peptides that orient the side chain differently depending on the peptide bound (Fig. 6A). As both peptides require the carboxyl side chain of Asp-80 for binding, they could interact with the SH3 domain of MLK3 in competitive fashion as confirmed by competitive ELISA (Fig. 7). Additionally, mutation of a residue conserved among SH3 domains, Y52A, disrupts binding of the MLK3 SH3 domain to the MIP and NS5A peptides (data not shown). Finally, the shift in the n-Src loop required for MIP binding (Fig. 5) likely interferes with the binding of NS5A, further explaining the competition between the two peptides (Fig. 7).</p><p>These findings led us to consider earlier reports of protein–protein interactions via the SH3 domain and specifically whether there is other evidence of noncanonical interactions. Previous reports suggest that two proteins, HPK1 and GCK, interact with the SH3 domain of MLK3. However, as they were assumed to interact with the SH3 domain via their PXXP motifs, only proline-rich stretches were subjected to further analysis (42, 46). In the literature, only one known protein–protein interaction has been reported to occur with the SH3 domain of MLK3 through a noncanonical PXXP motif. The SH3 domain of MLK3 can bind a region located between the leucine zipper and CRIB motif in an intramolecular fashion, resulting in autoinhibition of the kinase domain (43). However, the exact site of this interaction on the SH3 domain still remains to be determined. Our discovery of a noncanonical binding site on the surface of MLK3 SH3 that can accept motifs devoid of any proline residues could shed new light on the mechanism of ligand binding to the SH3 domain of MLK3. Based on findings of phage-displayed peptide ligands corresponding to cellular proteins (5), we suspect that a cellular protein exists that binds the MLK SH3 domain in a manner equivalent to MIP and are actively attempting to discover it. Finally, the presence of a unique binding site makes MLK3 SH3 even more attractive as a potential drug target, offering a new site for design or discovery of a specific inhibitor.</p><!><p>Two plasmids, gifts from Dr. Gerardo Morfini, University of Illinois at Chicago, encoding a GST protein fused to either the WT or mutant Y52A of the SH3 domain of MLK3 (UniProt entry Q16584; aa 43–104), respectively, between BamHI and EcoRI sites, were transformed into the Escherichia coli strain Rosetta(DE3)pLysS. DNA fragments encoding the SH3 domain of MLK1 (UniProt entry P80192; aa 52–116), MLK2 (UniProt entry Q02779; aa 16–81), and MLK4 (UniProt entry Q5TCX8; aa 38–102) were commercially prepared as gBlocks® Gene Fragments (Integrated DNA Technologies, Inc.). All coding regions were codon-optimized for E. coli expression. DNA fragments were cleaved with BamHI-HF® and EcoRI-HF® (New England Biolabs) and subsequently ligated into pGex-2T vector. The resulting DNA constructs, pGex-MLK1 SH3, pGex-MLK2 SH3, and pGex-MLK4 SH3 (verified by sequencing) were transformed into E. coli strain BL21(DE3).</p><p>For all constructs, the cells were initially grown at 37 °C in 2× YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl/liter), which was supplemented with 50 μg/ml carbenicillin (CB) and 12.5 μg/ml chloramphenicol, and then diluted (1:100) into fresh 2× YT medium supplemented only with 50 μg/ml CB. Protein expression was induced with 0.5 mm isopropyl β-d-thiogalactopyranoside (IPTG) when the culture reached an A600 nm of 0.6, and cells were incubated overnight at 27 °C at 250 rpm. Cells were harvested by centrifugation, and the pellets were resuspended in 1× bind/wash buffer (Novagen), which was supplemented with cOmpleteTM EDTA-free Protease Inhibitor Mixture (Roche Applied Science), and then lysed by sonication. Subsequent purification steps were carried out using GST·BindTM resin (Novagen) following the manufacturer's protocol. Following the elution step, the proteins were exchanged into 20 mm Tris, 150 mm NaCl, pH 8.0, using ZebaTM Spin Desalting Columns (Thermo Fisher Scientific, Inc.) and then concentrated with Amicon® Ultra centrifugal filters (EMD Millipore) and stored at −80 °C. To confirm the purity, the samples were resolved by SDS-PAGE and detected with Coomassie Brilliant Blue staining.</p><!><p>Screens for peptide ligands were performed following a protocol published previously (11). The purified target protein, GST-MLK3 SH3-WT, was prebound to GSH magnetic beads (Pierce, catalogue number 88821), and nonspecific binding sites were blocked with phosphate-buffered saline (PBS; 137 mm NaCl, 3 mm KCl, 8 mm Na2HPO4, 1.5 mm KH2PO4) containing 3% (w/v) bovine serum albumin (BSA). To isolate potential peptide ligands, the targets were incubated with a 12-mer combinatorial phage-displayed peptide library, ANL7 (51). After three rounds of affinity selection, individual phage clones were isolated, and their binding was evaluated via phage ELISA (11). Positive clones were then sequenced.</p><!><p>To evaluate the phage-displayed MIP (NH2-AIRINPNGTWSRQAETVES-COOH; insert (underlined) + linker region), isolated from ANL7 library (51), both WT and modified sequences (see Fig. S2) were cloned into a phage display vector, SAM (51), as an N-terminal fusion to the pIII capsid protein of M13 bacteriophage. All constructs were generated via Kunkel mutagenesis, described in detail elsewhere (11), and facilitated type 3 (pentavalent) display. Phage-displayed recombinants were evaluated for their binding to GST-MLK3 SH3 via phage ELISA as described elsewhere (11).</p><!><p>All constructs were generated via Kunkel mutagenesis (11, 57, 58) and facilitated type 3 (pentavalent) display. The single-stranded DNA template was generated using a modified bacteriophage M13 vector (SAM) containing an amber stop codon (TAG) within the linker upstream of the N terminus of pIII (51). The oligonucleotides used for generation of all clones were synthesized by Integrated DNA Technologies, Inc. and are shown in Table S1. All constructs were verified by DNA sequencing, and phage clones were evaluated for their binding to the target (GST-MLK3 SH3) via phage ELISA (11). A truncated version of MIP (ΔA1) was used as a negative control (see Fig. S2).</p><!><p>All peptides were synthesized at the Research Resource Center, University of Illinois at Chicago, and were of >90% purity as verified by MS. Two versions of MIP, unlabeled (AIRINPNGTWSRQAETVES) and biotinylated at the C terminus (AIRINPNGTWSRQAETVES-K-biotin), were used to determine the peptide's IC50. Two versions of NS5A-derived peptide (UniProt entry W8GG88; aa 2320–2331), unlabeled (KKAPTPPPRRRR-GGG) and biotinylated at the C terminus (KKAPTPPPRRRR-GGG-K-biotin), were used to determine the peptide's IC50. The biotinylated NS5A and unlabeled MIP peptide were used in competition ELISA. The biotinylated MIP, MIP-R12A (AIRINPNGTWSAQAETVES-K-biotin; as negative control), and NS5A peptide were used in specificity ELISA to determine whether MIP and NS5A can cross-react with MLK1–4.</p><!><p>To determine the IC50 value by competition, the biotinylated form of MIP (AIRINPNGTWSRQAETVES-K-biotin; 100 μl, ≈2 μm) was captured in wells of a Nunc MaxiSorpTM flat-bottom 96-well microtiter plate (Thermo Fisher Scientific), which had been previously coated with NeutrAvidinTM (100 μl, 7.5 μg/ml; Thermo Fisher Scientific) and blocked with 3% skim milk in PBS. Separately, the GST-MLK3 SH3 fusion protein (5 μg/ml) was preincubated (1–1.5 h) with increasing concentrations (≈0.01–100 μm) of unlabeled MIP (AIRINPNGTWSRQAETVES) or unlabeled NS5A (KKAPTPPPRRRR-GGG-K) as competitor and then allowed to interact for 45 min with biotinylated MIP prebound on the ELISA plate. After three washes with PBS containing 0.1% Tween 20 (PBST), retention of the SH3 domain fusion protein in wells was detected with an anti-GST antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare; 45 min; 1:5000 in PBST). Following three washes with PBST, the chromogenic substrate solution 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Pierce); 10 ml of 50 mm sodium citrate, pH 4.0; and 100 μl of 3% H2O2 were added to the plate (100 μl/well). The absorbance was measured at 405 nm with a POLARstar OPTIMA microtiter plate reader (BMG Labtech). This process was then repeated by testing the binding of the GST-MLK3 SH3 fusion protein to immobilized NS5A (KKAPTPPPRRRR-GGG-K-biotin; 100 μl, ≈2 μm) in the presence of unlabeled MIP or NS5A competitor.</p><!><p>Two DNA fragments encoding the SH3 domain of MLK3 (UniProt entry Q16584), MLK3 SH3(41–105) and MLK3 SH3(44–103), and four fragments encoding SH3 domain-MIP fusions, MLK3 SH3(41–105)-MIP(1–19), MLK3 SH3(41–105)-MIP(1–13), MLK3 SH3(41–105)-MIP(1–13)W10A, and MLK3 SH3(41–105)-MIP(1–13)R12A, were commercially prepared as gBlocks Gene Fragments. One fragment encoding SH3 domain fused to an NS5A (UniProt entry A0A076MER2) peptide, MLK3 SH3(41–105)-NS5A(2325–2332), was created by amplifying MLK3 SH3(41–105) with T7 promoter primer and a reverse primer (see Table S1). The subsequent insert contained MLK3 SH3(41–105) with the NS5A fragment (aa 2325–2332) at the C terminus. All coding regions were codon-optimized for E. coli expression. DNA fragments were cleaved with NdeI and BamHI-HF (New England Biolabs) and subsequently ligated into a modified version of the pET14b expression vector where the N-terminal His6 tag is followed by a SUMO protease cleavage site. The resulting DNA constructs (verified by sequencing) were transformed into E. coli strain BL21 C41(DE3). Cells were grown at 37 °C in 2× YT medium, which was supplemented with either 50 μg/ml CB or 100 μg/ml ampicillin, protein expression was induced with 0.1–0.3 mm IPTG at an A600 nm of 0.6–0.7, and cells were cultured overnight at 22 °C. Cells were harvested by centrifugation; washed with 200 mm KCl, 50 mm Tris, pH 7.4; and lysed by sonication in 25 mm Tris, pH 7.4, 200 mm KCl, 10 mm MgCl2, 10% glycerol, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride (PMSF). Clarified supernatant was loaded onto a 5-ml HisTrapTM HP Ni-Sepharose column (GE Healthcare), and the column was washed with 25 mm Tris, pH 7.4, 500 mm NaCl supplemented with 10 and 25 mm imidazole. Protein was eluted in the same buffer, which was supplemented with 250 and 500 mm imidazole, and the fractions were pooled together. The His6-SUMO tag was cleaved with SUMO protease while dialyzing against 25 mm Tris, pH 7.4, 500 mm NaCl, 10 mm imidazole, and the tag was removed by loading the sample back onto a nickel column. Collected fractions containing purified protein were concentrated and injected onto an S-200 size-exclusion column (GE Healthcare) equilibrated with 25 mm Tris-HCl, pH 7.4, 500 mm NaCl. To confirm the purity, collected samples were analyzed by SDS-PAGE and detected with Coomassie Brilliant Blue staining. All purified proteins were concentrated and stored at −80 °C.</p><!><p>All crystals were grown at 20 °C using the hanging-drop vapor-diffusion method. Crystals of MLK3 SH3(41–105)-MIP(1–19) were grown using a reservoir condition containing 0.1 m NaH2PO4, 0.1 m KH2PO4, 0.1 m MES, pH 6.5, 1.5 m NaCl. Drops of 4 μl were set up at a 1:1 ratio of protein (9 mg/ml) to reservoir. These crystals grew as long pyramids, ∼250 μm in length and 50 μm at the base. Prior to data collection, crystals of MLK3 SH3(41–105)-MIP(1–19) were soaked in the reservoir solution containing 30% ethylene glycol for cryoprotection. To grow crystals of MLK3 SH3(44–103), drops of 3 and 4 μl were set up, respectively, at 2:1 and 1:1 ratios of protein (15.7 mg/ml) to reservoir solution (1.4 m sodium malonate, pH 6.5). Cube-shaped crystals appeared within 2–3 days. Prior to data collection, crystals of MLK3 SH3(44–103) were soaked in mother liquor with 30% ethylene glycol as cryoprotectant.</p><p>Crystals of MLK3 SH3(44–103)-NS5A(2325–2332) at 3.6 mg/ml were set up in 2-μl drops at a 1:1 ratio with a mother liquor of 0.1 m KH2PO4, 0.1 m NaH2PO4, 0.1 m MES, pH 6.5, 2.5 m NaCl. Crystals grew over approximately 2 months and developed as clusters of square pyramids. Crystals were cryopreserved prior to data collection with 30% glycerol.</p><!><p>Diffraction data for MLK3 SH3–MIP, MLK3 SH3–NS5A, and apo MLK3 SH3 were obtained at the Life Sciences Collaborative Access Team ID beamline at the Advanced Photon Source, Argonne National Laboratory (wavelength, 0.979 Å; temperature, 100 K) (refer to Table 1 for data collection and refinement statistics). Data processing was executed using XDS (59). The structure of MLK3 SH3–MIP was solved by molecular replacement using MOLREP (60) with the available structure of the MAP3K10 SH3 domain (PBD code 2RF0) as the search model. Likewise, the apo MLK3 SH3 and MLK3 SH3–NS5A structures were solved using MOLREP using the MIP-bound structure as the model. Refinement was accomplished with REFMAC5 (61). Because the MLK3 SH3–NS5A data indicated the C2 space group with a unique β angle close to 120° (i.e. 120.006°), we also analyzed the data using phenix.xtriage, which confirmed C2 as the correct choice of space group (62). All structural figures were generated with MacPyMOL (PyMOLTM Molecular Graphics System, version 1.6, Schrödinger, LLC).</p><!><p>To determine oligomeric state of several MLK3 SH3 fusion proteins, samples were injected into a Superdex 200 10/300 GL size-exclusion chromatography column (GE Healthcare) in two independent experiments. Two samples, MLK3 SH3(41–105)-MIP(1–19) and MLK3 SH3(41–105), at 2 mg of protein/200-μl injection, were analyzed in the first experiment (Fig. S3). Three additional samples of MLK3 SH3 domain-MIP fusion proteins were analyzed in a second experiment by injecting 750 μg of protein in 200 μl: MLK3 SH3(41–105)-MIP(1–13), MLK3 SH3(41–105)-MIP(1–13)W10A, and MLK3 SH3(41–105)-MIP(1–13)R12A (Fig. S4). Prior to the injections, the column was equilibrated with 25 mm Tris-HCl, pH 7.4, 500 mm NaCl.</p><!><p>Competition ELISA was performed to determine whether MIP and NS5A peptides compete in binding the MLK3 SH3 domain and to calculate the relative binding strength (i.e. IC50) of the NS5A peptide. Biotinylated NS5A peptide (KKAPTPPPRRRR-GGG-K-biotin; 100 μl, ≈ 2 μm) was captured in wells of a Nunc MaxiSorp flat-bottom 96-well plate, which had been coated with NeutrAvidin (100 μl, 7.5 μg/ml), and blocked with 3% skim milk in PBS. Separately, the GST-MLK3 SH3(43–104) domain fusion protein (5 μg/ml) was preincubated (1–1.5 h) with increasing concentration (0.02–100 μm) of unlabeled MIP as competitor and then allowed to interact for 1 h with biotinylated MIP prebound on the ELISA plate (Fig. S7). The assay was then completed as described above for IC50 determination of MIP.</p><!><p>To determine whether MIP and NS5A can cross-react with other members of the MLK subfamily, SH3 domains of MLK1, MLK2, and MLK4 were purified as GST fusion proteins as described above. To determine their binding properties, 100 μl (≈2 μm) of biotinylated peptides, MIP (AIRINPNGTWSRQAETVES-K-biotin), MIP-R12A (AIRINPNGTWSAQAETVES-K-biotin; as negative control), and NS5A (KKAPTPPPRRRR-GGG-K-biotin), were first captured in wells of a Nunc MaxiSorp flat-bottom 96-well plate coated with NeutrAvidin (100 μl, 7.5 μg/ml) and then blocked with 2% skim milk in PBS. The GST-MLK1 SH3, GST-MLK2 SH3, GST-MLK2 SH3, and GST-MLK4 SH3 domain fusion proteins (0.15 μm) were incubated with each of the target peptides, MIP, NS5A, or MIP-R12A, for 1–1.5 h. The rest of the assay was performed as described above for IC50 determination of MIP.</p><!><p>The coordinates for apo SH3, SH3–MIP complex, and SH3–NS5A complex have been deposited in the PDB under accession codes 5K28, 5K26, and 6AQB, respectively.</p><!><p>M. E. K. and S. L. K. formal analysis; M. E. K. and J. E. M. validation; M. E. K., S. L. K., S. K., and J. E. M. investigation; M. E. K., S. L. K., A. L., and B. K. K. writing-original draft; M. E. K., S. L. K., S. K., J. E. M., A. L., and B. K. K. writing-review and editing; S. L. K. data curation; S. L. K. visualization; S. K., A. L., and B. K. K. resources; A. L. and B. K. K. conceptualization; A. L. and B. K. K. supervision; A. L. project administration; B. K. K. funding acquisition.</p><!><p>This work was supported by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust (to B. K. K.), National Institutes of Health Grant R01EB013685 (to A. L.), and National Institutes of Health T32 training support from NIDCR Grant DE018381 (to S. L. K.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</p><p>This article contains Figs. S1–S7 and Table S1.</p><p>The atomic coordinates and structure factors (codes 5K28, 5K26, and 6AQB) have been deposited in the Protein Data Bank (http://wwpdb.org/).</p><p>Src homology 3</p><p>mixed-lineage kinase</p><p>MLK3 SH3–interacting peptide</p><p>nonstructural 5A</p><p>root mean square deviation</p><p>Protein Data Bank</p><p>c-Jun N-terminal kinase</p><p>extracellular signal-regulated kinase</p><p>Cdc42/Rac interactive binding</p><p>hepatitis C virus</p><p>hematopoietic progenitor kinase 1</p><p>germline center kinase</p><p>GSH S-transferase</p><p>amino acids</p><p>carbenicillin</p><p>isopropyl β-d-thiogalactopyranoside</p><p>horseradish peroxidase</p><p>small ubiquitin-like modifier.</p>

PubMed Open Access

Improved synthesis of 6-epi-dictyostatin and antitumor efficacy in mice bearing MDA-MB231 human breast cancer xenografts

Structure-activity studies centered on the naturally occurring antitumor agent dictyostatin have recently identified several highly active epimers and analogs. From these compounds, 6-epi-dictyostatin was selected for scaleup preparation and evaluation in animals. Here we describe a new total synthesis that produced more than 30 mg of 6-epi-dictyostatin. The compound was found to have potent antitumor activity in SCID mice bearing MDA-MB231 human breast cancer xenografts.

improved_synthesis_of_6-epi-dictyostatin_and_antitumor_efficacy_in_mice_bearing_mda-mb231_human_brea

<p>Since the approval of paclitaxel in the early 1990's and docetaxel in the early 2000's, interest in compounds that bind to and stabilize microtubules has continued to grow.1 Recently, a semisynthetic derivative of epothilone B, ixabepilone, was approved by the FDA for use either alone or in combination to combat certain types of breast tumors.2 Other potent microtubule stabilizing agents, including members of the discodermolide/dictyostatin class, have also generated significant interest as drug candidates.3</p><p>Dictyostatin is a rare macrocyclic lactone isolated from marine sponges4,5 that has recently become more available through total synthesis.6-10 Members of the dictyostatin family strongly inhibit cancer cell growth, have high affinities for the taxoid binding site, and competitively inhibit the binding of paclitaxel and epothilone B to microtubules.4,9,11-13 Among several potent analogs, including 7-epi-dictyostatin, 16-normethydictyostatin and 15(Z),16-normethyldictyostatin, 6-epi-dictyostatin 1 was the most potent inhibitor of binding of epothilone B to microtubules. It was also the most potent inhibitor of HeLa cell growth, and its activity was not affected by mutations that induce paclitaxel resistance in the taxoid binding site.9,13</p><p> </p><p>Accordingly, 6-epi-dictyostatin 1 was selected for a scaleup synthesis and in vivo evaluation of its antitumor properties. Here we report a new synthetic approach to the dictyostatin family that produced more that 30 mg of 6-epi-dictyostatin 1. In the first animal tests of any dictyostatin, epimer 1 showed high antitumor activity in SCIDa mice bearing xenografts of the MDA-MB231 human estrogen receptor negative breast cancer cell line. 6-epi-Dictyostatin 1 was significantly better than paclitaxel in inhibiting tumor growth.</p><p>We adopted the retrosynthesis plan for 6-epi-dictyostatin shown in Figure 1. Compared to the original fluorous mixture synthesis of 1,12 the new route is more convergent because it features fully elaborated bottom 5, middle 6 and top 3 fragments. It also introduces a new strategy for uniting the bottom and middle fragments by a silicon-tethered ring-closing metathesis (RCM) reaction14 of 4 to form the critical C10-C11 Z-alkene.</p><p>The C1-C10 fragment 5 was synthesized through five steps, starting from readily available intermediate 7,15 as shown below. Deprotection of TBS ether 7 with HF·pyr and Dess-Martin oxidation16 of the resulting alcohol provided the aldehyde 8 in 91% yield over two steps. Addition of vinyl magnesium bromide, Dess-Martin oxidation, and Corey-Bakshi-Shibata (CBS)17 reduction gave an inseparable mixture of two C9-epimers 5 (α) and 9 (β) in a 5:1 ratio favoring the needed alcohol 5 (74% yield, three steps).</p><p> </p><p>The sequence of fragment couplings and macrolactonization is summarized in Scheme 1. The C11-C17 alcohol 6 was treated with BuLi/dimethyldichlorosilane, followed by addition of imidazole and the C1-C10 alcohol epimer mixture of 5 and 9. This provided another inseparable C9 mixture of the silylketals 4 and 10 in 85% yield, again in about 5:1 ratio. The RCM reaction of this silylketal mixture was mediated by a Grubbs-Hoveyda II catalyst to provide a third inseparable C9 mixture of the 8-membered disiloxanes in 57-69% yield. This mixture was deprotected with dichloroacetic acid and the crude product was purified by silica gel chromatography. This time, the C9 epimers separated, and the target diol 11 (C9S) was isolated in 45% yield alongside 7% of the C9R-epimer 12. Protection of the diol 11 as a TBS ether followed by treatment with DDQ gave the alcohol 13 in 62% yield over two steps. The alcohol 13 was oxidized by using the Dess-Martin periodinane to the aldehyde 2, which was coupled with the phosphonate 3 to provide the enone 14 possessing the full C1-C26 carbon skeleton of 6-epi-dictyostatin (89% yield over two steps).</p><p>The C17-C18 alkene of the enone 14 was reduced using a Stryker reagent ([Ph3PCuH]6)18 to give a saturated ketone, then the C21 PMB group was removed with DDQ to provide the β-hydroxyketone 15 in 68% over two steps. The 1,3-syn-reduction of the hydroxyketone 15 with NaBH4 and Et2BOMe19 and then selective protection of the C19 hydroxy group of the resulting diol with TBSOTf furnished the alcohol 16 (74% yield over two steps).8,20</p><p>In the usual end game, the C1 methyl ester was hydrolyzed by using KOH to produce a seco-acid, which was used for the next reaction without further purification. The macrolactonization of the seco-acid using the Yamaguchi reagent (2,4,6-trichlorobenzoyl chloride)21 gave a mixture of the C2 E/Z isomers of the TBS-protected macrolactone in a varying ratio, but the use of a Shiina reagent (2,6-methylnitrobenzoyl anhydride)22 in toluene suppressed the isomerization of the C2Z-alkene (1:13, E/Z). Finally, global deprotection with HCl provided 33 mg of 6-epi-dictyostatin in 45% yield over three steps.23</p><p>6-epi-Dictyostatin 1 and paclitaxel were each administered intravenously in three doses of 20 mg/kg/dose spaced 7 days apart to ten C.B-17 SCID female mice bearing established MDA-MB231 human breast cancer xenografts. Mean tumor volumes (Figure 2) and body weight loss (Figure 3) were periodically measured. One of the mice receiving 6-epi-dictyostatin 1 was accidentally injured then euthanized between days 14 and 17, while the other nine mice in that group continued to be followed.</p><p>Mean tumor volumes in the mice treated with 6-epi-dictyostatin 1 were significantly smaller than those in the control and vehicle-treated groups beginning on day 7 of treatment. And beginning on day 10, they were also smaller than those in the paclitaxel-treated group (Figure 2). Tumor regression was observed in six of the nine 6-epi-dictyostatin 1-treated mice on day 14 of study, and these tumors continued to regress until day 28 of study. In the remaining three 6-epi-dictyostatin 1-treated mice, tumor volumes did not increase until day 28, when tumor regrowth was observed. Tumors continued to grow in all the paclitaxel-treated mice, albeit at a slower rate than that observed for the tumors in the control and vehicle-treated groups. Paclitaxel-treated mice were euthanized between days 24 and 28 (Figure 2).</p><p>Body weight loss (Figure 3) was less than 10% in the 6-epi-dictyostatin 1-treated mice and their body weights were significantly lower than those in the other treatment groups. This difference in body weights may in part be due to the lack of tumor growth in the 6-epi-dictyostatin 1-treated mice.</p><p>Tumor doubling times, median optimal %T/C and median optimal %T/V for the various treatment groups are presented in Table 1. Tumors in the 6-epi-dictyostatin 1-treated mice did not double in volume at 28 days. The mean tumor doubling times for the paclitaxel-treated mice were significantly longer than the tumors in the control and vehicle-treated groups. Both the median optimal %T/C (day 14) and median optimal %T/V (day 17) were approximately 30% for the paclitaxel-treated mice, and 13% for the 6-epi-dictyostatin 1-treated mice, a significant difference.</p><p>In conclusion, we successfully executed an improved synthesis of 6-epi-dictyostatin 1 that yielded quantities sufficient for animal antitumor studies. 6-epi-Dictyostatin 1 was more effective than paclitaxel in mouse xeongraft studies, and excellent efficacy was observed at a dose that did not cause significant weight loss in the animals. Studies are ongoing to determine tissue distribution, metabolism of 1, and pharmacodynamic effects in tumor and normal tissue. The present results suggest that dictyostatins such as 1 hold promise as new microtubule-stabilizing chemotherapeutic agents.</p>

PubMed Author Manuscript

Immunohistochemical techniques for the human inner ear

In this review, we provide a description of the recent methods used for immunohistochemical staining of the human inner ear using formalin-fixed frozen, paraffin and celloidin-embedded sections. We also show the application of these immunohistochemical methods in auditory and vestibular endorgans microdissected from the human temporal bone. We compare the advantages and disadvantages of immunohistochemistry (IHC) in the different types of embedding media. IHC in frozen and paraffin-embedded sections yields a robust immunoreactive signal. Both frozen and paraffin sections would be the best alternative in the case where celloidin-embedding technique is not available. IHC in whole endorgans yields excellent results and can be used when desiring to detect regional variations of protein expression in the sensory epithelia. One advantage of microdissection is that the tissue is processed immediately and IHC can be made within 1 week of temporal bone collection. A second advantage of microdissection is the excellent preservation of both morphology and antigenicity. Using celloidin-embedded inner ear sections, we were able to detect several antigens by IHC and immunofluorescence using antigen retrieval methods. These techniques, previously applied only in animal models, allow for the study of numerous important proteins expressed in the human temporal bone potentially opening up a new field for future human inner ear research.

immunohistochemical_techniques_for_the_human_inner_ear

Introduction<!>Microdissected auditory and vestibular endorgans<!>Formalin-fixed cryostat sections obtained from microdissected endorgans<!>IHC in paraffin-embedded sections of the human inner ear<!>IHC and IF in whole auditory and vestibular endorgans<!>IHC in celloidin-embedded sections<!>Formalin-fixed cryostat sections<!>Paraffin-embedded tissue sections<!>Immunofluorescence in whole auditory and vestibular endorgans<!>IHC in celloidin-embedded tissue sections<!>Immunofluorescence in celloidin-embedded tissue sections<!>Discussion<!>Advantages<!>Disadvantages<!>Advantages<!>Disadvantages<!>Advantages<!>Disadvantages<!>Advantages<!>Disadvantages<!>Alternative human inner ear tissue sources and new imaging methodologies<!>Immunohistochemistry, scanning thin-sheet laser imaging microscopy (STSLIM) and scanning electron microscopy in decellularized human cochlea<!>Further IHC studies in the human temporal bone (celloidin-embedded sections)<!>Conclusions and future directions

<p>The traditional human temporal bone processing at the turn of the twentieth century employed the celloidin-embedding and serial-sectioning methods to evaluate predominantly light microscopic histopathology for clinic–pathologic correlations (Gulya 2007). Earlier studies utilized cytologic descriptions and graphic reconstructions of the cochlea (Guild 1921). Temporal bone science has advanced such that we are now entering a phase of methodological integration, whereby the same temporal bone can be used for light microscopy, transmission electron microscopy (TEM), immunohistochemistry (IHC), non-radioactive in situ hybridization, DNA and proteomics analysis (Aarnisalo et al. 2010; Kong et al. 1998; Merchant et al. 2008; Ishiyama et al. 2009, 2010; Lopez et al. 2005a, b, 2007; Markaryan et al. 2008a, b, c, 2009a, b, c, 2010a, b; Nguyen et al. 2014; Richard et al. 2015; Schrott-Fischer et al. 1994, 2002a, b, 2007; Wackym et al. 1990). The study of the human inner ear has lagged behind other areas of pathology, in large part due to the inaccessibility of the membranous labyrinth. The temporal bone contains delicate structures, the membranous labyrinth and cochlea, encased in the very dense temporal bone. Furthermore, the tissues are not available for study during a person's lifetime except in specialized cases, such as vestibular endorgans removed by ablative surgery in patients diagnosed with intractable Meniere's disease or temporal bone tumors. Thus, there are great technical difficulties in processing the temporal bone in a manner that preserves the morphology while allowing for IHC.</p><p>The methodologies used to process human inner ear tissue for IHC and applications have been previously revised by Arnold (1988), Bauwens et al. (1990), Fish et al. (2000), Tian et al. (1999). In the present review, the advantages and disadvantages of the several methodologies available to date to temporal bone investigators for immunohistochemical studies in the human inner ear are discussed. There are two major tissue processing methods available for studies of formalin-fixed postmortem human temporal bones: (1) microdissection of the auditory and vestibular endorgans from temporal bones which yields specimens that can be processed for cryosections, embedded in paraffin or used as whole-mount preparations, and (2) celloidin embedding of temporal bones that have been decalcified, a method that has been widely employed by most archival human temporal bone banks (Schuknecht 1993).</p><!><p>Formalin-fixed microdissected auditory and vestibular endorgans obtained from human temporal bone specimens have been successfully used to study the immunolocalization of aquaporins, several basement membrane proteins and cochlin in the vestibular endorgans and the cochlea (Lopez et al. 2007; Ishiyama et al. 2009, 2010; Calzada et al. 2012a, b). Using the microdissection technique, the three cristae ampullares, maculae utricle and saccule, as well as the organ of Corti, are removed from the otic capsule of the temporal bone and separately processed for either light or transmission electron microscopic examination (Wright and Meyerhoff 1989).</p><p>This technique is advantageous because the dissected tissues can be sectioned serially at any thickness to facilitate detailed cytological examination of specimens, from as thin as 0.1 μ to as thick as 20 μ. Another advantage of temporal bone microdissection over celloidin-embedded inner ear tissue is that each dissected endorgan can be properly oriented and processed along the optimal tissue plane, which differs depending on the particular endorgan. Illustrative of this problem (when using the traditional temporal bone processing), only a small fraction (8 %) of the utricle is sectioned in a perpendicular plane, predominantly in the superior pole (Merchant 1999). Thus, by individually dissecting each organ, the specimen can be properly oriented prior to sectioning, allowing accurate cross sectioning perpendicular to the plane for purposes of morphometric studies of specific structures. Previous studies from our laboratory have demonstrated the use of microdissected specimens from postmortem human temporal bones for design-based stereology (Gopen et al. 2003; Ishiyama et al. 2004; Lopez et al. 2005b).</p><p>A third advantage of microdissection is that minimal decalcification is required, allowing for excellent morphological preservation and accurate cytological identification in postmortem specimens. Using the standard procedure for the morphological study of the human temporal bone as established by Schuknecht (1993), the decalcification step for the human temporal bone requires that the specimen remains in ethylenediaminetetraacetic acid (EDTA) for up to 9 months. Temporal bone fixation, microdissection, tissue sectioning and immunostaining can be made within a week. Shorter postmortem times are associated with higher degrees of preservation of the sensory epithelia (se) morphology (Ishiyama et al. 2009). An additional advantage is the ability to apply unbiased stereology in immunostained endorgans (Lopez et al. 2005a). Additionally, it is also possible to use vestibular endorgans microdissected from human temporal bones to extract mRNA to evaluate the expression of muscarinic acetylcholine receptors (Wackym et al. 1996) or mu-opioid receptor mRNA expression by non-radioactive in situ hybridization (Nguyen et al. 2014).</p><!><p>Formalin-fixed cryostat sections of microdissected vestibular endorgans are routinely used for immunohistochemical studies of the inner ear of numerous animal models. Swartz and Santi (1999) used perilymphatic fixation, decalcification in 10 % EDTA for 3 days, and frozen sections for the study of tenascin-C immunoreactivity (IR) in the chinchilla inner ear. This methodology can also be applied in the human inner ear (Lopez et al. 2005a, 2007; Ishiyama et al. 2009). In the human inner ear, the feasibility and utility of cryosections for IHC have been demonstrated with immunolocalization of several neurotransmitters and peptides (Ishiyama et al. 1994; Kong et al. 1998; Popper et al. 2002), aquaporins (Lopez et al. 2007), basement membrane proteins (Ishiyama et al. 2009) and cochlin (Calzada et al. 2012a, b). Surgically obtained cochlear and vestibular endorgans can also be processed into cryosections and used for IHC (Calzada et al. 2012a, b; Ishiyama et al. 2010; Liu et al. 2009, 2015, 2016; Khalifa et al. 2003). Khalifa et al. (2003) and Rask-Andersen et al. (2000 and Rask-Andersen et al. (2016) used fresh surgically acquired human inner ear specimens to localize several proteins by IHC in the cochlea and spiral ganglia neurons. Of note, the morphological preservation even in postmortem vestibular and auditory endorgans is of sufficient quality to allow for cytological regional identification of IR patterns (Ishiyama et al. 2009). For example, aquaporin 4 is preferentially expressed in the basal portion of the vestibular supporting cells, while aquaporin 6 is expressed in their apical portion (Lopez et al. 2007).</p><!><p>Paraffin-embedded sections of the human inner ear are obtained from formalin-fixed (10–20 %) and decalcified temporal bones or obtained from formalin-fixed microdissected auditory or vestibular endorgans. Once the inner ear tissue is fixed in formalin, dehydrated in ascending ethylic alcohols (30–100 %), cleared with 100 % xylene and embedded in 100 % paraffin, it is possible to obtain 5- to 20-μm-thick sections for IHC.</p><p>IHC in paraffin-embedded sections has been conducted in the gerbil cochlea to study the distribution of dystroglycan, an extracellular matrix (ECM) protein, using a combination of microdissection and EDTA (48 h) with excellent morphology and retention of antigenicity (Heaney et al. 2002). Cunningham et al. (2001) described an alternative for IHC of human temporal bones that used a decalcification method with a microwave oven process. A good signal-to-noise ratio of the immunostaining of Na+K+-ATPase in the human organ of Corti, as well as acceptable morphological preservation, was demonstrated. Keithley et al. (2000) and Shi et al. (1991, 1992a, b) used paraffin-embedded sections of the human temporal bone and antigen retrieval methodology to localize neurofilaments, peripherin and subunit-1 of cytochrome oxidase (Keithley et al. 1995, 2000, 2001). A modified paraffin-embedding protocol was used for the immunolocalization of prestin and neurofilament antibodies in the human cochlea (Takahashi et al. 2010). Twenty percent formalin was used for fixation, followed by EDTA decalcification for 9 months. Proteinase K (0.2 mg/mL) for 5 min was used for tissue permeabilization. Carbonic anhydrase isoenzyme-IR (Yamashita et al. 1992), laminin-IR (Yamashita et al. 1991) and cytokeratins-IR (Anniko et al. 1989a, b) were detected in paraffin-embedded human fetal inner ear tissue sections. Human temporal bone sections embedded in polyester wax have been used for IHC (Merchant et al. 2006). However, tissue preservation and section quality were suboptimal for histopathology or IHC studies (O'Malley et al. 2009a, b). Thus, paraffin embedding is a good alternative for histology and IHC, when human inner ear tissue embedded in celloidin is not available.</p><!><p>Immunostaining of whole endorgans and surface (whole mount) preparations of auditory and vestibular endorgans has widely been used for anatomical and quantitative studies or cell regional distribution (Goran 1968; Rosenhall 1972; Rosenhall and Rubin 1975). Histochemical staining of actin with fluorescence-labeled phalloidin has been used to identify vestibular and auditory hair cell stereocilia and the apical portion of supporting cells. Whole endorgan staining of nerve fibers has been possible using antibodies against neurofilaments and markers that identify specific neuronal populations, i.e., calbindin, calretinin or nerve terminals, i.e., synaptophysin (Nadol et al. 1993). The organization of nerve terminals in the human cochlea was recently investigated using antibodies against several proteins present in the synaptic terminals (Viana et al. 2015): CTBP2 (to identify synaptic ribbons), anti-neurofilaments (nerve axons), anti-myosin VIIa (hair cell identification) and choline acetyltransferase to identify efferent axons. Viana et al. (2015) used triple and quadruple-IF staining to reveal the pattern of afferent and efferent innervation in human postmortem material and showed evidence that cochlear synaptopathy in the absence of hair cells loss may be an important factor in human presbycusis.</p><p>The whole immunostained endorgan has been embedded in plastic to obtain thin sections (0.5–1 μm) and also has been used for design-based unbiased stereology (Lopez et al. 2005a). Whole-mount immunostained specimens have also been used to conduct TEM (Nadol et al. 1993; McCall et al. 2009; Taylor et al. 2015).</p><!><p>A large number of archival human temporal bone specimens collected over the past 85 years are available in the USA and Europe for histopathological studies of the auditory and vestibular system (Schuknecht 1993; Merchant et al. 1993, 2008). Currently there are only three active temporal bone laboratories in the USA that continue to embed human inner ear tissue in celloidin (Chole 2010). The traditional method of temporal bone processing uses formalin as fixative (10 %, neutral-buffered formalin). Before decalcification, the temporal bones (immersed in formalin) are stored in a cold room or at 4 °C in the refrigerator, for 1–3 weeks. Decalcification step with EDTA (0.27–0.48 M, with or without 1 % formalin, pH 7.2–7.3) takes up to 9 months. Temporal bone specimens are then dehydrated in graded ascending ethylic alcohol and embedded in celloidin over a 3-month period. Detailed procedures of temporal bone collection, fixation, decalcification and celloidin embedding were described by Merchant (2010). Horizontal sections of celloidin-embedded temporal bones are obtained with a sliding microtome. The specimens are serially sectioned at 20 μm, and every tenth section is stained with hematoxylin and eosin. The celloidin-embedding method yields superb morphological and histopathological preservation of the human inner ear that can be used for IHC (Ganbo et al. 1997, 1999; Cunningham et al. 2001; Keithley et al. 1994, 2000; Markaryan et al. 2011; Ying and Balaban 2009; Ahmed et al. 2013; Balaker et al. 2013; Nguyen et al. 2014; Vorasubin et al. 2016). It is important to have in consideration the length of fixation specially for IHC. Excessive cross-link of proteins by the fixative will affect the binding of the antibody. Antigen retrieval steps discussed in this review can help to restore immunogenicity.</p><p>Several laboratories have described methods for celloidin removal and antigen retrieval steps for IHC (Shi et al. 1992a, b, 1993, 1998; O'Malley et al. 2009a). These two steps are necessary to expose the antigens and allow primary antibody binding. Antigen retrieval technique was first described by Battifora and Kopinski (1986) and was designed to increase epitope antigenicity for immunohistochemical studies. Once celloidin is completely removed from the tissue section, it is immersed in a heavy metal solution and heating in the microwave oven (Shi et al. 1992a, b, Appendix). The result is a significant increase in antigenicity and binding of proteins (O'Rourke and Padula 2016).</p><p>Thick celloidin-embedded sections (20–30 μm) of single temporal bone are kept immersed in 80 % ethylic alcohol for subsequent histological and/or immunohistochemical analysis including proteomics (Tian et al. 1999; Aarnisalo et al. 2010; Markaryan et al. 2010a, b). Cytologic quantification of the cochlea is feasible when postmortem time is sufficiently short (Schuknecht 1993). Detailed techniques for celloidin removal from temporal bone sections have been described in detail (O'Malley et al. 2009a). O'Malley et al. (2009a, b) used clove oil, acetone, ether-alcohol and methanol saturated with sodium hydroxide. The first three substances resulted in incomplete removal of celloidin, affecting the quality of immunostaining. Methanol–sodium hydroxide method completely removed celloidin and produced good immunostaining with six antibodies: prostaglandin D synthase, Na+K+-ATPase, aquaporin 1, connective tissue growth factor, tubulin and 200kd neurofilaments. We used sodium ethoxide to remove celloidin from the sections and obtain very good immunoreactive signal with almost no background (Calzada et al. 2012a, b; Ahmed et al. 2013; Balaker et al. 2013; Nguyen et al. 2014; Vorasubin et al. 2016).</p><p>In this review, we describe the current protocols for IHC using formalin-fixed frozen, paraffin-embedded and celloidin-embedded tissue sections of the human inner ear. The three types of tissue processing are useful for the identification of different types of antigens by IHC. We then contrast the advantages and disadvantages of these methodologies. Details of the various protocols used in this review are presented in "Appendix" section. IHC staining and IF staining in human inner ear tissue using these protocols are described below.</p><!><p>Figure 1 shows immunofluorescence (IF) micrographs using formalin-fixed frozen sections obtained from microdissected auditory and vestibular endorgans harvested from postmortem temporal bones. A wide variety of intracellular and extracellular proteins were detected using this protocol ("Appendix" section). IF of cryostat sections of the cochlea using neurofilaments antibodies demonstrated sensitivity to detect nerve fibers running throughout the modiolus, reaching the base of the inner and outer cochlear hair cells (Fig. 1a). Neurofilament-IF was also seen the soma of spiral ganglia neurons (Fig. 1b). Satellite cells that surround spiral ganglia neurons were Na+K+-ATPase-IF (Fig. 1c). Blood vessels within the cochlea were identified with the use of antibodies against alpha-smooth muscle actin (Fig. 1d). In the crista ampullaris, neurofilament-IF identified nerve fibers of varied diameter within the stroma and running throughout the sensory epithelium. Phalloidin-rhodamine visualized the apical portion of supporting cells and hair cells stereocilia (Fig. 1e). In the macula utricle, antibodies against collagen IV identified the perineural basement membrane associated with nerve fibers that run within the stroma and also delineated the basement membrane underneath the se (Fig. 1f). Connexin (Cx)-26-IF was localized in the supporting cells of the macula utricle (Fig. 1g). Figure 1h shows aquaporin-4-IF (red color) and GFAP-IF (green color) in the supporting cells of the macula utricle se. IF for the inward rectifier K+ channel 4.1 was detected in the stria vascularis of the cochlea (Fig. 1i). von Willebrand A domain-related protein (WARP)-IF was detected in small- and large-size blood vessels of the cochlea (Fig. 1j). Figure 1k shows cytochrome-C-IF in the hair cells cytoplasm. Cx-30-IF was seen in the se of the crista ampullares (Fig. 1l) and cochlea (Fig. 1l′).</p><!><p>Figure 2 shows IHC staining in paraffin-embedded tissue sections of microdissected macula utricle and vestibular ganglia. Figure 2a shows cochlin-IR in a cross section of the macula utricle. Cochlin-IR was seen in the basement membrane of the se and ECM of the stroma. The se was non-IR. Figure 2b shows superoxide dismutase-2 (SOD-2)-IR in a cross section of the vestibular ganglia. SOD-2-IR was seen in the cytoplasm of the vestibular ganglia neurons (punctated distribution resembling mitochondria). Figure 2c shows WARP-IR in a consecutive section of the vestibular ganglia. WARP-IR was confined to the blood vessels. Vestibular ganglia neurons and nerve fibers were non-IR. Paraffin-embedded microdissected specimens yielded good IHC, and numerous antibodies have been tested using this type of tissue sections (Table 1).</p><!><p>Figure 3a–c shows a whole-mount preparation of the organ of Corti immunostained with antibodies against myosin VIIa to visualized outer and inner hair cells. Figure 3a shows a low magnification view from the mid-apical organ of Corti. Higher magnification demonstrates myosin VIIa-IF in the hair cells (Fig. 3b in red color). Simultaneous visualization with Na+, K+-ATPase antibodies shows IF in the inner and outer sulcus cells (Fig. 3b in green color). In another specimen, hair cells and their stereocilia were identified with myosin VIIa antibodies and phalloidin Oregon green (Fig. 3c, red and green color, respectively). Actin was seen at the junctional complexes along the circumference of supporting cells. Figure 3d shows neurofilament-IF in nerve fibers of the cochlea from the hook region to the apical portion. Figure 3e shows a higher magnification view from the apical portion from Fig. 3d. Immunofluorescent nerve fiber processes that course radially toward the organ of Corti were easily identified. Figure 3f shows a whole-mount preparation of the saccule subjected to IF staining with antibodies against calmodulin. Vestibular hair cells were well visualized (Fig. 3f). Neurofilaments-IF identified nerve fibers and terminals in a whole-mount preparation of the utricle (Fig. 3g). Figure 3h shows another macula utricle stained by double labeling with calmodulin and neurofilaments antibodies that identify hair cells and the innervating vestibular nerve fibers, respectively.</p><!><p>Using the IHC protocol for celloidin-embedded sections ("Appendix" section), we were able to localize several proteins in the human inner ear. Figure 4a–b″ shows β2-laminin-IR and collagen IV-IR in the cochlea and vestibular endorgans. These celloidin-embedded immunostained sections were counterstained with hematoxylin (purple color). In the cochlea, β2-laminin-IR (dark amber color) was found in perivascular basement membranes. Reissner's membrane was also β2-laminin-IR, while the tectorial membrane was negative (Fig. 4a). Of note, Reissner's membrane and the tectorial membrane morphology were well preserved. Figure 4a′ shows the β2-laminin-IR around the stria vascularis blood vessels. This celloidin section was not counterstained with hematoxylin to demonstrate the specific β2-laminin-IR and the lack of background. Figure 4a″ shows perivascular β2-laminin-IR in the spiral ganglia from another temporal bone celloidin section. Figure 4a‴ shows β2-laminin-IR in the basement membrane of the se of the saccular macula. Perivascular β2-laminin-IR was seen around blood vessels within the stroma. Figure 4b shows collagen IV-IR in the vasculature of the spiral ligament and spiral prominence. Figure 4b′ shows collagen IV-IR in the basement membrane of the organ of Corti and in the spiral limbus. Figure 4b″ shows collagen IV-IR around the spiral ganglia neurons. Figure 4c, d shows SOD-2-IR and NF-IR in the cytoplasm of spiral ganglia neurons. Figure 4e shows acetylated tubulin-IR in pillar and Deiter's cells of the organ of Corti.</p><!><p>Colocalization of prestin and acetylated tubulin identifies outer hair cells (green) and supporting cells (red), respectively (Fig. 5a); it was possible in celloidin-embedded sections of the human cochlea. DAPI in blue color allows the identification of cell nuclei. Laser confocal microscopy was feasible in this type of tissue, with superb signal-to-noise ratio. Figure 5b shows a stack of images (0.5 μm) obtained with the confocal microscope; the creation of this stack using ImageJ (public software) allows the 3D view of hair cells (green) and supporting cells (red) with prestin and acetylated tubulin antibodies, respectively. DAPI (blue) stain allows the identification of cell nuclei (Fig. 5b). The exposure of celloidin-embedded sections to UV light before IF quenches auto-fluorescence effectively ("Appendix" section).</p><!><p>In this review, we show the results from the application of IHC using formalin-fixed frozen and paraffin sections obtained from microdissected human auditory and vestibular endorgans. Both tissue processing methods demonstrated excellent preservation of antigenicity and morphology. Whole mount (surface preparations) of the cochlea and vestibular endorgans was also useful to detect regional expression of several proteins. IHC and IF were also successfully performed in celloidin-embedded sections. The advantages and disadvantages for each type of tissue processing methods are discussed below.</p><!><p>The use of cryostat sections obtained from microdissected auditory and vestibular endorgans for IHC has several advantages. Each endorgan can be properly oriented and thin sections can be obtained. Processing of microdissected specimens has a much shorter time requirement compared to the preparation of celloidin-embedded specimens. Since little or no decalcification is needed, fixation and tissue sectioning can be as short as 1 week. For the cochlea, it is recommended to remove as much connective tissue and surrounding bone as possible and then place the otic capsule containing the cochlea into EDTA for 3 weeks. The tissue embedding media (i.e., Tissue Tek) used to obtain cryostat sections are soluble to water, and it is easily removed before IHC of IF.</p><p>One major advantage of formalin-fixed frozen sections of the cochlea or vestibule is the possibility of multi-screening of several antigens using double or triple labeling (Table 1). The use of multiple cellular markers allows the study of multiple specific proteins in the normal and pathological inner ear. Formalin-fixed human and animal inner ear frozen sections can be used as a positive control to test antibodies in cases where paraffin or celloidin sections do not show the expected protein expression. Normative tissue when collected within 3- to 6-h postmortem can be used to compare immunostaining with pathological tissue obtained from ablative surgery (i.e., labyrinthectomy). Formalin-fixed frozen sections are also a good reference for testing labile antigens to be investigated in valuable celloidin-embedded tissue sections. As revised by Shi et al. (1998) for any antibody tested, knowledge of immunolocalization of the antigen in formalin-fixed frozen fresh tissue is valuable as a gold standard. Otherwise, frozen tissue sections should be stained simultaneously with paraffin or celloidin sections to validate IHC or IF methods.</p><p>We have previously reported that microdissected vestibular endorgans can be used for stereology quantification (Gopen et al. 2003; Ishiyama et al. 2004; Lopez et al. 2005a). Microdissected crista ampullaris or utricle were subjected to whole-mount IHC as described on whole-mount methods ("Appendix" section), and then, the immunoreacted vestibular endorgans were immediately dehydrated, properly oriented (in the desired plane) and embedded in plastic. Two-micron-thick serial sections of the whole organ were obtained and counter-stained with toluidine blue. Design-based stereology was then applied (Lopez et al. 2005a). We have also used formalin-fixed microdissected vestibular endorgans embedded in paraffin for molecular biological studies (Pagedar et al. 2006). Other laboratories have used this type of tissue processing for proteomics (Robertson et al. 2006). Using a similar methodology, aquaporin 1, 4 and 6 (Lopez et al. 2007; Ishiyama et al. 2010), collagen IV, nidogen, β-laminin, α-dystroglycan and tenascin-C (Ishiyama et al. 2009) were detected in cryostat sections of microdissected auditory and vestibular endorgans. More recently, we have been able to implement nonradioactive in situ hybridization protocols to investigate the mRNA expression of the mu-opioid receptor in formalin-fixed cryostat sections of the human cochlea and protein localization by IHC using celloidin-embedded tissue sections (Nguyen et al. 2014).</p><!><p>Some structures are lost during auditory and vestibular endorgan microdissection, for example the tectorial and Reissner's membrane, the endolymphatic sac and the labyrinth membrane that surrounds the vestibular endorgans. Storage of the cryosections requires an ultra-low temperature refrigerator (−80 °C) and the need of a backup system. The quality of staining may decrease by prolonged storage of the sections. However, uncut tissue can be kept frozen. Training is required to learn the audio-vestibular endorgan microdissection. Antigen retrieval treatment may need to be applied to the formalin-fixed frozen sections in the case of antigens sensitive to formalin fixation. The microwave heating treatment may affect the integrity of the fragile tissue attached to the glass slide.</p><!><p>One of the advantages of whole-mount (surface preparations) specimens is that regional distribution of expression of antigens can be investigated. Whole-mount preparations are especially useful in the cochlea where there is standardization of regional classification and known differential expression of proteins regionally based. Whole-mount preparation is an ideal preparation for confocal studies (Viana et al. 2015). Whole endorgans can be embedded in plastic after IHC or IF and image documentation. Thin plastic serial sections can be used for unbiased stereology and quantification (Lopez et al. 2005a). Nadol et al. (1993) used microdissection and preembedding immunostaining and TEM to visualize synaptophysin.</p><!><p>Penetration of the reagents (PBS, blocking solution), including the antibody of interest, can be a problem, especially with respect to the crista ampullaris. This is notable in the identification of mesenchymal cells and the relative diminished penetration into the basal portion of supporting cells. For vestibular endorgans, the penetration of reagents and antibodies is limited to 5–10 μ in the epithelial portion, and the compactness of the stromal tissue underneath the se can be a limiting factor for access of antibodies. Increasing the concentration of Triton-X100 can allow for increased permeabilization of the tissue.</p><!><p>There are several advantages of the paraffin-embedded method. Paraffin embedding requires minimum equipment, and most of the equipment is standard in a traditional histopathology laboratory. Additionally, paraffin-embedding process is relatively quick and requires relatively less training than celloidin embedding and sectioning. Tissue samples embedded in paraffin can be used many years after they were obtained and can be kept as a paraffin tissue bank. Paraffin-embedded tissue sections are resistant to microwave oven heating during the antigen retrieval method.</p><p>IHC in paraffin-embedded tissues is used routinely for pathological specimens, and numerous antibodies can be tested for diagnostic purposes. In some cases, the antibodies are specifically designed to identify antigens in formalin-fixed paraffin-embedded specimens. Indirect IHC methods or fluorescence-labeled secondary antibodies can be used in this type of tissue. In Zehnder et al. (2005), a mid-modiolar section was stained with hematoxylin and eosin, and the remaining sections were used for IHC of α2, α3 and α5 collagen IV in Alport syndrome. The temporal bones had been in formalin for several years prior to processing. Paraffin-embedded human temporal bone specimens were also used to conduct proteomics and IHC for cochlin expression (Robertson et al. 2006). In that study, the temporal bones had been in 10 % formalin for several years and then later decalcified in EDTA and embedded in paraffin. Eight-micron sections were made and immunostained in the traditional manner.</p><!><p>Alcohols and xylenes are used for dehydration and paraffin infiltration, and heating (55–60 °C) may affect antigen conformation. Also, the degree of shrinkage is high in paraffin-embedded tissue (from 30 to 40 %). This shrinkage produces alterations of the tissue (Tang and Nyengaard 1997; Philipp and Ochs 2013), specially in the delicate cochlea and vestibular endorgans. The preservation of delicate structures like the Reissner's membrane and the tectorial membrane is not feasible. The use of paraffin-embedded tissue for quantitative unbiased stereology is not recommended as the cutting process can cause tissue loss, e.g., lost caps (physical removal of nucleolar fragments by the cutting blade). Finally, paraffin must be completely removed from the tissue to avoid false-positive reactions.</p><!><p>Most of the human temporal bone banks have a collection of hematoxylin and eosin sections, from which there are remaining 9 out of 10 sections unstained that can be used for different types of staining, including IHC, histochemistry, molecular biology or proteomics (Jokay et al. 1998; Kammen-Jolly et al. 2001; Khetarpal 2000; Robertson et al. 2001, 2006; Merchant et al. 2008; Aarnisalo et al. 2010; Markaryan et al. 2008a, b, c, 2009a, b, c, 2010a, b, 2011; Maekawa et al. 2010; Nelson and Hinojosa 2014). The tectorial membrane and Reissner's membrane are generally well preserved, which often is not the case in paraffin-embedded or microdissected specimens. Celloidin-embedding process causes minimum shrinkage of the tissue (Gardella et al. 2003). Another major advantage is the availability of panoramic views of the inner ear. For example, mid-modiolar temporal bone sections include the cochlea and vestibule, as well as the surrounding and supporting structures, i.e., middle ear, vasculature as well as neuronal components (spiral and vestibular ganglia as well as nerve fibers), and the endolymphatic sac. This allows for the localization of the different inner ear components (Table 1). In addition, there are temporal bones available from different ages from embryonic to very old age.</p><p>The use of celloidin-embedded sections for IHC has yielded important information regarding the expression of proteins relevant to inner ear structure or homeostasis. Using the preparative methods for IHC described previously (O'Malley et al. 2009a, b) adopted to our laboratory protocol, we have been able to detect Tom20 (translocase of the outer mitochondrial membrane-20) (Balaker et al. 2013), the glutamate transporter (GLAST) (Ahmed et al. 2013), the mu-opioid receptor (Nguyen et al. 2014) and recently neuroglobin (Vorasubin et al. 2016) in celloidin-embedded sections of the human cochlea. The protocol for IF in celloidin-embedded tissue sections described in this review manuscript allows for colocalization of several proteins in a regular fluorescent microscope or confocal microscope (Fig. 5a, b).</p><!><p>Decalcification and celloidin embedding of a temporal bone take 1 year. The sectioning of the specimens requires a sliding microtome that uses large-size blades that need to be sharpened frequently. For celloidin-embedded specimens, the orientation of cutting is crucial and histological and IHC staining requires special training. Celloidin, a sulfated and nitrated cellulose, is expensive and requires long infiltration times and is technically much more difficult to work with than paraffin wax or microdissection. Some histochemical staining that requires fresh-frozen fixed or unfixed tissue is not feasible because of dehydration and celloidin embedding. The use and storage of celloidin require experienced personnel, given that it is explosive when not properly handled and stored. Celloidin embedding is not ideal if there is a time limit for production of sections, or if there is not experienced personnel. It is noteworthy that we could not detect nidogen and aquaporins 1, 4, and 6 in celloidin-embedded specimens. However, we have successfully detected these proteins by IF using cryostat sections from microdissected auditory and vestibular endorgans obtained postmortem (Ishiyama et al. 2009; Lopez et al. 2007). These results suggest that prolonged decalcification, celloidin embedding and removal may affect the conformation of the antigen.</p><p>Celloidin needs to be removed completely to allow antigen exposure and antibody binding when using either indirect IHC methods (HRP + DAB) or IF. Prolonged decalcification and the type of decalcifying agent may cause loss of antigens. Table 1 lists several antigens identified in celloidin-embedded sections (this manuscript and previous reports by other researchers). Lack of detection by IHC of a given antigen in celloidin-embedded sections should be confirmed using frozen or paraffin sections.</p><!><p>Human inner ear tissue (cochlea and vestibular endorgans) obtained from ablative surgery has been useful to investigate the expression of several proteins by IHC, mRNA (Ishiyama et al. 2010; Calzada et al. 2012b; Liu et al. 2015, 2016), and ultrastructural organization of the auditory and vestibular sensory epithelium (Ishiyama et al. 2007; McCall et al. 2009; Liu et al. 2015; Taylor et al. 2015). Studies of Cx distribution in the human cochlea: IF and confocal microscopy analysis of Cx proteins in the human cochlea have shown that Cx26 and Cx30 proteins are co-expressed (Liu et al. 2009). Using super-resolution structured illumination fluorescent microscopy (SR-SIM) in fresh human cochlea obtained from surgery, Liu et al. (2016) found that Cx26 and Cx30 are expressed in the lateral wall in separate plaques. As described by Liu et al. (2016), SR-SIM provides structural information below the diffraction limit by superimposing various grid orientations of excitation light on the specimen to generate raw fluorescence images that are reconstructed into high-resolution derivatives. The volume resolution of three-dimensional SIM is approximately eightfold higher than that of a conventional microscopy (Liu et al. 2016; Schermelleh et al. 2008). Recently, scanning thin-sheet laser imaging microscopy (STSLIM) was used in decellularized human inner ear to investigate temporal bone histopathology (Shane et al. 2014) and the organization of the ECM in the cochlea (Santi et al. 2016). We summarize results from these studies below.</p><!><p>The application of recent imaging methodologies to the study of the anatomy of human temporal bones has revealed the organization of the ECM and the presence of novel structures. Santi and Johnson (2013) reported on the ECM of the cochlea and vestibular system after decellularization in mouse rat and human at the light microscopic level. To visualize the ECM of the cochlea, sodium dodecyl sulfate (SDS) was used to lyse and remove all cells in fresh, unfixed cochleas. Then, the cochleas were immersed in SDS for 1 week at 4 °C and one more week room temperature. Using a microtome/microscope called STSLIM, Santi and Johnson (2013) examined complete serial sections (approximately 300) of the cochlea. This methodology revealed a new structure on the apical surface of the basilar membrane. Liu et al. (2015) showed that this structure was immunoreactive for laminin and type IV collagen supporting the role of this structure as a component of the ECM. Recently, Santi et al. (2016) examined the anatomy of the mouse and human cochlea ECM following decellularization using scanning electron microscopy.</p><!><p>Studies on the identification of several proteins in the human inner ear by IHC and molecular biological techniques are continually expanding. Aggrecan (extracellular component of cartilage), S100 and connective tissue growth factor were detected in the human temporal bones from individuals diagnosed with DFNA9 (McCall et al. 2011). Changes in neurofilaments and myelin protein zero antibodies were detected in the cochlea of a patient with hearing loss caused by the p.L114P COCH mutation (Burgess et al. 2016). Recent studies that apply IHC in human inner ear celloidin-embedded sections have shown the presence of specific markers for macrophages/microglia: CD163+, Iba1+ and CD68+ (O'Malley et al. 2015). Nadol et al. (2014) identified B (CD20) and T cells (CD3) as well as macrophages (CD68) in temporal bones from individuals that received cochlea implants. Jung et al. (2016) identified CD45 in celloidin sections of an individual diagnosed with recent onset of Cogan's syndrome. Cochlin-IR was detected in the middle ear of normal and DFNA-9 affected human middle ear (Robertson et al. 2014).</p><!><p>For all methods described, a longer postmortem time of collection is associated with poor morphology. IHC and confocal studies with triple localization can be made in the three types of tissue sections. Formalin-fixed cryostat sections obtained from microdissected endorgans can be used for IHC and non-radioactive in situ hybridization. Microdissected auditory and vestibular endorgans can be used for morphological, quantitative stereology, IHC and IF studies.</p><p>Paraffin-embedded tissue sections can be used for IHC, but there is some loss of antigenicity due to heat and xylene used to embed and remove the paraffin. Some of the structures such as Reissner's membrane may be lost in processing. Formalin-fixed celloidin-embedded human temporal bone sections have been collected and archived for decades and represent a superb source of specimens to study inner ear diseases. Celloidin-embedded tissue sections can be treated with antigen retrieval protocols to be successfully used for IHC. By quenching of auto-fluorescence of celloidin-embedded sections, we have successfully developed a protocol for colocalization using double fluorescence staining ("Appendix" section). The methods of temporal bone processing are not exclusive; for bilateral disease processes, one temporal bone can be processed in the traditional manner (formalin-fixed celloidin-embedded) and the other can be processed using the microdissection technique. Celloidin-embedded specimens are the preferred methodology to identify new disease entities without previous histopathology (e.g., new hereditary hearing loss syndromes). Frozen or paraffin sections can be used as a pre-screening test for IHC, before celloidin tissue sections are used for this purpose.</p><p>In the last decade, an improvement in methodologies specifically designed to prepare human and animal inner ear tissue has helped to show the similarities and differences in the expression of several key proteins expressed in the inner ear. There are still ways to improve temporal bone processing for cellular and molecular biological investigations. For example, there is the need to test contemporary antigen retrieval methods (Shi et al. 2011) using buffers with different pH and periods of microwave heating (Gu et al. 2016). Post-processing methods like CLARITY (Poguzhelskaya et al. 2014) to visualize inner ear tissue in situ. A combination of fast temporal bone harvesting, processing and the use of higher quality of molecular biological reagents (antibodies and mRNA probes) will further contribute to the understanding of the human inner ear cellular composition, and how the inner ear of animal models can help to the design of new therapies for the treatment of disabling inner ear disorders.</p>

PubMed Author Manuscript

Modular Microcarrier Technologies for Cell-based Bone Regeneration

A variety of materials-based approaches to accelerate the regeneration of damaged bone have been developed to meet the important clinical need for improved bone fillers. This comprehensive review covers the materials and technologies used in modular microcarrier-based methods for delivery of progenitor cells in orthopaedic repair applications. It provides an overview of the field and the rationale for using microcarriers combined with osteoprogenitor cells for bone regeneration in particular. The general concepts and methods used in microcarrier-based cell culture and delivery are described, and methods for fabricating and characterizing microcarriers designed for specific indications are presented. A comprehensive review of the current literature on the use of microcarriers in bone regeneration is provided, with emphasis on key developments in the field and their impact. The studies reviewed are organized according to the broad classes of materials that are used for fabricating microcarriers, including polysaccharides, proteins and peptides, ceramics, and synthetic polymers. In addition, composite microcarriers that incorporate multiple material types or that are mineralized biomimetically are included. In each case, the fabrication, processing, characterization, and resulting function of the microcarriers is described, with an emphasis on their ability to support osteogenic differentiation of progenitor cells in vitro, and their effectiveness in healing bone defects in vivo. In addition, a summary of the current state of the field is provided, as are future perspectives on how microcarrier technologies may be enhanced to create improved cell-based therapies for bone regeneration.

modular_microcarrier_technologies_for_cell-based_bone_regeneration

Microcarriers in orthopaedic regenerative medicine<!>Microcarriers as vehicles for cell culture and delivery<!>Microcarrier Fabrication, Processing, and Characterization<!>Polysaccharide-composite microcarriers<!>Protein- and peptide-composite microcarriers<!>Ceramic and ceramic-composite microcarriers<!>Synthetic Polymer-composite Microcarriers<!>Summary and future perspectives

<p>The need for new materials and methods to accelerate and improve bone healing outcomes has stimulated a broad field of research aimed at potentiating bone regeneration.1,2 Although many bone fractures heal adequately without intervention, there is an unacceptably large number of cases each year in which bone heals sub-optimally, or in some cases fails to heal, which can lead to serious morbidity and chronic non-union.3–5 In these cases, more advanced therapeutic approaches are needed, and there has been a strong emphasis on developing new, more potent bone fillers and engineered bone constructs that can regenerate bone even in challenging situations.3 The development of engineered bone has shown promise in creating bulk tissue constructs that can rapidly fill a defect and support surrounding tissue.2 However, these approaches are hampered by the need for invasive surgery, a lack of new bone ingrowth, and potential compliance mismatch with surrounding tissues.3,6</p><p>The drawbacks of monolithic, macroscale engineered constructs have motivated the search for alternative strategies to treating large bone defects. In particular, there have been increasing efforts to develop modular approaches, in which discrete biomaterial or engineered tissue units are combined to create bone fillers that can be handled as slurries or pastes.6,7,8,9 The concept behind such semisolid systems is that these fillers can have transient fluidic properties sufficient for conformably filling irregularly-shaped cavities, while also allowing immobilization in the defect and providing support of the surrounding tissues as regeneration occurs. These bone-replacement materials are generally designed to be osteogenic in nature, such that they engender biomimetic mineralization of calcium phosphate minerals or allow the subsistence of cells that are capable of fulfilling this role. In addition, such modular systems can be designed for minimally invasive delivery, which has the potential to reduce residual pain, adverse scarring, and the risk of infection.</p><p>The field of orthopaedic repair has progressed as new biological approaches to tissue regeneration have been developed and proven effective. Over the past two decades, the subfield of orthobiologics has evolved to encompass a wide range of biological and bioactive materials that promote healing of musculoskeletal tissues.10 In addition to autografts and allografts, therapies involving growth factor delivery,4,5,11 marrow-4,10 and plasma-concentrates,4,5,10 and transplantation of progenitor cells10 are being developed and tested in the clinic. These approaches require specialized biomaterials designs and chemistries to promote the desired bioactivity and resulting biological responses. In particular, the delivery of cells presents a challenge in ensuring the viability, maintenance, and stimulation of differentiated function that is required for an effective cell-based therapy. In response to these needs, a variety of cell-based approaches to orthobiologic bone repair have been developed, using a wide range of materials.13 In addition, a diverse set of cell types, sources, and formulations have been applied to potentiate the regeneration of bone.</p><p>The development of microcarriers for cell delivery combines the discrete modular approach to tissue engineering with the bioactive strategies of orthobiologics. The use of such engineered particulate materials is a promising strategy to create functional bone fillers. While there has been considerable effort to develop therapies based on macroscale particles with diameters in the millimeter range,13,14 there has been less emphasis on establishing microscale approaches. Engineered microcarriers have the advantage that they can be designed to be conducive to cell attachment, to promote cell differentiation, and to degrade at a controlled rate. Their shape and size can be tailored to accommodate a range of cell densities and to form injectable microcarrier populations for downstream injectability. The composition and properties of such a carrier material can also be tailored to facilitate delivery, distribution, and immobilization of the microcarrier phase. These features make microcarrier-based delivery systems attractive for bone regeneration applications.</p><!><p>Cellular therapies typically require large numbers of cells and the production of this biomass can be a barrier to creating an effective therapy. The high surface area to volume ratio afforded by the use of microcarrier-based cell culture, relative to standard two-dimensional culture in flasks or dishes, makes it attractive for generating large cell populations. A large surface area allows proliferation and expansion of cells adhered to the microcarrier surface. In some applications, biochemicals secreted by the adhered cells are harvested for therapeutic or other uses.1,16 In other applications, the cells themselves are harvested by enzymatically releasing them from the microcarrier surface.3 In addition, the use of suspension culture techniques, typically in stirred bioreactors, allows efficient delivery of nutrients and controlled diffusion gradients. Such culture systems have been used for over 50 years for a variety of cell types of commercial interest in the biotechnology industry.16 However, microcarriers have not been as extensively used in the field of tissue engineering, despite their advantages in sustaining high volume cell growth.</p><p>Mesenchymal stromal cells (MSC) are adult progenitor cells that have been used widely in regenerative medicine. They can be harvested from a variety of tissues, including bone marrow and fat, and therefore offer a relatively accessible cell source for tissue engineering applications.1,10 There is a broad literature on the isolation, expansion, characterization, and use of MSC for therapeutic purposes, though their exact mechanisms of action remain the subject of investigation. It is clear that these cells can secrete substances that potentiate regeneration through paracrine mechanisms,11,12 and it has also been shown that they can have immunomodulatory effects.17,18 The range of their differentiation capacity is still a subject of debate; however, it is generally accepted that MSC can act as progenitors for bone, cartilage, and adipose tissues.1,19 In particular, it is well established that MSC form mineralized tissues when exposed to the appropriate environment, both in vitro and in vivo.13,19 Therefore, MSC are a valuable cell source in bone regeneration applications, particularly in situations where native progenitor cells are depleted and/or cannot be mobilized to the site of injury. In these instances, transplantation of MSC offers a way to enhance bone tissue repair. However, there is a need for improved methods to culture, differentiate, and deliver MSC for therapeutic purposes.</p><p>The combination of MSC and microcarrier culture is a promising way to culture, differentiate, and deliver potent progenitor cells in large numbers. Careful selection and design of the materials used to fabricate the microcarrier substrate offers a mechanism for controlling cell proliferation and differentiation. Furthermore, the modular microcarrier format allows delivery of the cultured cells while still attached to a supportive and potentially bioactive substrate. The implanted cells and microcarriers can be designed to degrade at a controlled rate post-transplantation, thereby releasing the attached cells and allowing new tissue to replace the bone filling implant. Advances in biomaterials science have made it possible to control the properties of the microcarrier such that the desired biological functions are promoted over time. Therefore, microcarrier-mediated MSC delivery offers the possibility to tailor the spatial and temporal behavior of advanced bone graft materials.</p><p>The treatment of large and challenging bone defects using this strategy is particularly promising because these indications require a volumetric bone filler that has osteogenic properties. The microcarrier format allows large numbers of cells to be grown and differentiated under controlled conditions. It is likely that regeneration of larger defects will require the transplantation of billions and perhaps trillions of cells, and use of microcarriers in suspension culture and bioreactor systems facilitates scale-up and production of well-characterized batches for such therapeutic use. Furthermore, microcarrier-bound cells can be delivered directly to bone defects while leaving their cell-matrix contacts intact. The microcarrier matrix can therefore be designed to promote osteogenic differentiation, and this stimulus can be maintained both prior to and after cell transplantation. Importantly, the packed bed geometry of microcarrier populations facilitates diffusion and perfusion to supply nutrients to the cells post-transplantation, and therefore may perform better than other approaches in ischemic situations.</p><p>The following sections of this review provide a description of the materials and strategies that have been used to create microcarriers for cell-based regeneration of bone. It focuses on approaches designed for MSC and osteoblast culture and delivery, with an emphasis on the materials used to create microcarriers and their function in directing osteogenic processes. These materials are used alone and in combination to mimic key aspects of bone composition and function, while also providing a supportive and instructive substrate for living cells. In addition, this paper covers the fabrication and processing of microcarriers, and how they are cultured and delivered for bone regeneration applications. An impressive variety of approaches to modular microcarrier-enabled bone regeneration have been developed. However, these technologies have yet to have an impact in the clinic. This review is aimed at describing the current state of knowledge in this still growing field by summarizing the approach and main findings of the studies that have been done to date, and the impact that these findings may have on future studies and translation of microcarrier-based bone regeneration technologies to the clinic.</p><!><p>Many microcarrier types are produced by simple emulsification, which offers the advantages of bulk processing and relatively high throughput. In general, batch emulsification involves mixing two immiscible liquids to create a dispersion containing a separated colloidal phase within a bulk continuous phase. The mixing rate and properties of the colloidal and bulk phases can be varied to control the size and size distribution of the resulting particles, and emulsification has proven well-suited to creating polymer and polysaccharide particles on the nano- and micro-meter scale. Water-in-oil emulsion techniques are often used to suspend a polymer solution within a processing oil, creating a spherical discrete phase within the larger continuous, nonpolar phase. This discrete phase can be easily settled out of suspension and is often extracted with the aid of secondary interface-stabilizing surfactants.</p><p>Microfluidic flow-focusing droplet generation is a modified version of bulk emulsification aimed at tight control of particle size. In these microfluidic systems, an aqueous polymer solution and typically a nonpolar oil or other fluid are co-extruded in a specific geometrical arrangement to produce consistently-sized droplets.18 The size of these droplets can be altered through inputs of the junction geometry for the intersecting fluids and the flow rates of each fluid. However, these systems are limited in the viscosity of the polymer solutions that can be used, and microcarriers are necessarily created one at a time, which can prolong production times. One variation of the microfluidic flow-focusing technique uses a gas stream in the place of an oil phase. Such methods have been found particularly useful in the formation of ceramic particles.8,21–26 Once particles have been formed and crosslinked, they are washed, dried, and sintered at elevated temperature.</p><p>Coacervation-based production methods for microcarriers are typically similar in general set-up to batch emulsification techniques (Fig. 1). The term, coacervation, is often used in reference to complex coacervation processes involving the mixture of two distinct solutions of a cationic macromolecular species and an anionic macromolecular species. This generally induces conglomeration and precipitation of a concentrated colloidal phase. Simple coacervation, which is more common to the process of microcarrier fabrication, refers to the phase separation of a macromolecular solution from a more dilute continuous phase to produce a relatively concentrated particulate colloidal phase. This process is often triggered by a change in temperature or pH, and has been used to create microcarriers for cell culture. Coacervate particles, like water-in-oil emulsified particles, can be collected by centrifugation and subsequently processed to stabilize and modify their morphology.27</p><p>Post-fabrication processing of microcarriers often includes crosslinking of the matrix to impart stability, reduce swelling, and resist dissolution. A variety of chemical, thermal and photonic crosslinking methods can be applied to microcarriers. For osteogenic applications, covalent crosslinking using glutaraldehyde or other aldehydes is often used to chemically bind primary amine groups between macromolecules.9 Carbodiimide-based crosslinking of amines using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is also a commonly used stabilization method.28 In specific instances, other crosslinkers may be of use, for example the use of divalent cations to stabilize alginate-based materials.24 Similarly, formed and stabilized microcarriers can be characterized using a variety of standard techniques. The size and morphology of microcarriers can be measured using optical microscopy-based imaging software or laser diffractometry-based dimensional estimation equipment.2,3,29 The composition of microcarriers can be determined using X-ray diffraction or Fourier transform infrared analysis.2,6,30</p><p>Biomineralization is another common post-processing technique for microcarriers intended for bone regeneration applications.31–35 Calcium mineral can be incorporated directly into the matrix of microcarriers to better simulate the composition of native bone.2,3,29 In addition, incubation of microcarriers in high calcium concentration solution like SBF or Ringer's solution can be used to induce the deposition of a mineral phase.31–35 Control of the composition and thermodynamics of these techniques allows the tailoring of the mineral phase of microcarriers to promote desired functionality. Calcium phosphate mineral is often used because of its chemical similarity to carbonated apatite, a calcium-deficient form of hydroxyapatite that makes up as much as 65 wt% of adult bone.36 Tricalcium phosphate is often added to microcarrier matrices,37–39 and this mineral phase can be transformed into hydroxyapatite through preferential dissolution of less stable calcium phosphate minerals and their recrystallization into more stable forms, in a process governed by the Ostwald-Lussac rule of stages.40 Calcium carbonate-based minerals can be used in a similar manner, and under controlled conditions can be transformed into calcium phosphate mineral phases that resemble those in biological hard tissue.32,41 Furthermore, the inclusion of specific protein and polysaccharide materials in combination with such mineral phases is often applied to mimicking the physiological environment of bone.2,3,6,29,42</p><!><p>Polysaccharide materials are generally abundant and well characterized, and they have been used extensively to produce microcarriers. Commercially available products such as the cellulose-based Cytopore16 and dextran-based Cytodex lines43 are polysaccharide microcarriers with varying size, composition, and porosity. Although Cytopore beads are generally more suited to the culture of Chinese hamster ovary (CHO) epithelial cells, baby hamster kidney fibroblasts (BHK-21), HeLa cervix cancer epithelial cells, and Vero kidney epithelial cells, the collagen-coated Cytodex line has been used in a number of bone progenitor cell studies.44 Cytodex beads made in the 150–250 μm range have been used in several studies to expand and osteogenically stimulate osteoblasts and osteoblast precursor cells for greater alkaline phosphatase (ALP) activity and mineral deposition.45–48</p><p>Chitosan is a common polysaccharide biomaterial that has been tested as a base constituent for the construction of osteogenic microcarriers (Fig. 2). Porous microspheres of chitosan doped with up to 30% hydroxyapatite have been successfully prepared and characterized.50 A relationship was established between the percentage of added hydroxyapatite and the microcarrier size. Pure chitosan microcarriers had diameters of 220–250 μm, which increased with hydroxyapatite addition to a diameter of 400–530 μm at 30% hydroxyapatite. This positive trend in size was accompanied by a negative trend in water sorption, which dropped from as much as 400% in pure chitosan to below 100% with mineral addition. Chitosan microcarriers have also been created using either coacervate precipitation or emulsion crosslinking, followed by immersion in simulated body fluid to create a coating of carbonated hydroxyapatite.27 The biological effects of these chitosan-based modules (350–710 μm diameter) were evaluated using MC3T3-E1 pre-osteoblast cells. Cell proliferation was found to be markedly higher on the coacervate-precipitated carriers, relative to emulsion-crosslinked carriers, potentially due to the rougher surface topography of the former. Mineral coating further enhanced proliferation. Interestingly, mineral coating increased ALP activity of emulsion-crosslinked carriers, but did not have the same effect on coacervate-precipitated carriers, though the latter generally exhibited higher ALP activity. Collagen deposition increased after mineral coating in both microcarrier types, though uncoated coacervate-precipitated microcarriers showed more deposition than emulsion-crosslinked counterparts. These studies suggest that both surface chemistry and roughness, as well as the presence of a mineral phase can be used to enhance the osteogenic capacity of microcarriers.</p><p>Pullulan is a natural polysaccharide polymer that has been used widely in the food and pharmaceutical industries and is being investigated for medical applications.51 This material has been used to create porous microcarriers (150 μm average diameter), which were then seeded with SaOS-2 osteosarcoma cells and cultured in either static or dynamic suspension culture conditions.52 Pure pullulan microcarriers were compared to those with a mineral surface coating or coated with silk fibroin, as well to silica glass control microcarriers. Cell viability after seeding was generally below 50% for the pullulan-based carriers, and was only slightly higher on glass controls. Pure and silk-fibroin-coated microcarriers showed upregulation of ALP activity in both static and dynamic culture, while mineralized carriers showed the lowest activity, possibly due to the inhibitory effect that increased calcium ions may have on the ALP enzyme. Dynamic culture did not produce marked changes in ALP activity in pullulan-based carriers. However, there was a strong increase in ALP activity on glass microcarriers in dynamic culture, relative to static controls. This study suggested that pullulan-based carriers can support osteogenic differentiation, though response to dynamic culture was highest on very stiff silica substrates.</p><!><p>Microcarriers created for use in osteogenic applications are very often composed of a collagen-derived base material (Fig. 3) supplemented with calcium-based compounds to further enhance functionality. These two components roughly mimic the composition of the native bone extracellular matrix, which is essentially a mineralized matrix of collagen.36 Gelatin is a mixture of peptides derived from the hydrolysis of collagen, and is therefore often used as a microcarrier base material. Collagen and collagen peptides have the advantage that cells can recognize, bind to, and degrade these materials, giving them enhanced biological functionality relative to most synthetic materials. These proteins and similar protein-based materials can be extracted from animal tissues using a variety of techniques, which impart specific physical and biological properties on the resulting matrix. Similarly, there are a variety of forms of calcium phosphate compounds that have been used to augment protein-based microcarriers. The selection of base material and filler can be leveraged to control cell function, including for osteoconductive, osteoinductive, and osteogenic purposes.</p><p>Gelatin-based microcarriers are available commercially. Cultispher-S microcarriers are composed of highly crosslinked porcine gelatin,9,19 and are designed with high porosity for cell loading. It was found that rat MSC exhibited markedly higher levels of the osteogenic markers osteocalcin and alkaline phosphatase when cultured on Cultispher-S microcarriers, compared to conventional tissue culture polystyrene.53 When tested in vivo, it was shown that MSC cultured on Cultispher-S carriers resulted in improved trabecular bone formation relative to cell-free microcarriers in long bone defects,53 and similar effects were observed upon application to periodontal defects.54 Earlier studies also investigated microcarriers composed principally of collagen Type I (Cellagen™) for the expansion of human osteoblasts. It was demonstrated that osteoblasts cultured on collagen microcarriers in spinner flask culture displayed a higher proliferative capacity over a 15-day period, when compared to cells cultured on tissue culture polystyrene. Spinner culture on collagen microsphere substrates was also shown to dramatically upregulate osteocalcin levels relative to monolayer cultures.43</p><p>In experimental systems, gelatin-based microcarriers have been augmented with various forms of calcium phosphate mineral. Gelatin mixed with tricalcium phosphate (TCP) was used to create microcarriers by emulsification in olive oil,28 followed by crosslinking using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) chemistry. Subsequently, the microcarriers were incubated in Ringer's solution for seven days to convert TCP to calcium-deficient hydroxyapatite via hydrolysis, as in a preliminary exploratory study.37 The resulting microcarriers (100–300 μm diameter) were seeded with SaOS-2 osteosarcoma cells and were used in static and dynamic suspension culture studies, with Cytodex-3 microcarriers serving as controls. In static conditions, cells attached to the gelatin-HA microcarriers but no statistical increase in cell number was observed over three days of culture, while cell number on Cytodex-3 microcarriers increased approximately 4-fold over the same time period. In contrast, under dynamic culture conditions, cell proliferation was clearly enhanced on gelatin-HA microcarriers, compared to Cytodex controls. This study illustrates that interactions between the cell substrate and the culture conditions that can lead to differences in cell behavior, and in particular highlights the benefit of using gelatin-based microcarriers in dynamic suspension culture.</p><p>Other studies have examined the mode of incorporation of calcium phosphate minerals into gelatin-based materials. Microcarriers formed of apatite and gelatin were fabricated through a sol-gel process by mixing gelatin with calcium hydroxide and subsequent reaction with phosphoric acid.2 The resulting apatite-gelatin microspheres (112 μm average diameter) were crosslinked using EDC chemistry. The sol-based emulsification method was found to impart a finer, nanoscale distribution of the calcium orthophosphate apatite crystal phase within the continuous gelatin phase, when compared to microcarriers made by direct mixing of pre-formed apatite powder into gelatin solution. Culture of MG-63 human osteosarcoma cells on the sol-formed microcarriers showed that they supported cell proliferation and osteogenic function. In a related study, apatite mineral was formed inside glutaraldehyde-crosslinked gelatin nanoparticles (230–250 nm diameter) created using a "miniemulsion" technique.6 This method of biomimetic mineralization promoted the constrained formation of a single crystalline hydroxyapatite phase throughout the gelatin spheres. These gelatin-CaP particles were too small to support the culture of mammalian cells, though the same technique could potentially be used for larger particles. These studies are important because they demonstrate control of the micro- and nano-scale structure of mineral in gelatin materials, which can be tailored to mimic native bone mineral.</p><p>Gelatin-based microcarriers have also been used in other formats aimed at regenerating bone. Addition of gelatin microspheres (20–100 μm diameter range) to MSC cultures in attachment-inhibiting cultureware demonstrated that aggregates of cells and microspheres formed over time.42 The presence of microspheres in the aggregates resulted in higher cell viability and production of alkaline phosphatase, a marker of osteogenic differentiation. It was proposed that the inclusion of gelatin particles increased the porosity of the aggregates, and therefore enhanced nutrient and oxygen delivery. In a separate study, direct precipitation of a mixed solution of gelatin and calcium phosphate was used to fabricate small (<10 μm diameter), spherical, and highly porous particles that were conducive to attachment, viability, and proliferation of human osteoblast-like G-292 cells.14 Implantation of composites of these microspheres and cells in a critical size rat calvarial defect model resulted in strongly improved bone regeneration at four and eight weeks, relative to clinically-used fibrin glue and a commercially available bone filler material (Osteoset® calcium sulfate mini-beads). In another study, gelatin microspheres (50–100 μm diameter) were incorporated into injectable co-N-isopropylacrylamide macromer materials designed for in-situ gelling.55 The gelatin phase acted as a substrate for cell attachment and as an enzymatically-degradable porogen. Testing in a rat critical size cranial defect model revealed a greater level of mineralization and bony bridging in microsphere-containing samples, relative to controls. These studies highlight the potential improvement in bone regeneration achieved when combining osteogenic cells and a rationally designed carrier material, relative to current clinical options.</p><p>Other forms of collagen peptides and preparations have also been combined with calcium mineral to form microcarriers. Hydroxyapatite particles dispersed in reconstituted fibrous collagen Type I were prepared by emulsification in olive oil and crosslinked with glutaraldehyde.3 Increased stirring speed during emulsification resulted in a decrease in average microcarrier diameter, which ranged from 220–1040 μm. A further decrease in microcarrier diameter could be achieved by the addition of a surfactant during emulsification, down to a range of 75–300 μm average diameter. Primary rat osteoblast cells proliferated and increased their production of alkaline phosphatase when cultured on these microcarriers. In a follow-on study, the ultrastructure of the matrix in similar microcarriers was examined.29 The hydroxyapatite was distributed evenly in the matrix and did not hinder collagen fibril formation. Primary rat osteoblasts cultured on these microcarriers under osteogenic conditions proliferated and maintained metabolic activity, and also mineralized the underlying collagen-HA matrix. In a third study, similar microcarriers were made to have bone-mimetic composition of 35:65 wt% collagen:hydroxyapatite at an average diameter of 500 μm.56 The collagen-HA microcarriers were compressed into discs and were press-fit into a critical size cranial defect in the rat, without the addition of exogenous cells. After 16 weeks if implantation, the collagen-HA microcarriers were completely resorbed and showed good bone ingrowth, whereas pure hydroxyapatite control implants were not completely resorbed.</p><p>Collagen microcarriers incorporating tricalcium phosphate (TCP) and calcium phosphate cements are of interest because of their similarity to bone matrix and ability to support osteoblast function.39 Microcarriers made from CPC with a range of particle sizes became more spherical and cell-adhesive when collagen was added to the matrix.38 It was shown that nanoscale calcium phosphate cement particles (2.4 nm average diameter) had the effect of increasing ALP activity when using Saos-2 osteosarcoma cells, whereas cell proliferation decreased on microcarriers relative to on silica glass controls. In a follow-on study, similarly-formulated composite collagen and calcium-deficient hydroxyapatite microcarriers were implanted in femoral defects in a rabbit model.57 Compared to injectable calcium phosphate cement, both with and without the addition of collagen, the microcarriers produced a ten-fold increase in bone ingrowth at 3 months. These results again emphasize that the geometry and composition of microcarriers can be used to tailor their function in bone regeneration applications.</p><p>Sol-gel preparation can also be used to create apatite-loaded collagen microcarriers, in a process analogous to that developed for gelatin.2 Pure collagen and collagen-apatite microcarriers (166 μm average diameter) were made using peptides derived from collagen sponge material using emulsification and subsequent crosslinking with EDC-NHS chemistry.58 Apatite was then precipitated within the collagen microcarrier matrix as homogeneously-dispersed nanocrystals, to a loading of approximately 30 wt%. Pure collagen microcarriers were somewhat smaller (120 μm average diameter), again reflecting a relationship between mineral loading and particle size. Rat bone marrow-derived MSC cultured on these collagen-apatite microcarriers proliferated more rapidly compared to those on pure collagen microcarriers. In addition, MSC grown on collagen-apatite microcarriers expressed higher levels of the osteogenic marker ALP, relative to MSC cultured on pure collagen microcarriers or on standard tissue culture plates. These findings reinforce the use of a defined mineral phase in enhancing the function of peptide- and protein-based microcarriers.</p><p>Protein-based microcarriers designed to fully envelop progenitor cells for delivery in bone regeneration applications have also been developed. Composites of collagen Type I and the polysaccharide agarose were used to prepare microbeads (30–150 μm diameter range) by emulsification, with human MSC embedded directly within the microbead matrix.59 3D microcarrier culture resulted in increased bone sialoprotein production and calcium deposition, relative to 2D controls. Similar microbeads (90–290 μm diameter range) were also fabricated using a collagen-chitosan matrix with embedded human MSC (Fig. 4).60 High cell viability was supported in these microcarriers and osteogenic capacity was demonstrated. Populations of microbeads could be concentrated into a paste for delivery through a standard 25G needle without loss of cell viability. Implantation of collagen-chitosan microbeads containing bone marrow mononuclear cells supplemented with purified MSC in an ectopic site in the rat resulted in markedly enhanced bone formation, relative to controls.61 An extension of this work involved augmenting the collagen-chitosan matrix with exogenous hydroxyapatite, and examining bone regeneration in an orthotopic, critical size cranial defect model in the mouse.62 This study showed the value of pre-differentiating MSC prior to implantation, and showed that such microbeads could be used to conformally fill and heal a critical size defect.</p><p>Silk fibroin has been investigated in a variety of regenerative medicine applications,18,20,52 including for the production of osteogenic microcarriers. Gelatin-fibroin microcarriers were prepared at a 30:70 wt% gelatin:fibroin composition, both with and without mineralization using calcium phosphate.18 MSC attached and spread on these microcarriers and proliferated more robustly on gelatin-fibroin substrates than on Cytodex-3 controls, but it was observed that mineralization greatly decreased cell proliferation. In contrast, ALP activity was strongly upregulated on gelatin-fibroin microcarriers that had been mineralized with calcium phosphate. The effect of composition on the function of gelatin-fibroin was further examined using microcarriers (300–400 μm diameter range).20 The seeding efficiency and subsequent proliferation of rat MSC seeded on these microcarriers decreased with increasing silk fibroin content. However, a 75:25 wt% fibroin:gelatin composition was found to be the most osteogenic in terms of osteopontin expression, relative to control Cultispher-S microcarriers. These studies suggest that mineralization of non-collagenous matrices is also beneficial in achieving osteogenic differentiation of progenitor cells.</p><!><p>Bioactive glasses are a class of calcium sodium phosphosilicate ceramics that have been used widely in bone healing applications.5,11,63 Their use as microcarriers typically involves pre-coating or otherwise incorporating a calcium phosphate mineral phase. Optimization of the microcarrier coating procedure has shown that extended sequential exposure to Tris-HCL followed by tissue culture medium results in a more functional coating, with a transition over time from amorphous calcium phosphate to carbonated hydroxyapatite, and a resulting positive effect on the osteogenic differentiation of MSC.64 It has also been suggested that carbonated hydroxyapatite coatings preferentially adsorb fibronectin and other cell-binding proteins, which may enhance biological function.7,65 Microspheres (100–150 μm diameter) created using bioactive glass have been studied for orthopaedic use as solid microparticles, hollow microspheres, and as part of polymer composite microspheres that were coated with mineral through submersion in simulated body fluid.66–67 Cell culture studies showed that bone marrow stromal cells attached to the microcarriers and produced extracellular matrix that could be mineralized. This process also produced larger cell-microcarrier aggregates. Similarly mineralized bioactive glass microshells were shown to support osteogenic differentiation of rat MSC, as indicated by alkaline phosphatase, collagen type I, and osteopontin expression.49 Mineral deposition by rat MSC was also demonstrated on such microspheres using scanning electron microscopy.13,69 Composite microspheres of bioactive glass and poly(lactic-co-caprolactone) incubated in simulated body fluid (SBF) for one week exhibited complete CaP mineral coverage along with inducing significantly higher ALP activity than purely poly(lactic-co-caprolactone) microspheres on day 14 and 21 of culture in osteogenic media.35 Other work on phosphate-based bioactive glass microspheres has suggested that the release of ions is important to their stimulation of bone regeneration, as well as the need to allow for appropriate degradation rates to support collagen deposition and matrix remodelling.11,63,70</p><p>Calcium titanium phosphate (CTP) microcarriers are a material variation on bioactive glasses.8,21–23 Particles of CTP (600 μm average diameter) were compared to polystyrene microcarriers (200 μm average diameter) in terms of the support and differentiation of rat MSC.8 Attachment efficiency of MSC to CTP microspheres was lower than that on corresponding polystyrene microcarriers, though the cells proliferated over two weeks in culture on both substrates. However, ALP and osteocalcin secretion was shown to be higher on CTP microcarriers, relative to polystyrene controls. Another formulation of composite titanium phosphate glass microcarriers (50–100 μm diameter range) has also been compared to similarly-sized silica glass microspheres.71–72 These microcarriers supported the proliferation of MG-63 osteosarcoma cells in static and spinner flask culture over one week. In a similar study using human MSC, it was observed that CTP microcarriers potentiated BMP expression, relative to silica glass, and in particular that osteopontin was highly upregulated over time on CTP microcarriers. These studies suggest that the chemical structure of calcium titanium phosphate microcarriers is conducive to the osteogenic differentiation of bone progenitor cells.</p><p>Hydroxyapatite is a ceramic of particular interest in orthopaedic regeneration applications because it closely mimics the mineral phase of native bone.2,11,36 This mineral has been incorporated as an additive in microcarriers based on a variety of materials, and has also been used as the primary constituent in microparticles designed for cell delivery. Hydroxyapatite microspheres (400–550 μm diameter range) were shown to support the adhesion, proliferation, and osteoblastic lineage of human MG-63 osteosarcoma cells (Fig. 5).22,26 The effect of the density and porosity of hydroxyapatite particles was examined using microcarriers (200–700 μm diameter range) designed for the delivery of goat MSC.73 Larger, microporous, MSC-seeded carriers resulted in no visible bone formation when implanted subcutaneously in athymic mice, and produced only a vascularized fibrous tissue. Smaller, dense microcarriers exhibited higher cell attachment efficiency, and resulted in trablecular-like bone formation in a subcutaneous site. The effect of surface topography on cell seeding was examined using spherical, hollow hydroxyapatite microcarriers (360 μm average diameter) seeded with murine osteoblasts.74 These microcarriers supported cell attachment and proliferation both on the outer concave and inner convex surfaces. These findings emphasize the importance of microcarrier morphology and topography on the response of seeded progenitor cells.</p><p>Tricalcium phosphate (TCP) is a ceramic with similar composition to hydroxyapatite, but which typically has a lower Ca:P ratio than native hydroxyapatite. TCP can be converted to hydroxyapatite by sintering and chemical processing,28,31,37–39,57 and the latter is generally found to be more osteoconductive.75 Microcarriers (100–250 μm diameter range) have been formed out of composites of HA and TCP and were seeded with bone marrow-derived MSC.76 Implantation of MSC-seeded HA-TCP microcarriers into both calvarial and mandibular defects in the mouse resulted in significant new bone formation by six weeks. In contrast, unseeded HA-TCP microparticles implanted into similar defect sites resulted in poor bone formation even at very long time points. Perforated HA-TCP microcarriers (370 μm average diameter) were also prepared and used as a culture substrate for human adipose-derived MSC.77–78 Calcium deposition increased on these microcarriers over time in response to osteogenic stimulation in the culture medium, although the response was not as strong as from cells grown on tissue culture plastic. Implantation of unseeded versions of these HA-TCP microcarriers in rabbit calvarial defects resulted in the production of mature bone within the microcarrier cavities and lamellar osteons over six weeks. These observations suggest that in these composite microcarriers the HA component nucleates mineralization, while the degradation of the TCP phase provides space for new tissue formation.</p><p>Ceramic materials have also been augmented with other components to enhance the bone regeneration capability of microcarriers. Strontium-doped hydroxyapatite microspheres (520 μm average diameter) were combined with an injectable, in-situ crosslinkable RGD-alginate carrier gel to treat critical size femoral defects in the rat.24 Micro-computed tomography and histological analysis indicated more robust bone formation in strontium-augmented implants, relative to strontium-free controls. Microcarriers augmented with strontium also degraded more quickly, leaving less residual material in the implant site. The result was increased collagen deposition and new bone formation in the interstitial space between microcarriers. Composite TCP-alginate microcarriers (100–500 μm diameter range) were fabricated and increasing TCP content was correlated with an increase in diameter.31 These carriers were subsequently incubated in SBF and their pore size could be modulated through control of the freezing temperature during lyophilization, with increasing freezing temperature resulting in larger pores. Using MC3T3-E1 pre-osteoblast cells, it was shown that cell proliferation and ALP activity under osteogenic conditions were similar on TCP-alginate microcarriers, relative to tissue culture plastic controls, suggesting that such microcarriers support the osteoblastic differentiation of progenitor cells.</p><!><p>Polystyrene is widely used in tissue cultureware because it can be plasma treated to promote cell attachment.79 This polymer can also be used to fabricate spherical microparticles, which have found broad utility in the biotechnology field as microcarriers.80–81 Uncoated and collagen-coated SoloHill® polystyrene microcarriers (125–210 μm diameter range) were seeded with human MSC derived from bone marrow, placenta, or embryonic stem cells.17 Similarly, untreated or collagen-coated polystyrene tissue culture plates served as controls. It was observed that MSC cultured on collagen-coated microcarriers exhibited markedly higher ALP activity, collagen secretion, and calcium deposition compared to culture plate controls, even in the absence of osteogenic stimulation by growth factors. Both disruption of cytoskeletal actin using latrunculin B and inhibition of actomyosin contraction using blebbistatin reduced the osteogenic response, suggesting that in this system the microcarriers induced osteogenesis in MSC through enhancing cytoskeletal tension.</p><p>Polylactic acid (PLA) has been used widely for regenerative medicine applications, including for orthopaedic repair (Fig. 6).32–34 Microspheres of PLA used for bone regeneration generally incorporate a ceramic phase to enhance osteogenic properties. The addition of calcium phosphate mineral can also mediate the production of acidic degradation products of PLA, and can act as a template for further biomineralization. Hollow spheres (500–1000 μm diameter range) were fabricated from a composite of PLA and calcium carbonate (CaCO3) via emulsion.32 Subsequent immersion in SBF for one week resulted in production of a carbonated hydroxyapatite layer, as demonstrated by x-ray diffraction analysis. Similar porous PLA microspheres (110–250 μm diameter) were produced and treated in sodium hydroxide solution to hydrolyze surface functional groups.33–34 Subsequent mineralization studies in SBF showed that pre-hydrolized microcarriers mineralized to a greater degree than untreated controls, and that the degree of mineralization increase with time after day 5. Seeding of human osteoblast-like MG-63 cells on mineralized microcarriers showed high cell viability (>80%) over five days of culture.</p><p>Composite poly(lactide-co-glycolide) (PLGA) microcarriers made with 50 wt% hydroxyapatite exhibited increased attachment efficiency of mouse OCT-1 osteoblast-like cells with increasing sodium hydroxide treatment.82 Cells proliferated over time in culture and alkaline-treated microcarriers were shown to be more osteogenic than untreated controls. PLGA microcarriers have also been used in dynamic culture systems for SaOS-2 human osteosarcoma cells.83 While rotating dynamic culture decreased the proliferation rate, it also increased ALP activity in this cell line. Surface-mineralization of PLGA microcarriers also increased the attachment efficiency of rat osteoblast cells, relative to untreated controls.84 Similarly, surface mineralization was shown to increase bone regeneration by these cell-seeded PLGA microcarriers when implanted into subcutaneous sites in athymic mice for 6 weeks. PLGA-based microcarriers have additionally been shown to generate nearly complete bone restoration in rat critical-size calvarial defects after 12 weeks of implantation.41</p><!><p>A wide variety of microcarriers have been developed with the target of augmenting bone regeneration. The studies reviewed in this paper are characterized by the rational combination of materials and chemistries to achieve desired physical and biological functions. Polysaccharides, proteins, peptides, ceramics, and polymers all have applied in this way. Composites of these materials are often used in an effort to harness and combine the desired properties of the individual components to create a more functional matrix. In some cases, these materials directly mimic the composition of biological bone tissue. In other cases, they are designed to promote cell attachment and function for the purpose of potentiating the biological response. Direct addition of calcium phosphate compounds or promotion of a biomineralization is a key strategy in creating many of these microcarrier types, since this mineral phase is a major component of bone tissue and has been demonstrated to have osteogenic effects on progenitor cells. Overall, there has been a remarkable diversity in the approaches taken to creating microcarriers for orthopaedic applications, and to designing their composition for enhanced biological function.</p><p>The use of cell-based approaches to orthopaedic tissue regeneration is more complex than purely materials-based approaches. However, only cells can produce new bone and therefore if appropriate cell types are not available at the site of injury, they must either be recruited endogenously or delivered exogenously. In large and ischemic bone defects, recruitment of progenitor cells is impaired, and cell delivery has the potential to greatly augment the healing process. Microcarrier-based strategies to cell therapy have the advantage that cells are delivered on a substrate that can be designed to direct their differentiation and function. In addition, the microcarrier material acts as a space-filling extracellular matrix that can have biological and mechanical function. Importantly, populations of microcarriers can often be delivered as a moldable paste or putty, and therefore can conformally fill defects. In most cases, these microcarrier-based approaches do not provide load-bearing mechanical stability. However, cell-seeded biomaterial microcarriers offer new and potentially improved options to fill bone defects that may be superior to current approaches in healing challenging cases.</p><p>The literature reviewed in this paper emphasizes the materials used to create microcarriers, and how the properties of the materials affect cell function. Materials chemistry is an important component of cell-matrix interactions, since cells bind directly to the microcarrier substrate and receive both physical and biological signals via that binding. Similarly, the topography of the substrate can be an important determinant of cell function, particularly in the case of progenitor cells such as MSC. Surface roughness, geometry, and porosity all have been demonstrated to affect cell differentiation, and these features can be designed into microcarriers in a variety of ways. Similarly, mechanical stiffness of materials is known to be a strong determinant of progenitor cell fate, particularly in orthopaedic applications. In addition, microcarrier materials can be designed to be more or less resistant to degradation in the physiological environment, which enables control over the dynamics of resorption and replacement of microcarriers by new biological tissue. Taken together, there are a wide range of options of materials design parameters that can be employed to make microcarriers optimally functional and effective in bone regeneration applications.</p><p>While remarkable progress has been made in the design, fabrication, characterization, and application of microcarriers for orthopaedic applications, there are exciting opportunities for further improvement of these technologies. New materials and composites are being developed that may facilitate tailoring of the cell-instructive properties of microcarriers. In particular, a better understanding and tighter control over material mechanical properties may allow more targeted cell differentiation. Materials can also be designed to change their properties dynamically over time or upon application of a specific stimulus, so that integration into the host is enhanced. Immobilization and release of bioactive factors is an area that has also been extensively studied in regenerative medicine, but has not been applied widely for microcarrier-based applications. Growth factors, function-specific ligands, gene delivery vectors, and a variety of other stimuli can be incorporated into and presented by the microcarrier matrix. Genetic modification of cells to be better suited for microcarrier delivery, and to be more effective upon delivery, is another exciting avenue that has not been extensively pursued in the field of microcarrier technology. Finally, there is a growing recognition that control of the biological response, and in particular the inflammatory and immunological response, is a key to achieving functional tissue regeneration. There remains great potential in using rationally-designed microcarriers for the delivery and directed differentiation of progenitor cells, with the goal of rapidly and effectively regenerating bone tissue.</p>

PubMed Author Manuscript

Genetic Code Expansion in Zebrafish Embryos and Its Application to\nOptical Control of Cell Signaling

Site-specific incorporation of unnatural amino acids into proteins provides a powerful tool to study protein function. Here we report genetic code expansion in zebrafish embryos and its application to the optogenetic control of cell signaling. We genetically encoded four unnatural amino acids with a diverse set of functional groups, which included a photocaged lysine that was applied to the light-activation of luciferase and kinase activity. This approach enables versatile manipulation of protein function in live zebrafish embryos, a transparent and commonly used model organism to study embryonic development.

genetic_code_expansion_in_zebrafish_embryos_and_its_application_to\noptical_control_of_cell_signalin

<p>While early examples of the incorporation of unnatural amino acids into proteins and their application in cell-free and cell-based environments involved the synthesis of chemically misacylated tRNAs,1 recent genetic code expansion approaches through the addition of engineered, orthogonal tRNA/tRNA synthetase pairs to the endogenous protein-biosynthetic machinery provide powerful tools for the study and manipulation of protein function in biological systems.2 For example, photocaged amino acids enable precise regulation of protein function with light,3 amino acids bearing bio-orthogonal chemical handles allow for selective protein labeling and imaging in living cells,4 and amino acids containing biophysical probes facilitate studies of protein structure and function.2b In order to expand the genetic code, an orthogonal aminoacyl tRNA synthetase/tRNACUA pair is added to the biosynthetic machinery of cells to incorporate an unnatural amino acid at a desired position of a protein in response to an amber stop codon.2 The PylRS system from Methanosarcina species (M. barkeri or M. mazei) is a versatile tool for genetic code expansion.5 It requires a pyrrolysyl tRNA synthetase (PylRS) and its cognate tRNAPylCUA (PylT), and it shows excellent orthogonality to the endogenous tRNA/tRNA synthetase pairs in bacterial, yeast, and mammalian cells. It is a natural amber suppressor, and the synthetase active-site has a large hydrophobic pocket that can be engineered to accept a wide range of substrates.6 While site-specific incorporation of unnatural amino acids has been performed in metazoans,7 previous experiments were limited to reporter genes and no expression of functional proteins that affect animal physiology has been reported. Here, we describe the genetic code expansion of zebrafish embryos with four unnatural amino acids, and its application to the optical control of protein function in live animals.</p><p>Zebrafish are a commonly employed model organism for vertebrate development,8 disease modeling,9 and drug discovery.10 The ex vivo development and transparency of the embryo make it an excellent system for the application of noninvasive optical tools, including light-activated antisense agents,11 thereby providing insight into gene regulatory processes and networks with spatial and temporal resolution. Moreover, microinjection of mRNA into the 1-cell stage embryo is a standard and rapid approach for delivery of exogenous genes that can be readily adapted to encode for any gene product and provides homogeneous protein expression in zebrafish.12 Taken together, these distinct advantages over other model organisms make the zebrafish an ideal system for a wide range of biological studies.13</p><p>Light regulation of protein activity in zebrafish has been reported using natural photoreceptor domains;14 however, the genetic encoding of photocaged amino acids will further expand the optogenetic toolbox and will enable a rational design of light-activated proteins based on their function or structure. For example, photocaged lysine analogs have been applied to the optical control of protein localization,15 kinase function,16 and CRISPR/Cas9 gene editing17 in human cells. By genetically encoding a photocaged lysine using the PylRS system in zebrafish embryos, we demonstrate the consequences of optical control of MEK activation at different stages in development. Temporal control of kinase function led to the identification of a critical time window for activity of the MEK/ERK pathway in order to establish dorsal/ventral polarity in the early embryo.</p><p>We first tested incorporation of unnatural amino acids using a Renilla luciferase (Rluc) reporter assay. Wild-type Rluc was active in zebrafish embryos (Figure S1) and was used as a positive control. We predicted that incorporation of an unnatural amino acid at a leucine residue (L95) located at the surface of Rluc would not interfere with Rluc function, thereby generating a highly specific reporter for amber codon suppression (Figure S2). Thus, L95 was mutated to a TAG codon to probe read-through during translation. Wild-type M. barkeri PylRS (WTRS) mRNA, Rluc-L95TAG mRNA, and PylT RNA were synthesized through in vitro transcription. The purity of PylT was confirmed by agarose gel (Figure S3). WTRS mRNA, Rluc-L95TAG mRNA, and PylT RNA were injected together with the unnatural amino acid (UAA) into zebrafish embryos. After 48 h, zebrafish lysate was collected for luciferase assays and a 217- and 161-fold increase of Rluc activity was observed in the presence of the UAAs 1 and 2, respectively (Figure 1c). Negligible Rluc activity in the absence of the UAA demonstrated the excellent fidelity of the PylRS system in zebrafish embryos, as none of the common 20 amino acids were recognized as substrates. The effect of PylT on incorporation efficiency was further explored, and we found that chemically synthesized PylT showed similar efficacy compared to in vitro transcribed PylT (Figure S4). However, when we tested in vitro transcribed PylT without a CCA tail, significantly lower efficacy was noted (Figure S4). The CCA tail is a conserved sequence at the 3′ end of the tRNA, which is acylated with the amino acid. Although this sequence can be added by CCA-adding enzymes in cells,18 our results suggested that the direct addition of a CCA tail on the PylT improved incorporation efficiency, and thus was used in all subsequent experiments. We also explored the effect of different PylT concentrations on incorporation efficiency, and 2 ng were found to be critical for high incorporation efficiency (Figure S5). This is in agreement with previous findings of the PylT amount possibly being a limiting factor for genetic encoding of UAAs.19</p><p>Inspired by the success of using wild-type PylRS for incorporating 1 and 2, which could be applied in both protein labeling and protein activation experiments,20 we examined if mutant PylRS enzymes can be employed in embryos to incorporate more structurally complex amino acids. To this end, we synthesized mRNA of HCKRS21 and OABKRS,22 which have been shown to incorporate 3 and 4, respectively. We performed injections as described above, and observed a 70- and 34-fold increase of Rluc activity in the presence of 3 and 4, respectively (Figure 1c). No toxicity was observed for any of the four UAAs (Figure S6). Taken together, these results demonstrate successful genetic encoding of four different UAAs in zebrafish embryos.</p><p>The photocaged lysine 3 has previously been applied to control protein function in mammalian cells using 365, 405, and 760 nm (two-photon) irradiation.21 With successful genetic encoding of 3 in zebrafish, we tested if protein function could be manipulated with light in developing embryos. As an initial proof-of-concept, we utilized firefly luciferase (Fluc) with a TAG amber codon at position lysine 206, because installation of 3 blocks Fluc activity until light exposure.21 In order to create an internal control for incorporation efficiency, we fused Rluc to the C-terminus of Fluc-K206TAG (Figure 1d). To this end, Fluc-K206TAG-Rluc mRNA was injected, together with HCKRS mRNA, PylT, and 3, into zebrafish embryos. After 48 h, injected embryos were either briefly irradiated at 365 nm or kept in the dark. Embryo lysate was subsequently collected, and a luciferase assay was performed for both Fluc and Rluc. Excellent optical OFF to ON switching of Fluc function was observed, with negligible background activity before irradiation (Figure 1e). Normalization of Fluc activity to Rluc activity, as a TAG codon suppression control, revealed a 26-fold increase of Fluc activity upon light triggering. This result shows that light activation of protein function can be achieved in live zebrafish embryos with an expanded genetic code.</p><p>We then sought to apply genetic code expansion in zebrafish to an enzyme with endogenous function in order to demonstrate its utility in altering embryonic development. Incorporation of photocaged amino acids into proteins enables precise dissection of signaling pathways with light, and this approach has been applied to study the dynamics of MEK/ERK signaling in mammalian cells.16 While optical activation on the second to minute time scale in mammalian cells provided further insight into adaptive behavior of the MEK/ERK network in single cells, in the context of zebrafish biology, kinase signaling pathways are important regulators throughout embryogenesis.23 The MEK/ERK pathway is a well-known downstream target of Fibroblast Growth Factor (FGF) signaling and plays an important role in mesendoderm induction and dorsoventral patterning of the zebrafish embryo. FGF signaling induces expression of chordin and noggin, secreted inhibitors of ventralizing bone morphogenetic proteins resulting in dorsalization (Figure 2a).24 An inhibitor-based chemical approach has previously been used for perturbation of the MEK/ERK pathway during zebrafish development;25 however, pharmacological inhibitors only allow for the deactivation of kinase function, not activation, and their specificity is often limited. We reasoned that optical activation of the MEK/ERK pathway in zebrafish provides an innovative tool to study its role, as site-specific incorporation of the caged amino acid 3 conveys complete kinase specificity. By substituting the critical lysine 97 with 3, the caging group blocks the ability of the enzyme to correctly position ATP in the MEK1 active site (Figure 2b). We first confirmed the incorporation of 3 into MEK1, and subsequent decaging through UV exposure, by MS/MS analysis of recombinantly expressed MEK1 protein in E. coli (Figure S7 and Figure S8). We then generated mRNA of constitutively active MEK1 (caMEK1, containing S218D and S222D mutations) and confirmed that injection of caMEK1 led to dorsalized embryos at 10 hpf (Figure 2d,e), as previously reported.25 We further generated caMEK1-K97TAG mRNA and injected it into zebrafish embryos, together with HCKRS mRNA, PylT, and 3. We detected full-length MEK1 by Western blot in the presence of 3 but not in the absence of 3, suggesting successful incorporation into caMEK1 at position K97 and generation of the photocaged enzyme in live animals (Figure S9). When these embryos were left in the dark, they developed normally, indicating that caged MEK1 was inactive (Figure 2d). To activate caged MEK1 at different developmental stages, we irradiated embryos for 30 s at 2, 5, or 8 h postinjection. Light activation of MEK1 can efficiently increase ERK phosphorylation at all three time points (Figure 2c). Embryos irradiated at 2 and 5 h showed an elongated phenotype at 10 hpf (Figure 2d,e). However, the majority of embryos irradiated at 8 h appeared normal at 10 hpf (Figure 2e), indicating that active MEK was not able to efficiently trigger an elongated phenotype after 8 hpf. As a control, embryos that were injected with caMEK1-K97TAG mRNA, HCKRS mRNA, and PylT, but not 3, developed normally in both the presence and absence of UV irradiation (Figure S10).</p><p>We then tested if optical activation of the MEK/ERK pathway resulted in a change at the gene expression level. We probed expression of the brachyuary homolog a (ta) gene, a well-known downstream target of the FGF/MEK/ERK pathway.26 At shield stage (6 hpf), embryos that were exposed to UV light showed broader expression of ta in the margin when compared to embryos that were kept in the dark (Figure 3a). In some instances, ta expression was detected at the animal pole of the embryos, a pattern that is similar to embryos injected with constitutively active MEK1. At bud stage (~10 hpf), the expression of the ta was also wider along the notochord in light-activated embryos compared to embryos that were not irradiated (Figure 3b). We also probed expression of the chordin (chd) gene, a marker for dorsalized embryos that is known to be induced following activation of the FGF/Ras/MAPK pathway.27 As expected, embryos that were exposed to UV light showed expanded expression of chd at shield stage, compared to embryos that were kept in the dark (Figure S11).</p><p>Taken together, the observed ta and chd expression patterns in response to optical MEK1 activation and the time-resolved phenotypic studies shown in Figure 2d–e demonstrate that the MEK/ERK pathway influences dorsal/ventral patterning in zebrafish development before 8 hpf, thereby providing support for early intervention with pharmacological MEK inhibitors for related congenital defects in humans, such as cardio-faciocutaneous syndrome.25,28</p><p>In conclusion, we incorporated four unnatural amino acids into proteins in zebrafish embryos through genetic code expansion using injection methods that are applicable to many zebrafish studies. We demonstrated light activation of enzymatic function, specifically luciferase activity, through site-specific incorporation of a photocaged unnatural amino acid in live embryos. We then applied this methodology to the temporal activation of the MEK/ERK pathway in zebrafish and identified a time window for MEK activity that can influence dorsoventral patterning. Besides controlling protein function with light, other potential applications of unnatural amino acids in live zebrafish embryos include small molecule triggered protein activation, site-specific labeling of proteins with fluorescent and biophysical probes, and probing protein interactions through covalent bond formation with electrophilic or photo-cross-linking groups. The zebrafish is a well-established model organism for human development and disease, and we anticipate that the ability to genetically encode a 21st amino acid will become a powerful tool to manipulate and study protein function in animals.</p>

PubMed Author Manuscript

Label-free chronopotentiometric glycoprofiling of prostate specific antigen using sialic acid recognizing lectins

In recent decades, it has become clear thatmost of human proteins are glycosylated and that protein glycosylation plays an important role in health and diseases. At present, simple, fast and inexpensive methods are sought for clinical applications and particularly for improved diagnostics of various diseases, including cancer. We propose a label- and reagent-free electrochemical method based on chronopotentiometric stripping (CPS) analysis and a hangingmercury drop electrode for the detection of interaction of sialylated protein biomarker a prostate specific antigen (PSA) with two important lectins: Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). Incubation of PSA-modified electrode with specific SNA lectin resulted in an increase of CPS peak H of the complex as compared to this peak of individual PSA. By adjusting polarization current and temperature, PSA-MAA interaction can be either eliminated or distinguished from the more abundant PSA-SNA complex. CPS data were in a good agreement with the data obtained by complementary methods, namely surface plasmon resonance and fluorescent lectin microarray. It can be anticipated that CPS will find application in glycomics and proteomics.

label-free_chronopotentiometric_glycoprofiling_of_prostate_specific_antigen_using_sialic_acid_recogn

Introduction<!>Chemicals<!>Lectin microarrays (LMA)<!>Surface plasmon resonance (SPR)<!>Electrochemical measurements<!>Chronopotentiometric analysis<!>Adsorption/desorption behaviour of PSA<!>Evaluation of lectin binding specificity by other methods<!>Conclusions

<p>Prostate cancer (PCa) is one of the leading issues concerning men worldwide. According to the statistics, PCa is the third most common cause of death of cancerous diseases among males in European Union [1]. The main problem behind PCa diagnostics is that at an early stage of the disease only mild symptoms are present, or its symptoms may resemble those of benign prostate hyperplasia (BPH). There were also cases reported, when metastatic PCa caused no symptoms at all [2]. Therefore it is often diagnosed only when it reaches a more developed stage and is more difficult to cure. The main diagnostic tool currently used for PCa diagnostics is determination of a prostate specific antigen (PSA) level in a blood serum. Elevated PSA level in patients with PCa is a result of a leakage from disrupted prostate cells, rather than its increased expression [3]. A concentration cut-off point to distinguish between healthy individuals and people with a PCa risk was considered to be 4 ng·mL−1, nonetheless this limit has lately been reconsidered by many authors to 2 ng·mL−1 due to the fact that almost 15% of patients diagnosed with PCa had PSA level lower than 4 ng·mL−1 [4]. Such cases even prompted The US Preventive Services Task Force to issue a recommendation not to use PSA for routine screening of PCa due to its low sensitivity, specificity and prognostic value [5]. This has led to a quest for finding new biomarkers, more specific to PCa, rather than for disease of prostate in general (i.e. BPH).</p><p>Since aberrant glycosylation occurs in many intracellular signalling pathways andmay eventually lead to the development of cancer, various glycoforms of proteins are often proposed as novel biomarkers [6]. Altered glycosylation patterns were also recognized in tumours that expressed rapid growth or were metastasizing into other parts of organism. This indicates that analysis of various glycoforms of biomarkers may not only be used for disease diagnostics, but also for prognostic purposes to detect aggressive forms of cancer [7]. Until now, there are only 9 protein biomarkers approved by theUS Food and Drug Administration (FDA) for clinical use as cancer biomarkers and all of them are glycosylated [8].</p><p>Composition of PSA glycan is well known, however study of glycan changes as a result of PCa is more challenging. PSA protein from healthy donor is reported to contain fucosylated complex biantennary N-glycan with one or two terminal sialic acids attached [9]. Several research groups study glycan changes of PSA from cancerous origin [7,9–12]. An overexpression of α(1,6)-fucosyltransferase resulting in an increased α1,6-fucosylation of PSA was reported to be a sign of an aggressive form of PCa and thus being a perspective prognostic marker [7,13]. Monitoring of an increased branching (i.e. presence of tri- and tetraantennary glycans) is also a valuable predictive marker with a potential to predict a castration resistant PCa [14]. Another important cancer-related modification of PSA glycan is aberrant sialylation. N-acetylneuraminic acid (Neu5Ac) is a predominant sialic acid found in humans. Terminal Neu5Ac present on the PSA glycan is in healthy individuals linked to galactose predominantly via α(2,6)-glycosidic linkage. Contrary to this, presence of α(2,3)-glycosidic linkage of Neu5Ac to Gal is significantly increased in prostate cancer [15]. Since PSA samples from patients with BPH do not show this increase, monitoring of altered sialylation pattern is a promising tool to distinguish between PSA of cancerous and non-cancerous origin [16].</p><p>The main approaches for glycan analyses are robust and reliable methods such as mass spectrometry in conjunction with liquid chromatography or capillary electrophoresis [17]. Nevertheless, these methods require an experienced operator and are highly time-consuming. An alternative way for glycan analyses are different lectin-based methods, such as enzyme-linked lectin binding assay (ELLBA), fluorescent microarray or lectin-based biosensors and biochips [18]. Number of studies are focused on preparation of a sensitive biosensor for analysis of biological molecules using various approaches [19]. Alterations in glycan composition occur not only during cancer development, but also in various autoimmune diseases (systemic sclerosis, rheumatoid arthritis etc.) or even as a result of an aging process [20,21]. Electrochemical platforms of detection provide several advantages, such as a minute sample consumption, low detection limits, low price and a possibility to miniaturize such devices. Jolly et al. [22] combined an aptamer detection with a molecular imprinted polymer to develop a biosensor resistant to non-specific interactions able to detect PSA down to 1 pg·mL−1. Tzouvadaki et al. reported a memristive aptasensor capable of detecting 0.7 fg·mL−1 of PSA – among the most sensitive electrochemical biosensor for PSA detection published [23]. However, as previously stated, determining only the concentration of PSA marker is not sufficient for a definite PCa confirmation, further investigation of aberrant glycosylation changes is necessary to determine the origin of a biomarker. Pihikova et al. described an impedimetric biosensor for quantification of PSA and its subsequent glycoprofiling, both on the same interface [24].When detecting various interactions, such as protein-protein or glycan-protein interactions, it is often necessary to modify electrode surfaces, i.e. using self-assembled monolayers (SAMs).</p><p>Palecek's group in Brno invented (i) new methods of chemical modification of glycans and glycoproteins by complexes of six-valent osmium (binding to 1,2 diglycols), followed by electrochemical [25] and/or immunochemical assays [26] and (ii) new approaches in electrochemical analysis of non-conjugated proteins [6,27–31] based on the ability of proteins to catalyse hydrogen evolution at mercury electrodes. In constant current chronopotentiometric stripping (CPS) proteins produce the so-called peak H [6]. Using this peak, the structure-sensitive analysis has been developed, capable to detect small changes in protein structures resulting e.g. from a single amino acid exchange in a mutated protein [6,29]. Moreover, it has been shown that the same principles can be applied to study interactions of proteins with DNA, such as the specific binding of tumour suppressor protein p53 to DNA [28].</p><p>Recently, Ostatna et al. [31] extended these efforts for studies of lectin-glycoprotein interactions. They used CPS and a bare hanging mercury drop electrode (HMDE) to investigate interactions of Concanavalin A lectin (Con A, from Canavalia ensiformis) with mannosylated glycoproteins [31] and showed that, based on changes in peak H, free lectins and free glycoproteins can be distinguished from the lectin-glycoprotein complexes and the time course of the complex formation can be followed.</p><p>In this study, we attempted to apply this methodology for analysis of sialylated protein biomarker PSA and its interactions with two important lectins: (i) Sambucus nigra agglutinin-I (SNA, specific for Neu5Ac(α2–6)Gal) and (ii) and Maackia amurensis agglutinin (MAA, specific for Neu5Ac(α2–3)Gal).We show that CPS analysis is able to recognize specific binding of PSA to SNA fromits less abundant interaction with MAA.</p><!><p>KH2PO4, K2HPO4, NaH2PO4 and Na2HPO4, hydrochloric acid, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), Maackia amurensis agglutinin (MAA, Neu5Ac(α2–3)Gal-specific, 130 kDa, 2 subunits, pI 4.7, a glycoprotein without Cys residues) were purchased from Sigma Aldrich (USA). Prostate specific antigen (PSA, a serine protease, human kallikrein 3, pI 7.26, a glycoprotein containing single complex N-type glycan and 10 Cys residues) (98%) from a human seminal fluid was purchased from Fitzgerald, USA. Sambucus nigra agglutinin-I (SNA, Neu5Ac(α2–6)Gal-specific, 140 kDa, 4 subunits, pI 5.4–5.8, a glycoprotein containing 8 Cys residues) was purchased from EY Labs, USA. Biotinylated lectins (Maackia amurensis and Sambucus nigra agglutinin) and carbo-free blocking solution for lectin microarray experiments were purchased from Vector Laboratories (USA). A CF647-streptavidin fluorescent label was purchased from Biotium (USA). All solutions were prepared in 0.055 μS ultrapure deionized water and were subsequently filtered prior to use using 0.2 μm sterile filters.</p><!><p>LMA experiments were run using SpotBot3 Microarray Protein edition (Arrayit, USA) on epoxide coated slides Nexterion E (Schott, Germany) using a previously optimized protocol and scanned using InnoScan710 scanner (Innopsys, France) at the wavelength of 630 nm [21]. The slide image was evaluated using the Mapix 5.5.0 software.</p><p>Fluorescent protein microarray experiment was performed using 10 mM K-phosphate pH 7.0 as a printing and washing buffer and containing a 10× diluted carbo-free blocking solution (VectorLabs, USA) as a blocking buffer. Shortly, six different concentrations of diluted PSA (including a 1 mg·mL−1 stock solution) were spotted using SpotBot3 Microarray Protein edition (Arrayit, USA) on epoxide coated slides Nexterion E (Schott, Germany) using a previously optimized protocol [21]. Spotting temperature was set to 10 °C and humidity to 60%. Subsequently, the slide was blocked using a blocking buffer at room temperature for 1 h, rinsed under a gentle stream of a printing buffer and drained. Then, 100 μL of 25 μg·mL−1 biotinylated lectin (SNA and MAA respectively) in a binding buffer was applied to the slide surface and incubated for 1 h. After lectin incubation, the slide was incubated with the Biotium CF647-streptavidin solution (1 μg·mL−1 in a printing buffer) for 15 min. After a washing procedure, the slide was scanned using an InnoScan710 scanner (Innopsys, France) at a wavelength of 635 nm. The slide image was evaluated using the Mapix 5.5.0 by evaluation of the intensity of fluorescence and intensity of all independent array spots on the array (normalized to the background).</p><!><p>For the SPR measurements, a carboxymethyldextran hydrogel (CMD) modified gold chip (12 × 12 × 0.3 mm, 50 nm Au thickness, medium density, Xantec Bioanalytics, Germany)was used. The chip was activated using EDC/NHS (1+1 ratio of 0.2 M EDC and 0.05 M NHS, respectively) and subsequently PSA was covalently immobilized on the chip surface from a stock solution with a concentration of 0.33 mg·mL−1 (11.6 μM) for 10 min at a flow rate of 5 μL·min−1. After washing step, MAA and SNA lectins as binding analytes were injected on a chip in five different concentrations (prepared by dilution from their 0.33 mg·mL−1 stock solutions). After each binding step, the chip surface was regenerated by 20 mM HCl. The sensorgram was recorded and evaluated using SPR Autolink software 1.1.7 (Reichert, USA). Surface coverage of bound PSA, as well as the ratio of SNA/MAA lectin binding was obtained using a SPR machine (SR7000DC, Reichert, USA) operated with an autosampler. All proteins were dissolved in 10 mM K-phosphate buffer pH 7.0 prepared from ultra-pure deionized water (0.0055 μS).</p><!><p>Electrochemical measurements were performed on an Autolab analyser (PGSTAT30, EcoChemie the Netherlands) connected with VA-Stand 663 (Metrohm Switzerland) with a three-electrode system. HMDE(0.4 mm2) as aworkingelectrode,Ag|AgCl|3MKCl as a reference one and Pt wire as an auxiliary electrode were used in a standard thermostated cell open to air. 1 μM PSA (if not stated otherwise) was adsorbed at the working electrode from 5 μL of 50 mM Na-phosphate, pH 7.0 at open current circuit for 60 s without stirring (Schematic 1A) to reach full electrode coverage. The HMDE modified by PSA was incubated in 1 μM lectin in 50mMNa-phosphate, pH7.0 at open current circuit for 120 s under stirring at 1500 rpm (Schematic 1B). The protein-modified electrode was washed and transferred into a blank background electrolyte followed by recording of chronopotentiogram(Schematic 1C) or C-E curve by alternating current voltammetry (a.c. voltammetry) in phase-out mode at 20 °C (if not stated otherwise). A.c. voltammetry: scan rate 7.5 mV/s, frequency 223 Hz, amplitude 50 mV. CPS measurements: stripping current Istr − 45 μA (if not stated otherwise). Experiments were replicated at least 3 times for each measurement.</p><p>Denaturation of 12.6 μM PSA in sodium-phosphate buffer, pH 7 solution with 7 M urea was performed overnight at 4 °C. The protein solution was then diluted by the background electrolyte to the final protein 1 μM concentration and immediately measured; the final non-denaturing concentration of urea was 560 mM.</p><!><p>We have shown that CPS in combination with a high electron-yield electro-catalytic process is convenient for protein analysis [6]. Such analysis is based on the ability of some amino acid residues to catalyse hydrogen evolution on mercury-containing electrodes [6,32]. Our approach to study PSA-lectin interactions was based on our previous paper dealing with lectin-ovalbumin interactions [31]. In difference to that paper, in which the Concanavalin A-ovalbumin was prepared in a test-tube and then measured at the electrode, here we interacted the surface-attached PSA with a lectin in solution. This arrangement is more convenient for sensing of differences in PSA glycosylation, but it strongly depends on orientation of the PSA molecules on the electrode surface. Earlier we showed that HMDE could be easily modified with thiols and particularly with dithiols, such as DTT [27]. The thiol SAM protects the surface-attached protein from the denaturing effect of the electric field in the vicinity of the negatively charged electrode surface. PSA molecule contains 10 Cys residues, all of them involved in disulfide binding. Among these residues eight are located close to the surface of the PSA molecule in the side opposite to the glycan moiety (Fig. 1). Considering the PSA structure and high affinity of sulphur to mercury, we decided to use bare HMDE because we expected that the four cystine dithiols will strongly bind to mercury electrode surface, while the glycan will be available for its interactions with the environment, capable to form specific complexes with lectins.</p><p>1 μM PSA was adsorbed on HMDE from a 5 μL drop for tA 60 s. After a gentle rinse, the PSA-modified HMDE was transferred into a solution of 1 μM MAA or 1 μM SNA lectin in 50 mM sodium phosphate, pH 7 and incubated for 2 min. After this incubation the PSA-modified electrode was transferred into a blank electrolyte followed by CPS analysis at Istr − 45 μA at 20 °C (Schematic 1). PSA alone yielded two small peaks H at −1.86 V and −1.89 V vs. Ag|AgCl|3 M KCl (Fig. 2A), respectively, suggesting that PSA was on the HMDE surface in its native form(denatured PSA yielded more than 4 times higher peak H than that of native PSA, Fig. 2B). Under these conditions MAA lectin (not containing any Cys residues) produced no peak H (Fig. 2A). In contrast SNA lectin yielded a small peak at −1.90 V vs. Ag|AgCl|3 M KCl (Fig. 2A). MW of these two lectins are similar, but SNA contains 8 Cys residues. These SNA Cys residues are most likely less available for the electrocatalysis and might be more distant from the HMDE surface as PSA molecule, which is about 5 times smaller (Fig. 2A). After the incubation of PSA-modified HMDE with SNA lectin, peak Hof PSA (peak potential of −1.86 V vs. Ag|AgCl|3-M KCl) increased more than twice (Fig. 2A). Similarly, specific interaction of ovalbumin with Concanavalin A in solution resulted in an increase of the ovalbumin peak H [31]. This phenomenon may be explained by the change in the PSA orientation on the HMDE surface after the lectin binding to PSA N-glycan, or by a conformational change in PSA after this biorecognition process. Peak H height of PSA after SNA binding was about 25% lower than that of urea-denatured PSA (Fig. 2B), suggesting that not all electroactive residues of PSA were involved in catalytic hydrogen evolution reaction after the biorecognition. In contrast, peak H of PSA alone was almost the same as that obtained after PSA incubation with MAA lectin (Fig. 2A). These results can be due to almost no binding of MAA to the surface-attached PSA as compared to the specific SNA binding to PSA. Electrochemical data (Fig. 2) appeared thus in a good agreement with impedimetric assay [24] and the lectin microarray results (see below).</p><p>In CPS the rate of potential changes increases non-linearly with a current density and may reach very high polarization rates [6]. By using high negative intensity Istr, the exposure time of molecules attached to the surface may be reduced to milliseconds. Exposure to the electric field can induce denaturation of the surface-attached protein [6,27,33] as well as disintegration of the DNA-protein [28] or lectin-glycoprotein complexes [31]. Susceptibility of proteins and their complexes to the effects of an electric field at negative potentials decreases with decreasing temperature [6,28,33].We studied effect of the stripping current (Istr) intensity on the CPS responses of PSA and PSA after the interaction with MAA and SNA lectins at a constant temperature (Fig. 2C) and effect of temperature at a constant current (Fig. 2D). Fig. 2C and D are showing a structural transition of PSA alone between −40 and −45 μA at 20 °C and at constant current −45 μA between 25 and 30 °C. These results are in a good agreement with the results obtained for other proteins [6, 33], where structural changes between native and denatured forms of proteins were caused by an external electric field [33]. PSA with MAA and PSA alone displayed almost the same curves, suggesting very weak or no interaction between PSA and MAA (Fig. 2C). On the other hand, incubation of PSA with SNA resulted in an increase of peak H at Istr–45 μA and at more negative Istr intensities (Fig. 2C). This increase can be explained by partial protein unfolding after PSA-SNA interaction. At less negative Istr intensities, where the surface-attached PSA is denatured (due to the prolonged electric field effects at negative potentials) PSA alone and PSA-SNA complex yielded almost the same peak H areas (Fig. 2C). It cannot be excluded that, in addition to the protein denaturation, disintegration of the PSA-SNA complex took place under these conditions at the electrode surface. The observed dependence of peak H on Istr (Fig. 2C) is supported by the results of temperature dependence at constant Istr of −45 μA (Fig. 2D), which shows a structural transition between 25 and 30 °C in PSA and PSA-MAA but not in PSA-SNA in agreement with our assumption that protein unfolding resulted already from the interaction of SNA with the surface-attached PSA.</p><!><p>Electrochemical double layer capacitance is a sensitive indicator of adsorption processes [34]. Protein adsorbed on an electrode surface displaces solvent molecules and ions and reduces the capacity because of higher dielectric permittivity of solvent compared to protein solution. We measured a dependence of capacity (C) of PSA-modified HMDE electrochemical double layer on polarization potential E (C-E curves). PSA lowers the C in the potential range of about–0.1 to–1.7 V vs. Ag|-AgCl|3 M KCl (Fig. 3A), giving two peaks: first at −0.57 V and second (less developed) at −1.56 V vs. Ag|AgCl|3 M KCl. Presence of the first one is caused by the protein reorientation on the surface after Hg-S(Cys) bond reduction at a potential of about −0.6 V vs. Ag|AgCl|3 M KCl. Signals of proteins at negative potentials, far from the potential of the zero charge (p.z.c) are markedly influenced by processes linked to denaturation of the surface-attached protein. We therefore considered E-C values obtained close to p.z.c., which are better comparable to the CPS results at high -Istr. Fig. 3B shows that at −0.3 V vs. Ag|AgCl|3 M KCl, there is an observable increase in the ΔC correlating to an increasing concentration of PSA (from 50 to 750 nM). For higher PSA concentrations, no further increase was observed, suggesting that we already obtained a full surface coverage for this protein.</p><p>To obtain more information about arrangement of PSA and lectins layers at the electrode surface, we measured their specific capacity. We did not observe any peak around −0.6 V vs. Ag|AgCl|3 M KCl on C-E curves of free MAA lectin, while SNA yielded the peak much smaller than that of PSA alone (Fig. 3A). C-E curves of both lectins significantly differed from that of PSA at potentials more negative than −1.2 V vs. Ag|AgCl|3 M KCl. We incubated the PSA-modified electrode with lectin in solution (50 mM Na-phosphate, pH 7) and transferred the electrode into the blank electrolyte. In this way we obtained C-E curves, which were at potentials close to p.z.c., similar to that of free PSA (Fig. 3A), suggesting that PSA remained at electrode surface even after the interactions with the lectins.</p><p>Furthermore, we studied the effect of the lectin concentration on peak H of surface-attached PSA (Fig. 3C). Additions of SNA lectin up to the 250 nM concentration significantly increased the peak H area until reaching almost 1.5-fold area of this peak as for PSA alone. Further increase in the SNA concentration resulted in only slight increase. Peak area almost twice as much as for PSA alone or PSA-MAA, respectively, was obtained for 3 μM SNA concentration. At full surface coverage of PSA, we were able to selectively distinguish between specific and nonspecific lectin recognition for lectin concentrations above 250 nM.</p><p>CPS as a simple and inexpensive method shown to be a useful tool for the in situ glycoprofiling of widely used PSA biomarker without previous release of the intact glycan (using PNGase F). It is of great importance to switch from the study of only the protein part to the study of their glycan part as well, especially for this biomarker, since its glycosylation is changed during the development of PCa. The main advantage of this method is the reproducibility, which (due to atomically smooth HMDE surface) is much better than that obtained with most of the popular solid electrodes [35,36]. Slightly higher concentrations of PSA are needed for this analysis than contained in human sera (present prostate cancer diagnostic test), this approach can be used for PSA isolated from urine or seminal fluid where the PSA concentration can be relatively high (~1 mg·mL−1) [37,38].</p><!><p>Binding of SNA and MAA lectins to PSA was evaluated also by standardly used methods like SPR and LMA, respectively. For calculation of the total amount of bound protein during SPR measurements, the conversion 1 μRIU=1 pg ·mm−2 (according to the manufacturer) was applied. Surface coverage of PSA on a SPR chip was calculated as ΓPSA = (212 ± 60) pg·mm−2 i.e. 30% of a theoretical surface coverage of a full protein monolayer. SPR analysis further revealed that MAA lectin from 25 nM protein solution was bound to immobilized PSA with ΓMAA = 0.55 pg·mm−2, while lectin SNA with much higher surface coverage of ΓSNA = 10.1 pg·mm−2. SPR assays thus showed that the amount of MAA lectin bound to PSA was only 5.4% of the amount of SNA lectin attached to PSA. Similar results were obtained by fluorescent lectin microarray experiment (Fig. 4) using lectins in a concentration range of PSA from 317 nM to 7.69 μM with a response ratio observed for MAA bound to PSA compared to SNA attached to PSA of (7.6 ± 0.1)%. These data well-correlated with electrochemical results (Fig. 2).</p><!><p>We studied chronopotentiometric and a.c. voltammetric behaviour of the PSA and its interactions with lectins capable to recognize PSA glycans occurring in healthy people or in patients with prostate cancer. We show for the first time that CPS analysis in combination with HMDE can be used to distinguish specific interaction of SNA lectin with PSA (occurring in healthy men) from the PSA interaction with lectin MAA, specific for prostate cancer (PCa) PSA. Earlier we showed that proteins are not significantly denatured when adsorbed at mercury electrode under solution conditions close to physiological but the surface-attached protein can be denatured due to prolonged exposure to the electric field effects at negative potentials [6,33]. In CPS using high current densities, this exposure can be limited to miliseconds, preventing thus the denaturation of the surface-attached protein. Here we found conditions under which the surface-attached PSA was not denatured in CPS experiments and showed that as a result of PSA-SNA interaction some protein unfolding was taking place in the PSA-SNA complex under conditions at which the PSA alone remained native.</p><p>For several reasons, in this paper no attempt was made to develop a sensor. At this stage we prefer to get more data about lectin-glycan interactions and about chemical modification and electrochemical behaviour of glycoproteins in our further work. Moreover, we are aware that HMDE has some unique properties, but it is not the best electrode for sensor development. On the other hand, it has been shown that the HMDE can be substituted by solid amalgam electrodes in the analysis of nucleic acids [36], proteins [6,39] and glycans [40] and we wish to test this possibility in a near future. Knowing that the problem of improving specificity of biomarkers used in cancer and other diseases is very difficult and urgent we believe that attempts to apply electrochemical methods for this purpose are promising and desire increased attention of electrochemists and their involvement in the interdisciplinary research.</p>

PubMed Author Manuscript

Versatile peptide macrocyclization with Diels-Alder cycloadditions

Macrocyclization can improve bioactive peptide ligands through preorganization of molecular topology, leading to improvement of pharmacologic properties like binding affinity, cell permeability and metabolic stability. Here we demonstrate that Diels-Alder [4+2] cycloadditions can be harnessed for peptide macrocyclization and stabilization within a range of peptide scaffolds and chemical environments. Diels-Alder cyclization of diverse diene-dienophile reactive pairs proceeds rapidly, in high yield and with tunable stereochemical preferences on solid-phase or in aqueous solution. This reaction can be applied alone or in concert with other stabilization chemistries, such as ring-closing olefin metathesis, to stabilize loop, turn, and \xce\xb1-helical secondary structural motifs. NMR and molecular dynamics studies of model loop peptides confirmed preferential formation of endo cycloadduct stereochemistry, imparting significant structural rigidity to the peptide backbone that resulted in augmented protease resistance and increased biological activity of a Diels-Alder cyclized (DAC) RGD peptide. Separately, we demonstrated the stabilization of DAC \xce\xb1-helical peptides derived from the ER\xce\xb1 -binding protein SRC2. We solved a 2.25 \xc3\x85 co-crystal structure of one DAC helical peptide bound to ER\xce\xb1, which unequivocally corroborated endo stereochemistry of the resulting Diels-Alder adduct, and confirmed that the unique architecture of stabilizing motifs formed with this chemistry can directly contribute to target binding. These data establish Diels-Alder cyclization as a versatile approach to stabilize diverse protein structural motifs under a range of chemical environments.

versatile_peptide_macrocyclization_with_diels-alder_cycloadditions

INTRODUCTION<!>RESULTS AND DISCUSSION<!>Diels-Alder Cycloadditions Form Chemically Stable Peptide Macrocycles with Enhanced Protease Resistance and Biological Activity<!>Diels-Alder Cycloadducts Preferentially Form with endo Stereochemistry and Stabilize Peptide Conformation<!>Diels-Alder Peptide Cycloadditions Operate in Aqueous Solution and Stabilize \xce\xb1-Helices<!>Diels-Alder Cycloadduct Geometry, Alone or in Tandem with other Chemistries, Differentially Affects Peptide Helicity and Activity<!>X-ray Crystal Structure of a DAC Stapled SRC2 Peptide- ER\xce\xb1 Complex Demonstrates Cycloadduct Stereochemistry and Contribution to Target Binding<!>CONCLUSION

<p>Peptides represent powerful starting points for the development of bioactive ligands. Short linear peptide sequences mediate protein-protein interactions (PPIs) by adopting diverse secondary structures, including helix, sheet, loop and turn motifs1–2. Removal from their native protein context often abrogates structural rigidity, leading to reduced binding affinity and pharmacologic properties. Therefore, numerous chemical strategies have been developed to synthetically stabilize peptide secondary structure and retain or augment pharmacologic utility. Most of these chemistries capitalize on alkylation or acylation reactions on naturally-occurring nucleophilic residues such as cysteine and lysine; these approaches have been applied to stabilize helix, loop, and sheet motifs3–8. A key limitation with many of these chemistries is their incompatibility with layering multiple stabilizing chemistries. Another strategy is incorporation of non-natural amino acids that enable metal-catalyzed bond formation. The most well-studied of these strategies include olefin metathesis of terminal alkene containing groups as well as [3+2] Huisgen ligation of alkyne/azide pairs9–14. These 'stapled' peptides show increased favorable properties including structural stability, binding affinity, cellular uptake, and in vivo pharmacokinetics for direct or allosteric targeting of PPIs15–21. Notably, compounds in this class have entered clinical trials for various indications22. While these chemistries can successfully stabilize peptide secondary structure, they are often incompatible with diverse structures, other natural and non-natural functionalities and aqueous conditions necessary for proper folding of specific peptide and protein scaffolds. From a practical standpoint, the transition-metal catalyzed reactions require expensive and toxic catalysts that must be removed during purification. Therefore, new strategies to control secondary and tertiary structure, ideally with wide functional group and reaction condition tolerance, would greatly expand the synthetic arsenal for developing peptide-based chemical probes.</p><p>A notable carbon-carbon bond forming reaction that has enjoyed widespread synthetic application is the Diels-Alder [4+2] cycloaddition. First reported in 1928, this reaction is employed by nature and chemists alike to construct complex molecules23–25. Several aspects of this transformation make it attractive for synthesis, including a wide range of diene-dienophile pairs, high regio- and chemoselectivity in many applications, simultaneous introduction of multiple stereocenters and compatibility with a range of reaction conditions26. Indeed, pioneering studies demonstrated that aqueous conditions are not only tolerated, but can enhance reaction rates27. While these qualities overlap with those desired for selective modification of biomolecules, beyond its limited use in intermolecular ligations28–32, this chemistry has not been applied for site-specific stabilization of peptides or proteins. Here we report the application of Diels-Alder carbon-carbon bond forming reactions for the macrocyclization of diverse turn, loop and α-helix peptide motifs.</p><!><p>We began our study of Diels-Alder cyclized (DAC) peptides with turn and loop motifs, including an anti-migratory RGD peptide, analogs of which are undergoing clinical testing in oncology33–34. Diene-dienophile functional groups span a wide range of structure and reactivity, thus we chose the moderately activated diene 2,4-hexadiene and the well-characterized maleimido dienophile32. Model peptides of various length and sequence were synthesized containing orthogonally-protected cysteine (tBuS-) and lysine (Mmt-) side chains, with the former invariably in the i-position, and the latter in the i+4, i+5 and i+7 positions. Sequential on-resin cysteine deprotection and hexadiene alkylation with 1-bromo-2,4-hexadiene proceeds quantitatively (Figure 1A,B; Scheme S1). We also synthesized the corresponding diene-containing amino acid, Fmoc-Cys(2,4-hexadiene)-OH, which is compatible with direct incorporation by SPPS (Fig. 1A; Scheme S2, Supplementary Spectroscopic Data). Subsequent on-resin lysine deprotection and acylation with N-maleimido-glycine on an RGD model peptide resulted in formation of two distinct peaks on LC-MS (1 and 1a) with the same mass and ~1 minute retention time shift, suggesting formation of a cyclized product eluting earlier on the C18 column (Figure 1A,B). This product appeared rapidly, with an approximate 2:1 ratio of 1a to 1 within 5 minutes of dienophile introduction; near-complete conversion to 1a was observed upon extended incubation on resin (16 hr, Figure 1B). Kinetic reaction monitoring confirmed rapid conversion of later-eluting 1 to 1a, as well as minor product 1b, with nearly 95% formation within 1 hour (Figure 1C). This pattern was observed across diverse intervening sequences and diene-dienophile spacing (Table 1, Table S1), with high yields of ~85–95% conversion to earlier-eluting, presumably cyclized species. In each scaffold studied, additional earlier-eluting minor isomeric species (≤10% relative to major product) were observed ~4 minutes before the linear peptide (Figure 1B; Figure S1).</p><p>All peptides contain N-terminal acetyl and C-terminal amide groups. "Hex" indicates 2,4-hexadiene alkylation; "Mal" indicates a maleimido-glycine modified residue. Each compound number represents the non-cyclized (X) and cyclized isomers (Xa, Xb…) as determined by separation on LC-MS. *Ratios reported as major:minor product(s) were quantified by integrated LC-MS peak area. Percent conversion (% conv.) refers to the percent yield from non-cyclized to cyclized products.</p><p>A limited screen of reaction conditions revealed that mild heating on resin in DMSO was an optimal condition to maximize cyclic conversion, but also resulted in increased ratio of major to minor isomeric products (vide infra, Table S2), which may be due to de-aggregation of the protected peptide on resin. We also found that other less reactive dienophiles such as acryloyl and crotonyl groups react with hexadiene in yields comparable to maleimide (Figure S2). These viable alternative dienophiles are also compatible with direct incorporation during SPPS and offer increased diversity of cyclic structures35. Similar reaction profiles were observed for peptides produced by on-resin diene incorporation or direct use of Fmoc-Cys(2,4-hexadiene)-OH, as well as upon incubation in aqueous solution following peptide cleavage (Figure S3). These data confirm the compatibility of Diels-Alder peptide cyclization in diverse organic and aqueous chemical environments.</p><!><p>To verify 1 and 1a represented non-cyclized and cyclized products, respectively, we capitalized on the maleimide dienophile uniquely present in the non-cyclized starting material. On-resin incubation of the 1 and 1a crude mixture with excess 2-mercaptoethanol (βME), which should only react with linear maleimide-containing peptide, resulted in complete consumption of 1, yielding a species with the mass of the βME conjugate addition product (Scheme S3; Figure S4). By contrast, 1a levels remained constant, consistent with the loss of the α,β-unsaturated electrophile after Diels-Alder cyclization. Likewise, prolonged exposure of purified 1a to βME did not result in any addition product, suggesting formation of a stable DAC-RGD peptide macrocycle (Figure 1D).</p><p>The RGD motif has seen considerable therapeutic development as an anti-migratory agent targeting aggressive cancers34, 36. Like other peptides, in vivo use of native RGD shows limited efficacy due to cleavage by circulating proteases, whereas head-to-tail cyclized analogs demonstrate increased protease resistance and in vivo activity37. To test whether DAC peptides similarly display differential protease susceptibility, we exposed cyclic 1a and linear analog 1wt to an in vitro trypsin sensitivity assay. Native peptide 1wt was rapidly degraded within two hours (Figure 1E), whereas DAC 1a showed an extended half-life, with 30% remaining intact after 4 hours. To determine if this effect was applicable to other scaffolds and proteases, we subjected the major cyclic isomer of i, i+7 DAC 4a and linear analog 4wt to chymotrypsin. As with the RGD scaffold, the linear species 4wt was degraded more rapidly than 4a (Figure 1E). Consistent with the increased stability of the cyclized RGD analog, compound 1a was significantly more effective at blocking wound closure, an integrin-mediated phenotype, relative to the linear control peptide 1wt (Figure 1F; Figure S5). Taken together, these results confirm that Diels-Adler cyclization can affect the structure and biological function of loop peptide ligands.</p><!><p>To characterize a putative Diels-Alder cycloadduct and its resulting effects on peptide conformation, 4a and precursor peptides 4wt and 4hex were subjected to a series of NMR experiments. 1H-NMR of 4a confirmed the presence of two vinylic protons, absent in 4wt and distinct from those of the diene in 4hex (Figure 2A) or α,β-unsaturated maleimido protons. The observed doublet of triplets pattern for the vinylic protons in 4a results from small 3J and 4J coupling constants of the unsaturated system, indicative of near-90° dihedral angles between neighboring vinyl and allyl protons. TOCSY experiments permitted complete assignment of cyclized and non-cyclized peptides (Supplementary Spectroscopic Data). Direct through-bond coupling was observed between 4a vinylic protons and all protons within the putative cycloadduct (Figure 2B; Figure S6A), supporting the formation of a fused bicyclic adduct. The combination of TOCSY and NOESY experiments resolved the connectivity of the cycloadduct in 4a and revealed extensive through-space interactions between its protons (Figure 2B; Figure S6B). Most notably, interactions between H3/H6 allylic protons and H4/H5 succinimidyl bridgehead protons suggest the cycloadduct in 4a is the endo stereoisomer (Figure 2B).</p><p>NMR results further revealed substantial perturbation to the cyclic 4a structure relative to linear analogs. Following hexadiene incorporation, only modest changes in chemical shifts were observed for cysteine and nearby residues, with virtually no change in 3J-coupling constants between backbone amide and alpha protons (Figure 2C; Figure S6C–F). Conversely, 4a displays marked changes in chemical shifts and backbone 3J-coupling constants for several residues, most notably the tyrosine and alanine adjacent to the cyclizing residues (Figure 2C; Figure S6C–F). These alterations likely result from both cyclization-induced limits on degrees of freedom, as well as emergent intramolecular interactions such as hydrogen-bonding between amide and carbonyl oxygen atoms found on the cycloadduct and peptide.</p><p>To further investigate the structure of this i, i+7 DAC peptide, we carried out molecular dynamics (MD) studies of 4wt and 4a. Briefly, models were built in MOE and run in the NAMD2 simulation package (see Methods for additional details)38–39. Trajectory analysis using VMD revealed linear 4wt displayed no persistent structure, while cyclized 4a displayed a highly stabilized extended loop structure over 50 ns simulations (Figure 2D)40. RMSD measurements of the respective backbones confirm a substantial decrease in 4a conformational flexibility relative to linear 4wt (Figure 2E; average backbone RMSD = 1.07 Å and 2.69 Å, respectively), which also translated to reduced motion in the side chain dynamics of 4a (Figure 2E, Videos S1 and S2).</p><!><p>Helical peptides represent the dominant structural feature that mediates biomolecular interactions1. We therefore asked if Diels-Alder cyclization strategies can be leveraged to stabilize α-helical peptide conformations from diverse peptide sequences. We first tested whether a conformationally-restricted and less reactive furan diene could cyclize on a p53-derived helical sequence16. Like Fmoc-Cys(2,4-hexadiene)-OH, we found that Fmoc-protected (2-furanyl)alanine (denoted as AFur) could be directly incorporated during SPPS (Figure 3A,B). Notably, the Diels-Alder cyclization reaction using AFur and KMal at previously established i, i+7 positions in the p53-derived sequence proceeded slowly on solid support, yielding a dominant linear species that could be purified by HPLC (Figure 3A,B). Incubation of the isolated linear peptide 5 in physiological PBS buffer, however, resulted in the rapid appearance of earlier-eluting cyclized species and a minor fraction of peptide dimers (Figure 3B). Michael-addition trapping experiments confirmed that the putatively cyclized compounds 5a, 5b and 5c lacked the reactive maleimide, which contrasted with the isolated linear peptide 5 (Figure 3C; Figure S7). Circular dichroism spectroscopy of the linear control p53 sequence and all three isolable cyclized species confirmed increased α-helical character for the dominant DAC peptide 5a relative to the minor products 5b and 5c (Figure 3D). These data are reminiscent of other studies reporting that stereochemical yield is correlated with global stability of the peptide fold41, here suggesting that the aqueous chemical environment may aid in the selection of favorable adducts that promote global helix stabilization.</p><!><p>To further explore the potential to stabilize diverse helical conformations, we synthesized a series of Diels-Alder stabilized, α-helical peptides derived from the co-activator protein SRC2. A short helical stretch within this protein contains a conserved LXXLL motif that engages a hydrophobic groove in estrogen receptor-α (ERα Figure 4A)42–44. We synthesized several i, i+4 'DAC-stapled' peptides, which were tolerant of direct 2-furanylalanine incorporation during SPPS. Unlike the p53-derived sequence, both SRC2 peptides 6 and 7 cyclized on resin rapidly and in high yield following maleimide incorporation (Figure 4B, Table S3). We characterized the two isolable cyclized species 6a and 6b, and the one dominant compound 7a by CD spectroscopy. The dominant species 6a and 7a were significantly more helical than either the unmodified wildtype SRC2 peptide or minor 6b compound (Figure 4C). Previous attempts to stabilize the SRC2 LXXLL motif have shown that hydrophobic residues that engage ERα can be replaced by a hydrocarbon staple without significant loss in binding affinity45. Therefore, we sought to test whether we could synthesize a double-stapled SRC2 ligand containing both a hydrocarbon staple installed by ring closing metathesis of olefin containing amino acids in concert with Diels-Alder cyclization of a diene-dienophile pair (Figure 4A,D). Direct incorporation of olefin-containing S5 amino acids and furan yielded a clean peptide precursor, 8pre, that was readily cyclized by Grubbs-I catalyst (8rcm; Figure 4D,E). Subsequent introduction of the maleimide dienophile resulted in immediate appearance of an earlier eluting tandem stapled peptide, which was the stoichiometric product after mild heating for 4 hr on resin (Figure 4E, Table S3). CD characterization of this bicyclic peptide 8a revealed classic α-helical minima at 208 and 222 nm (Figure 4F). 1H-NMR analysis confirmed the presence of Diels-Alder cycloadduct vinylic protons and a single allylic proton in all major DAC SRC2 peptide products, and, in the case of 8a, peaks corresponding to the RCM alkene product were observed (Figure S8, Supplementary Spectroscopic Data).</p><p>To probe the structure activity relationships of these DAC-SRC2 peptides we performed competitive fluorescence polarization binding assays with ERα. Both the single DAC-stapled 7a and tandem stapled 8a peptides competed a fluorescein-linked SRC2 peptide off of ERα with IC50s of approximately 0.5 and 2.5 μM respectively (Figure 4G, Table S4). Intriguingly, the dominant, more helical isomer 6a demonstrated 4-fold improved activity compared to the less helical, minor product 6b (Figure 4G, Table S4). These results echo those observed for the p53 sequence, where stereochemical yield is correlated with overall peptide fold stabilization, and in the case of SRC2 improved biochemical potency.</p><!><p>To determine the structure of the Diels-Alder adduct in 6a and its role in promoting the interaction with ERα, we solved the X-ray structure of 6a and estradiol co-crystallized with residues 300–500 of the ERα ligand binding domain (LBD) at 2.25 Å resolution (Figure 5A; Table S5). The Y537S mutant of ERα was used as it stabilizes the agonist conformation of the receptor and aids in crystal formation. An FO-Fc difference map using wild-type SRC2-bound ERα confirmed the unambiguous presence of estradiol bound in the core of the LBD, and DAC-stapled 6a bound in the canonical activating function 2 (AF2) cleft of ERα (Figure 5A; Figure S9A,B). The Diels-Alder adduct was highly ordered and permitted unequivocal confirmation that the more stabilizing and dominant stereochemical product in the context of SRC2 is that of the endo isomer (Figure 5B). The endo bicycle presents a convex hydrophobic surface that directly contacts ERα residues Val355, Ile358 and Leu539 that form the hydrophobic shelf adjacent to the cleft that binds the LXXLL motif (Figure 5C). Residues in 6a that are derived from Leu690 and Leu694 in SRC2 retained their canonical contacts and were deeply engaged with a network of hydrophobic residues in the AF2 groove (Figure 5C, bottom). Additional orienting contacts are mediated by His691 to the edge opposite the Diels-Alder adduct, as well as a conserved hydrogen bond between Glu542 of ERα that caps the N-terminus of the peptide helix (Figure S9C). At the C-terminus the last ordered residue present in the DAC-peptide structure, Q695, folds back on the peptide to satisfy a hydrogen bond to the i-2 amide carbonyl, effectively capping the helix (Figure S9C). Taken together, these structural data confirmed that the unique Diels-Alder chemical and stereochemical composition can stabilize an active helical conformation and directly contribute to target engagement, as evidenced here by forming a molecular "clasp" around the core and edge of the ERα AF2 cleft.</p><!><p>Backbone- and side chain-directed peptide cyclization strategies can effectively improve desirable properties for chemical probes or therapeutics. Our study establishes several emergent and improved capabilities offered by the incorporation of Diels-Alder cycloadditions to the toolbox of reactions used for peptide macrocyclization and stabilization. First, we demonstrated that a range of reactive diene-dienophile pairs can be applied to diverse peptide sequences in both organic and aqueous solvents. This opens up many possibilities to tailor the macrocyclization motif itself to improve not only the inherent structure of the peptide, but also physicochemical properties and target binding. This aspect was highlighted by our observation that the preferred endo DAC stapled isomer of SRC2 displays augmented helical stabilization and directly contributes to ERα binding. Specifically, the convex hydrophobic surface of the adduct presented by the endo isomer rests on a hydrophobic surface adjacent to the AF2 pocket. Future exploration of this interaction could probe whether alternative Diels-Alder adducts could be developed that contribute specific, polar contacts to the binding surface. This potential to access unique adduct structures and conformations is especially noteworthy given the physicochemical differences relative to existing, predominantly hydrophobic staples in use. Indeed, it is under-appreciated that while added hydrophobicity can improve target binding affinity, this may come at the expense of increased off-target interactions. The range of possible Diels-Alder cycloadducts may enable design of and selection for macrocycles that temper these contributions. Structural studies of both the stabilized loop and helical peptides confirmed that the expected endo Diels-Alder adduct was the major product in these contexts. Furthermore, we established that mild heating on-resin in organic solvents can drive the stereoselective formation of the endo isomer across diverse peptides. Based on the evidence here, we expect that this will be general for many peptides, however future studies are warranted to determine if there are conditions that will bias toward the formation of the exo isomer if desired.</p><p>Another compelling aspect of the DAC peptide strategy is that following installation of reactive diene and dienophile pairs, the macrocyclization reaction occurs spontaneously. This contrasts with many side-chain crosslinking approaches that require metal catalysts and the ensuing limitations on reaction conditions, cross-reactivity with other functional groups present in target peptides, and practical considerations of cost and reagent removal during synthesis and purification. Diels-Alder macrocyclizations are therefore essentially "reagentless," and are instead only impacted by the chemical environment and reaction conditions. This suggests cyclization or intramolecular crosslinking could be performed on native protein folds, or aid in proper folding, alone or in concert with other chemistries. As was seen for DAC-p53 peptide 5, an aqueous reaction environment not only promoted spontaneous cyclization, but also preferentially formed macrocyclic isomer 5a with the greatest helicity. Indeed, this implies the context specific contributions of different Diels-Alder adducts and even stereochemical products of unique reactive pairs could be screened for inherent stabilization and extrinsic effects on the activity of stabilized peptides.</p><p>A major goal in developing new strategies to chemically modify and stabilize peptides is to permit increased access to unique and larger peptide structures, including those containing multiple secondary structural elements. Our data suggests that the current and future Diels-Alder cyclization pairs will contribute to this work in two ways. First, the stabilization of larger peptide and proteomimetics may require properly folded structures in order to template ligation reactions or other chemical modifications. In these cases, the ability to operate directly in physiological environments, which we demonstrated here for both loop and helical DAC peptides, will make the Diels-Alder cyclization an attractive choice compared to other chemistries that cannot operate on unprotected peptides and proteins, or in aqueous environments. Another avenue to stabilize larger, conformationally complex peptide structures is to incorporate multiple stabilizing elements, but finding compatible biorthogonal chemistries that can operate in concert has remained a challenge. Here we demonstrated the facile double stapling of an SRC2 helical peptide that contained RCM and DAC stapling residues adjacent to one another. Despite this proximity, these two chemistries faithfully permitted dual macrocyclization directly on-resin. We envision that future exploration of Diels-Alder stabilization of secondary and tertiary structural elements with other natural and non-natural stabilizing elements will permit improved access to hyperstable peptide and protein structures. Toward this goal, we established Diels-Alder reactions between diverse diene-dienophile pairs, and future work is warranted to explore additional combinations that are tailored for specific functional group compatibility, secondary structures, reaction conditions and ligand physicochemical properties. In summary, we believe the application of Diels-Alder as a biocompatible protein chemistry offers abundant opportunities for peptide macrocycle and protein domain construction.</p>

PubMed Author Manuscript

Self-assembled molecular nanowires on prepatterned Ge(001) surfaces

It is a long-standing goal to fabricate conductive molecular nanowires (NWs) on semiconductor surfaces.Anchoring molecules to pre-patterned surface nanostructures is a practical approach to construct molecular NWs on semiconductor surfaces. Previously, well-ordered inorganic Ge NWs were deduced to spontaneously grow onto Pt/Ge(001) surfaces after annealing at an elevated temperature. In this work, we further demonstrate that organic 7,7,8,8-tetracyanoquinodimethane (TCNQ) molecular NWs can selfassemble onto the atomic NWs on Pt/Ge(001) surfaces. The outer nitrogen atoms in TCNQ molecules hybridize with under-coordinated Ge atoms in Ge NWs with an energy release of $1.14 eV per molecule, and electrons transfer from Ge NWs to the frontier orbitals of anchored TCNQs resulting in a negatively charged state. This largely tailors the electronic configurations of TCNQs and Pt/Ge(001) surfaces, enhancing the electron transport along the dimer row direction. The TCNQ molecular NWs coupled with the Ge NWs represent an exemplary showcase for the fabrication of molecular NWs on semiconductor surfaces.

self-assembled_molecular_nanowires_on_prepatterned_ge(001)_surfaces

Introduction<!>Methods<!>Results and discussion<!>Conclusions

<p>Molecular electronics have been attracting much attention due to the fact that commercially utilized silicon-based electronic devices are approaching their physical limits. [1][2][3] Organic molecules can be utilized to design molecular devices. [4][5][6] Onedimensional (1D) molecular nanowires (NWs) may serve as conductive channels for molecular devices. Therefore, the fabrication of molecular NWs on semiconductor surfaces becomes important for designing high-performance molecular electronics.</p><p>To date, various strategies have been proposed to fabricate molecular NWs on semiconductor substrates. 7 Among them, self-assembly may be the simplest yet effective approach. For example, the self-assembly of molecular NWs can occur on a predened position on Si(001) platforms via a radical chain reaction mechanism. 8 Molecules with terminal C^C, C]C, and C]O groups can easily react with the dangling bonds on silicon surfaces, where the dangling bonds could be created by the removal of H atoms on H-terminated silicon surfaces using an STM tip. These predesigned dangling bonds serve as a starting point for the subsequent radical chain propagation. The reactions involve the breakage of the double or triple bond, thereby resulting in a radical intermediate with a Si-C bond and a C-centred radical. Subsequently, the newly formed radical intermediate is capable of abstracting H atoms from its neighbouring Si atoms, producing a new dangling bond on the nearby Si atom, which continues to interact with another molecule. This series of reactions sets off a chain reaction on hydrogenated silicon surfaces, leading to the growth of molecular NWs on Si(001) and Si(111) surfaces. [8][9][10][11][12] From the examples stated above, we notice that dangling bonds play critical roles in the self-assembly of molecular NWs on semiconductor surfaces. The presence of dangling bonds is a prerequisite to fabricate covalently adsorbed molecular NWs onto semiconductor surfaces. It was reported that atomic NWs, extending for hundreds of nanometres, can be self-organized on Pt/Ge(001) surfaces. 13 These NWs are constructed of undercoordinated Ge atoms, possibly suggesting the presence of dangling bonds on NWs. In this regard, they are expected to be good platforms for fabricating molecular NWs, because the dangling bonds on Ge NWs on Pt/Ge(001) surfaces can serve as potential points for the assembly of molecular NWs. Moreover, an appropriate space should be provided to accommodate the molecular NWs. On the Pt/Ge(001) platform, Ge NWs are spaced by either 1.6 nm or 2.4 nm. In our experimental observations, we indeed found that 7,7,8,8-tetracyanoquinodimethane (TCNQ) molecular NWs can be self-organized on Ge NWs on Pt/ Ge(001) surfaces. This observation of TCNQ molecular NWs is an exemplary showcase for the fabrication of molecular NWs on semiconducting surfaces.</p><!><p>Low-temperature scanning tunnelling microscopy (LT-STM) characterization was carried out in a custom-designed Unisoku LT-STM system with the base pressure maintained in a low 10 À10 Torr range at 77 K, using the constant current mode with a commercial Pt-Ir tip. The bias voltages were applied to the sample. Ge(001) was cut from one-side-polished n-type wafers (commercially available from AXT Inc.). Samples were ultrasonically cleaned in propanol for 15 min several times and then dried under nitrogen gas. Subsequently, the prepared sample was xed onto a Mo sample holder. Pristine Ge(001) surfaces with c(4 Â 2) and p(2Â 2) periodicities were prepared through several cycles of 500 eV Ar + ion sputtering and annealing at around 1100 K. Pt atoms were then resistively evaporated onto dimerized Ge(001) surfaces at about 873 K for 10 min. TCNQ molecules (98%, Aldrich) were deposited on Pt/Ge(001) surfaces at 300 K using a Knudsen cell at 393 K. The calculation details are presented in the ESI. † [14][15][16][17][18][19][20][21][22][23][24][25]</p><!><p>Noble metals (i.e., Au and Pt) are known to trigger the selfassembly of atomic NWs on Ge(001) surfaces. 13,[26][27][28][29] DFT calculations revealed that self-organized NWs on Pt/Ge(001) surfaces are constituted by under-coordinated Ge atoms (Fig. 1b, S1a and b †), suggesting the presence of dangling bonds on the NWs. 30 As aforementioned, dangling bonds on semiconductor surfaces can capture molecules by chemically bonding with them, and then setting off the self-assembly of molecular NWs on the semiconductor surfaces. TCNQ molecules, as archetypal electron acceptors, are widely utilized to prepare charge transfer complexes, showing potential in engineering molecular electronics. [31][32][33][34] The electronic properties of TCNQ are mainly determined by the population of the lowest unoccupied molecular orbital (LUMO) (Fig. 1f). Here, the cyano groups in TCNQ are expected to chemically react with under-coordinated Ge atoms in the NWs on Pt/Ge(001) surfaces, abstracting electrons from the Ge atoms in the NWs. Moreover, Fig. 1e indicates that TCNQ is a planar molecule with a width of 4.0 Å and a length of 8.3 Å. The Ge NWs in Fig. 1a are spaced by 1.6 or 2.4 nm, providing adequate room to accommodate TCNQs. The Ge dimers in the NWs are spaced by 5.4 Å, further suggesting that the outer nitrogen atoms, separated by 4.0 Å (d N1-N2 ¼ d N3-N4 ¼4.0 Å), in a TCNQ molecule possibly react with Ge atoms. As expected, TCNQ molecular NWs were experimentally observed alongside the Ge NWs on Pt/Ge(001) surfaces (see Fig. 1c and d). Furthermore, individual TCNQ molecules can adsorb in another orientation, marked with white dotted rectangles in Fig. 1d, perpendicular to the TCNQ molecular NWs.</p><p>Fig. 2a shows that the TCNQs anchor onto the Ge NWs with their cyano groups located above the Ge NWs, generating a at adsorption geometry with respect to the Pt/Ge(001) surface, and Ge NWs could be observed adjacently about 1.6 nm away. DFT calculations indicate that TCNQ molecules prefer to chemically bond to the Ge NWs instead of physisorption. The calculation results in Fig. 2b suggest that the nitrogen atoms on the le side of the TCNQ (i.e., N1 and N2) form covalent bonds with the Ge1 atoms in NWs, and the remaining nitrogen atoms (i.e., N3 and N4) on the right side (see the blue arrow in Fig. 2c) are situated in the trench between two Ge NWs. The formation of Ge-N bonds causes the rupture of the Ge1-Ge2 dimer in Ge NWs. The Ge1 atom chemically interacts with the N1 and N2 atoms, and the Ge2 atom, marked with dotted circles, moves downwards to react with Pt atoms. No covalent bond is formed between the Ge2 atom and TCNQ molecule. Furthermore, the N3 and N4 atoms, indicated with a blue arrow, slightly move downwards due to the presence of dangling bonds of the Ge atoms (red circles in Fig. 2c) near Pt atoms. The conjugated p-system extending across the TCNQ makes it a planar and rigid molecule in the gas-phase state. The adsorbed TCNQ becomes exible if electrons are transferred to the cyano groups since the peripheral carbon atoms of TCNQ turn into the nonplanar sp 3 hybridization. 35 Moreover, the LUMO of a TCNQ is constructed of antibonding p orbitals gathering around the double bonds in TCNQ; therefore, the electron lling of the LUMO can weaken these double bonds and make TCNQ exible. 35 The negatively charged TCNQ molecular NWs are no longer rigidly planar, accounting for the bending of anchored TCNQs.</p><p>The chemical adsorption of TCNQs onto the Ge NWs on Pt/ Ge(001) surfaces is an exothermic reaction with an energy release of 1.14 eV per molecule. Energy variation during this process is calculated using the formula: E ads ¼ E[TCNQ/Ge(001)] À E(TCNQ) À E[Ge(001)], where E[TCNQ/Ge(001)], E(TCNQ) and E[Ge(001)] are the energies of the anchored TCNQ on Pt/Ge(001) surfaces, the isolated TCNQ molecule, and the Pt/Ge(001) surfaces, respectively.</p><p>The DFT-simulated occupied state STM in Fig. 2d presents well-ordered TCNQ molecular NWs propagating along the Ge NWs on Pt/Ge(001) surfaces. Two TCNQs are 8.1 Å apart alongside NWs, double the distance of the neighbour Ge dimers on Ge(001)c(4 Â 2) surfaces, which is consistent with the experimental value of $8.1 Å (Fig. 2a). As aforementioned, the TCNQ chemisorption leads to the Ge atom rearrangement in Ge NWs. Given that the Ge NWs near the TCNQ NWs (see Fig. 2a) are not fully occupied in the experiment, the slight rotation of anchored TCNQs in experiment may be attributed to the effect from the nearest neighbour Ge NWs. Moreover, a single TCNQ can be adsorbed on either side of Ge NWs; however, TNCQs in Fig. 2a appear on the same side. The nearest distance between two TCNQs is about 3.5 Å, suggesting a pronounced van der Waals interaction responsible for the low entropy conguration.</p><p>To further investigate the charge transfer between the TCNQ molecular NWs and Ge NWs, the band structures and charge density differences are illustrated in Fig. 3. Fig. 3a shows the band structure of the dimerized Ge(001) surface (see the atomic structure in Fig. S1e and f †), and this surface is semiconducting with a bandgap around the Fermi level, E F . The red bands in Fig. 3b refer to the Pt/Ge(001) substrates, and the navy blue energy levels correspond to isolated TCNQs (see the atomic structure in Fig. S1c and d †). One can note that the Pt modied Ge(001) surfaces behave as a metal since the S1 and S2 bands pass through the Fermi level (Fig. 3b), and the energy band dispersions perpendicular to the Ge dimer row direction are weaker than the bands parallel to the Ge dimer row direction, indicating that the NWs are more conducting. Moreover, compared to S1 and S2, the bands S3 and S4 across the Fermi level are more dispersed around the Fermi level aer being chemically bonded with TCNQ molecular NWs, as displayed in Fig. 3c. More dispersive bands typically suggest smaller effective masses and higher carrier mobility; therefore, the conductivity along the NW direction is enhanced due to the presence of the TCNQ molecular NWs. Additionally, no band passes through the Fermi level along the X-S and Y-G paths perpendicular to the direction of NWs, possibly suggesting the suppression of conductivity perpendicular to the TCNQ molecular NWs. Moreover, the TCNQ molecular energy level in Fig. 3b is isolated without the chemical interactions between TCNQs and the Pt/Ge(001) surfaces (see the structural model in Fig. S1c and d †). The molecular energy level of TCNQ in close vicinity to the Fermi level is considered as the LUMO. It is mentioned in Fig. 2b that the Ge dimers of NWs on the Pt/Ge(001) surfaces are rearranged aer the formation of TCNQ molecular NWs. Therefore, compared to Fig. 3b, the electronic band structures of TCNQ molecular NWs (Fig. 3c) are largely modied. The pronounced dispersions of the HOMO and LUMO of TCNQs reect hybridization between TCNQ and Pt/ Ge(001) surfaces (Fig. S2 †). In particular, the LUMO of TCNQ presents a concentrated distribution from 0.1 to 0.5 eV (Fig. 3c) below the Fermi level aer chemically reacting with Ge NWs on Pt/Ge(001) surfaces, suggesting substantial electron charge transfer from Ge NWs to TCNQs. This charge transfer is further visualized in Fig. 3d. The Bader charge analysis suggests that each Ge in Ge-N bonds donates about 0.6 electrons to the anchored TCNQ. Notably, the charge transfer occurs between Ge NWs and TCNQs and between the Ge atoms near the Pt arrays and TCNQs indicated by the black arrows in Fig. 3e. The electron transfer of the Ge atoms near the Pt arrays should be partially responsible for the bending of anchored TCNQs on Ge NWs because of the Coulomb interactions. In general, the Bader charge analysis indicates the transfer of a total of 1.1 electrons from the substrate to each anchored TCNQ. A closer inspection of the whole charge transfer pattern in Fig. 3d indicates that the electron accumulated region resembles the LUMO of TCNQs (Fig. 1f). This further conrms the electron transfer to the LUMO of TCNQs, generating negatively charged TCNQ molecular NWs. To sum up, the adsorption of TCNQ molecular NWs causes pronounced alterations in the electronic and geometrical congurations of both TCNQs and Pt/Ge(001) surfaces.</p><!><p>In this work, the deposition of TCNQs onto Ge NWs on Pt/ Ge(001) surfaces at room temperature results in the selfassembly of TCNQ molecular NWs. The Ge NWs can serve as fences on Pt/Ge(001) surfaces, providing adequate space for the growth of TCNQ molecular NWs. The cyano groups in TCNQs can chemically react with the under-coordinated Ge NWs, generating nearly at TCNQ molecular NWs along the Ge NWs. Electrons transfer from the Ge NWs to the TCNQs, making the TNCQ molecular NWs negatively charged. Our work not only provides an effective approach for fabricating molecular NWs on prepatterned semiconductor surfaces but also offers important insights into tailoring the surface properties of semiconductor surfaces.</p>

Royal Society of Chemistry (RSC)

Synthesis of Enantioenriched Allylic Silanes via Nickel-Catalyzed Reductive Cross-Coupling

An asymmetric Ni-catalyzed reductive cross-coupling has been developed to prepare enantioenriched allylic silanes. This enantioselective reductive alkenylation proceeds under mild conditions and exhibits good functional group tolerance. The chiral allylic silanes prepared here undergo a variety of stereospecific transformations, including intramolecular Hosomi-Sakurai reactions, to set vicinal stereogenic centers with excellent transfer of chirality.

synthesis_of_enantioenriched_allylic_silanes_via_nickel-catalyzed_reductive_cross-coupling

<p>Organosilanes are valuable organic materials with applications in medicinal chemistry,1 materials science,2 and as reagents for organic synthesis.3 In particular, chiral allylic silanes are versatile synthetic reagents that engage in a variety of highly stereoselective reactions. For example, the Hosomi-Sakurai reaction4 is a powerful method for C–C bond formation that provides homoallylic alcohols with excellent transfer of chirality when enantioenriched allylic silanes are used.5,6 Despite the utility of this and related transformations, the enantioselective preparation of chiral allylic silanes often requires multistep sequences, or the incorporation of specific functional groups to direct the formation of the C(sp3)-Si bond. Here we describe a Ni-catalyzed asymmetric reductive cross-coupling to directly prepare enantioenriched allylic silanes from simple, readily available building blocks (Figure 1). The resulting chiral allylic silanes undergo a variety of post-coupling transformations with high levels of chirality transfer.</p><p>Chiral allylic silanes are most commonly prepared through diastereoselective or stereospecific transformations,7 which include the Claisen rearrangement of vinyl silanes,8 bis-silylation of allylic alcohols,9 silylene insertion of allylic ethers,10 and the alkenylation of 1,1-silaboronates.11 In addition, several enantioselective transition metal-catalyzed reactions have been developed, including the hydrosilylation of dienes,12 the silylboration of allenes13, the insertion of metal carbenoids into Si-H bonds,14 and conjugate addition15 and allylic substitution16 reactions. Asymmetric transition metal-catalyzed cross-coupling, in which the critical silicon-bearing C(sp3) stereogenic center is established in the C–C bond forming step, represents an alternative and highly modular approach to chiral allylic silanes. Indeed, the first synthesis of an enantioenriched chiral allylic silane was the Pd-catalyzed asymmetric cross-coupling between α-(trimethylsilyl)benzylmagnesium bromide and 1-bromo-1-propene reported by Kumada and coworkers in 1982.5a However, this method employs Grignard reagents as coupling partners, which are not stable to long-term storage and decreases the functional group compatibility of the reaction. We envisioned that a Ni-catalyzed asymmetric reductive alkenylation would address this limitation,17 in that the required (chlorobenzyl)silanes are bench stable compounds and these reactions typically exhibit good functional group tolerance. Thus, a Ni-catalyzed reductive alkenylation could provide chiral allylic silanes that were not readily accessible by other methods.</p><p>Our investigations began with the coupling between (E)-1-(2-bromovinyl)-4-methoxybenzene (1) and (chloro(phenyl)methyl)trimethylsilane (2a) using chiral bis(oxazoline) ligand L1, which was optimal in our previously developed enantioselective reductive alkenylation reaction (Table 1).14 A screen of reaction parameters revealed that when the reaction is conducted at 5 °C with with N-methyl-2-pyrrolidone (NMP) as the solvent,18 allylic silane 3a was formed in low yield, but with high enantioselectivity (entry 1). We hypothesized that the presence of the bulky silyl group impeded the oxidative addition of 2a to the Ni catalyst. The addition of cobalt(II) phthalocyanine (CoPc), a cocatalyst that enables the Ni-catalyzed cross-coupling of benzyl mesylates by facilitating alkyl radical generation,19 doubled the yield of 3a (entry 3). The yield of 3a increased when excess vinyl bromide was used (entries 3–5); since the use of 2.0 equiv 1 proved most generally robust across a range of substrates, these conditions were used to evaluate the scope of the reaction (see Tables 2 and 3). A screen of other bis(oxazoline) ligands, e.g. L2 and L3, determined that L1 provided the highest enantioselectivity (entries 6–7). The use of isolated complex L1·NiCl2 gave 3a in comparable yield to the in situ generated catalyst (entry 8). Control experiments confirmed that NiCl2(dme), ligand, and Mn0 are required to form 3a.15 To demonstrate scalability, the cross-coupling was conducted on a 6.0 mmol scale, delivering 1.3 g of 3a in 74% yield and 97% ee.</p><p> </p><p>With the optimized conditions in hand, the scope of the silane was investigated (Table 2). Whereas strained silacy-clobutane20 3b was prepared in good yield and excellent ee, the corresponding triethylsilane 3d was formed in poor yield, presumably due to the increased steric encumbrance at silicon. Substrates bearing either electron-withdrawing or electron-donating groups on the arene cross-coupled with universally high ee; however, in some cases the yield was diminished due to instability of the products (3e, 3g). The presence of an ortho substituent on the arene also decreased the yield of the cross-coupling product (3j).</p><p>The reaction tolerates a diverse array of functional groups on the alkenyl bromide partner (Table 3),21 including aryl boronates (6c), esters (6b, 6h), imides (6m), amides (6n), alkenyl silanes (6o), and alkyl halides (6f). For greasy substrates, m-methoxy silane 2h was used as the coupling partner to facilitate product purification (6o–6u). Alkyl-substituted alkenyl bromides performed comparably to styrenyl bromides; however, a limitation of the reaction is that Z-alkenes and tri- and tetra-substituted alkenyl bromides failed to react. By changing which enantiomer of L1 is employed, diastereomeric polyenes 6t and 6u were prepared, although the yield is decreased with the mismatched (3S,8R)-L1 catalyst. Finally, alkenyl bromides bearing furan (6q), thiophene (6r), pyridine (6k), pyrimidine (6l), and indole (6e) heterocycles could be cross-coupled, giving the corresponding allylic silanes in high ee. Functional groups that were not well tolerated include aldehydes and nitriles.22</p><p>Although halide electrophiles were the primary focus of this study, oxygen-based electrophiles were also evaluated. We were pleased to find that mesylate 7 provided 3a in 45% yield and 92% ee; the lower yield was due to incomplete conversion of the starting material (Scheme 1a). Enol triflate 8 underwent cross-coupling to afford 6a in 57% yield, again with excellent enantioselectivity (Scheme 1b). Although the yields were modest, we note that these reactions were conducted under the conditions developed for the organic halides with minimal re-optimization.</p><p>This reductive cross-coupling provides rapid access to functionalized chiral allylic silanes that are useful in a variety of synthetic transformations.23,24 For example, allylic silanes 6i and 6j, which contain pendant acetals, undergo stereospecific TiCl4-mediated intramolecular cyclization to form the 5- and 6-membered rings 9 and 10, respectively (Scheme 2a). The observed absolute and relative stereochemistry is consistent with an anti-SE' mode of addition, which gives rise to the trans-substituted 5-membered ring and the cis-substituted 6-membered ring.25,26,27 Either the 2,3-cis or 2,3-trans tetrahydrofurans can be prepared by Lewis-acid mediated cyclizations of alcohol 6g or chloride 6f, respectively; both proceed with excellent transfer of chirality (Scheme 2b).5b,28 The utility of the method was further demonstrated in a concise enantioselective synthesis of (+)-tashiromine (Scheme 2c).29 Sodium borohydride reduction of imide 6m provided aminal 13, which upon exposure to formic acidcyclized to form bicycle 14 in 93% yield as a 3.8:1 mixture of diastereomers.30 The major diastereomer was isolated in 57% yield and 97% ee. Ozonolysis and reduction of the amide provided (+)-tashiromine.</p><p>In summary, a highly enantioselective cross-coupling reaction has been developed for the preparation of chiral allylic silanes. The reactions proceed under mild conditions and tolerate a variety of functional groups. The enantioenriched allylic silanes undergo several stereospecific transformations with high transfer of chirality, which we anticipate will prove useful in an array of synthetic contexts.</p><p> ASSOCIATED CONTENT </p><p>Experimental procedures, characterization and spectral data for all compounds, and crystallographic data (CIF). This material is available free of charge via the Internet at http://pubs.acs.org.</p>

PubMed Author Manuscript

Au(<scp>iii</scp>)-aryl intermediates in oxidant-free C–N and C–O cross-coupling catalysis

Au(III)-aryl species have been unequivocally identified as reactive intermediates in oxidant-free C-O and C-N cross coupling catalysis. The crystal structures of cyclometalated neutral and cationic Au(III) species are described and their key role in 2 electron-redox Au(I)/Au(III) catalysis in C-O and C-N cross couplings is shown. Nucleophiles compatible with Au-catalyzed cross couplings include aromatic and aliphatic alcohols and amines, as well as water and amides.

au(<scp>iii</scp>)-aryl_intermediates_in_oxidant-free_c–n_and_c–o_cross-coupling_catalysis

Introduction<!>Results and discussion<!>Conclusions

<p>In the last few years, the use of gold in homogeneous catalysis has experienced increasing attention and progress. Although typically regarded as superior Lewis acids for the activation of multiple C-C bonds towards nucleophiles, 1 gold complexes have recently found new patterns of reactivity, namely 2-electron redox processes applied to cross-coupling transformations. 2 In spite of the growing number of examples in this eld, the access to key Au(III) intermediates has been limited to harsh sacricial oxidants, such as I 3+ derivatives or F + sources (Scheme 1a), 3 as well as using highly electrophilic aryldiazonium salts under photoredox conditions or via light-driven radical chain reactions. 4 On the other hand, the straightforward pathway through the oxidative addition of C sp 2 -X and C sp 3 -X bonds (X ¼ halide) to Au(I) has generally been regarded as highly reluctant, 5 markedly differing from other transitionmetal chemistry. 6 However, recent in-depth organometallic investigations have dismissed this conception with remarkable achievements. The rst evidence for the intramolecular oxidative addition of aryl halides to gold(I) complexes was disclosed by the Bourissou group in 2014,7 showing that phosphinechelation assistance is key to delivering the C sp 2 -X bond in close proximity to the gold center, thus promoting the oxidative addition even at room temperature for X ¼ I. The same group later took advantage of the ability of carborane diphosphines to chelate gold(I) with small P-Au-P bite angles, which render a preorganized architecture closer in energy to the ensuing square-planar geometry of the oxidative addition product. By means of this strategy, intermolecular oxidative addition of aryl iodides and strained C-C bonds under mild conditions was accomplished. 8 Also in 2014, the Toste group substantiated the ability of gold to perform the elementary steps of organometallic cross-coupling chemistry, including oxidative addition, with the rst example of a Au(I)-catalyzed C-C bond formation without the requirement of external oxidants. 9 A tethered Au(I) aryl complex featuring an allyl bromide moiety allowed to support this mechanism via oxidative addition under intramolecular conditions (Scheme 1b).</p><p>In our group we envisioned that a coordinating environment could favor the oxidative addition of Au(I) salts to aryl halides and the following reactivity towards nucleophiles. This strategy, based on attaching a macrocyclic appendage to aryl halide substrates bearing three available nitrogen coordination sites, had actually proved extremely benecial in the isolation and exhaustive characterization of square pyramidal aryl-Cu(III) and aryl-Ag(III) complexes in the context of 2e À redox M(I)/M(III) cross coupling catalysis. 10 Hence, using this approach we were able to describe the rst oxidant-free gold(I)-catalyzed halide exchange and C sp 2 -O bond forming reactions (Scheme 1c), additionally transferring this novel chemistry to more easily available substrates such as 2-(2-halophenyl)pyridines. 11 In this work we present the expansion of the nucleophile scope from halides and phenols to amines and amides, which stands as the rst example of a gold(I)-catalyzed C-N bond formation, resembling the well-known Cu-based Ullmanntype 12 or Pd-based Buchwald-Hartwig cross-coupling catalysis. 13 Furthermore, the straightforward synthesis and crystallographic characterization of the hitherto new (N,C)-cyclometalated Au(III) complexes 3a and 3b via the oxidative addition of a C sp 2 -I bond is also herein described. Their competency as intermediate species in catalytic C-O and C-N couplings is also demonstrated, therefore conrming the redox Au(I)/Au(III) mechanistic cycle previously postulated.</p><!><p>In the course of our investigations into the oxidant-free gold(I)catalyzed halogen exchange we found that the applicability of the system could be extended to C-O coupling reactions using sodium p-chlorophenolate and, interestingly, sodium methoxide. The latter required its conjugate acid (MeOH) as solvent to proceed, presumably due to the low solubility of the salt in CH 3 CN. This prompted us to study the related transformations with other alkoxides and protic solvents. In a similar manner, EtONa was reacted with 2-(2-bromophenyl)pyridine 1a-I in ethanol (110 C, [Au(NCMe)IPr]SbF 6 as catalyst) and aer 24 h a moderate 56% yield of the desired coupling product 1ac was obtained, which could be increased up to 78% aer 48 h (Table 1, entry 6). This can be attributed both to the lower acidity of EtOH compared to MeOH and the larger steric hindrance of the ethoxide. The preference for smaller and more acidic alkoxides was conrmed by carrying out the reaction with MeONa in EtOH, whereby 1ab was the major product. This trend could be further extrapolated to more sterically demanding alkoxides such as 2-propoxide (1ad) and tert-butoxide (1ae) giving yields of 21% and 4%, respectively (Table 1, entries 7 and 8).</p><p>The use of sodium hydroxide as a nucleophile (in H 2 O solvent) deserves a special mention. Although in terms of reactivity one might anticipate a similar behavior to sodium methoxide in MeOH (Table 1, entry 3) to readily provide 2-(pyridin-2-yl)phenol 1af, this was only formed in a 34% yield (Table 1, entry 9). On the contrary, the diaryl ether homocoupling product 1ag was the major product (26% yield). A simple explanation for this outcome involves the deprotonation in basic media of the early-stage generated phenol, which subsequently acts as the preferred nucleophile in the coupling reaction with 1a-Br starting material. It should be noted that this transformation using NaOH as a base was performed in aqueous media, in the absence of a phase-transfer reagent and without the addition of a co-solvent. In an effort to gain a better understanding of this reactivity, we carried out the same reaction using NaOMe instead of NaOH, and the yield of phenol 1af increased up to 52% (Table 1, entry 10). These results suggest that control over the selectivity and yield of each product might be achieved by the appropriate modication of the reaction conditions. Notably, the combination of sodium hydroxide and methanol led to the complete formation of the methoxy insertion product 1ab (Table 1, entry 2).</p><p>Overall, we have shown that steric and pH effects play a crucial role in the Au(I)-catalyzed ether and phenol formation, with smaller and more basic alkoxides leading to better results. This methodology allows for the synthesis of 2-(pyridin-2-yl) phenol 1af in water and 2-(2-methoxyphenyl)pyridine 1ab and 2-(2-ethoxyphenyl)pyridine 1ac in moderate to excellent yields, and represents the rst example of the Au-catalyzed crosscoupling of aliphatic alcohols to aryl halides. Moreover, gold promotes the coupling of water to form phenols, and the coupling of linear aliphatic alcohols to form ethers, thus presenting a complementary methodology to the Cu-based C-O cross-couplings for these challenging nucleophiles. 14 In light of these promising results, we then sought to expand the Au-catalyzed C-heteroatom reaction scope towards C sp 2 -N bond formation through the combination of aryl halides and N-nucleophiles, typically relying on two different approaches: (a) Cu-catalyzed Ullmann-type and (b) Pd-catalyzed Buchwald-Hartwig coupling reactions. As far as we know, this reactivity has only been successfully transferred to nickel. 15 We initially selected p-nitroaniline, given its major acidity compared to other amines (pK a ¼ 20.9 in DMSO), as the nucleophile to be reacted with 1a-Br in the presence of 10 mol% [Au(NCMe)-IPr](SbF 6 ) at 110 C (Table S1 †). Regardless of the base used, in all cases the starting material was recovered. Thus, aerwards, optimization of the solvent was investigated, whereby we obtained a signicant and encouraging 39% yield of the desired product in DMSO and with KOt-Bu as a base. The same conditions employing the more reactive 1a-I substrate resulted in almost quantitative yields. The nal optimization set the preferred experimental conditions at 110 C, 24 h and an excess of the nucleophile and 3 equivalents of base for further substrate scoping. It is worth pointing out the high-performance of KOt-Bu as a base without competing with p-nitroaniline as the nucleophile in the coupling reaction, as foreseen from the C-O bond forming results (Table 1, entry 8). Detailed NMR and X-ray crystallography analysis of the isolated product conrmed the expected structure. Remarkably, a gold-free blank experiment was performed and the reaction did not proceed (0% yield). The sluggishness of the reaction when 1a-Br is used instead of 1a-I is in good agreement with a rate-limiting oxidative addition step.</p><p>Then the scope of the optimized protocol was examined using different amines and amides. Cyclic aromatic and aliphatic amines and amides (imidazole and 2-hydroxypyridine, Scheme 2, products 1am and 1ai) afforded the best results, most likely owing to their superior acidity (pK a ¼ 18.6 and 17.0 in DMSO, respectively). 16 Good outcomes were obtained for primary aliphatic amines (Scheme 2, products 1ak and 1al), while secondary amines were less prone to arylation, with moderate and low yields for piperidine (1ap) and diethylamine (1aq), respectively. Likewise, benzamide was arylated in a moderate yield (59%, 1aj). Essentially, acidity and steric hindrance seem to be the basis of the observed reactivity trend for the aryl iodide 1a-I. We then turned our attention to the electronic properties of the para-substituents on the aniline ring. In contrast to the excellent results provided by p-nitroaniline, aniline did not exceed 50% yield, while p-methoxyaniline was poorly arylated (17%, 1ao). This observation, together with the data collected for the other N-nucleophiles, indicates sensitivity to less acidic and sterically hindered substrates, which is translated into a decrease in yields. In this system, the Au-based methodology for the arylation of aliphatic amines is superior to Cu-based Ullmann-type couplings. 14 C-N cyclometalated gold(III) complexes have been the subject of deep investigation, ever since the rst report of neutral AuCl 2 (ppy) containing a 2-phenylpyridine-type ligand by Constable and co-workers. 17 This family of complexes has shown potential anticancer activity 18 and photophysical properties, 19 and their use has beneted from their tolerance to both air and water. The methods to prepare them require either transmetallation from toxic organomercury derivatives or formal C-H auration with gold(III) tri or tetrahalide salts. 20 Furthermore, an alternative approach, employing aryldiazonium salts and Au(I) complexes under visible light photoredox conditions, has been developed very recently. 4c Nevertheless, since their synthesis had not been previously realized via the oxidative addition of C sp 2-X bonds, we deemed it worthy to investigate this possibility using the 2-(2-halophenyl)pyridine substrates.</p><p>Inspired by the work of Bourissou and co-workers, 7 we rst reacted 2-(2-iodophenyl)pyridine 1a-I and AuI in dichloromethane at room temperature. However, the complete recovery of the starting materials was observed, even aer changing the solvent to toluene and o-xylene and heating up to 130 C. A cationic [Au(NCCH 3 )IPr]SbF 6 gold(I) source gave identical results, regardless of the reaction conditions. This observation suggests the equilibrium displacement towards more stable reagents (see the ESI, † Section 1.6, for details). To tackle the aforementioned difficulties, we envisaged to block the rotation of the pyridine chelating group by incorporating an extra ring into the substrate, aiming at enhancing the stability of the desired product. Consequently, 10-iodobenzo[h]quinolone 2a-I was prepared following a two-step procedure starting from benzo[h]quinolone (see the ESI †). Gratifyingly, oxidative addition of the C Ar -I bond proceeded readily with AuI at 60 C for 18 h to give the cyclometalated Au(III) complex 3a as a red powder (Scheme 3). Complex 3a withstands air and water indenitely and is only soluble in CH 2 Cl 2 , CHCl 3 and DMSO. X-ray-quality crystals were obtained by gently stirring 2a-I and AuI in CH 2 Cl 2 at room temperature for 4 days, followed by slow evaporation of the solvent. The solid-state structure of the complex displays a Au-C bond length of 2.055( 7 With neutral 3a in hand, we next investigated the abstraction of iodide using 1 equivalent of AgSbF 6 in order to generate a more soluble and reactive cationic complex (Scheme 4). The reaction was carried out in acetonitrile due to its coordinating properties, but no changes were detected aer vigorously stirring for 3 hours at room temperature. At this point we hypothesized that the medium-strength Lewis base character of acetonitrile cannot efficiently stabilize a three-coordinate gold(III) species, and consequently a stronger Lewis base was employed. Upon the addition of pyridine (1.1 equiv.), 3a was immediately consumed and the solution turned bright yellow Scheme 2 Substrate scope of the C-N bond forming reactions. with the formation of an abundant grey precipitate (AgI). Complex 3b was isolated as an orange powder aer ltration and solvent removal (91% yield). 3b is also air-and moisturestable and can be stored on the benchtop without noticeable decomposition. The ESI-HRMS mass spectrum of this complex shows one major peak at m/z 580.9782 corresponding to the mass of [(2a)AuI(Py)] + , the cationic fragment of 3b. Lowtemperature 1 H-NMR (248 K) allowed the structural characterization of 3b in solution (see the ESI †). Crystals of this complex, grown from the slow diffusion of diethyl ether in a CH 3 CN solution of the compound, conrmed the removal of the iodide trans to the Au-C bond, as expected from the larger trans effect (Scheme 4).</p><p>In our previous studies on gold(I)-catalyzed halogen exchange and C-O bond formation we proposed a mechanism operating through the general two-electron-based Au(I)/Au(III) cycle (Scheme 6), 11 albeit the high-valent [Au(2a)(IPr)X] (X ¼ halide or phenolate) species could not be detected. In this regard we reasoned that the cationic Au(III) complex 3b might be helpful in trying to unveil the involvement of aryl-Au(III) species in this oxidant-free transformation. First, 1 equivalent of complex 3b was subjected to the same conditions as used for the p-chlorophenolate insertion with 2-(2-bromophenyl)pyridine 1a-I and the desired 10-(4-chlorophenoxy)benzo[h]quinoline 2aa was obtained in 86% yield (Scheme 5a), validating the feasibility of aryl-Au(III) species in Au(I)-catalyzed cross couplings. Nonetheless, an analogous experiment starting from 10-bromobenzo[h]quinolone 2a-Br and catalytic amounts of 3b (10 mol%) provided almost 2 catalytic turnovers (16% yield of product 2aa, Scheme 5b). Therefore, 10 mol% of the N-heterocyclic carbene donor ligand IPr$ (IPr ¼ 1,3-bis(diisopropylphenyl)imidazol-2-ylidene) employed in the catalytic transformations was also added, and we were pleased to nd that virtually quantitative yields of 2aa were achieved (Scheme 5b). Equal behavior was found with the p-nitroaniline insertion to 2a-I (Scheme 5c), overall unequivocally substantiating the implication of aryl-Au(III) species as competent catalytic intermediates. 3b rapidly generates the neutral complex [Au(2a)(Nuc)X] (X ¼ Br, I) in the presence of a nucleophile (Scheme 6). Then, the IPr$ carbene exchanges one of the anionic ligands to form the less favored [Au(2a)(IPr)Nuc] intermediate, which rapidly reductively eliminates the crosscoupling product (2ax) and gold(I) as [Au(X)IPr]. 11 Strikingly, 1 H-NMR inspection of the crude product proved that all the Au(I) remained in solution in this resting state form. On the other hand, attempts to isolate the [Au(2a)(IPr)Nuc] species generated in solution when IPr$ is added were fruitless, supporting their short-lived nature in the catalytic cycle.</p><!><p>In conclusion, we have developed the rst examples of gold(I)catalyzed C-N cross-coupling reactions in the absence of sacricial oxidants, in parallel to the well-established Cu-and Pd-catalyzed methodologies, and extended the previously described C-O coupling catalysis with phenols to aliphatic alcohols and water. This system allows entry to different arylamine, arylamide, phenol and aryl-ether products under practical synthetic laboratory conditions, with absolute tolerance for both air and water. In either case, the acidity of the nucleophile is at the basis of the reactivity observed. Moreover, we have synthesized novel neutral and cationic C-N cyclometalated Au(III) complexes through mild oxidative addition of a C sp 2 -I bond to gold(I) iodide, and presented conclusive evidence of their competence in the C-O and C-N coupling transformations. To the best of our knowledge, this represents the rst example in which the intermediacy of Au(III) species in an oxidant-free 2-electron coupling processes is demonstrated, clarifying the proposed mechanism operating via oxidative addition and reductive elimination steps. Future work is directed towards investigating other suitable chelating groups for a more versatile system, with special interest in removable directing groups.</p>

Royal Society of Chemistry (RSC)

Speeding up biomolecular interactions by molecular sledding

Numerous biological processes involve association of a protein with its binding partner, an event that is preceded by a diffusion-mediated search bringing the two partners together. Often hindered by crowding in biologically relevant environments, three-dimensional diffusion can be slow and result in long bimolecular association times. Similarly, the initial association step between two binding partners often represents a rate-limiting step in biotechnologically relevant reactions. We demonstrate the practical use of an 11-a.a. DNA-interacting peptide derived from adenovirus to reduce the dimensionality of diffusional search processes and speed up associations between biological macromolecules. We functionalize binding partners with the peptide and demonstrate that the ability of the peptide to onedimensionally diffuse along DNA results in a 20-fold reduction in reaction time. We also show that modifying PCR primers with the peptide sled enables significant acceleration of standard PCR reactions.

speeding_up_biomolecular_interactions_by_molecular_sledding

Introduction<!>Results and discussion

<p>The crowded intracellular environment presents many challenges for basic molecular processes to occur. Non-specic interactions between proteins hinder diffusional mobility and increase the time needed for binding partners to nd each other and associate. 1 Nature displays several examples in which the dimensionality of search processes is reduced to speed up association times. 2 For example, binding partners of certain classes of cell-surface receptors associate with lipid membranes and utilise two-dimensional diffusion to promote association. 3 Many DNA-interacting proteins nd specic sequences or lesions in large amounts of nonspecic DNA by performing onedimensional random walks along the DNA. 4 Every time such a protein associates with DNA, it transiently diffuses along the duplex and thus drastically increases the number of sampled DNA positions per unit of time. It then dissociates from the DNA, undergoes three-dimensional (3D) diffusion through solution to rebind at an entirely different region and again searches a stretch by one-dimensional (1D) diffusion. The combination of 3D and 1D searches gives rise to a drastic increase in the effective bimolecular association rate constant of the protein with its target. 5,6 An example of a naturally occurring system in which 1D diffusion along DNA is used to speed up association between two proteins is found in adenovirus. 7,8 During viral maturation, a large number of proteins within a single viral particle need to be proteolytically processed by the adenovirus protease (AVP) before infection of a cell. 9 Tight packing of protein and DNA within the viral particle makes regular 3D diffusion as a mechanism for the protease to travel from one target to the other impossible. Recent work has shown that the AVP protein 10 recruits the short 11-a.a. pVIc peptide (GVQSLKRRRCF), 11 itself a proteolytic product in early maturation, and uses it to slide along the DNA inside the viral particle and thus effectively reduces the search space for the protease from three dimensions to one. 8</p><!><p>In this work we demonstrate that the ability of the pVIc peptide to slide along DNA can be used to speed up a much broader class of biomolecular processes than just those occurring in vivo and that it can be used to dramatically improve the speed of common laboratory reactions (Fig. 1). First, as a proof of principle, we couple each of the two binding partners in a canonical biotin-streptavidin association to the pVIc 'molecular sled' and show that association proceeds more than an order of magnitude faster in the presence of DNA (Fig. 2a). Fluorescence Resonance Energy Transfer (FRET) 12 was used to monitor the time dependence of the bimolecular association. For simplicity, we refer to the functionalised biotin and streptavidin as binding partners B and S, respectively. Binding partner B is formed by reacting a maleimide-functionalised biotin with the cysteine Cys10 of Cy3-labelled pVIc in a Michael-addition reaction (see ESI, Fig. S1 †). The maleimide and biotin units are connected via a high-molecular weight polyethylene glycol (PEG) linker resulting in a total molecular weight for binding partner B of 6.7 kDa. This high molecular weight reduces its diffusional mobility and allows us to more easily gain access to the timescale of association. Binding partner S is prepared by forming a complex between a Cy5-labelled tetrameric streptavidin and an unlabelled biotin-pVIc conjugate (see ESI †). The ability of both B and S to 1D diffuse along DNA was conrmed on a single-molecule level using Total Internal Reection Fluorescence (TIRF) microscopy (Fig. 2b, see ESI † for experimental conditions and notes). We estimated the binding times s 1D y 0.3 s and the 1D diffusion coefficient D 1D y 3 Â 10 4 nm 2 s À1 . Using these values, we can calculate that S and B are able to explore a DNA segment of length L 1D ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2D 1D s 1D p y130 nmy400 bp before dissociating and returning to solution.</p><p>Binding partners B and S were combined in aqueous solution at nal concentrations of 150 nM and 37.5 nM, respectively, and ensemble FRET between the Cy3 donor and Cy5 acceptor uorophores was measured (Fig. S2 †). Fig. 2c shows the time dependencies of bimolecular association in the presence of 2686 bp long double-stranded DNA (dsDNA) at different concentrations. Addition of the DNA up to 1 pM did not have a signicant effect on the reaction rate, whereas DNA concentrations of higher than 10 pM resulted in a clearly discernable reduction of the reaction time. For a DNA concentration of 300 pM, already aer 15 s, 99% of the maximum FRET efficiency was achieved.</p><p>Fig. 3a shows the reaction times as derived from the FRET traces for different DNA concentrations and lengths. For each length, varying from 2686 to 15 base pairs (Table S1 †), the association times decrease by up to 20-fold at higher concentrations of added DNA. Interestingly, the critical concentration for reaction speed up differs for the different DNA lengths: longer DNA fragments are required at lower concentrations than short DNA molecules to achieve the same catalytic effect. This behaviour can be explained by the fact that the critical number of reaction partners associated with DNA is reached at higher DNA concentrations for short fragments and at low DNA concentrations for longer pieces of DNA. Thus, the main parameter that governs the kinetics of reaction is the total base pair concentration, a unit that describes the total length of DNA per unit volume. This notion is validated by plotting the reaction time against DNA base pair concentration (Fig. 3b), showing the curves cluster together in three distinct regions. These regimes can be understood in terms of the density of binding partners trapped on the catalytic DNA molecules. At low base pair concentrations, the amount of DNA available per binding partner is too low to trap a noticeable fraction of the binding partners and inuence the overall reaction rate. In the optimal regime, around 0.1 to 10 mM of base pairs, the binding partners have high probability to be trapped by DNA where they can nd each other by 1D diffusion. At base pair concentrations higher than 100 mM, the probability for binding partners B and S to bind to the same DNA molecule diminishes, resulting in a deceleration of the association.</p><p>In an alternative mechanism to explain the increased association rates, DNA-bound binding partners bound to the same DNA molecule could be brought into proximity of each other by bending and looping of the DNA duplex. In such a mechanism, the binding partners would rely on the conformational exibility of the DNA and use the duplex as a scaffold to bring binding partners together. In order to exclude this pathway, we conducted a series of experiments with DNA of four different lengths (50, 100, 150 and 300 bp), which were chosen such that the corresponding DNA looping probability differed signicantly from one another. 13,14 Under the low-salt buffer conditions used in this study, DNA molecules of 50 and 100 bp can be regarded as stiff rods whose folding onto itself is excluded (DNA persistence length is estimated to be 250 bp at 2 mM NaCl). 15 In case looping was the main mechanism for reaction speed-up one would expect a considerably lower reaction acceleration in case of 50 and 100 bp long DNA as compared to 300 bp, which is long enough to form loops. However, in all cases we observed the same 20-fold reaction speed-up (Fig. 3a and b), conrming that association is not mediated by DNA bending onto itself.</p><p>Using a similar reasoning, one could argue that the conformational exibility of the long PEG linkers attached to both binding partners allows those binding partners that are statically but distally bound to the same DNA to associate without the need for sliding. Fig. 3b shows, however, a 10-fold increase in reaction time at a reaction stoichiometry as low as 1 binding partner per 1000 base pairs, clearly an average molecular separation too high to be bridged by the binding partners statically bound to the same DNA. From the considerations above, one can conclude that sliding along DNA, and not just static binding, is responsible for the increase in association rate.</p><p>An understanding of the origin of the reaction acceleration effect can be obtained from our recent work in which we formulated a kinetic model for a system with linear sinks (i.e. DNA) that can intermittently trap molecules present in a solution and serve as an assembly line for 1D diffusing molecules. 16 Our model semiquantitatively predicts the experimentally observed speed-up in the presence of DNA molecules of different lengths and concentrations. Moreover, according to our simulations, the relative contribution of the 1D reaction pathway in the optimum speed-up regime can be as high as 90%. In this work, we concluded that although association of the binding partners on DNA without 1D sliding does play a role, the primary contributor to the reaction acceleration is a 1D sliding mechanism. This model also shows that in the case of extremely short DNA molecules (15 bp and 50 bp), the reaction acceleration cannot be explained by 1D sliding alone due to the sizes of the binding partners being comparable to the dimensions of the DNA. Instead, reaction acceleration is introduced by the high diffusional mobility of the short DNA duplexes and their ability to electrostatically capture the cationic peptides. 16 As a next step, we set out to use our method to speed up a standard polymerase chain reaction (PCR) by reducing the time needed for pVIc-coupled primers to anneal to the template DNA. The exponential amplication of DNA during PCR can be divided into three distinct steps. 17 The rst step is the melting of the double-stranded DNA template (Fig. 4a), followed by primer annealing and elongation with the polymerase. During the annealing step, primers need to nd and hybridise to their complementary target sequence on a template. During this annealing step, the DNA will consist of a mixture of denatured and double-stranded regions, providing a large variety of structures for the pVIc-primers to interact with and potentially move along, resulting in a reduction of the time needed for the primer to locate and bind to its target sequence.</p><p>We covalently coupled (see ESI †) the pVIc peptide to the 5 0 ends of a pair of PCR primers (primer set I, Table S2, Fig. S3 and S4 †) designed to amplify a 807 bp stretch from a linear doublestranded 1970 bp-long template and used real-time PCR (qPCR) experiments with SYBR Green I uorescence to report on the kinetics of amplicon formation 18 (Fig. 4b and S5 †). The correct length of the PCR product was conrmed by agarose gel electrophoresis (Fig. S6 †). Similar results were obtained for PCR experiments employing a different pair of primers (Table S2 and Fig. S7 †) and a longer 8669 bp-long circular template M13KO7 (Fig. S8 †). The kinetics of amplicon formation were quantied in an unbiased manner by employing a PCR threshold cycle analysis (see ESI, Fig. S9 †). Remarkably, the PCR reaction containing the pVIc-conjugated primers displayed a signicant reduction in the number of cycles needed, suggesting the use of a molecular sled as a viable approach to speed up the overall reaction time of PCR. In our experiments we were able to shorten the PCR reaction time by 15-27%. To ensure that the increase in speed is not caused by a nonspecic electrostatic association between the four positively charged amino acids in the sliding peptide and the negatively charged DNA backbone, we repeated the same PCR experiments using primers conjugated to a scrambled peptide (S-peptide, SFRRCGLRQVK) containing the same residues in a random order, which presumably affects the sliding behaviour of the peptide yet preserving the net charge. Our qPCR data reveals that use of primers conjugated with this scrambled peptide does not result in a decrease of the number of PCR cycles required for amplication (Fig. 4b, 'S-peptide').</p><p>The performance of the S-peptide-modied primers is very similar to the unmodied primers. Furthermore, we observed a signicant reduction in the number of PCR cycles when using a truncated pVIc variant containing only the last six amino acids of pVIc, four of which are positively charged and are sufficient to support sliding along DNA (Fig. 4b, K-peptide, KRRRCF, Fig. S10 †). Finally, we studied the behaviour of the primer modications under different conditions by varying the annealing time t A and primer concentration C primer (Fig. S8 †). In case of the most stringent conditions (short annealing time, low primer concentration) the effect of the sliding peptides was the most pronounced.</p><p>To exclude a scenario in which the acceleration effect could originate from the enhanced primer-template binding due to cationic nature of the peptides, we compared the melting temperatures T m of the modied and unmodied primers that were used in the PCR experiments. When using short complementary oligonucleotides, and thus excluding sliding contributing to affinity, the measured T m values of the peptidefunctionalised primers were identical to those of the nonfunctionalised ones (Fig. S11 †). This observation excludes an enhanced stability of binding to DNA in the PCR reactions because of the peptide.</p><p>The use of chimeric molecules, where the desired functions of parent moieties are combined within one molecule is a wellestablished approach in biotechnology. In PCR, for example, attempts have been made to increase the affinity of primers and polymerases to DNA by functionalising primers with DNAintercalating molecules 19,20 and expressing the polymerases with an additional cationic peptide motif in the sequence. [21][22][23][24] The enhancement of molecular activity in these cases arises from the increase of the attractive electrostatic and intermolecular forces between the desired molecule and DNA. Another approach that uses the same concept of chimeric molecules is DNA-templated synthesis, where the binding partners are conjugated to single-stranded DNA oligonucleotides and are physically brought into proximity of one another by hybridising them to a DNA template. [25][26][27] In our study, however, the mechanism of activity enhancement is different from these approaches: as opposed to increasing the affinity between the binding partners by prolonging the dissociation time, we aimed to speed up association by addition of a different reaction pathway -1D diffusion along DNA. The reduction of search dimensionality makes the binding partners nd each other faster and, thus, results in the overall reaction acceleration.</p>

Royal Society of Chemistry (RSC)

Thermoregulatory and metabolic responses of Japanese quail to hypoxia

Common responses to hypoxia include decreased body temperature (Tb) and decreased energy metabolism. In this study, the effects of hypoxia and hypercapnia on Tb and metabolic oxygen consumption (V\xcc\x87o2) were investigated in Japanese quail (Coturnix japonica). When exposed to hypoxia (15, 13, 11 and 9% O2), Tb decreased only at 11% and 9% O2 compared to normoxia; quail were better able to maintain Tb during acute hypoxia after a one-week acclimation to 10% O2. V\xcc\x87o2 also decreased during hypoxia, but at 9% O2 this was partially offset by increased anaerobic metabolism. Tb and V\xcc\x87o2 responses to 9% O2 were exaggerated at lower ambient temperature (Ta), reflecting a decreased lower critical temperature during hypoxia. Conversely, hypoxia had little effect on Tb or V\xcc\x87o2 at higher Ta (36\xc2\xb0C). We conclude that Japanese quail respond to hypoxia in much the same way as mammals, by reducing both Tb and V\xcc\x87o2. No relationship was found between the magnitudes of decreases in Tb and V\xcc\x87o2 during 9% O2, however. Since metabolism is the source of heat generation, this suggests that Japanese quail increase thermolysis to reduce Tb. During hypercapnia (3, 6 and 9% CO2), Tb was reduced only at 9% CO2 while V\xcc\x87o2 was unchanged.

thermoregulatory_and_metabolic_responses_of_japanese_quail_to_hypoxia

1. Introduction<!>2.1 Experimental animals<!>2.2 Surgical preparation<!>2.3 Body temperature and metabolism<!>2.4 Lactate measurements<!>2.5 Experimental protocols<!>2.5.1 Protocol 1: Graded hypoxia<!>2.5.2 Protocol 2: Blood lactate<!>2.5.3 Protocol 3: Chronic hypoxia<!>2.5.4 Protocol 4: Graded Ta<!>2.5.5 Protocol 5: Graded hypercapnia<!>2.6 Data analysis<!>3.1 Normoxic Tb and metabolic rate<!>3.2 Protocol 1: Effect of graded hypoxia<!>3.3 Protocol 2: Blood lactate during hypoxia<!>3.4 Protocol 3: Effect of chronic hypoxia<!>3.5 Protocol 4: Effect of graded Ta<!>3.6 Relationship between Tb and metabolic responses to hypoxia<!>3.7 Protocol 5: Effect of graded hypercapnia<!>4. Discussion<!>4.1 Methodological considerations<!>4.2 Effect of ambient temperature<!>4.3 Relationship between decreases in Tb and metabolic rate during hypoxia<!>4.4 Acclimation to hypoxia<!>

<p>A common response to hypoxia exposure among vertebrates and invertebrates is to reduce O2 demand. Reduced body temperature (Tb) and the associated metabolic depression protect against hypoxia by reducing metabolic O2 consumption and, in many species, lowering Tb also enhances O2 loading at gas exchangers by increasing the O2 affinity of respiratory pigments (Steiner and Branco, 2002; Bicego et al., 2007). Both decreased Tb and metabolic depression have been shown to increase survival in hypoxia (Steiner and Branco, 2002).</p><p>Thermoregulatory and metabolic responses to hypoxia have been extensively studied in mammalian and ectothermic models (Hill, 1959; Dupre et al., 1988; Wood, 1991; Gautier, 1996). Frappell et al. (1992) observed decreases in oxygen consumption (V̇o2) in 26 of 27 mammalian species studied, with most species showing a concomitant decrease in Tb. Reduced Tb via behavioral thermoregulation has even been recorded in the protozoan Paramecium caudatum during hypoxia (Malvin and Wood, 1992). In mammals, the response to hypoxia is most dramatic in newborn and small species (Hill, 1959; Frappell et al., 1992; Mortola, 1996, 1999).</p><p>Despite overwhelming evidence for each response, the relationship between changes in Tb and metabolic rate during hypoxia remains unclear. In mammals, it is frequently reported that the decrease in Tb is a direct result of a decrease in metabolic rate (i.e., reduced heat production), with the decrease in metabolic rate always observed first (Hill, 1959; Gautier and Bonora, 1994; Gautier, 1996). On the other hand, there have been several studies in which the decrease in Tb reportedly precedes the decrease in metabolic rate. For example, the little brown bat (Phyllostomus discolor) decreases Tb at 12% O2 despite no measurable reduction in metabolic rate at this level of hypoxia (Walsh et al., 1996). Similarly, the golden-mantled ground squirrel (Spermophilus lateralis) exhibits an immediate decrease in Tb followed by a delayed suppression of metabolic rate upon exposure to hypoxia (Barros et al., 2001). This suggests that increased heat loss (thermolysis) may also be an important mechanism for the decrease in Tb in some mammals. The decrease in Tb could, in turn, depress metabolic rate directly (i.e., Q10 effect), although active suppression of metabolism may also contribute to metabolic depression in hypoxia (Barros et al., 2001).</p><p>It is generally accepted that the decrease in Tb during hypoxia is a regulated process (i.e., anapyrexia; Steiner and Branco, 2002; Bicego et al., 2007). In behaviorally thermoregulating ectotherms, this is reflected in a change in preferred Tb (Wood, 1991; Tattersall and Boutilier, 1997), although there is also evidence for a shift in the temperature at which lizards begin evaporative cooling during hypoxia (Dupre et al., 1986). In mammals, the regulated decrease in Tb is evidenced by a shift in the thermoneutral zone to lower Ta during hypoxia (Dupre et al., 1988; Barros et al., 2001). This shift reduces metabolically expensive processes such as shivering and non-shivering thermogenesis at low Ta, which also explains why metabolic depression is exaggerated at low Ta (Gautier, 1996). The mechanism by which hypoxia initiates this process is largely unknown, but reduced oxidative metabolism in the central nervous system (CNS) (Steiner and Branco, 2002) and/or changes in the thermal sensitivity of preoptic neurons during hypoxia (Tamaki and Nakayama, 1987) have been implicated. Indeed, nitric oxide, serotonin and dopamine all appear to be important modulators of hypoxic anapyrexia within the preoptic area (Branco et al., 2006).</p><p>The effects of hypoxia on Tb and metabolism have been studied far less in birds than mammals, and many of the previous reports have yielded conflicting results. Body temperature has been reported to decrease during hypoxia in most avian species studied to date, including the Japanese quail (studied at Ta = 5°C; Weathers and Snyder, 1974), bobwhite quail (Boggs and Kilgore, 1983), greylag goose (Scott et al., 2008), bar-headed goose (Scott et al., 2008), burrowing owl (Boggs and Kilgore, 1983; Kilgore et al., 2008), house sparrow (Tucker, 1968) and rufous-collared sparrow (Novoa et al., 1991), although a study on the rosy finch and house finch reported no change in Tb during hypoxia (Clemens, 1988). The pekin duck also appears to decrease Tb in hypoxia (Faraci et al., 1984; Scott et al., 2008), but this has not been consistently observed (Kiley et al., 1985, Bouverot and Hildwein, 1978). In contrast, V̇o2 has only been shown to decrease during hypoxia in the Japanese quail (studied at Ta = 5°C; Weathers and Snyder, 1974) and, at least in one study, the rufous-collared sparrow (Castro et al., 1985; but see Novoa et al., 1991). Rather, many avian species do not change V̇o2 during hypoxia, including the bobwhite quail (Boggs and Kilgore, 1983), burrowing owl (Boggs and Kilgore, 1983; Kilgore et al., 2008) and several small passerines (Novoa et al., 1991) or actually increase V̇o2 during hypoxia, including the greylag goose (Scott et al., 2008), bar-headed goose (Black and Tenney, 1980; Scott et al., 2008), house sparrow (Tucker, 1968), and the rosy and house finches (Clemens, 1988). Pigeons and pekin ducks have variously been shown to have either no change (duck: Kiley et al., 1985, Bouverot and Hildwein, 1978; pigeon: Bouverot et al., 1976) or an increase in V̇o2 during hypoxia (duck: Black and Tenney, 1980; Scott et al., 2008; pigeon: Barnas et al., 1986). Since factors such as the level of hypoxia, Ta and duration of the hypoxic exposure have been shown to influence the magnitude of the changes in Tb and metabolic rate in mammals (Gautier, 1996; Barros et al., 2001), some of this intraspecific and interspecific variation could reflect differences in experimental design. However, it is difficult to address these questions because of the limited data available for any one species.</p><p>The purpose of this study was to investigate the effects of hypoxia on Tb and metabolic rate in Japanese quail (Coturnix japonica). Several levels of hypoxia were investigated to determine the severity of hypoxia required to elicit a response, and a week-long chronic hypoxia exposure was used to determine whether these responses exhibit plasticity. Quail were also exposed to hypoxia at several Ta to test the hypothesis that there would be a shift in the thermoregulatory set point similar to that observed in mammals. Finally, several levels of hypercapnia were studied to determine whether another respiratory stimulant elicits similar changes in Tb and/or metabolic rate in this species.</p><!><p>Fertile Japanese quail (Coturnix japonica) eggs were purchased from a commercial supplier (Boyd's Birds, Pullman, WA, USA) and were hatched and raised to adulthood in-house. Adult birds were maintained on a 12:12 light cycle and provided food and water ad libitum. A total of 26 adult males, between 11 and 24 weeks of age, were used in these experiments; individuals were generally used in more than one experimental protocol (see below) with a minimum of one week between protocols. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Bates College.</p><!><p>At least two weeks prior to experimentation, a temperature transponder (E-mitter G2; Respironics, Bend, OR) was surgically implanted into the abdominal cavity of each quail. Quail were anesthetized with isoflurane, first in a closed box and then maintained via nose cone (2.5% isoflurane, balance O2). The transponder was then placed into the abdominal cavity through a ventral midline incision. Carprofen (30 mg kg−1 i.m., Rimadyl, Pfizer, Exton, PA, USA) was administered post-operatively as an analgesic (Hocking et al., 2005).</p><!><p>Metabolic rate was measured using open-system respirometry. A cylindrical, opaque respirometer chamber (15.2 cm diameter, 14.6 cm height) was housed in an incubator (Model 818, Precision, Winchester, VA, USA) to maintain constant ambient temperature. Mixtures of N2, O2, and/or CO2 were forced through the chambers at a flow rate of 1 liter/min using precision rotameters (7300 and 7400 series, Matheson, Montgomeryville, PA); flow rate (STPD) was continuously monitored with a mass flowmeter (G265, Qubit Systems, Kingston, ON, Canada) and recorded to a computer. At this flow rate, it took less than ten min to switch from 21% O2 to 9% O2.</p><p>The fractional concentrations of O2 and CO2 entering (Fio2 and Fico2) and exiting (Feo2 and Feco2) the chamber were measured using a gas analyzer (PowerLab ML206, ADinstruments, Colorado Springs, CO, USA); air was dried (Drierite, W.A. Hammond Drierite Co., Xenia, OH, USA) prior to analysis. Ambient temperature (Ta) was measured via a T-type thermocouple situated within the respirometer chamber, just below the lid; unless otherwise noted, Ta was 23°C for all measurements. Ta, gas flow rate, and O2 and CO2 concentrations were recorded to computer during each experiment (PowerLab/8sp, Chart 5.2 ADinstruments). Body temperature (Tb) was simultaneously monitored by telemetry (ER-4000 energizer receiver and VitalView 4.1 software; Respironics, Bend, OR, USA).</p><!><p>To measure blood lactate, the toe nail (claw) was visualized to determine the location of the blood vessel. The claw was then clipped so as to release a few drops of blood. Blood was collected directly onto test strips and analyzed for lactate concentrations using a blood lactate analyzer (Lactate Pro, Arkray, Kyoto, Japan).</p><!><p>Metabolic rate, body temperature, and/or lactate were measured during five different experimental protocols. Each quail was exposed to the respirometry chamber on at least two occasions prior to the study (~60 min per exposure). Animals were not fasted prior to experimentation. When a protocol required the same individual to be studied on multiple days (see below), the individual was studied at approximately the same time each day; all measurements were made during the light period of the light cycle. Barometric pressure averaged 754 ± 5 (S.D.) mmHg across all protocols</p><!><p>On the day of study, each quail was weighed and placed in the respirometer chamber (Ta = 23°C). Quail remained at normoxia (21% O2, 0% CO2) for 100 min to establish a baseline and were then switched to one of five test gases for 60 min: 21% (time control), 15%, 13%, 11%, and 9% O2. Only one test gas was used each day, so each individual was studied on five consecutive days; the order in which each quail was exposed to the five test gases was randomly assigned. A total of 16 quail were used in this protocol.</p><!><p>Quail were placed in a respirometer chamber and exposed to normoxia for 90 min, then exposed to normoxia (21% O2) or hypoxia (11% or 9% O2) for 30 additional min. At the end of this exposure, quail were quickly removed from the chamber and sampled for blood lactate concentrations. Each quail was exposed to only one test gas per day, and the order of the test gases was assigned randomly. Six quail were used in this protocol.</p><!><p>As in protocol 1 (see section 2.5.1), quail were placed in a respirometer chamber to measure Tb and metabolic rate. After 100 min of normoxia (21% O2), the test gases were switched to 9% O2 for 60 min. Quail were then placed in large acrylic chambers for one week at 10% O2; quail were individually caged and provided food and water ad libitum. Chambers were opened briefly (<10 min) each day to clean cages and replenish food and water. After seven days, Tb and metabolic rate measurements were repeated as described above. Ten quail were used in this protocol.</p><!><p>On the day of study, each quail was placed in the respirometer chamber at one of eight ambient temperatures: all sixteen quail used in this protocol were studied at 13°, 18°, 23°, 28°, and 33°C, and six of these were also studied at 30° and 36°C to better estimate the thermoneutral zone. As described in protocol 1 (see section 2.5.1), quail remained at normoxia (21% O2) for 100 min to establish a baseline before being switched to 9% O2 for 60 min. Only one Ta was used each day, so each individual was studied on 5–7 consecutive days; the order of exposure to each Ta was randomly assigned. To verify that the observed changes in Tb and metabolic rate reflected the effect of hypoxia versus the duration of time spent in the respirometer chamber, six quail were also studied for 160 min in normoxia at 13°C and 33°C (i.e., time controls).</p><!><p>As in protocol 1 (see section 2.5.1), quail were placed in a respirometer chamber to measure Tb and metabolic rate. Quail remained in normoxic normocapnia (21% O2, 0% CO2) for 100 min and were then switched to one of four test gases for 60 min: 0%, 3%, 6% and 9% CO2. Only one test gas was used each day, so each individual was studied on four consecutive days; the order in which each quail was exposed to the four test gases was randomly assigned. Ten quail were used in this experiment.</p><!><p>Body temperature was recorded every five min for the final 30 min of baseline and throughout the 60 min of the test gas exposure. Metabolic O2 consumption (V̇o2) and CO2 production (V̇co2) were calculated based on inspired and expired gas levels averaged over 30 s and sampled every 10 min during the final 30 min of baseline and during exposure to the test gas; reported V̇o2 and V̇co2 are corrected to standard temperature and pressure (STPD). At the most severe levels of hypoxia, V̇co2 could be affected by lactate production (see section 3.3). Preliminary analysis showed that V̇o2 and V̇co2 yielded similar results for all experimental conditions, so only V̇o2 is reported in the figures. Tb and V̇o2 are reported both as the minimum value achieved during the 60 min of test gas exposure ("minimum Tb" or "minimum V̇o2") and as the corresponding change from baseline, where baseline represents the average Tb or V̇o2 for the final 30 min of the 100-min baseline (i.e., normoxic and normocapnic) exposure. Normalization to baseline reduces the effects of day-to-day variation that might result from factors such as acclimation to the chamber over repeated measurements or time since the last meal.</p><p>The effects of graded hypoxia and hypercapnia on Tb, V̇o2 and/or lactate were analyzed using one-way repeated measures ANOVA followed by Newman-Keuls post hoc tests. The effects of chronic hypoxia on Tb and V̇o2 responses to hypoxia were analyzed by paired t-tests, and by two-way repeated measures ANOVA with Bonferroni post hoc tests (comparing all time points to baseline) to further analyze the time course of changes in Tb and V̇o2 during acute hypoxia. Two-way repeated measures ANOVA followed by Student-Newman-Keuls post hoc tests were used test the effect of hypoxia on Tb and V̇o2 at different Ta; only a subset of quail were studied at two additional Ta (30 and 36°C), so separate paired t-tests were used to test the effects of hypoxia on Tb and V̇o2 at these Ta. Where appropriate, one-way repeated measures ANOVA and/or one-sample t-tests were used to determine whether there was a significant change in Tb or V̇o2 with time in 21% O2 trials (i.e., time controls). Linear regression was used to determine whether hypoxia-induced changes in Tb were related to changes in V̇o2. Statistical tests were run using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA) or SigmaStat 3.11 (Systat Software, Inc., San Jose, CA, USA). The threshold of significance was set at P < 0.05 for all tests. Values in text are means ± SEM, unless otherwise noted.</p><!><p>The resting Tb and metabolic rate for adult quail breathing 21% O2 are reported in Table 1. Since individuals were studied multiple times, the average value for each individual was computed prior to calculating the group mean. Although Tb was fairly consistent across days within an individual (coefficient of variation (CV) = 0.005±0.002), individuals showed considerable day-to-day variation in V̇o2 (CV = 0.22±0.09). The average Tb (41.2°C) was similar to previous reports for this species (e.g., 40.8°C; Bavis and Kilgore, 2001), whereas average V̇o2 (4.3 mL O2 min−1 100g−1) tended to be greater than previously reported (e.g., 3.1 mL O2 min−1 100g−1; Bavis and Kilgore, 2001). Ten individuals were also studied at least twice after an additional 60 min in the respirometer chamber (i.e., total exposure 160 min) (Table 1). For these individuals, there were no significant differences between 100 min and 160 min measurements for Tb (P=0.38), V̇o2 (P=0.10), V̇co2 (P=0.08) or respiratory quotient (P=0.64).</p><!><p>Body temperature was analyzed both as the minimum value achieved during the 60 min of test gas exposure ("minimum Tb") and as the change from baseline. In both analyses, Tb consistently decreased during moderate/severe hypoxia. When compared to the minimum Tb observed during an equivalent duration at 21% O2 (Tb=41.0±0.1°C), Tb was reduced during hypoxia (P<0.001), but only at 11% (Tb=40.7±0.1°C; P<0.05) and 9% (Tb=40.3±0.2°C; P<0.05) O2 (Fig. 1A); Tb at 9% was significantly lower than at 11% (P<0.001). Although the minimum Tb of quail maintained at 21% was also slightly lower than baseline in this group of quail (ΔTb=0.2°C, P<0.001), the decrease was greater at 11% (Tb=0.5°C, P<0.05) and 9% (ΔTb=0.9°C, P<0.05) (Fig. 1B). In contrast, no significant reduction in Tb was observed at 15% or 13% O2 compared to 21% O2 (Figs. 1A, B; both P>0.05).</p><p>The rate of oxygen consumption tended to decrease during hypoxia as well. Although minimum V̇o2 did not vary with inspired O2 (P=0.07, Fig. 1C), there was a trend for V̇o2 to be lower in hypoxia. Indeed, there was a significant overall effect of hypoxia on V̇o2 when normalized to baseline (P=0.03, Fig. 1D). Post hoc tests were unable to distinguish which groups varied (all P>0.05), but V̇o2 appeared to be reduced at 11% and 9% O2 compared to at 21% O2. Indeed, V̇o2 was significantly reduced during 9% O2 in subsequent experiments (e.g., Fig. 4 and Fig 5, see below). Similar to Tb, minimum V̇o2 at 21% O2 during the 60-minute test gas exposure was lower than during baseline (P<0.001).</p><!><p>A decrease in V̇o2 during hypoxia could reflect an overall decrease in energy metabolism. Alternatively, the decrease in aerobic metabolism could be offset by an increase in anaerobic metabolism. To test this possibility, blood lactate levels were measured during 21%, 11% and 9% O2. Overall there was an increase in blood lactate compared to normoxia (P=0.003, Fig. 2). Post hoc analysis revealed that blood lactate levels in quail exposed to 9% O2 increased by 186% over the normoxic values (P<0.05), but no increase in lactate concentration was observed at 11% O2 (P>0.05).</p><!><p>To determine whether chronic hypoxia alters the Tb or metabolic responses to hypoxia in quail, Tb and V̇o2 responses to 9% O2 were measured in a group of ten quail before and after 7 days at 10% O2; Tb data were unavailable for three of the quail following chronic hypoxia because of technical problems, so n=7 for Tb and n=10 for V̇o2. Tb decreased compared to baseline during acute hypoxia both before and after acclimation, but the average decrease after chronic hypoxia was 51% less (P=0.01, Fig. 3A). To determine whether this difference was an artifact of the rate at which Tb decreased versus the actual Tb achieved, we examined the time course for the change in Tb during hypoxia (Fig. 4). There was a significant statistical interaction between treatment and time (P<0.001). Normoxic Tb was not altered by acclimation (41.4±0.1°C before and after acclimation) and the decrease in Tb was noticeable within 15–20 min of switching to hypoxia in each group. However, Tb began to level off within 40–45 min in acclimated quail (i.e., after acclimation) while it continued to decrease until 50–55 min in the unacclimated condition (i.e., before acclimation). Since Tb stabilized in the recording period for both groups, higher hypoxic Tb in acclimated quail was not simply due to a slower decrease in Tb.</p><p>There was no change in baseline V̇o2 (4.2±0.3 and 3.7±0.3 mL O2 min−1 100g−1 for before and after acclimation, respectively; P=0.12), or in the V̇o2 response to acute hypoxia following acclimation (P=0.52; Fig. 3B). V̇o2 decreased within 20 min of the onset of acute hypoxia and remained fairly constant (on average) between min 20 and 60 of the exposure (Fig. 4B).</p><!><p>The effect of hypoxia on Tb varied with ambient temperature (Fio2 × Ta, P< 0.001). Body temperature was lower during 9% O2 compared to 21% O2 at the five Ta (13°, 18°, 23°, 28°, and 33°C) at which all quail were studied (all P<0.001; Fig. 5A), but this difference tended to be greater at low Ta. Tb during normoxia was slightly higher at 33°C than at lower Ta, but remained consistent for all other Ta studied. In contrast, Tb tended to decrease as Ta decreased during hypoxia, which equates to a larger decrease in Tb (versus normoxia) at lower Ta. Conversely, the effect of hypoxia on Tb appeared quite small at higher Ta. Six quail were studied at ambient temperatures of 30° and 36°C (Fig. 5A). Although these data were not included in the overall statistical analysis discussed above, the effects of hypoxia on Tb were tested by separate paired t-tests at 30° and 36°C for this subset of quail. Hypoxia had no effect on Tb at 36°C (P=0.73), the highest Ta studied; in contrast, hypoxia significantly reduced Tb at 30°C (P=0.02).</p><p>The effect of inspired O2 on V̇o2 also varied with Ta (Fio2 × Ta, P<0.001). V̇o2 decreased during 9% O2 compared to 21% O2 at the five Ta (13°, 18°, 23°, 28°, and 33°C) at which all quail were studied (all P≤0.02, Fig. 5B). During normoxia, V̇o2 increased as Ta decreased. A similar trend was seen during hypoxia, but the increase was less dramatic with V̇o2 only increasing at Ta<23°C. When additional Ta (30° and 36°C) are considered, the threshold at which V̇o2 begins to increase (i.e. lower critical temperature) occurs somewhere between 30 and 33°C in normoxia whereas it appears to occur at approximately 23°C in 9% O2. As with Tb, hypoxia had no apparent effect on V̇o2 in the subset of quail studied at 36°C (paired t-test, P=0.32), the highest Ta studied (Fig. 5B); V̇o2 was reduced by hypoxia at 30°C in this same subset of quail (P=0.01).</p><p>As reported above, there was a decrease in minimum V̇o2 compared to baseline in quail maintained at 21% O2 for an additional 60 min post-baseline in protocol 1 (see section 2.5.1). To ensure that the decrease in metabolic rate observed during hypoxia was not due to a time-dependent decrease in baseline V̇o2, several quail (n=6) were maintained in normoxia at 13°C and 33°C for 160 min (i.e., the equivalent of the entire protocol). No change in V̇o2 from baseline was observed at either Ta (both P>0.9; Fig. 6). As expected, V̇o2 was greater at 13°C than at 33°C. No change in Tb was observed from baseline at 33°C (P=0.08), but a gradual decrease was observed at 13°C (P<0.001). However, the greatest decrease in Tb during this time control experiment was 0.2°C compared to the average 0.7°C decrease observed at 9% O2 (Fig. 5A).</p><p>Given that Tb decreased as Ta decreased in hypoxia (Fig. 5A), the relationship between Ta and V̇o2 during hypoxia is influenced by the direct effects of Tb depression on V̇o2 (i.e., Q10 effect). To look at the Tb-independent effects of Ta and hypoxia on V̇o2, we corrected V̇o2 for the change in Tb by assuming a constant Q10 of 3 (Fig. 5C); a Q10 of 2–3 is typical of biological reactions and has been used by others to investigate the temperature-independent effects of hypoxia on metabolic rate in ground squirrels (Barros et al., 2001). Specifically, we corrected the hypoxic V̇o2 for each quail to the normoxic Tb of the same individual at the corresponding Ta. Analyzed this way, the effect of inspired O2 on V̇o2 still varied with Ta (Fio2 × Ta, P=0.002). However, V̇o2 was only reduced by hypoxia at 13°, 18°, 23 and 28°C (and in the subset of quail studied at 30°C; paired t-test, P=0.03), and not at 33°C (nor in the subset of quail studied at 36°C; paired t-test, P=0.33). Temperature-corrected V̇o2 increased as Ta decreased at Ta<28°C but was not affected by changes in Ta between 28 and 33°C. Thus, after controlling for the direct effects of Tb depression on V̇o2, the lower critical temperature occurred somewhere between 23 and 28°C in hypoxia, which is lower than observed in normoxia (i.e., between 30 and 33°C).</p><!><p>To further investigate the relationship between the Tb and metabolic responses to hypoxia, we combined data for all 23 quail studied at Ta = 23°C without prior exposure to chronic hypoxia into a single regression analysis. There was no relationship between the decrease in V̇o2 and the decrease in Tb during exposure to 9% O2 (r2=0.01, P=0.65). Indeed, individuals with the least (ΔTb=0.1°C) and greatest (ΔTb=2.6°C) decreases in Tb had similar decreases in V̇o2 relative to baseline (ΔV̇o2 ~ −60%).</p><!><p>Body temperature decreased during hypercapnia (P<0.001). Post hoc analysis revealed that minimum Tb decreased significantly at 9% CO2 compared to 0% CO2 (P<0.05, Fig. 7A); Tb was unaffected by 3% or 6% CO2. Similar results were observed for the change in Tb from baseline, with a significantly larger decrease in Tb at 9% CO2 than at any other level of inspired CO2 (Fig. 7B). V̇o2 was unaffected by the level of inspired CO2 (all P>0.05; Figs 7C, D).</p><!><p>Mammals generally reduce O2 demand in hypoxia by decreasing both their body temperature and their energy metabolism (Wood, 1991; Frappell et al., 1992; Gautier, 1996; Mortola, 1996, 1999; Steiner and Branco, 2002; Bicego et al., 2007), but equivalent responses have not been consistently reported in birds. The present study demonstrated both reduced Tb and reduced metabolic rates in adult Japanese quail acutely exposed to moderate-severe hypoxia (Fio2≤11%). These responses were relatively small compared to those observed in small mammals and were most evident at low Ta. In addition, our results indicate that the Tb response is modifiable by chronic exposure to hypoxia (i.e., exhibits phenotypic plasticity). These data confirm and extend an earlier report by Weathers and Snyder (1974) who observed decreases in Tb and V̇o2 during hypoxia in Japanese quail when studied at low ambient temperatures (5°C).</p><!><p>Quail were allowed 100 min to adjust to the respirometry chambers before switching to the test gas condition. Despite this, Tb and V̇o2 tended to decrease during the subsequent 60 min even when kept at 21% O2 (e.g., Fig. 1B,D and Fig 7B,D); this gradual decrease was not immediately obvious during data collection. The apparent time-dependent decrease in Tb and V̇o2 may be overestimated here since the minimum value recorded over the 60-min test gas exposure is being compared against baseline values that represent an average of multiple time points. Indeed, V̇o2 did not decrease during normoxia in the time control experiments presented in Figure 6. However, the quail in Figure 6 had been studied up to ten previous times (i.e., these experiments were completed after those presented in Fig. 1 and Fig 7), and these repeated measurements may have caused the quail to settle more quickly after being handled and placed into the respirometer chamber. Moreover, Tb did decrease slightly over time in these time control experiments (Fig. 6A). Thus, quail may require more than 160 min to adjust to the chamber after handling in order to obtain stable measurements, or may require multiple chances to become accustomed to handling and the experimental apparatus. As such, the effects of hypoxia and/or hypercapnia on Tb and V̇o2 are most appropriately evaluated with reference to quail maintained under normoxic and normocapnic conditions (i.e., time controls).</p><p>We sampled gases exiting the respirometer chamber and calculated V̇o2 at ten minute intervals during the test gas exposure. Although it was not possible to observe the activity level of the birds directly, it was obvious based on fluctuations in Feo2 and Feco2 that individuals were active at some time points. Moreover, Tb and/or V̇o2 are expected to change gradually after the onset of hypoxia or hypercapnia until a new steady-state is established (e.g., Fig. 4), and the specific timing of these changes may differ among individuals. Therefore, with the exception of the time course data in Figure 4, we used the minimum Tb and V̇o2 observed for each quail during the 60-min test gas exposure for statistical analyses; this typically occurred between the 40 and 60 minute time points. This approach reduced confounding variation in Tb and V̇o2 contributed by periodic activity and thus improved our ability to detect differences between experimental conditions.</p><!><p>The effects of hypoxia on Tb and V̇o2 were most apparent at lower Ta in Japanese quail, as previously reported in mammals (Gautier, 1996). In mammals, this reflects reduced shivering and nonshivering thermogenesis associated with a shift in the thermoneutral zone to lower Ta (Dupre et al., 1988; Barros et al., 2001). Previous work demonstrated that shivering thermogenesis is also attenuated by hypoxia in the pigeon (Gleeson et al., 1986a,b; Barnas and Rautenberg, 1990), suggesting that the thermoneutral zone may be altered by hypoxia in birds as well. The present study supports this hypothesis. In normoxia, the threshold Ta at which V̇o2 began to increase (i.e., lower critical temperature, LCT) occurred between 30° and 33°C, similar to a previous report of 29°C for this species (Marjoniemi, 2000). When exposed to 9% O2, however, the LCT occurred closer to 23°C. In mammals, the shift in the LCT is less apparent after correcting for the direct effects of depressed Tb on V̇o2 (i.e., Q10 effect) (Barros et al., 2001), but the LCT appears to be reduced somewhat (perhaps by 2–10°C) in quail exposed to 9% O2 even after temperature correction. The upper critical temperature was not determined in the present study, so it is unclear whether this represents a widening of the thermoneutral zone or a shift of the entire thermoneutral zone to lower Ta.</p><p>Interestingly, quail exhibited a decrease in metabolic rate during hypoxia at Ta within lower end of the normoxic thermoneutral zone (e.g., 33°C). Thus, inhibition of shivering and nonshivering thermogenesis below the LCT is not the only mechanism for reducing metabolic expenditure in this species (see section 4.3, below). It should be noted, however, that the effects of hypoxia on Tb and V̇o2 are diminished at higher Ta and that no effect was observed in quail studied at 36°C, a temperature also within the normoxic thermoneutral zone for this species. To the extent that other species have been studied within or above their thermoneutral zones, this observation may help to explain some of the discrepancies in Tb and V̇o2 responses to hypoxia (or lack thereof) reported for birds.</p><p>In mammals, shifts in the lower critical temperature (Dupre et al., 1988; Barros et al., 2001) and preferred Ta (Gordon and Fogelson, 1991) have been interpreted as a change in the thermoregulatory set point during hypoxia. Thus, the decreased Tb during hypoxia is a regulated event (i.e., anapyrexia) rather than hypothermia. Taken together, the results of the present study indicate that quail, and perhaps all birds, exhibit hypoxia-induced anapyrexia as well. It is somewhat surprising, however, that Tb continued to fall in hypoxic quail as Ta decreased below the LCT (e.g., 13 vs. 18°C, Fig. 5A); this also appears to be the case in the rat (Dupre et al., 1988) and the golden-mantled ground squirrel (Barros et al., 2001). In hypoxia, therefore, it appears that increased heat production below the LCT is not adequate to maintain a constant Tb. This is different than what is generally observed in torpid birds and mammals, for example, where Tb stabilizes once the new LCT is reached even as Ta continues to fall (e.g., Wolf and Hainsworth, 1972; Buck and Barnes, 2000). V̇o2 did not increase significantly at Ta between 18 and 13°C in the Japanese quail at 9% O2, so the ability to increase V̇o2 may have been compromised by inadequate O2 delivery or, alternatively, this may reflect the direct effects of falling Tb on V̇o2 (i.e., Q10 effect). Our data support the latter hypothesis. Specifically, V̇o2 increased progressively as Ta decreased below the LCT after correcting for the effects of hypoxia-induced Tb depression, and the slope of this response approximates that observed in normoxia (Fig. 5C). A similar pattern was observed in ground squirrels exposed to hypoxia, with little apparent change in V̇o2 or V̇co2 as Ta decreased unless these values were corrected for the falling Tb (Barros et al., 2001). Thus, the further decrease in Tb as Ta falls may serve to reduce energetic expenditure (and conserve O2) at very low Ta.</p><!><p>Changes in Tb and energy metabolism clearly influence one another in endotherms. Indeed, Tb depression during hypoxia will decrease metabolic rate through the Q10 effect, all else being equal. As previously discussed, we temperature-corrected the V̇o2 data for hypoxic quail to their normoxic Tb to estimate the Tb-independent effect of hypoxia on V̇o2 at various ambient temperatures. The actual Q10 for metabolic rate is not known for Japanese quail, so we assumed a constant Q10 of 3 (cf. Barros et al., 2001). Because of the relatively small Tb depression exhibited by quail at 9% O2, this operation had only a modest effect on V̇o2 (e.g., +11% at 13°C and +5% at 33°C). Even so, corrected V̇o2 no longer differed between normoxia and hypoxia at 33°C (Fig. 5C); there was also no effect of hypoxia on either the corrected or uncorrected V̇o2 at 36°C, which is noteworthy since Tb was not depressed at this Ta. These results must be interpreted cautiously since we used an assumed value for Q10, but they suggest that the passive effects of lower Tb on energy metabolism can completely explain the hypoxia-induced decreases in V̇o2 observed within the normoxic thermoneutral zone. This may also be the case in mammals: correction of V̇o2 to normoxic Tb (Q10=3) appears to explain most (if not all) of the hypoxia-induced reduction in V̇o2 observed in ground squirrels at thermoneutrality (Barros et al., 2001). Corrected V̇o2 was still significantly reduced by hypoxia at Ta below the normoxic LCT in quail; however, it is likely that metabolic depression below the nomoxic LCT reflects the combined effects of Tb depression and the downward shift of the LCT (and associated decrease in shivering/nonshivering thermogenesis at a given Ta). Importantly, these results do not preclude an important role for active metabolic suppression in the thermoregulatory and metabolic responses of Japanese quail to hypoxia. In ground squirrels, for example, it has been suggested that metabolic suppression facilitates the decline in Tb soon after the onset of hypoxia (Barros et al., 2001).</p><p>Despite the obvious interaction between Tb and V̇o2, there was no apparent relationship between the magnitudes of the decreases in Tb and V̇o2 during hypoxia in Japanese quail (section 3.6, above). This is consistent with several other studies on birds which note decreases in Tb with no decrease, and perhaps even an increase, in V̇o2 during hypoxia (Boggs and Kilgore, 1983; Kilgore et al., 2008; Scott et al., 2008). Thus, the decrease in either Tb or V̇o2 during hypoxia is not solely dependent on a decrease in the other. Moreover, metabolic heat production during hypoxia may be underestimated by measuring V̇o2 – anaerobic mechanisms may maintain metabolic heat production (at least temporarily) even as V̇o2 falls. Although direct calorimetry is needed to fully address this issue, the observed increase in blood lactate levels during exposure to 9% O2 supports this hypothesis. Blood lactate levels also increase in rats during acute hypoxia (7% O2) (Bicego et al., 2002); interestingly, there was no correlation between blood lactate levels and Tb depression in the rat, suggesting that there is no direct relationship between anaerobic heat production and Tb depression in that species. These data suggest that increased thermolysis is principally responsible for the reduced Tb during hypoxia in Japanese quail, and perhaps in other birds.</p><p>Although not specifically investigated here, two major routes of thermolysis are likely involved in hypoxia-induced reduction of Tb in quail: (1) peripheral vasodilation and (2) convective and evaporative cooling associated with increased ventilation. First, a recent study by Scott et al. (2008) demonstrated increased bill surface temperature during hypoxia in the pekin duck, greylag goose and bar-headed goose, suggesting that birds increase blood flow to poorly insulated regions of the body (i.e., thermal windows) during hypoxia to promote heat loss. A similar strategy has been proposed for the golden-mantled ground squirrel (Tattersall and Milsom, 2003). In the latter species, surface temperatures of various body regions (e.g., feet, ears, nose) increase during the onset of hypoxia and then return toward normal, presumably once the new Tb set point is achieved (Tattersall and Milsom, 2003). Together, these data indicate that birds can regulate peripheral blood flow to reduce Tb during hypoxia independent of changes in metabolic rate.</p><p>Second, the hypoxic ventilatory threshold (i.e., where ventilation increases by at least 10%) occurs at an inspired partial pressure of O2 (Po2) of 90–100 Torr in adult, male Japanese quail (R.W. Bavis & D.L. Kilgore, Jr., unpublished data). This threshold is equivalent to approximately 13% O2, with ventilation expected to increase by ~30% and ~50% at 11% O2 and 9% O2, respectively, almost exclusively through an increase in respiratory frequency (R.W. Bavis & D.L. Kilgore, Jr., unpublished data). Thus, respiratory heat loss could contribute to the progressive decrease in quail Tb observed at FIo2<13% O2. Consistent with this hypothesis, Tb also decreased during exposure to hypercapnia, a powerful respiratory stimulant that is not consistently reported to reduce Tb or V̇o2 in mammals (Mortola and Lanthier, 1996). Interestingly, we observed moisture inside the respirometry chamber after exposure to moderate-severe hypercapnia, implying increased respiratory water loss. However, the present study cannot rule out the possibility that hypercapnia itself induces an anapyrexic response (Bicego et al., 2007).</p><p>On the other hand, increased ventilation will also increase heat production by the respiratory muscles due to their increased metabolic demands. The additional metabolic costs of the respiratory muscles could partially offset, or perhaps even exceed, temperature-dependent decreases in V̇o2 and/or active metabolic depression elsewhere in the body. Indeed, variation in the energetic costs of the cardiorespiratory responses to hypoxia, and the associated heat production, may contribute to variation in the Tb and metabolic responses to hypoxia within and among bird species, as well as to differences between birds and mammals. Given that most endothermic vertebrates exhibit decreased Tb during acute hypoxia, however, it appears that hypoxia-induced thermolysis generally exceeds the increased heat production associated with cardiorespiratory responses to hypoxia.</p><!><p>Japanese quail were better able to maintain Tb during acute hypoxia following acclimation to chronic hypoxia. This result is similar to that of Weathers and Snyder (1974) for quail acclimated to simulated high altitude (6100 m) for six weeks and studied at 5°C. The present study, however, demonstrates that this plasticity is observable in as little as one week, and that this effect is evident at relatively mild Ta. Contrary to the earlier study, acclimation had no effect on V̇o2 in normoxic or hypoxic conditions; Weathers and Snyder (1974) observed a considerably greater V̇o2 in acclimated quail under normoxic conditions, although this difference tended to decrease as inspired Po2 decreased. It is possible that longer durations of hypoxia have greater effects on metabolic rates.</p><p>The ability to maintain Tb better following acclimation likely reflects increased capacity to deliver O2 to the tissues responsible for anapyrexia. In birds, similar to mammals, chronic hypoxia tends to increase parabronchial ventilation (i.e., increased ventilatory chemosensitivity; Powell et al., 2000), although the time course of these changes are not known for the Japanese quail. Blood O2 carrying capacity also increases, both due to progressive increases in hematocrit and hemoglobin concentration in Japanese quail (Jaeger and McGrath, 1974; Weathers and Snyder, 1974). Importantly, these changes begin within the first week of chronic hypoxia (Jaeger and McGrath, 1974) and, therefore, may contribute to changes in the thermoregulatory response to hypoxia observed in the present study. It is also possible that chronic hypoxia alters the hypoxic sensitivity of CNS structures mediating anapyrexia, but this hypothesis awaits further study.</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

Design, synthesis and evaluation of carbazole derivatives as potential antimicrobial agents

AbstractFive series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5–2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure–activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.

design,_synthesis_and_evaluation_of_carbazole_derivatives_as_potential_antimicrobial_agents

Introduction<!><!>Chemistry<!>Procedure to synthesise compound 2a<!>Procedure to synthesise compound 2 b<!>General procedure to synthesise compounds 2c–k<!>General procedure to synthesise compounds 4a–l<!>General procedure to synthesise compounds 8a–l<!>N2,N2-Dimethyl-6-(9-methyl-9H-carbazol-3-yl)-3,6-dihydro-1,3,5-triazine-2,4-diamine (8a)<!>6-(9-Ethyl-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8 b)<!>6-(9-Benzyl-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8c)<!>N2,N2-Dimethyl-6-(9-(4-methylbenzyl)-9H-carbazol-3-yl)-3,6-dihydro-1,3,5-triazine-2,4-diamine (8d)<!>6-(9-(2-Fluorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8e)<!>6-(9-(2,4-Dichlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8f)<!>6-(9-(2-Chlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8 g)<!>6-(9-(3-Chlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8 h)<!>6-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8i)<!>6-(9-(4-Bromobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8j)<!>4-((3-(4-Amino-6-(dimethylamino)-2,5-dihydro-1,3,5-triazin-2-yl)-9H-carbazol-9-yl)methyl)benzonitrile (8k)<!>6-(6-Bromo-9-phenyl-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-1,3,5-triazine-2,4-diamine (8 l)<!>General procedure to synthesise compounds 9a–k<!>(E)-2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9a)<!>(E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9b)<!>(E)-2-((9-Benzyl-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9c)<!>(E)-2-((9-(4-Methylbenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9d)<!>(E)-2-((9-(2-Fluorobenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9e)<!>(E)-2-((9-(2,4-Dichlorobenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9f)<!>(E)-2-((9-(2-Chlorobenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9 g)<!>(E)-2-((9-(3-Chlorobenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9 h)<!>(E)-2-((9-(4-Chlorobenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9i)<!>(E)-2-((9-(4-Bromobenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9j)<!>(E)-2-((9-(4-Cyanobenzyl)-9H-carbazol-3-yl)methylene)hydrazine-1-carboximidamide (9k)<!>General procedure to synthesise compounds 10a–b and 11<!>(E)-2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazine-1-carbothioamide (10a)<!>(E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)hydrazine-1-carbothioamide (10 b)<!>(E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)hydrazine-1-carboxamide (11)<!>General procedure for the synthesis of compounds 12a–12d<!>(E)-N'-((9-Benzyl-9H-carbazol-3-yl)methylene)isonicotinohydrazide (12a)<!>(E)-N'-((9-(4-Methylbenzyl)-9H-carbazol-3-yl)methylene)isonicotinohydrazide (12 b)<!>(E)-N'-((9-(2-Fluorobenzyl)-9H-carbazol-3-yl)methylene)isonicotinohydrazide (12c)<!>(E)-N'-((9-(2,4-Dichlorobenzyl)-9H-carbazol-3-yl)methylene)isonicotinohydrazide (12d)<!>Evaluation of antibacterial activity in vitro<!>MTT assay<!>Docking study<!>Inhibition of DHFR activities in vitro<!>Chemistry<!><!>Evaluation of in vitro antimicrobial activities<!><!>Evaluation of in vitro antimicrobial activities<!>Cell viability<!><!>Cell viability<!>Docking analysis<!><!>Inhibition studies of compound 8f with DHFR<!><!>Conclusions<!>Disclosure statement

<p>The steady increase of microbial pathogens that do not respond to conventional treatments presents a significant threat to global public health. Fungal and bacterial infections are becoming progressively more resistant towards currently available antimicrobial medicines, such as antibiotics. Therefore, there is an urgent requirement for new drugs that target these pathogens. Without effective antimicrobials for prevention and treatment of infections, medical procedures such as organ transplantation, chemotherapy, diabetes management and routine surgery (e.g. caesarean sections or hip replacements) have a significantly higher risk of morbidity and mortality1. The scale of the threat of antimicrobial resistance has led to the development of numerous strategies to conserve and make more effective use of existing antibiotics and to promote the development of novel antimicrobials2. In 2010, the Infectious Diseases Society of America (IDSA) launched the 10 × 20 initiative that called for the development of 10 novel, safe and effective systemic antibiotics by 20203.</p><p>Carbazole alkaloids have attracted considerable attention in medicinal chemistry because they exhibit a broad spectrum of biological and pharmacological activities, including antibacterial, antituberculous, antitumour, antioxidant and anti-inflammatory properties4–13. In our previous studies, guanidines exhibited significant antibacterial activity and triazine ring-containing compounds displayed hypoglycaemic, antitumour and antibacterial activities14,15. Dihydrotriazine compounds have a higher hydrophobicity compared with guanidine compounds and several molecules containing this scaffold have been reported as antibacterial agents16–18. Recent studies have identified dihydrotriazine-containing compounds as inhibitors of dihydrofolate reductase (DHFR), a ubiquitous enzyme that catalyses the conversion of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, which is involved in metabolic reactions such as purine and thymidine nucleotide biosynthesis19,20. DHFR is required by all organisms to grow and multiply; however, selective inhibitors of microbial enzymes have been utilised as therapeutic agents.</p><p>Here, we report the design and synthesis of five series of novel carbazole derivatives (Figure 1), totaling 30 compounds and the subsequent in vitro evaluations of their antibacterial and antifungal activities. Several different substituents were systematically introduced onto the carbazole ring and their effects on the overall antimicrobial activity were investigated.</p><!><p>The structures of the target compounds.</p><!><p>All reagents were obtained commercially and were used without further purification. Solvents were dried according to standard procedures. Reactions were monitored by thin-layer chromatography (TLC) on silica gel plates. Melting points were determined in open capillary tubes and were uncorrected. 1H NMR and 13C NMR spectra were measured on an AV-300 (Bruker, Switzerland), and all chemical shifts were given in ppm relative to TMS. Mass spectra were measured on a HP1100LC (Agilent Technologies). High-resolution mass spectra were measured on an MALDI-TOF/TOF mass spectrometer (Bruker Daltonik, Germany).</p><!><p>To a solution of carbazole (1.03 g, 6.16 mmol) in DMC (10 ml), DABCO (0.069 g, 0.62 mmol) was added and the resulting solution was heated to 90–95 °C for 24 h to give compound 2a21.</p><!><p>A solution of potassium hydroxide (1.21 g, 21.56 mmol) and bromoethane (1.01 g, 9.24 mmol) in acetone (20 ml) was added to carbazole (1.03 g, 6.16 mmol) and the mixture was stirred at room temperature for 2 h to give compound 2b22.</p><!><p>To a solution of carbazole (1.67 g, 9.00 mmol) in DMF (20 ml) followed by addition of NaH (400 mg, 16.50 mmol) in small portions at 0 °C, over 5 min, benzyl halide (9.90 mmol) was added drop wise at 0 °C, over 5 min, the mixture was stirred at room temperature for 8–18 h to give compounds 2c–k23.</p><!><p>In a three-necked round-bottomed flask, POCl3 (700 mg, 4.56 mmol) was added dropwise to DMF (400 mg, 5.42 mmol) while stirring at 0 °C. The mixture was then stirred at room temperature for 1 h. A solution of 9-substituted carbazole (1.93 mmol) was slowly added to this mixture over 5 min at 45 °C, and then the temperature was raised to 95 °C for 8–18 h to yield compounds 4a–l23.</p><!><p>A mixture of the required compound 4 (2 mmol) and metformin hydrochloride (2 mmol) in glacial acetic acid (20 ml) was heated under reflux at 120 °C for 4–8 h (completion of the reaction was monitored by TLC). The solvent was then removed under reduced pressure. The crude products were purified by silica gel column chromatography using dichloromethane:methanol (20:1) as the eluent.</p><!><p>White powder; yield 52.3%; m.p. 267.3–268.9 °C. IR (KBr) cm−1: 3300, 3134 (NH2, NH), 1606 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.88 (br s, 2H, NH2), 8.26–8.11 (m, 2H, Ar-H and NH), 7.76–7.46 (m, 5H, Ar-H), 7.25 (t, 1H, J = 7.6 Hz, Ar-H), 5.99 (s, 1H, CH), 3.90 (s, 3H, CH3), 3.08 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 157.59, 155.85, 141.05, 140.75, 131.06, 126.01, 123.96, 121.75, 121.63, 120.13, 119.01, 118.02, 109.39 (2 C), 62.77, 36.93 (2 C), 29.12. HRMS (MALDI) calcd for C18H20N6 [M + H]+: 321.1822, found: 321.1806.</p><!><p>White powder; yield 61.6%; m.p. 256.9–259.8 °C. IR (KBr) cm−1: 3356, 3132 (NH2, NH), 1598 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.80 (br s, 2H, NH2), 8.25–8.12 (m, 2H, Ar-H), 7.68 (dd, 2H, J = 15.0, 8.4 Hz, Ar-H), 7.51 (ddd, 2H, J = 16.2, 8.3, 1.5 Hz, Ar-H), 7.24 (t, 1H, J = 7.4 Hz, Ar-H), 5.98 (s, 1H, CH), 5.76 (s, 1H, NH), 4.48 (q, 2H, J = 7.1 Hz, CH2), 3.08 (s, 6H, CH3), 1.31 (t, 3H, J = 7.0 Hz, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 157.66, 155.83, 139.95, 139.66, 131.06, 126.02, 123.98, 121.95, 121.82, 120.27, 118.98, 118.21, 109.34 (2 C), 62.76, 37.06, 36.94 (2 C), 13.67. HRMS (MALDI) calcd for C19H22N6 [M + H]+: 335.1979, found: 335.1967.</p><!><p>White powder; yield 46.1%; m.p. 237.1–238.8 °C. IR (KBr) cm−1: 3440, 3240 (NH2, NH), 1606 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.83 (br s, 2H, NH2), 8.27–8.16 (m, 2H, Ar-H), 7.70 (dd, 2H, J = 11.5, 8.4 Hz, Ar-H), 7.55–7.44 (m, 2H, Ar-H), 7.25 (dtd, 4H, J = 8.1, 6.5, 4.4 Hz, Ar-H), 7.16 (dd, 2H, J = 8.0, 1.7 Hz, Ar-H), 5.97 (s, 1H, CH), 5.76 (s, 1H, NH), 5.70 (s, 2H, CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 157.62, 155.85, 140.60, 140.29, 137.65, 131.37, 128.59 (2 C), 127.28 (2 C), 126.71, 126.20, 124.18, 122.01, 121.92, 120.30, 119.35, 118.29, 109.84 (2 C), 62.76, 45.67, 36.96, 33.9. HRMS (MALDI) calcd for C24H24N6 [M + H]+: 397.2135, found: 397.2134.</p><!><p>White powder; yield 58.8%; m.p. 237.2–239.8 °C. IR (KBr) cm−1: 3350, 3037 (NH2, NH), 1609 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.88 (s, 1H, NH2), 8.82 (s, 1H, NH2), 8.32–8.14 (m, 2H, Ar-H), 7.69 (dd, 2H, J = 12.8, 8.2 Hz, Ar-H), 7.49 (dd, 2H, J = 19.7, 8.4 Hz, Ar-H), 7.25 (t, 1H, J = 7.4 Hz, Ar-H), 7.07 (d, 4H, J = 4.7 Hz, Ar-H), 5.97 (s, 1H, CH), 5.77 (s, 1H, NH), 5.65 (s, 2H, CH2), 3.07 (s, 6H, CH3), 2.22 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 158.06, 156.35, 141.06, 140.77, 136.92, 135.05, 131.69, 129.58 (2 C), 127.20 (2 C), 126.63, 124.63, 122.47, 122.40, 120.75, 119.77, 118.79, 110.33 (2 C), 63.34, 55.44, 45.95, 37.41, 21.09. HRMS (MALDI) calcd for C25H26N6 [M + H]+: 411.2292, found: 411.2299.</p><!><p>White powder; yield 40.1%; m.p. 201.2–205.0 °C. IR (KBr) cm−1: 3423, 3144 (NH2, NH), 1603 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.90 (s, 1H, NH2), 8.84 (s, 1H, NH2), 8.30–8.16 (m, 2H, Ar-H), 7.68 (dd, 2H, J = 13.2, 8.3 Hz, Ar-H), 7.56–7.43 (m, 2H, Ar-H), 7.33–7.22 (m, 3H, Ar-H), 7.03 (td, 1H, J = 7.4, 1.6 Hz, Ar-H), 6.88–6.78 (m, 1H, Ar-H), 5.97 (s, 1H, CH), 5.77 (s, 3H, NH and CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 161.69, 158.44, 157.61, 155.96, 140.57, 140.28, 131.43, 129.50 (d, 1 C, J = 8.1 Hz), 128.71 (d, 1 C, J = 4.2 Hz), 126.23, 124.57 (d, 1 C, J = 3.4 Hz), 124.22, 124.04, 122.08 (d, 1 C, J = 2.3 Hz), 120.28, 119.50, 118.34, 115.65, 115.37, 109.75, 62.92, 62.01, 56.01, 36.95. HRMS (MALDI) calcd for C24H23FN6 [M + H]+: 415.2041, found: 415.2037.</p><!><p>White powder; yield 40.1%; m.p. 255.2–257.1 °C. IR (KBr) cm−1: 3240, 3060 (NH2, NH), 1603 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.93–8.72 (br s, 2H, NH2), 8.35–8.18 (m, 2H, Ar-H), 7.75 (d, 1H, J = 2.1 Hz, Ar-H), 7.68–7.17 (m, 7H, Ar-H), 6.33 (d, 1H, J = 8.4 Hz, CH), 5.97 (s, 1H, NH), 5.76 (s, 2H, CH2), 3.08 (s, 6H, CH3). 13C NMR (75 MHz, DMSO-d6, ppm) δ 157.55, 155.97, 140.52, 140.25, 133.76, 132.77, 132.69, 131.74, 129.10, 128.50, 127.69, 126.43, 124.41, 122.17, 120.43 (2 C), 119.76, 118.48, 109.63 (2 C), 62.92, 45.32, 43.47, 36.95. HRMS (MALDI) calcd for C24H22Cl2N6 [M + H]+: 465.1356, found: 465.1359. Purity: 97.41% by HPLC (A: 0.1% FA in H2O; B: 0.1% FA in CH3CN, graded: 20–100%), tR 12.273 min, λ: 250 nm.</p><!><p>White powder; yield 37.6%; m.p. 240.7–243.2 °C. IR (KBr) cm−1: 3396, 3211 (NH2, NH), 1603 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.86 (s, 1H, NH2), 8.81 (s, 1H, NH2), 8.38–8.20 (m, 2H, Ar-H), 7.70–7.39 (m, 6H, Ar-H), 7.37–7.23 (m, 2H, Ar-H), 7.11 (dd, 1H, J = 8.8, 6.3 Hz, Ar-H), 6.36 (d, 1H, J = 8.0 Hz, CH), 5.98 (s, 1H, NH), 5.84–5.75 (m, 2H, CH2), 3.09 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 157.99, 156.37, 141.07, 140.80, 134.95, 132.24, 132.07, 130.10, 129.52, 127.97, 127.59, 126.86, 124.86, 122.57, 122.54, 120.90, 120.13, 118.95, 110.13 (2 C), 63.36, 55.43, 44.22, 37.42. HRMS (MALDI) calcd for C24H23ClN6 [M + H]+: 431.1745, found: 431.1733.</p><!><p>White powder; yield 54.9%; m.p. 207.0–209.2 °C. IR (KBr) cm−1: 3313, 3029 (NH2, NH), 1600 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 8.87 (s, 1H, NH2), 8.81 (s, 1H, NH2), 8.34–8.16 (m, 2H, Ar-H), 7.71 (dd, 2H, J = 13.6, 8.3 Hz, Ar-H), 7.59–7.45 (m, 2H, Ar-H), 7.39–7.20 (m, 4H, Ar-H), 7.06 (dd, 1H, J = 6.3, 2.9 Hz, Ar-H), 5.97 (s, 1H, CH), 5.77 (s, 1H, NH), 5.74 (s, 2H, CH2), 3.12 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 158.06, 156.36, 140.96, 140.74, 140.69, 133.67, 131.94, 131.03, 127.78, 127.03, 126.80, 125.77, 124.82, 122.52, 122.46, 120.85, 120.02, 118.91, 110.23 (2 C), 63.34, 55.44, 45.55, 37.42. HRMS (MALDI) calcd for C24H23ClN6 [M + H]+: 431.1745, found: 431.1749.</p><!><p>White powder; yield 36.7%; m.p. 262.4–264.1 °C. IR (KBr) cm−1: 3373, 3059 (NH2, NH), 1603 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 9.00 (s, 1H, NH2), 8.94 (s, 1H, NH2), 8.36–8.14 (m, 2H, Ar-H), 7.69 (dd, 2H, J = 14.6, 8.2 Hz, Ar-H), 7.60–7.43 (m, 3H, Ar-H), 7.35 (dd, 2H, J = 8.6, 2.1 Hz, Ar-H), 7.29–7.13 (m, 3H, Ar-H and NH), 5.98 (s, 1H, CH), 5.72 (s, 2H, CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 158.02, 156.35, 140.95, 140.67, 137.15, 132.36, 131.92, 129.05 (4 C), 126.75, 124.73, 122.52, 122.46, 120.83, 119.96, 118.85, 110.26 (2 C), 63.32, 49.04, 45.47, 37.41. HRMS (MALDI) calcd for C24H23ClN6 [M + H]+: 431.1745, found: 431.1761.</p><!><p>White powder; yield 35.1%; m.p. 248.7–252.1 °C. IR (KBr) cm−1: 3311, 3054 (NH2, NH), 1603 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 9.06 (s, 1H, NH2), 8.99 (s, 1H, NH2), 8.34–8.13 (m, 2H, Ar-H), 7.68 (dd, 2H, J = 15.2, 8.3 Hz, Ar-H), 7.63–7.41 (m, 5H, Ar-H), 7.25 (t, 1H, J = 7.5 Hz, Ar-H), 7.17–7.06 (m, 2H, Ar-H and NH), 5.99 (s, 1H, CH), 5.69 (s, 2H, CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 157.53, 155.87, 140.47, 140.20, 137.10, 131.49(4 C), 128.91 (2 C), 126.28, 124.27, 122.04, 121.98, 120.37, 119.49, 118.39, 109.79 (2 C), 62.86, 45.04, 36.94 (2 C). HRMS (MALDI) calcd for C24H23BrN6 [M + H]+: 475.1240, found: 475.1230.</p><!><p>White powder; yield 52.9%; m.p. 234.1–235.4 °C. IR (KBr) cm−1: 3306, 3061 (NH2, NH), 1605 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 9.06 (s, 1H, NH2), 9.01 (s, 1H, NH2), 8.35–8.15 (m, 2H, Ar-H), 7.78–7.62 (m, 4H, Ar-H), 7.56 (d, 1H, J = 8.4 Hz, Ar-H), 7.51–7.43 (m, 1H, Ar-H), 7.36–7.23 (m, 3H, Ar-H), 6.01 (s, 1H, CH), 5.83 (s, 2H, CH2), 5.77 (s, 1H, NH), 3.09 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 158.06, 156.33, 143.92, 140.95, 140.66, 133.04 (2 C), 132.15, 127.98 (2 C), 126.82, 124.80, 122.58, 122.54, 120.87, 120.10, 119.11, 118.88, 110.59, 110.18 (2 C), 63.28, 55.41, 49.03, 37.42. HRMS (MALDI) calcd for C25H23N7 [M + H]+: 422.2088, found: 422.2077.</p><!><p>White powder; yield 41.4%; m.p. 221.5–224.8 °C. IR (KBr) cm−1: 3383, 3070 (NH2, NH), 1600 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 9.19 (s, 2H, NH2), 8.44 (d, 2H, J = 19.4 Hz, Ar-H), 7.64 (dd, 4H, J = 15.7, 8.1 Hz, Ar-H), 7.55 (d, 3H, J = 7.6 Hz, Ar-H), 7.39 (d, 1H, J = 8.4 Hz, Ar-H), 7.29 (t, 1H, J = 6.9 Hz, Ar-H), 6.05 (s, 1H, CH), 5.76 (s, 1H, NH), 3.08 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 158.01, 156.25, 141.08, 139.79, 136.62, 133.51, 130.71 (2 C), 129.41, 128.52, 127.02 (2 C), 125.85, 124.88, 123.47, 121.88, 119.35, 112.82, 112.18, 110.60, 63.04, 55.41, 49.03. HRMS (MALDI) calcd for C23H21BrN6 [M + H]+: 461.1084, found: 461.1066. Purity: 98.69% by HPLC (A: 0.1% FA in H2O; B: 0.1% FA in CH3CN, graded: 20–100%), tR 11.660 min, λ: 250 nm.</p><!><p>A mixture of the required compound 4 (2.3 mmol) and aminoguanidine hydrochloride (2 mmol) in ethanol (20 ml) in the presence of 5 drops of concentrated hydrochloric acid was stirred at 50–60 °C for 8–12 h. The solvent was then removed under reduced pressure. The crude products were purified by silica gel column chromatography using dichloromethane:methanol (10:1) as the eluent.</p><!><p>White powder; yield 52.3%; m.p. 199.1-200.8 °C. IR (KBr) cm−1: 3330, 3147 (NH2, NH), 1629 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.89 (br s, 1H, NH), 8.63 (s, 1H, CH = N), 8.33 (s, 1H, Ar-H), 8.18 (d, 1H, J = 8.0 Hz, Ar-H), 8.05 (d, 1H, J = 8.4 Hz, Ar-H), 7.91–7.46 (m, 6H, Ar-H and NH), 7.29 (d, 1H, J = 7.5 Hz, Ar-H), 3.94 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.41, 147.77, 141.81, 141.07, 126.20, 125.14, 124.36, 122.08, 121.96, 120.59, 120.36, 119.41, 109.53, 109.40, 29.16. HRMS (MALDI) calcd for C15H15N5 [M + H]+: 266.1400, found: 266.1405.</p><!><p>White powder; yield 59.2%; m.p. 199.4–200.5 °C. IR (KBr) cm−1: 3354, 3120 (NH2, NH), 1628 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.85 (br s, 1H, NH), 8.63 (s, 1H, CH = N), 8.33 (d, 1H, J = 1.9 Hz, Ar-H), 8.18 (d, 1H, J = 7.7 Hz, Ar-H), 8.03 (d, 1H, J = 8.6 Hz, Ar-H), 7.77–7.46 (m, 6H, Ar-H and NH), 7.27 (t, 1H, J = 7.3 Hz, Ar-H), 4.49 (q, 2H, J = 6.9 Hz, CH2), 1.33 (t, 3H, J = 7.1 Hz, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.42, 147.76, 140.83, 140.04, 131.54, 128.68, 126.26, 125.23, 124.44, 122.31, 120.72, 120.53, 119.42, 109.56, 109.40, 37.18, 18.68. HRMS (MALDI) calcd for C16H17N5 [M + H]+: 280.1557, found: 280.1548.</p><!><p>White powder; yield 63.1%; m.p. 271.0–272.4 °C. IR (KBr) cm−1: 3319, 3154 (NH2, NH), 1600 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.91 (br s, 1H, NH), 8.67 (s, 1H, CH = N), 8.34 (s, 1H, Ar-H), 8.21 (d, 1H, J = 7.7 Hz, Ar-H), 8.01 (d, 1H, J = 8.5 Hz, Ar-H), 7.84–7.65 (m, 5H, Ar-H and NH), 7.47 (t, 1H, J = 7.7 Hz, Ar-H), 7.24 (dt, 6H, J = 18.8, 7.5 Hz, Ar-H), 5.71 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.82, 148.14, 141.94, 141.09, 137.95, 132.14, 131.97, 129.05, 127.80, 127.21, 126.85, 125.85, 125.26, 122.87, 122.73, 121.10, 120.99, 120.21, 110.50, 110.33, 46.20. HRMS (MALDI) calcd for C21H19N5 [M + H]+: 342.1713, found: 342.1701.</p><!><p>White powder; yield 45.0%; m.p. 295.5–298.0 °C. IR (KBr) cm−1: 3382, 3134 (NH2, NH), 1629 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.82 (br s, 1H, NH), 8.59 (s, 1H, CH = N), 8.25 (s, 1H, Ar-H), 8.13 (d, 1H, J = 7.7 Hz, Ar-H), 7.94 (dd, 1H, J = 8.7, 1.6 Hz, Ar-H), 7.75–7.58 (m, 4H, Ar-H and NH), 7.40 (t, 2H, J = 7.7 Hz, Ar-H), 7.20 (t, 1H, J = 7.4 Hz, Ar-H), 7.01 (s, 4H, Ar-H), 5.59 (s, 2H, CH2), 2.14 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.46, 147.63, 141.47, 140.63, 136.53, 134.46, 129.14 (2 C), 126.81 (2 C), 126.37, 125.39, 124.78, 122.43, 122.30, 120.65, 120.54, 119.71, 110.09, 109.91, 45.56, 20.63. HRMS (MALDI) calcd for C22H21N5 [M + H]+: 356.1869, found: 356.1854. Purity: 96.74% by HPLC (A: 0.1% FA in H2O; B: 0.1% FA in CH3CN, graded: 20–100%), tR 11.533 min, λ: 250 nm.</p><!><p>White powder; yield 39.3%; m.p. 248.3–249.9 °C. IR (KBr) cm−1: 3356, 3163 (NH2, NH), 1637 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.88 (s, 1H, NH2), 8.67 (s, 1H, CH = N), 8.32 (s, 1H, Ar-H), 8.21 (d, 1H, J = 7.8 Hz, Ar-H), 8.02 (dd, 1H, J = 8.7, 1.6 Hz, Ar-H), 7.89–7.41 (m, 6H, Ar-H and NH), 7.37–7.19 (m, 3H, Ar-H), 7.04 (td, 1H, J = 7.4, 1.4 Hz, Ar-H), 6.89 (td, 1H, J = 7.7, 1.7 Hz, Ar-H), 5.77 (d, 2H, J = 2.2 Hz, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 161.68, 158.43, 155.40, 147.52, 141.38, 140.51, 128.75 (d, 1 C, J = 4.1 Hz), 126.40, 125.36, 124.93, 124.57 (d, 1 C, J = 3.3 Hz), 124.12, 123.92, 122.43, 122.30, 120.58 (d, 1 C, J = 8.3 Hz), 119.85, 115.67, 115.39, 109.89 (d, 1 C, J = 8.6 Hz), 65.01. HRMS (MALDI) calcd for C21H18FN5 [M + H]+: 360.1619, found: 360.1611.</p><!><p>White powder; yield 58.2%; m.p. 240.7–242.8 °C. IR (KBr) cm−1: 3397, 3146 (NH2, NH), 1629 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 12.12 (br s, 1H, NH), 8.71 (s, 1H, CH = N), 8.35 (s, 1H, Ar-H), 8.24 (d, 1H, J = 7.7 Hz, Ar-H), 8.11–7.66 (m, 5H, Ar-H and NH), 7.59 (d, 1H, J = 8.6 Hz, Ar-H), 7.54–7.41 (m, 2H, Ar-H), 7.29 (t, 1H, J = 7.3 Hz, Ar-H), 7.19 (dd, 1H, J = 8.3, 2.3 Hz, Ar-H), 6.41 (d, 1H, J = 8.4 Hz, Ar-H), 5.75 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.52, 147.63, 141.45, 140.55, 133.66, 132.83, 132.76, 129.13, 128.66, 127.72, 126.61, 125.56, 125.24, 122.63, 122.46, 120.74, 120.68, 120.12, 109.83, 109.73, 43.59. HRMS (MALDI) calcd for C21H17Cl2N5 [M + H]+: 410.0934, found: 410.0932.</p><!><p>White powder; yield 45.4%; m.p. 249.7–250.4 °C. IR (KBr) cm−1: 3386, 3159 (NH2, NH), 1629 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.88 (br s, 1H, NH), 8.70 (s, 1H, CH = N), 8.33 (s, 1H, Ar-H), 8.24 (d, 1H, J = 7.6 Hz, Ar-H), 8.00 (dd, J = 8.6, 1H, 1.7 Hz, Ar-H), 7.96–7.43 (m, 7H, Ar-H and NH), 7.37–7.24 (m, 2H, Ar-H), 7.18–7.06 (m, 1H, Ar-H), 6.51–6.40 (m, 1H, Ar-H), 5.77 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.88, 147.98, 141.96, 141.05, 134.82, 132.26, 130.10, 129.55, 127.95, 127.71, 126.99, 125.94, 125.55, 122.97, 122.83, 121.20, 121.10, 120.45, 110.29, 110.18, 44.30. HRMS (MALDI) calcd for C21H18ClN5 [M + H]+: 376.1326, found: 376.1322.</p><!><p>White powder; yield 41.5%; m.p. 256.1–257.5 °C. IR (KBr) cm−1: 3389, 3137 (NH2, NH), 1634 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.93 (s, 1H, NH), 8.68 (s, 1H, CH = N), 8.33 (s, 1H, Ar-H), 8.22 (d, 1H, J = 7.7 Hz, Ar-H), 8.09–7.58 (m, 6H, Ar-H and NH), 7.49 (t, 1H, J = 7.7 Hz, Ar-H), 7.29 (t, 4H, J = 6.0 Hz, Ar-H), 7.08 (dd, 1H, J = 6.6, 3.1 Hz, Ar-H), 5.75 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.89, 148.00, 141.82, 140.97, 140.57, 133.66, 131.02, 127.81, 127.07, 126.96, 125.94, 125.83, 125.46, 122.91, 122.77, 121.19, 121.07, 120.38, 110.43, 110.28, 45.59. HRMS (MALDI) calcd for C21H18ClN5 [M + H]+: 376.1326, found: 376.1319.</p><!><p>White powder; yield 38.7%; m.p. 239.5–240.8 °C. IR (KBr) cm−1: 3266, 3057 (NH2, NH), 1629 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 12.07 (br s, 1H, NH), 8.68 (s, 1H, CH = N), 8.35 (d, 1H, J = 5.4 Hz, Ar-H), 8.21 (d, 1H, J = 7.7 Hz, Ar-H), 8.01 (d, 1H, J = 8.3 Hz, Ar-H), 7.68 (m, 5H, Ar-H and NH), 7.47 (t, 1H, J = 7.7 Hz, Ar-H), 7.38–7.24 (m, 3H, Ar-H), 7.18 (d, 2H, J = 8.7 Hz, Ar-H), 5.72 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.44, 147.58, 141.38, 140.53, 136.56, 131.96, 128.66 (2 C), 128.62 (2 C), 126.48, 125.47, 124.96, 122.49, 122.35, 120.72, 120.62, 119.88, 110.02, 109.86, 45.08. HRMS (MALDI) calcd for C21H18ClN5 [M + H]+: 376.1326, found: 376.1329.</p><!><p>White powder; yield 41.1%; m.p. 239.5–240.7 °C. IR (KBr) cm−1: 3377, 3161 (NH2, NH), 1629 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.86 (s, 1H, NH), 8.67 (s, 1H, CH = N), 8.32 (s, 1H, Ar-H), 8.21 (d, 1H, J = 7.7 Hz, Ar-H), 8.09–7.98 (m, 1H, Ar-H), 7.97–7.56 (m, 5H, Ar-H and NH), 7.48 (dt, 3H, J = 7.0, 3.5 Hz, Ar-H), 7.29 (t, 1H, J = 7.5 Hz, Ar-H), 7.13 (d, 2H, J = 8.5 Hz, Ar-H), 5.72 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.43, 147.62, 141.39, 140.54, 136.98, 131.54 (2 C), 129.00 (2 C), 126.49, 125.48, 124.96, 122.49, 122.35, 120.71, 120.61, 120.46, 119.89, 110.02, 109.85, 45.16. HRMS (MALDI) calcd for C21H18BrN5 [M + H]+: 420.0818, found: 420.0829.</p><!><p>White powder; yield 45.7%; m.p. 240.6–243.9 °C. IR (KBr) cm−1: 3300, 3123 (NH2, NH), 1631 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.92 (br s, 1H, NH), 8.68 (s, 1H, CH = N), 8.33 (s, 1H, Ar-H), 8.22 (d, 1H, J = 7.7 Hz, Ar-H), 8.17–7.55 (m, 8H, Ar-H and NH), 7.48 (t, 1H, J = 7.7 Hz, Ar-H), 7.30 (dd, 3H, J = 7.9, 2.2 Hz, Ar-H), 5.85 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 155.31, 147.68, 143.30, 141.37, 140.50, 132.61 (2 C), 127.56 (2 C), 126.60, 125.57, 125.09, 122.54, 122.35, 120.71 (2 C), 120.05, 118.65, 110.15, 109.91, 109.79, 45.43. HRMS (MALDI) calcd for C22H18N6 [M + H]+: 367.1666, found: 367.1656.</p><!><p>A mixture of the required compound 4 (2 mmol) and either thiosemicarbazide hydrochloride or semicarbazide hydrochloride(2 mmol) in ethanol (20 ml) was stirred at 50–60 °C for 8–12 h in the presence of five drops of concentrated hydrochloric acid. The solution was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography using dichloromethane:methanol (80:1) as the eluent.</p><!><p>White solid; yield 60.5%; m.p. 222.4–223.8 °C. IR (KBr) cm−1: 3238, 3026 (NH2, NH), 1630 (C = N), 1120 (C = S). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.42 (s, 1H, NH), 8.59 (s, 1H, CH = N), 8.21 (d, 3H, J = 7.8 Hz, Ar-H), 8.08–7.89 (m, 2H, NH2), 7.62 (d, 2H, J = 8.6 Hz, Ar-H), 7.50 (t, 1H, J = 7.6 Hz, Ar-H), 7.25 (t, 1H, J = 7.4 Hz, Ar-H), 3.90 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 177.45, 143.65, 141.54, 141.02, 126.11, 125.11, 125.02, 122.19, 122.00, 120.47, 120.19, 119.30, 109.41, 109.36, 29.09. HRMS (MALDI) calcd for C15H14N4S [M + H]+: 283.1012, found: 283.1020.</p><!><p>White solid; yield 64.6%; m.p. 199.8–202.0 °C. IR (KBr) cm−1: 3280, 3043 (NH2, NH), 1594 (C = N), 1200 (C = S). 1H NMR (300 MHz, DMSO-d6, ppm) δ 11.40 (s, 1H, NH), 8.58 (s, 1H, CH = N), 8.27–8.13 (m, 3H, Ar-H), 8.04–7.91 (m, 2H, Ar-H), 7.64 (s, 1H, NH2), 7.61 (s, 1H, NH2), 7.53–7.44 (m, 1H, Ar-H), 7.24 (t, 1H, J = 7.4 Hz, Ar-H), 4.45 (q, 2H, J = 6.9 Hz, CH2), 1.32 (t, 3H, J = 6.8 Hz, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 177.45, 143.56, 140.52, 139.96, 126.12, 125.13, 125.08, 122.39, 122.21, 120.61, 120.24, 119.25, 109.39, 109.29, 37.10, 18.64. HRMS (MALDI) calcd for C16H16N4S [M + H]+: 297.1168, found: 297.1161.</p><!><p>White solid; yield 42.3%; m.p. 211.1–212.0 °C. IR (KBr) cm−1: 3343, 3049 (NH2, NH), 1691 (C = O), 1595 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 10.17 (s, 1H, NH), 8.51 (s, 1H, CH = N), 8.20 (d, 1H, J = 7.7 Hz, Ar-H), 8.01 (s, 1H, Ar-H), 7.86 (d, 1H, J = 8.5 Hz, Ar-H), 7.61 (dd, 2H, J = 8.4, 3.7 Hz, Ar-H), 7.47 (t, 1H, J = 7.7 Hz, Ar-H), 7.23 (t, 1H, J = 7.4 Hz, Ar-H), 6.52 (br s, 2H, NH2), 4.46 (q, 2H, J = 7.0 Hz, CH2), 1.32 (t, 3H, J = 6.9 Hz, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 157.03, 140.48, 140.05, 139.93, 126.01, 125.89, 124.46, 122.33, 122.22, 120.58, 119.22, 119.08, 109.31, 109.19, 37.06, 13.72. HRMS (MALDI) calcd for C16H16N4O [M + H]+: 281.1397, found: 281.1392.</p><!><p>The intermediate 4 (2 mmol) reacted with isonicotinic acid hydrazide (2 mmol) in the presence of catalytic amounts of hydrochloric acid (5 drops) in ethanol (20 ml) at 70 °C for 5 h. The solution was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography with (dichloromethane: methanol = 60:1).</p><!><p>Yellow solid; yield 80.0%; m.p. 198.0–199.5 °C. IR (KBr) cm−1: 3250, 3046 (NH), 1646 (C = O), 1601 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 12.02 (s, 1H, NH), 8.90–8.73 (m, 2H, Ar-H), 8.65 (s, 1H, CH = N), 8.55 (s, 1H, Ar-H), 8.31 (dd, 1H, J = 9.6, 6.5 Hz, Ar-H), 7.97–7.63 (m, 5H, Ar-H and pyridine-H), 7.48 (dd, 1H, J = 10.7, 4.7 Hz, Ar-H), 7.38–7.06 (m, 6H, Ar-H), 5.73 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 161.44, 150.29 (2 C), 141.50, 140.77, 140.70, 137.43, 128.59 (2 C), 127.32 (2 C), 126.70 (2 C), 126.40, 125.41, 125.04, 122.60, 122.25, 121.54 (2 C), 120.69, 120.29, 119.74, 110.03, 109.92, 45.81. HRMS (MALDI) calcd for C26H20N4O [M + H]+: 405.1710, found: 405.1719.</p><!><p>Yellow solid; yield 74.7%; m.p. 196.0–197.8 °C. IR (KBr) cm−1: 3320, 3060 (NH), 1647 (C = O), 1595 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 12.02 (s, 1H, NH), 8.80 (d, 2H, J = 5.0 Hz, Ar-H), 8.63 (s, 1H, CH = N), 8.54 (s, 1H, pyridine–H), 8.29 (d, 1H, J = 7.4 Hz, pyridine–H), 7.95–7.81 (m, 3H, Ar-H), 7.71 (dd, 2H, J = 23.9, 8.3 Hz, pyridine–H), 7.48 (t, 1H, J = 7.6 Hz, Ar-H), 7.27 (t, 1H, J = 7.4 Hz, Ar-H), 7.09 (s, 4H, Ar-H), 5.66 (s, 2H, CH2), 2.22 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 161.40, 150.29 (2 C), 150.21, 141.43, 140.72, 140.63, 136.52, 134.40, 129.13 (2 C), 126.73 (2 C), 126.37, 125.28, 124.95, 122.52, 122.19, 121.57, 120.69, 120.33, 119.68, 110.08, 109.97, 45.53, 20.58. HRMS (MALDI) calcd for C27H22N4O [M + H]+: 419.1866, found: 419.1854.</p><!><p>Yellow solid; yield 73.8%; m.p. 220.3–221.5 °C. IR (KBr) cm−1: 3366, 3050 (NH), 1651 (C = O), 1589 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 12.07 (s, 1H, NH), 8.82 (s, 2H, Ar-H), 8.68 (s, 1H, CH = N), 8.57 (s, 1H, pyridine–H), 8.26 (s, 1H, pyridine–H), 8.00–7.83 (m, 3H, Ar-H), 7.66 (dd, 2H, J = 26.4, 8.4 Hz, pyridine–H), 7.46 (t, 1H, J = 7.6 Hz, Ar-H), 7.35–7.16 (m, 3H, Ar-H), 7.00 (t, 1H, J = 7.2 Hz, Ar-H), 6.89 (t, 1H, J = 7.7 Hz, Ar-H), 5.74 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 161.68, 161.43, 150.29 (2 C), 150.14, 141.38, 140.70, 140.55, 129.53 (d, 1 C, J = 8.1 Hz), 128.66 (d, 1 C, J = 4.3 Hz), 126.44, 125.48, 125.05, 124.58 (d, 1 C, J = 3.4 Hz), 122.59, 122.24, 121.56 (2 C), 120.71, 120.28, 119.86, 115.67, 115.39, 110.03, 109.86, 40.23. HRMS (MALDI) calcd for C26H19FN4O [M + H]+: 423.1616, found: 423.1612.</p><!><p>Yellow solid; yield 64.2%; m.p. 209.1–210.3 °C. IR (KBr) cm−1: 3400, 3137 (NH), 1657 (C = O), 1597 (C = N). 1H NMR (300 MHz, DMSO-d6, ppm) δ 12.04 (s, 1H, NH), 8.88–8.73 (m, 2H, Ar-H), 8.64 (s, 1H, CH = N), 8.58 (d, 1H, J = 1.6 Hz, pyridine–H), 8.33 (d, 1H, J = 7.7 Hz, pyridine–H), 7.96–7.69 (m, 4H, Ar-H and pyridine–H), 7.68–7.17 (m, 5H, Ar-H), 6.42 (d, 1H, J = 8.4 Hz, Ar-H), 5.76 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) δ 161.38, 150.28 (2 C), 150.01, 141.34, 140.67, 140.50, 133.60, 132.73, 132.67, 129.09, 128.41, 127.68, 126.60, 125.74, 125.18, 122.67, 122.31, 121.54 (2 C), 120.84, 120.34, 120.07, 109.92, 109.73, 54.91. HRMS (MALDI) calcd for C26H18Cl2N4O [M + H]+: 473.0930, found: 473.0941.</p><!><p>The in vitro antimicrobial and antifungal activities of the synthesised compounds were evaluated to obtain the minimum inhibitory concentrations (MICs), using a 96-well microtiter plate and a serial dilution method. Gatifloxacin and moxifloxacin were used as positive controls and DMSO as a negative control. The micro-organisms used in the present study were two Gram-positive strains (Staphylococcus aureus 4220, Streptococcus mutans 3289), one clinical isolate of multidrug-resistant Gram-positive bacterial strain (Methicillin-resistant Staphylococcus aureus CCARM 3167), one Gram-negative strain (Escherichia coli 1924) and one fungus (Candida albicans 7535). The bacteria were grown to mid-log phase in Mueller-Hinton broth and diluted 1000-fold in the same medium. Stock solutions of the test compounds in dimethyl sulfoxide were prepared and then poured into 96-well plates. The final concentration of 0.5–64 µg/mL underwent a twofold serial dilution14,24. The bacteria were suspended and contained approximately 105 CFU/mL. These were applied to 96-well plates with a serial dilution and incubated at 37 °C for 24 h. The bacteria growth was measured from the turbidity at 630 nm using a microplate reader. All experiments were carried out in triplicate. Their MBC and MFC values were also determined (only for the compounds with MIC values of < 256 μg/mL).</p><!><p>Cell viability has been determined using the 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium Bromide (MTT, Sigma–Aldrich, Milan, Italy) assay. Cells have been seeded on 48 well plates and grown in complete medium. Before being treated, cells have been starved in serum-free medium for 24 h for allowing cell cycle synchronisation. 72 h after treatments, fresh MTT, re-suspended in PBS, was added to each well (final concentration (0.5 mg/mL). After 2 h incubation at 37 °C, cells have been lysed with DMSO, and then optical density was measured at 570 nm using a microplate reader. At least six doses of the studied compounds, solubilised in DMSO (0.1% final concentration), have been evaluated and each experiment has been performed in triplicate. The absorbance values have been used to determine the IC50 for each cell line using GraphPad Prism 5 Software (GraphPad Inc., San Diego, CA).</p><!><p>Preliminary docking was performed to evaluate whether binding to DHFR might account for the bactericidal effect of the compounds. All docking studies were carried out using Discovery Studio 2017 (Accelrys, San Diego, CA). Docking studies were performed according to our previous report25. The crystal structure data were obtained from the protein data bank (E. coli DHFR PDB_ID: 1RX7). Enzyme structures were checked for missing atoms, bonds and contacts. Hydrogen atoms were added to the enzyme structure. Water molecules and bound ligands were manually deleted. Then the protein was refined with CHARMm. The structures of compounds were sketched in 2D and converted into 3D using the DS molecule editor. Automated docking studies were carried out to investigate the binding mode of compounds 8b and 8f utilizing DS-CDOCKER protocol. The pose with the top CDOCKER_INTERACTION_ENERGY was chosen for analysing the binding features of compounds 8b and 8f with DHFR.</p><!><p>The inhibition of DHFR activities of compound 8f was measured using ELISA kits (Mlbio, Shanghai, China) under different concentrations (0, 0.1, 0.3, 1, 3, 10 µmol/L), according to the manufacturer's instructions.</p><!><p>The synthetic routes used for the construction of target compounds 8a–l, 9a–k, 10a–b, 11 and 12a–d are outlined in Scheme 1. A series of intermediates (2) was obtained from carbazole (1) via N-alkylation or N-arylation. Next, we performed a formylation reaction (Vilsmayer-Hack) on the intermediates (2) and commercially available compound 3 in the presence of POCl3 and DMF to obtain 9-substituted carbazole-3-carbaldehyde analogues 4 in good yields. Compounds 8a–l were prepared by the reaction of each of the series 4 compounds with metformin hydrochloride in acetic acid. Compounds 9a–k were synthesised via the reactions of each of the series 4 compounds with aminoguanidine hydrochloride in ethanol with a catalytic amount of concentrated hydrochloric acid. Compounds 10a–b and 11 were prepared by reacting compounds 4 with thiosemicarbazide and semicarbazide, respectively, in the presence of acetic acid in methanol under reflux. Next, we reacted compounds 4 with isonicotinic in the presence of acetic acid in alcohol to obtain compounds 12a–d.</p><!><p>Synthetic scheme for the synthesis of the target compounds. Reagents and conditions: (a) DMC, DABCO, 95 °C, 24 h (2a); KOH, 25 °C, 2 h (2b); NaH, DMF, 25 °C, 16 h (2c–k) (b) POCl3, DMF, 90 °C, 8–18 h (c) AcOH, 120 °C, 4–8 h (d) CH2CH3OH, HCl, 40 °C, 6 h (9a–k); CH3OH, AcOH, 68 °C, 4 h (10a–b,11) (e) CH2CH3OH, AcOH, 70 °C, 5 h.</p><!><p>The antimicrobial activities of the synthesised compounds were then tested against a series of pathogenic bacterial and fungal strains. Several different bacterial strains and one fungal strain were susceptible to most of the compounds tested with MICs in the range of 1–64 µg/ml. N-aryl carbazole derivatives containing an aminoguanidine or 1,4-dihydro-1,3,5-triazine moiety (8c–l and 9c–k) exhibited potent antibacterial activities with MICs ranging from 0.5 to 16 µg/ml. These compounds also exhibited antifungal activity against Candida albicans 7535 with MICs ranging from 1 to 32 µg/ml.</p><p>Compounds 8f, 8l, 9d and 9e exhibited strong antibacterial activity against two Gram-positive strains (methicillin-resistant Staphylococcus aureus (MRSA CCARM 3167) and Staphylococcus aureus RN4220) and one Gram-negative strain (Escherichia coli 1924) with MIC values of 0.5 or 1 µg/ml. Furthermore, the antibacterial activities of compound 8f against Gram-positive bacterial strains (S. aureus RN4220 and Streptococcus mutans 3289) were comparable to the positive control drugs gatifloxacin and moxifloxacin. In addition, compound 8f displayed considerable potency against MRSA CCARM 3167 (MIC of 0.5 μg/ml) that was two- and four-fold greater compared with moxifloxacin (MIC of 1 μg/ml) and gatifloxacin (MIC of 2 μg/ml) respectively. In addition, compound 8f demonstrated a strong inhibitory activity (MIC of 0.5 μg/ml) against E. coli 1924, which was four-fold greater than the activities of moxifloxacin (MIC of 2 μg/ml) and gatifloxacin (MIC of 2 μg/ml), respectively. In contrast, compound 8f showed a weaker activity against the fungus C. albicans 7535 compared with the positive controls.</p><p>Moreover, we determined the minimum bactericidal concentrations (MBCs) and minimum fungicidal concentrations (MFCs) for compounds whose MIC value less than 256 µg/ml against strains. MBCs and MFCs are defined as the lowest bactericidal or fungicidal concentration required to kill a particular bacterial strain or the fungal strain over a fixed incubation time. Most of the series 8 compounds displayed MBCs at 1–2 fold higher than their MICs against the strains tested. Compounds 9i and 9k also had four-fold higher MIC values compared with their MBCs against S. aureus RN4220 and MRSA CCARM 3167, respectively. The MBC/MIC ratio of compounds 9 g and 9j were ≥4 for S. mutans 3289. Compounds 9c, 9e, 9h and 9i demonstrated MFC values that were four-fold higher than their MICs against Candida albicans 7535. Notably, bacteria appeared to be less tolerant to the series 8 compounds compared with series 9 compounds. When we altered the phenyl ring substituents of compounds 8a–l, we observed a significant effect on the potency of their antimicrobial activities. The order of antibacterial activities were as follows: phenyl group > 2,4-dichloro-substitutions > 4-CH3(for compounds bearing an electron-donating group) > halogen substitutions > benzyl group > 4-CN (for compounds bearing an electron-withdrawing group) > alkyl group. Furthermore, bromo- and chloro-substitutions on the phenyl ring in compounds 8a–l were observed to improve their antifungal activity against C. albicans 7535. The antibacterial activity order of compounds 9a–k was similar to compounds 8a–l. Compounds in series 10a–b, 11 and 12a–d generally showed weak activity against all the strains tested in this study, with MICs greater than 256 µg/ml. As shown in Table 1, most of the compounds exhibited equivalent or higher potency (with MIC values in the range of 0.5 to 16 µg/ml) towards MRSA CCARM 3167 compared with the positive controls gatifloxacin and moxifloxacin (MICs of 2 and 1 µg/ml, respectively).</p><!><p>Inhibitory activities (MIC/MBC or MFC, μg/mL) of compounds 8a–l, 9a–k, 10a–b, 11 and 12a–d against various bacteria and fungi.</p><p>CCARM: Culture Collection Antimicrobial Resistant Microbes; KCTC: Korean Collection for Type Cultures.</p><p>aStaphylococcus aureus RN 4220.</p><p>bMethicillin-resistant S. aureus CCARM 3167.</p><p>cStreptococcus mutans 3289. dEscherichia coli KCTC 1924. eCandida albicans 7535.</p><p>fn.t.: Not test.</p><!><p>To summarise, our findings revealed that the compounds in series 8 exhibited significantly higher antimicrobial activities compared with the compounds in the other four series. This suggests that the presence of a dihydrotriazine moiety is critical to the potency of these carbazole derivatives. These results therefore provide further evidence to suggest that aryl groups and the dihydrotriazine moiety play critical roles in the activity of these carbazole compounds.</p><!><p>To determine whether the synthesised compounds were selectively toxic towards microbes and not human cells, we evaluated the cytotoxicity of six compounds in two gastric cancer cell lines (SGC-7901 and AGS) and a normal human liver cell line (L-02) using a standard technique. As shown in Table 2, the cytotoxicities of compounds 8b, 8d, 8f and 8k were lower than that of compounds 9b and 9e. Compounds 8b, 8d, 8f and 8k had no effect on human liver cell viability at their MICs and, in addition, their IC50 values were significantly higher than their MIC values. However, the IC50 values of the series 9 compounds were close to their MIC values, which make it likely that the promising antibacterial activity of these compounds is due to their cytotoxic properties.</p><!><p>Cytotoxic activity (IC50a, µg/mL) of compounds 8b, 8d, 8f, 8k, 9b and 9e against human cell lines.</p><p>aIC50 is the concentration required to inhibit the cell growth by 50%. Data represent the average of three independent experiments running in triplicate. Variation was generally between 5 and 10%.</p><p>bHuman gastric cancer cells.</p><p>cHuman normal hepatic cells.</p><!><p>Compounds in series 8a–l, containing a dihydro-1,3,5-triazine ring moiety, were generally found to have more potent antimicrobial effects but less cytotoxicity compared with the corresponding series 9a–l compounds that comprised an aminoguanidine moiety. Interestingly, our results also showed that compound 9 b was selectively more toxic to human cancer cells compared with normal human cells. The IC50 value is about three times higher in normal cells than AGS cell lines. Further studies are required to investigate if this compound has any potential as a new anticancer agent.</p><!><p>The potent and selective antimicrobial activities of the series 8 compounds prompted us to study the binding of these derivatives to their potential target, E. coli DHFR. In 3D binding mode, alkyl chains (ethyl and methylene) at the 9-position of carbazole possess considerable flexibility (Figures 2–4). As shown in Figure 3, the aryl group at the 9-position of carbazole in compound 8f inserted deeply into the active pocket of DHFR (composed of Ala7, Trp22, Leu28 and Phe31). Moreover, both the folic acid substrate and compound 8f have a nitrogen atom in their heterocyclic rings that forms a hydrogen bond with the E. coli DHFR residue Gly15. The hydrophobic interactions between compound 8f and residues in the DHFR active pocket were enhanced due to the presence of the carbazole moiety. In addition, the primary amine group at the N-3 position of the carbazole ring plays an important role in binding to the active site of E. coli DHFR. A salt bridge was formed between the primary amine and Glu17. To summarise, docking results suggested that 8f, the compound with the most therapeutic potential, has interacted with the critical active-site residues of E. coli DHFR.</p><!><p>Binding mode of folate inside the E. coli DHFR active site.</p><p>Binding mode of 8f inside the E. coli DHFR active site.</p><p>Binding mode of 8b inside the E. coli DHFR active site.</p><!><p>To investigate whether compound 8f can bind to and block the active site of DHFR, we performed in vitro enzyme assays to test the inhibitory effect of compound 8f (MIC of 0.5 μg/ml) on DHFR activity (Figure 5). At concentrations of 3 and 10 μmol/L, compound 8f decreased DHFR activity by 71% compared with the negative control. The results indicated that compound 8f exerts its antibacterial activity via binding to DHFR, which however might not be the only mechanism of action.</p><!><p>Inhibition of DHFR of compound 8f.</p><!><p>We have designed, synthesised and evaluated the antibacterial and antifungal activities of five series of novel carbazole derivatives. Compound 8f showed the most potential as a therapeutic agent, with an MIC of 0.5–2 µg/ml against selected bacterial strains. Furthermore, compound 8f also was the least cytotoxic to SGC-7901, AGS and L-02 cells. Compound 9d also exhibited strong antibacterial activity with the cancer therapeutic potential. Therefore, the clinical potential of carbazole derivatives 8f and 9d could be explored further for future applications in antitumour and antimicrobial therapies.</p><p>Docking simulation and in vitro enzyme activity assays suggested that binding to DHFR might account for the antimicrobial activity of the compounds. Further studies of the mechanisms of action of these compounds are currently underway in our laboratories and will be reported in due course.</p><!><p>We declare that we have no conflict of interest with respect to this study.</p>

PubMed Open Access

Extending the \xcf\x83-Hole Motif for Sequence Specific Recognition of the DNA Minor Groove

The majority of current drugs against diseases, such as cancer, can bind to one or more sites in a protein and inhibit its activity. There are, however, well known limits on the number of druggable proteins and complementing current drugs with compounds that could selectively target DNA or RNA would greatly enhance therapeutic progress and options. We are focusing on the design of sequence-specific DNA minor groove binders that, for example, target the promoter sites of transcription factors involved in a disease. We have started with AT specific minor groove binders that are known to enter human cells and have entered clinical trials. To broaden the sequence-specific recognition of these compounds, we have identified several modules that have H-bond acceptors that strongly and specifically recognize G\xe2\x80\xa2C base pairs. A lead module is a thiophene-N-alkyl-benzimidazole \xcf\x83-hole based system with terminal phenyl-amidines where the optimum compounds have excellent affinity and selectivity for a G\xe2\x80\xa2C base pair in the minor groove. Efforts are now focused on optimizing this module. We have previously optimized the alkyl group. In the work described here we are evaluating modifications to the compound aromatic system with the goal of improving GC selectivity and affinity as with the N-alkyl modifications. The lead compounds from these studies retain the thiophene-N-alkyl-BI module but have halogen substituents adjacent to an amidine group on the terminal phenyl-amidine. Other improved compounds in this set have modified amidines and conversion of the amidine to an imidazoline, for example, resulted in a strong binding compound with good specificity.

extending_the_\xcf\x83-hole_motif_for_sequence_specific_recognition_of_the_dna_minor_groove

Introduction<!>Materials<!>UV-vis Thermal Melting (Tm)<!>Biosensor-Surface Plasmon Resonance (SPR)<!>Circular Dichroism (CD)<!>Competition Electrospray Ionization Mass Spectrometry (ESI-MS)<!>Fluorescence Spectroscopy Binding Determinations<!>Molecular Dynamics (MD) Simulations.<!>Chemistry<!>DNA Thermal Melting: Screening for Relative Binding Affinity<!>Biosensor-SPR: Methods for Quantitative Binding<!>Circular dichroism (CD): Determination of DNA Binding Mode<!>Fluorescence Titrations: Binding affinity<!>Competition electrospray ionization mass spectrometry (ESI\xe2\x80\x93MS): A Direct Determination of Binding Stoichiometry and Binding Specificity with Relative Binding Affinity<!>Molecular Structure: Molecular Dynamics<!>Discussion

<p>There are intense research efforts underway to design new synthetic compounds that have excellent selectivity for targeting RNA or DNA sequences and/or structures1–11. Such compounds could have a major impact on the treatment of important for diseases that are the result of aberrant behavior of transcription factors. Because nucleic acids are at an early stage in the disease development process, targeting them has many advantages, especially since many transcription factors are classified as "undruggable"12. Relative to drugs that bind to protein sites, however, drugs that are specific for DNA are relatively rare and except for aminoglycosides, are even more rare with RNA. Small molecule binding sites in proteins are relatively well-understood but the number of druggable proteins is only a very small part of the genome13. With this limit on protein-drug binding sites, the interest in therapeutic agents that are specific for RNA or DNA has rapidly increased.</p><p>Duplex DNA sequences and sequence-dependent local microstructures offer useful and specific targets for binding small molecules3,5,9,10. Major non-duplex structural forms of nucleic acids, such as quadruplexes, also offer the possibility of selective binding interactions and a targeted therapeutic effect14,15. With RNA an enormous variety of different structural forms are known and being discovered, but the selection of specific target regions and structures that will have a therapeutic effect and finding compounds specific for the structure remains a major challenge1,2,4,6,13.</p><p>Our development efforts have focused on novel compounds to bind with high selectivity properties in the DNA minor groove. Results so far in several laboratories have found the strongest binding, optimum recognition of DNA and most specific synthetic compounds bind in the DNA minor groove. Compounds that have served as models for AT sequence-specific minor groove binding over an extended time period include pentamidine, Hoechst 33258, netropsin, DAPI, furamidine, and analogs of all of these compounds. Such compounds are classical minor groove agents, they are all highly AT sequence-specific binders, and they have been used in therapeutic applications or for biotechnology purposes such as cell staining16–26. The compounds generally have H-bond donor groups, such as benzimidazoles, amides, and amidines/guanidiniums, that interact with the A-N3 and T=O groups on the edges of A·T base pairs (bps) at the floor of the minor groove. The G-NH2 that projects into the groove interferes with the binding of these compounds both sterically and electronically. To recognize a G·C bp then it is necessary to incorporate H-bond accepter groups into designed, synthetic minor groove binders27,28. The GC recognizing compounds should have a shape that allows space for the G-NH2 H-bond, follows the curvature of the minor groove and can also recognize AT bps in the target sequence. Needless to say, the design and preparation of such compounds is a challenge and to date has only rarely been accomplished9,10,27–36.</p><p>To initiate a project to systematically develop compounds that could specifically recognize G·C bps, a focused library of approximately 200 heterocyclic, cationic minor-groove binders with H-bond acceptor groups was evaluated. This analysis produced several initial compounds that could bind to a single G·C bp in an AT context. Three new compounds that bound strongly and specifically to a single G·C bp flanked by A·T bps (Figure S1 in Supporting Information) were selected for the initial study. The accepter groups in these compounds are azabenzimidazole, pyridine and a thiophene-N-methylbenzimidazole (N-MeBI) σ-hole motif which is preorganized into a conformation to match the curvature of the minor groove7. We have recently explored changing the methyl substituent on the thiophene-N-MeBI unit in a successful project to enhance the binding selectivity of that module9. The idea behind these substituent changes was to better recognize microstructural variations in the minor groove in addition to sequence recognition9,37,38. The results with the modified compounds were quite successful, with a modest loss in affinity to single G•C sequences, but with a major gain in binding selectivity over closely related sequences. The sequences that were tested, for example, were a single G•C sequence with an AAAGTTT binding site, and for selectivity a pure AT-binding sequence with an AAATTT site and a GC containing the binding site, AAAGCTTT (Figure 1B). These binding sites were incorporated in hairpin DNA duplexes for binding analysis.</p><p>In this report, we have built on the results with the modified thiophene-N-MeBI module, which illustrated that modified structures could have significantly improved recognition properties. We have addressed three specific, important questions in this work: what is the effect on binding affinity and selectivity of (i) modifying the terminal amidine group, (ii) incorporating different halogen substituents at different positions in the aromatic systems, and (iii) changing the basic structure of the thiophene-N-MeBI aromatic system? Modifications to include different halogens are based on successful, previous results with a -Cl substituent9. The amidine structure modifications as well as replacing the N-alkyl-BI-thiophene group by other chemically and stereochemically similar structures (Figure 1) are exploratory in order to establish the limits on the thiophene-N-alkyl-BI chemical space. The results with these new compounds illustrate new methods of design in the thiophene-σ-hole system with excellent specificity for recognition of G•C bps can be obtained even without the thiophene or alkyl-BI groups.</p><!><p>In the DNA thermal melting (Tm) and circular dichroism (CD) experiments, the hairpin oligomer sequences were used (Figure 1B). In SPR experiments, 5′-biotin-labeled hairpin DNA oligomers were used. All DNA oligomers were obtained from Integrated DNA Technologies, Inc. (IDT, Coralville, IA) with reverse-phase HPLC purification and mass spectrometry characterization.</p><p>The buffer used in Tm and CD experiments was 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.4 (TNE 100). The biosensor-surface plasmon resonance (SPR) experiments were performed in filtered, degassed TNE 100 with 0.05% (v/v) surfactant P20.</p><!><p>DNA thermal melting experiments were performed on a Cary 300 Bio UV-vis spectrophotometer (Varian). The concentration of each hairpin DNA sequence was 3 μM in TNE 100 using 1 cm quartz cuvettes. The solutions of DNA and ligands were tested with a ratio of 2:1 [ligand] / [DNA]. All samples were increased to 95 °C and cooled down to 25 °C slowly before each experiment. The spectrophotometer was set at 260 nm with a 0.5 °C/min increase beginning at 25 °C, which is below the DNA melting temperature and ending above it at 95 °C. The absorbance of the buffer was subtracted, and a graph of normalized absorbance versus temperature was created using KaleidaGraph 4.0 software. The ΔTm values were calculated using a combination of the derivative function and estimation from the normalized graphs.</p><!><p>SPR measurements were performed with a four-channel Biacore T200 optical biosensor system (GE Healthcare, Inc., Piscataway, NJ). A streptavidin-derivatized (SA) CM5 sensor chip was prepared for use by conditioning with a series of 180 s injections of 1 M NaCl in 50 mM NaOH (activation buffer) followed by extensive washing with HBS buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% P20, pH 7.4). Biotinylated-DNA samples (AAATTT: 5'-biotin-CCAAATTTGCCTCTGCAAATTTGG-3'; AAAGTTT: 5'-biotin-CCAAAGTTTGCTCTCAAACTTTGG-3'; AAAGCTTT: 5'-biotin-CCAAAGCTTTGCTCTCAAAGCTTTGG-3') of 25–30 nM were prepared in HBS buffer and immobilized on the flow cell surface by noncovalent capture. Flow cell 1 was left blank as a reference, while flow cells 2–4 were immobilized separately by manual injection of biotinylated-DNA stock solutions (flow rate of 1 μL/min) until the desired amount of DNA response units (RU) was obtained (150–250 RU). Ligand solutions were prepared with degassed and filtered TNE 100 with 0.05% (v/v) surfactant P20 by serial dilutions from a concentrated stock solution. Typically, a series of different ligand concentrations (2 nM to 500 nM) were injected over the DNA sensor chip at a flow rate of 100 μL/min for 180 s, followed by buffer flow for ligand dissociation (600–1800 s). After each cycle, the sensor chip surface was regenerated with a 10 mM glycine solution (pH 2.5) for 30 s followed by multiple buffer injections to yield a stable baseline for the following cycles. The reference response from the blank cell was subtracted from the response in each flow cell containing DNA to give a signal (RU, response units) that is directly proportional to the amount of bound compound. The predicted maximum response per bound compound in the steady-state region (RUmax) was determined from the DNA molecular weight, the amount of DNA on the flow cell, the compound molecular weight, and the refractive index gradient ratio of the compound and DNA. RU was plotted as a function of free ligand concentration (Cfree), and the equilibrium binding constants were determined with a one-site binding model (K2 = 0). (1)r=(K1·Cfree+2K1·K2·Cfree2)/(1+K1·Cfree+K1·K2·Cfree2) where r represents the moles of bound compound per mole of DNA hairpin duplex, K1 and K2 are macroscopic binding constants (for a single-site or 1:1 model K2 = 0), and Cfree is the free compound concentration in equilibrium with the complex. RUmax in the equation was used as a fitting parameter, and the obtained value was compared to the predicted maximal response per bound ligand to evaluate the stoichiometry. Kinetic analysis was performed by globally fitting the binding results for the entire concentration series using a standard 1:1 kinetic model with integrated mass transport-limited binding parameters as described previously39,40.</p><!><p>Circular dichroism experiments were performed on a Jasco J-1500 CD spectrometer in 1 cm quartz cuvette at 25 °C. A buffer scan as a baseline was collected first in the same cuvette and subtracted from the scan of the following samples. The hairpin DNA sequence AAATTT: 5'-CCAAATTTGCCTCTGCAAATTTGG-3'; AAAGTTT: 5'-CCAAAGTTTGCTCTCAAACTTTGG-3' (5 μM), Figure 1B, in TNE 100 was added to the cuvette prior to the titration experiments, and then the compound was added to the DNA solution and incubated for 10 min to achieve equilibrium binding for the DNA-ligand complex formation. For each titration point, four spectra were averaged from 500 to 230 nm wavelength with a scan speed of 50 nm/min, with a response time of 1 s. Baseline-subtracted graphs were created using the KaleidaGraph 4.0 software.</p><!><p>Electrospray Ionization Mass Spectrometry (ESI-MS) analyses were performed on a Waters Q-TOF micro Mass Spectrometer (Waters Corporate, Milford, MA) equipped with an electrospray ionization source (ESI) in a negative ion mode. DNA sequences AAATTT (5'-CCAAATTTGCCTCTCGAAATTTGG-3'), AAAGTTT (5'-GCCAAAGTTTGCCTCTGCAAACTTTGGC-3') and AAAGCTTT (5'-CCAAAGCTTTGCTCTCAAAGCTTTGG-3'), for ESI-MS experiments were purified by dialyzing it in 50 mM ammonium acetate buffer (pH 6.7) at 4 °C with 3x buffer exchange. Test samples were prepared in 50 mM ammonium acetate with 10% v/v methanol at pH 6.7 and introduced into the ion source through a direct infusion at 5 μl/min flow rate. The competitive experiments were done by mixing a ligand and DNAs with different sequences at different ratios. The instrument parameters were typically as follows: capillary voltage of 2800 V, sample cone voltage of 30 V, extraction cone voltage of 1.0 V, desolvation temperature of 70 °C, and source temperature of 100 °C. Nitrogen was used as nebulizing and drying gas. A multiply charged spectra were acquired through a full scan analysis at mass range from 300–2500 Da and then deconvoluted to the spectra presented. MassLynx 4.1 software was used for data acquisition and deconvolution.</p><!><p>Fluorescence spectra were recorded on a Cary Eclipse Spectrophotometer, with excitation and emission slit width as determined depending on the concentrations of ligands. The free compound solutions at different concentrations were prepared in an appropriate buffer in TNE 100, and DNA sequence (AAATTT: 5'-CCAAATTTGCCTCTGCAAATTTGG-3'; AAAGTTT: 5'-CCAAAGTTTGCTCTCAAACTTTGG-3'; AAAGCTTT: 5'-CCAAAGCTTTGCTCTCAAAGCTTTGG-3') aliquots were added from a concentrated stock. All titration spectra were collected after allowing an incubation time of 10 min. DB2802 was excited at 342 nm and DB2803 was excited at 336 nm based on molecular absorbance from UV-vis spectroscopy. Emission spectra of these compounds were monitored from 200 nm wavelength range. All the fluorescence titrations were performed at 25 °C. Then the Fluorescence titration spectrums and fitting plots were made in Kaleidagraph 4.0 software to determine the KD value.</p><!><p>Structure optimization of DB2789 was performed by using DFT/B3LYP theory with the 6–31+G* basis set in Gaussian 09 (Gaussian, Inc., 2009, Wallingford, CT) with Gauss-view 5.0941. Partial charges were derived using the RESP fitting method (Restrained Electrostatic potential)42,43. AMBER 16 (Assisted Model Building with Energy Refinement) software suite was used to perform molecular dynamics (MD) simulations44. Canonical B-form ds[5'-CGAAAGTTTCG-3'][5'-CGAAACTTTCG-3'] DNA was built in Nucleic Acid Builder (NAB) tool in AMBER. AMBER preparation and force field parameter files required conducting molecular dynamics simulations for the DB2789 molecule by using ANTECHAMBER45. Specific atom types assigned for DB2789 molecule were adapted from the ff99 force field. Most of the force field parameters for DB2789 molecule were derived from the existing set of bonds, angles and dihedrals for similar atom types in parm99 and GAFF force fields46. Some dihedral angle parameters were obtained from previously reported parametrized data47,48. The molecular structure with specific atom types used for the DB2789 molecule is shown in Figure S3. Parameters of DB2789 in frcmod file are listed at the Table S1.</p><p>The AutoDock Vina program was used to dock the DB2692 in the minor groove of DNA to obtain the initial structure for the DB2789-DNA complex49. MD simulations were performed in explicit solvation conditions where the DNA-DB2789 complex was placed in a truncated octahedron box filled with TIP3P water using xleap program in AMBER. Sodium ions were used to neutralize the system. A 10 Å cutoff was applied on all van der Waals interactions. The MD simulation was carried out using the Sander module with SHAKE algorithm applied to constrain all bonds. Initially, the system was relaxed with 500 steps of steepest-descent energy minimization. The temperature of the system was then increased from 0 K to 310 K over 10 ps under constant-volume conditions. In the final step, the production run on the system was subsequently performed for 500 ns under NPT (constant-pressure) conditions.</p><!><p>Scheme 1 outlines the synthesis of the final diamidines 6a-h. The nitro derivatives 2a-e were obtained by reaction of the bromo derivatives 1a-c with different boronic acid esters under the Suzuki coupling conditions using palladium tetrakis triphenylphosphine as a catalyst in dioxane and potassium carbonate solution. These nitro compounds were reduced to the corresponding amines 3a-e using tin dichloride dihydrate in ethanol. The diamines were converted to the corresponding benzimidazole dinitriles 5a-g on reaction with different aldehydes with the aid of sodium metabisulphite in DMSO. The intermediate dinitriles were converted to the final diamidines through reaction with either lithium bis(trimethylsilyl)amide in THF followed by deprotection of the silylated intermediate using HCl gas in ethanol, or applying Pinner methodology where the dinitriles are suspended and stirred in ethanolic HCl to form the imidate ester hydrochloride that was allowed to react with different amines to afford the final diamidines 6a-h. The synthesis of the diamidine 11 is described in Scheme S1 (supplemental information). 4-Ethynylbenzonitrile 7 was allowed to react with the bromo derivative 8 under Sonogoshira coupling conditions using palladium (II)bis(triphenylphosphine) dichloride and copper iodide as catalysts in a mixture of THF and triethylamine to afford the aldehyde 9. This intermediate aldehyde was converted to the benzimidazole 10 and then to the final diamidine 11, as described in Scheme 1. Compound 18 was prepared as described in Scheme S2. The intermediate bromo derivative 14 was prepared by reaction of the iodo derivative 12 and the tin compound 13 utilizing Stille cross-coupling conditions using palladium tetrakis triphenylphosphine as a catalyst in dioxane. The bromo derivative 14 was allowed to couple with cyanophenylboronic acid to afford the mono nitrile intermediate 15 by applying Suzuki conditions. This nitrile was brominated using NBS in DMF to furnish the intermediate bromo compound 16. This bromo derivative was converted to the dinitrile intermediate 17 using Suzuki coupling conditions. The final diamidine 18 was prepared after reaction lithium bis(trimethylsilyl)amide with the dinitrile as described before. Scheme S3 describes the synthesis of the final diamidine 25, starting from the bromoisatin 19. 2-Acetylthiophene 20 and 19 were heated in ethanolic potassium hydroxide to furnish the quinoline carboxylic acid derivative 21. The carboxylic acid 21 and copper cyanide in NMP heated at reflux led to cyanation and decarboxylation, furnishing the cyanoquinoline derivative 22. Compound 22 was subjected to bromination using NBS, followed by Suzuki coupling to produce the dinitrile 24. This dinitrile was converted to the final amidine 25 using the lithium bis(trimethylsilyl)amide as described before. The synthesis of the diamidines 29a,b is described in Scheme S4. Suzuki coupling of the bromo derivative 26 with cyanophenylboronic acid followed by condensation with different amines to produce the dinitriles 28a,b. Pinner conditions were applied to this dinitrile to produce the final compounds 29a,b.</p><!><p>Changes in thermal melting temperature (Tm) of DNA provide a rapid way for initial screening of ligands for binding affinity with different DNA sequences (Table 1). Three related DNA sequences that we have successfully used in comparative studies9 were selected for testing (Figure 1B). In order to determine the binding selectivity of our compounds, we chose two quite similar sequences, AAATTT and AAAGCTTT, as the control sequences for the AAAGTTT target site.</p><p>The parent compound, DB2457, with an N-methyl substituent was previously reported7 and has preferential binding to the single G·C bp sequence and weaker binding to the pure AT and two G·C bps sequences (Table 1). A compound with N-isopropyl substituents, DB2708, has similar binding to AAAGTTT as DB2457, and significantly weaker binding to the pure AT sequence for improved selectivity as desired9. DB2759 with a -Cl substituted on the phenyl ring keeps strong binding to AAAGTTT while binding with the two control sequences drops to an undetectable amount under the standard conditions.</p><p>Based on the successful results with modified N-substituents in improving the binding selectivity, focused modifications of the thiophene compounds in other parts of the structure is attractive to search for improved affinity and selectivity. First, considering the improved selectivity of the -Cl derivative, DB2759, -F, -Br halogen modifications were made as well as changing the position of the -Cl substituent to the opposite phenyl ring (Table 1). Both -Cl and the -Br compounds bind with the AAAGTTT sequence strongly and with good selectivity compared to AAATTT and AAAGCTTT. Surprisingly, the -F substituent binds much weaker to AAAGTTT compared to other halogen compounds. We also tested two compounds with modifications on the amidine group, DB2786, and DB2787. The isopropyl substituent obviously improves the binding to AAAGTTT, while the imidazoline compound has similar binding to all three sequences as DB27089. In addition to N-isopropyl substituents, we also tested an N-Ph substituted compound, DB2740, and found that it has strong binding and good selectivity for the single G•C sequence. DB2788 with a -Cl substituent was also prepared based on the success of the substituent in previous studies. In this compound, however, the -Cl modification did not significantly increase the binding selectivity for the N-Ph substituted compound.</p><p>In order to explore the effects on binding from variations of the basic aromatic structure, we also introduced a C≡C bond linkage into the structure (DB2770) to change the connection between phenyl rings. The acetylene linkage, however, reduces the binding by a large amount. Changing the N-RBI into a pyridine group produces DB2655, which binds strongly to AAAGTTT but with lower selectivity than the BI compounds. Changing the N-RBI into a quinoline (DB2654) decreases the single G·C bp sequence binding affinity but maintains a high level of selectivity. In addition to the N-RBI module, the thiophene was modified to a thiazole ring (DB2647 and DB2650) and, surprisingly, the simple change to a thiazole causes the compounds to entirely lose the function of the thiophene σ–hole system with the consequence of very poor binding. In addition to the thiazole, the thiophene was replaced with benzothiophene, DB2802 and DB2803. For both methyl and isopropyl BI substituted compounds, the binding affinity to the target AAAGTTT sequence decreases with a decrease in binding selectivity. The N-Me derivative, DB2802, binds to the pure AT test sequence in a similar manner to the single G·C bp sequence.</p><!><p>A biacore sensorchip (CM5) functionalized with streptavidin was used to immobilize 5'-biotin labeled hairpin duplex DNA sequences (Figure 1B) in flow cells 2–4 and flow cell 1 was left as a blank, for background subtraction. With different compounds in the flow solutions, we were able to determine comparative binding constants for all the derivatives (Table 2). Sensorgrams were obtained and are shown for representative compounds with the different DNAs (Figure 2). With the AAATTT and AAAGCTTT sequences, most compounds showed significantly weaker binding than to AAAGTTT, with faster on and off rates. No kinetics and limited KD values could be accurately determined for the pure AT and the two G•C bps DNA sequences with most compounds. However, with the single G•C bp containing AAAGTTT sequence, excellent sensorgrams were obtained at low concentrations for most derivatives and representative SPR sensorgrams are shown in Figure 2. Kinetics fits with a one-site model were excellent and allowed a determination of KD values. Kinetics fits were generally required to determine KD values since many sensorgrams did not reach a steady-state level, especially at the lower concentrations.</p><p>While the parent compounds, DB2457 and DB2708, have strong, selective binding to the AAAGTTT sequence, it is necessary to increase the selectivity of new compounds to a higher level for biological applications. As previously reported, DB2759 with -Cl on the aromatic system binds to AAAGTTT with a 14 nM KD and is very selective with no detectable binding to AAATTT and AAAGCTTT9. To find the optimum halogen substituent, different halogen substituents were incorporated and tested in the aromatic systems: DB2798 (-F), DB2801 (-Br) and DB2789 with -Cl on the other phenyl of the aromatic system. The different modification positions of -Cl do not make a major difference in binding affinity when DB2789 and DB2759 are compared. The -Br modification maintains similar binding affinity and specificity to AAAGTTT as with the -Cl derivatives (Figure 2). It is interesting that the smaller, electron-withdrawing atom, F, causes the binding affinity to become 40 times weaker than the -Cl modified compound (KD = 632 nM vs 14 nM). The potential intramolecular hydrogen bonding between the adjacent -F and -H of the amidine group limits the rotation of amidine on the phenyl ring to adopt an appropriate angle for binding to DNA minor groove and significantly reduces the binding affinity of the amidine group with the adjacent T=O of DNA.</p><p>As previously reported, DB2740 with an N-Ph substituent binds strongly to AAAGTTT but with limited selectivity versus AAAGCTTT9. The above results suggested that a -Cl substituent would improve the selectivity of the N-phenyl compound. To evaluate this point, DB2788 was synthesized and it binds to AAAGTTT slightly weaker than DB2740, but with 10-fold better binding selectivity to AAAGCTTT. This result shows that a -Cl on the aromatic systems, adjacent to an amidine group, can increase the AAAGTTT binding selectivity of different thiophene-N-RBI type compounds.</p><p>To evaluate the effects of changes in the chemical space of the compounds, two groups were also introduced into the amidines of DB2708. DB2786 with isopropyl amidine groups represents the strongest binding (KD = 1 nM) to AAAGTTT in this series. Unfortunately, DB2786 loses binding selectivity with a 55 nM KD for AAAGCTTT. DB2787 with amidine replaced by imidazoline binds to AAAGTTT similar to the -Cl compound but with slightly worse selectivity (Figure 2). In order to confirm the importance of the thiophene-N-RBI in the core system of the original compounds. Five compounds with basic structure variations of the thiophene σ-hole system were prepared. With the introduction of an alkyne between thiophene and phenyl ring of DB2708, the binding to AAAGTTT dramatically decreases. The replacement of N-RBI with pyridine, DB2655 gives a strong binding compound but with poor selectivity. The replacement of N-RBI-phenyl with quinoline, DB2654, gives a compound with good selectivity but weaker binding affinity. Surprisingly, the thiazole-N-RBI substituents destroyed the binding ability for all three DNA sequences. This is a surprising and puzzling result given the success of thiazole in other minor groove binders50. These results indicate the thiophene, or related derivative such as benzothiophene, is necessary but can be combined with the other kinds of N containing heterocyclic groups for a 1, 4 N···S interaction to produce a σ-hole system.</p><!><p>CD titration experiments are an effective and convenient method of evaluating the binding mode and the saturation limit for compounds binding with DNA sequences. CD spectra monitor the asymmetric environment of the binding of the compound to DNA and therefore, can be used to obtain information on the binding mode51,52. No CD signals have been observed at 300–450 nm wavelength for the free compounds. However, in Figure 3 (left, middle), on the addition of the compounds into DNA, substantial positive induced CD signals (ICD) are seen in the absorption region between 300 and 450 nm. These positive ICD signals in this region indicate a minor groove binding mode by these ligands, as expected from their structures. When the Ligand/DNA ratio is titrated to 1:1, the saturated spectra indicate 1:1 binding between ligand and AAAGTTT. However, in Figure 3 (right), when we change sequence from AAAGTTT to AAATTT, the ICD is low and indicates weak binding between DB2788 and the pure AT sequence. In summary, as can be seen from Figure 3, DB2786 and DB2788 give CD spectra typical of complexes in the minor groove of the AAGTTT sequences with a 1:1 stoichiometry, in agreement with SPR results and modeling.</p><!><p>Fluorescence spectroscopy is an effective method to quantitatively measure the interactions between biomolecules and small molecules, such as a small organic molecule binding with DNA. If a change in the fluorescence intensity accompanies the binding of two species, this can be used to monitor the binding interaction and determine the stoichiometry of binding using equilibrium titration methods53,54.</p><p>In order to study the binding specificity of DB2802 and DB2803 with benzothiophene, which have non-optimum properties for SPR studies, fluorescence titration experiments were conducted. The fluorescence titration spectra and binding affinity fitting plots are shown in Figure S2 (Supporting Information). The fluorescence intensity of DB2802 increases on titration with AAATTT. For AAAGTTT and AAAGCTTT, the fluorescence intensities both decrease in the titration. From DNA thermal melting results, the binding affinity between DB2802 with AAATTT and AAAGTTT are similar and stronger than AAAGCTTT. DB2803 with an N-isopropyl group binds to AAAGTTT strongly compared to the pure AT and AAAGCTTT sequences. The results agree with our previous analysis of the N-isopropyl benzimidazole that showed strong binding to AAAGTTT with good selectivity. The above results indicate that the isopropyl group on benzimidazole matches the microstructure of AAAGTTT better than methyl group and increases the binding specificity for both the thiophene and benzothiophene groups.</p><!><p>Mass spectrometry is an excellent method for the evaluation of relative binding affinity and specificity as well as stoichiometry. It provides a good test of experimental results obtained from other methods, such as SPR, where macromolecule-ligand stoichiometry is obtained by fitting the signal at different concentrations8,55. For this series of compounds, we chose two representative thiophene-BI substituents, DB2787, and the parent compound DB2708, for the competition ESI-MS experiments. DB2787 binds to sequence AAAGTTT with 11 nM KD value and good selectivity. DB2708 binds to AAAGTTT with 4 nM KD value but the binding selectivity is less than DB2787. In Figure 4A, three free DNA peaks are shown for AAATTT (m/z = 7302), AAAGCTTT (m/z = 7921), and AAAGTTT (m/z = 8540). On addition of DB2787, the peak for AAAGTTT (m/z = 8540) decreases with the simultaneous appearance of a new peak at m/z = 9070 that is characteristic of a 1:1 AAAGTTT–DB2787 complex (Figure 4B). There is no appearance of any complex peak with the other DNA sequences. As shown in Figure 4C, the same three DNA sequences are used to test DB2708. On the addition of DB2708, the peak of AAAGTTT almost disappeared with a new peak at m/z = 9019 that is characteristic of a 1:1 AAAGTTT–DB2708 complex (Figure 4D). However, another small new peak at m/z = 8540 appeared and is consistent with a 1:1 AAAGCTTT-DB2708 complex. Since the binding affinity between the DNA sequence AAAGCTTT and DB2708 is 223 nM KD (AAAGTTT/AAAGCTTT binding ratio is 56), with the increasing titration of DB2708 (up to 4:1 ratio), the binding between DB2708 and AAAGCTTT can be observed. Moreover, DB2787 with excellent selectivity (AAAGTTT/AAAGCTTT binding ratio is 172) only shows the AAAGTTT complex under these conditions. When excess ligands were added in the DNA mixture solution, only 1:1 Ligand/DNA complex peaks were observed. These results confirm the 1:1 stoichiometry between ligands and DNA, which agrees with CD results.</p><!><p>To help better understand the structural basis of molecular recognition of DNA sequences with a single G·C bp in an AT context, molecular dynamics (MD) simulations for the -Cl substituted N-isopropyl compound, DB2789, with the [5'-CGAAAGTTTCG-3'][5'-CGAAACTTTCG–-3'] duplex sequence were conducted. The 500 ns MD simulation has been performed by using the Amber 16 software package in the presence of 0.15 M NaCl44. Force constants for DB2789 were determined as described previously and in the Methods Section and added to the force field for the simulations46,47. Three optimum H-bonds in the complex have been observed. Both amidine groups form -N–H to T=O H-bonds (Figure 5A) that are an average of 2.9 Å in length. The amidines also form frequent highly dynamic H-bonds to terminal water molecules that move in and out of the minor groove. These water molecules frequently also form H-bonds with A·T bps at the floor of the minor groove and help link the compound to the specific binding site in the groove and stabilize the complex. The third strong H-bond is from the central G-NH that projects into the minor groove to the unsubstituted imidazole N in the BI group of DB2789, Figure 5A, to account for much of the binding selectivity of DB2789. Additional selectivity in binding is provided by the -CH group of the six-member ring of BI that points into the minor groove. This -CH forms a dynamic close interaction with the -N3 of the dG base of the central G·C bp. Additional direct stabilizing interactions are formed by other -CH groups that point to the floor of the minor groove from the two phenyls of DB2789. These -CH groups form significant dynamic interactions with A-N3 and T=O groups on the bases at the floor of the minor groove. Although DB2789 is optimally oriented along the minor groove with appropriate twist to match the minor groove curvature, the bulky -Cl group at the -ortho position of the bottom amidine group restricted the degree of freedom of the rotation of the -amidine group (Figure 5B, D). On the other hand, the top amidine group shows dynamic amidine group rotations due to the constraint reduction (Figure 5C, E).</p><!><p>We are engaged in a project that first time is converting strictly AT specific heterocyclic diamidine DNA minor groove binders into compounds that can bind to mixed AT and GC sequences of DNA11. This involves the creation of a set of modules that can recognize a single G•C bp and combining the modules or truncated versions of them to recognize a range of complex sequences, such as promoter sites of transcription factors. The ability to target such sites could have a major impact on understanding of transcription factor function as well as therapeutic development. An example is the PU.1 transcription factor that is involved in the development of a number of cancers such as AML. There are no drugs effective against PU.1, and it has been classified as "undruggable". In cases like PU.1 it seems more effective to target the promoter sequence rather than the transcription factor for the treatment of AML56.</p><p>The first step in this project involved the design and synthesis of a broad array of heterocyclic diamidines that could recognize a single G•C bp in an AT context7,9,57 (Figure S1). This effort produced several compounds that were selective for the PU. 1 promotor target sequence. One of our lead compounds was based on a thiophene-N-methyl-benzimidazole σ-hole system that is preorganized to bind to the DNA minor groove (Figure 1).</p><p>Our goal in the research described in this report is to probe the limits on the thiophene-N-MeBI module to determine if there are improved structures for strong and specific binding to G•C bps. As noted in the Introduction, three specific, important questions are addressed: what is the effect on binding affinity and selectivity of (i) modifying the amidine groups, (ii) incorporating different halogen substituents in the aromatic systems, and (iii) modifying the basic structure of the thiophene-N-MeBI aromatic system? In the halogen series, our initial compound had a -Cl substituent substituted adjacent to the amidine on the phenyl connected to the N-alkyl-BI group. To determine if this is the optimum halogen, -Br and -F were substituted in the same position. In quantitative SPR studies, the -Br group performed identically to the -Cl with good binding and excellent selectivity for the single G•C bp sequence. This substituent diversity may be helpful in targeting different cell types. Somewhat surprisingly, the -F substituent gave very poor binding, which we attribute to its locking the adjacent amidine into an unfavorable orientation for H-bonding to DNA through H-bonding to an adjacent amidine -NH. Putting the -Cl on the phenyl at the other end of the compound gave both stronger binding and excellent specificity for the single G•C bp sequence. The benzimidazole was also modified with an N-phenyl substituent, which gave quite strong binding but poor selectivity for the two G•C bps DNA sequence. A -Cl was added to determine if its enhanced selectivity would be available in this system. The N-phenyl-Cl compound has somewhat weaker binding, as with other -Cl derivatives, but with improved selectivity, although not as much improvement as desired.</p><p>Modifying the amidines with isopropyl groups gave the strongest binding of any compound in the series to the single G•C bp sequence, but unfortunately, the binding to the two G•C bps DNA was also enhanced. Converting the amidine to an imidazoline slightly reduced the binding to the single G•C bp sequence but the selectivity for the single G•C bp sequence over 150. A major question was whether the thiophene-N-alkyl-BI module was required or whether other σ-hole structures with H-bond acceptors would recognize the single G•C bp sequence. The first compounds in these experiments were what we thought was a conservative substitution of thiazole for thiophene to make two isomeric compounds, DB2740 and DB2788. It was a surprise that neither of these compounds gave any detectible binding to any of the three test DNAs, especially, since the triazole group has performed well in other minor groove binders50. To maintain the σ-hole the N-alkyl-BI group was replaced with a pyridine, DB2655, and quinoline, DB2654. The pyridine had satisfactory binding to the single G•C bp sequence but poor selectivity for the two G•C bps sequence and especially for the pure AT sequence. The quinoline had significantly reduced binding to the single G•C bp sequence but no detectible binding to the AT or GC test sequences.</p><p>In conclusion, there are three lead compounds from this design, synthesis and biophysical studies of GC recognition agents. All three compounds have halogen substituents adjacent to one amidine. Either amidine seems to give excellent results for the single G•C bp sequence. All three compounds have KD values in the quite strong 10 – 15 nM range for the single G•C bp sequence with no detectible binding to the AT and GC test sequences. The twist of the amidine caused by the halogen reduces binding to all sequences, but the reduction with AT and GC sequences is much larger. We have previously noted the microstructural variations in the minor groove among the three sequences and this difference obviously works to significantly enhance the selectivity of compounds with the extra twist on the amidines or size of the N-BI substituent9. The next compound to consider and perhaps test in cells is the imidazoline, DB2787. It also has good binding to the single G•C bp sequence, excellent selectivity over the AT sequence, which is the natural binding sequence for these type heterocycle diamidines and related compounds. Relative to the halogen derivatives, the imidazoline has slightly reduced selectivity for the two G•C bps sequence. This project has now shifted toward an emphasis on recognizing two G•C bps in the same sequences as will be necessary for strong binding with selectivity to other binding sites on the PU.1 promoter as well as recognizing promoters for other transition factors such as HOX A958.</p>

PubMed Author Manuscript

Intrinsic ssDNA Annealing Activity in the C-Terminal Region of WRN\xe2\x80\xa0

Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3\xe2\x80\xb2 to 5\xe2\x80\xb2 exonuclease, 3\xe2\x80\xb2 to 5\xe2\x80\xb2 helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072\xe2\x80\x931150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.

intrinsic_ssdna_annealing_activity_in_the_c-terminal_region_of_wrn\xe2\x80\xa0

<!>Cloning and Expression of GST-WRN Fusion Proteins<!>ssDNA Annealing Activity<!>Gel Mobility Shift Assay<!>Gel Filtration Analysis<!>Mapping the ssDNA Annealing Activity of WRN<!>Fine-Mapping WRN ssDNA Annealing Activity to a 79-Amino Acid Region between the RQC and HRDC Domains<!>Amino Acids 1072\xe2\x80\x931150 Are Required for ssDNA Binding Activity<!>Oligomerization Status of WRN Fragments<!>DISCUSSION

<p>Werner syndrome (WS1) is a rare autosomal recessive disorder characterized by early onset aging and genomic instability (1). WS patients carry mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. There is growing evidence that WRN plays diverse roles in DNA repair, DNA replication, homologous recombination, and telomere maintenance (2, 3). WRN possesses 3′–5′ helicase, 3′–5′ exonuclease, DNA-dependent ATPase, and ssDNA annealing activities (4, 5). Recent studies also showed that WRN catalyzes DNA strand exchange (6). WRN helicase unwinds a wide variety of DNA substrates in vitro, including Holliday junction and forked DNA duplex structures (7). Additionally, unlike other RecQ helicases, WRN can unwind RNA–DNA heteroduplexes (8).</p><p>WRN is the only RecQ helicase with exonuclease activity, which allows it to digest dsDNA with 3′ to 5′ polarity. Preferred WRN DNA substrates include bubble structures, forked DNA duplexes, and Holliday junctions (33). The exonuclease domain is located in the N-terminal region and the helicase domain in the central region of the WRN protein. The C-terminal region includes the RQC (RecQ conserved) and the HRDC (helicase, RNase D Conserved) domains. The RQC domain contains a wing helix (WH) motif, which mediates binding to dsDNA and ssDNA. In addition, WRN contains a nucleolar-targeting sequence (10). In some WS cells, WRN is C-terminally truncated and lacks the nuclear localization signal (NLS). Although previous studies have not mapped any enzymatic function to the WRN C-terminal region, this region mediates both protein–protein interactions and DNA binding. WRN interacts physically and functionally with numerous proteins, including replication protein A (RPA), proliferating cell nuclear antigen (PCNA), Flap endonuclease 1 (Fen 1), DNA polymerase β, and telomere binding protein TRF2 (reviewed in ref 11).</p><p>Human cells possess five RecQ helicases: WRN, RecQ1 (RecQL), BLM, RecQ4, and RecQ5 (12). Defects in the activity or expression of BLM and RecQ4 proteins result in Bloom syndrome and Rothmund–Thomson syndrome, respectively. So far, no diseases have been associated with defects in RECQ1 and RECQ5. Helicase activity has been detected biochemically in all human RecQ homologues except RecQ4. All five human RecQ helicases have ssDNA annealing activity (6, 13–17), which in BLM and RecQ5β has been mapped to the poorly conserved C-terminal region. ATP suppresses the annealing activity of RecQ helicases. It has been proposed that ATP binding induces a conformational change in RecQ1, which, in turn, inhibits its ssDNA annealing activity (17).</p><p>This study demonstrates that the ssDNA annealing activity of WRN maps to the C-terminal 79 amino acid segment (aa1072–1150) between the RQC and the HRDC domains. Furthermore, the ssDNA annealing activity of WRN correlates with DNA binding and oligomerization status of the protein.</p><!><p>Recombinant GST-tagged-WRN fragments WRN1–120, WRN239–499, WRN500–946, WRN949–1092, and WRN1072–1432 were constructed as described previously (18, 19). Recombinant 6xHis tagged-WRN fragments, WRN500–1104 (20), WRN1072–1432, and WRN1–368, were cloned and expressed as described previously (18). Fragments of the WRN C-terminal domain were expressed using the Gateway cloning system (Invitrogen). Briefly, the WRN fragments were generated by PCR (20). The PCR product was verified by sequencing and cloned into the pENTR-TEV-D-TOPO vector followed by recombination into bacterial expression plasmid pDEST15. Plasmids were propagated in DH5α during cloning and BL21 cells during expression. Purification was performed as described in ref 21 with some modifications; GST fusion proteins were purified from cell extracts using GST beads and eluted with 10 mM glutathione or 100 units of acTEV protease (Invitrogen) in 100 μL of elution buffer (100 mM Tris at pH 8.0, 120 mM NaCl, 5% glycerol, 0.2% Triton X-100, 300 mM LiSO4, and 5 mM DTT). The cloned human WRN gene was kindly provided by Dr. Junko Oshima (University of Washington Medical School, Seattle, WA).</p><!><p>ssDNA annealing activity was measured using C80 and G80 oligonucleotides described previously (6), at a concentration of 0.1 nM each, one of which was 5′-32P-end-labeled. Reactions (20 μL) were carried out in 20 mM Tris/HCl at pH 7.5, 2 mM MgCl2, 40 μg/mL BSA, and 1 mM DTT for 15 min at 37 °C. Protein was added in 2-fold serial dilutions at concentrations ranging from 0.5 to 16 nM. Reactions were stopped by the addition of stop buffer (50 mM EDTA, 1% SDS and 50% glycerol) and immediately loaded onto 10% (w/v) native polyacrylamide gel. Gels were run in TBE for 2 h at room temperature at 200 V. Radiolabeled DNA was detected using the Typhoon Imager (GE Healthcare), and percentage of annealed oligonucleotide was quantified using ImageQuant software.</p><!><p>Radiolabeled oligonucleotide C80 (1.5 nM) was incubated with WRN in 40 mM TrisHCl at pH 7.5, 20 μg/mL BSA, 8% glycerol, 1 mM EDTA, and 10 mM NaCl for 20 min on ice. The protein–DNA complexes were resolved on a 4% (w/v) native polyacrylamide gel and run in TBE for 2 h at 4 °C at 200V.</p><!><p>A Superdex 200 10/30 GL (GE Healthcare) column was equilibrated in dilution buffer (20 mM Tris/HCl at pH 7.4, 2 mM MgCl2, 100 mM NaCl, and 1 mM DTT) and calibrated with the following protein standards: ferritin, catalase, adolase, albumin, ovalbumin, chymotrypsinogen A, ribonuclease A, and blue dextran 200 (GE healthcare). Samples were diluted to 150 μg/mL in 20 mM Tris/HCl at pH 7.5, 2 mM MgCl2, 100 mM NaCl, and 1 mM DTT and loaded onto the column. Proteins were separated using FPLC-AKTA express (GE healthcare). Column fractions were analyzed by SDS–PAGE followed by silver staining.</p><!><p>It has been suggested that ssDNA annealing activities map to the C-terminal regions of WRN and BLM proteins and demonstrated that truncation of the C-terminal HRDC domain inhibits the ssDNA annealing activity of BLM (13). Recent studies also showed that WRN and BLM have more efficient ssDNA strand annealing activity than Drosophila melanogaster RecQ5b, most likely because Drosophila's RecQ5b has a shorter C-terminal region than WRN or BLM (6). This possibility was tested by constructing and characterizing deletion/truncation mutants of WRN tagged with GST or 6X-Histidine (6X-His) (Figure 1A). Selection of WRN fragments was based on proteolytic studies of WRN domain boundaries (19). Initially, five GST- and two 6X-His-tagged WRN fragments were overexpressed, purified, and tested for ssDNA annealing activity (Figures 1 and 2). The purity of the fragments was assessed by silver stain (Figure 1B). The ssDNA annealing activity was significantly above background only for full-length WRN and the GST-WRN fragment containing the C-terminal region, GST-WRN1072–1432. GST was included in the assay as a negative control (Figure 2A, lane 8). This result indicates that the WRN region 1072–1432 (Figure 2A, lanes 2 and 7, and Figure 2B) includes amino acids that are required for ssDNA annealing activity. This region of WRN lies immediately after the RQC domain and includes two conserved motifs: the HRDC domain and an NLS. However, the HRDC domain is not well conserved among RecQ helicases and is present in only two of the five human RECQ helicases, WRN and BLM (reviewed in ref 22). When the WRN amino acid sequence was aligned with RecQ helicases from mouse, Xenopus laevis, and Caenorhabditis elegans using ClustalW (23), weak sequence similarity (17%) was detected in the C-terminal region (Figure 2C). This low level of sequence homology argues against strong evolutionary pressure for functional conservation in the C-terminal region of WRN and other RecQ helicases.</p><!><p>The region required for WRN ssDNA annealing activity was defined more precisely by constructing, overexpressing, and characterizing six additional GST-WRN truncation proteins (Figure 3A). The longest of these variants GST-WRN949–1432 includes the RQC domain, the region between the RQC and HRDC domain, the HRDC domain, and the NLS. Additional WRN variants were truncated C-terminally (aa949–1236), N-terminally (aa1072–1432; aa1151–1432), or both (aa1072–1369; aa1072–1150). The smallest WRN fragment (aa1072–1150) includes only the region between the RQC and HRDC domains. WRN942–1432 and WRN949–1236 fragments include the WH motif from the RQC domain, which has high DNA and protein binding capacities (11). The protein folding patterns of these WRN variants were predicted using the Profile Library Search engine FUGUE (24), and their purity was confirmed using SDS–PAGE followed by staining with SuperBlue reagent (Figure 3B).</p><p>ssDNA annealing assays were carried out with variable amounts of WRN and WRN C-terminal truncations (Figure 4). The results show that WRN1072–1150 is sufficient for ssDNA annealing activity (Figure 4B) and deletion of this region strongly inhibits ssDNA annealing function (Figure 4A, middle panel). WRN1072–1432 (Figure 4A, upper panel) and WRN1072–1369 (Figure 4A, bottom panel) fragments containing that region, but truncated at opposing termini, have annealing activities. The WH motif in the RQC domain together with the intact C-terminal domain had ssDNA annealing activity (Figure 4C, bottom panel), whereas the WRN949–1236 fragment covering the WH motif with the HRDC domain had a slightly inhibitory effect on ssDNA annealing activity (Figure 4C and D). These results suggest that the region required for the ssDNA annealing activity lies between amino acids 1072 and 1150 (Figure 4B and D).</p><p>Site-directed mutagenesis was used to identify specific amino acids responsible for the ssDNA annealing activity in WRN1072–1150. First, a computer algorithm ConSeq (25) was used to predict putative functionally important amino acids in this region. The amino acids identified were serine 1079, tyrosine 1112, lysine 1113, and lysine 1117. On the basis of the prediction of ConSeq, WRN1072–1432 fragments were generated with mutations of serine 1079 to alanine or leucine, tyrosine 1112 to alanine, lysine 1113 to alanine, and lysine 1117 to alanine. The serine 1079 to leucine variant was constructed because there is a naturally occurring single nucleotide polymorphism (SNP) variant in a human population in sub-Saharan Africa (25). ssDNA annealing assays showed that alanine-substituted and leucine-substituted WRN fragments were fully active (data not shown). Thus, additional studies are needed to identify specific residues or combinations of residues that are required for WRN ssDNA annealing activity.</p><!><p>E. coli and S. cerevisiae RecQ homologues preferentially bind ssDNA via their HRDC domains (26, 27). Previous studies indicate that the WRN C-terminal region binds dsDNA and has lower affinity to ssDNA (19). Here, binding of WRN truncations to an 80-mer ssDNA oligonucleotide (C80) was examined using gel mobility shift assay. The WRN1072–1432 fragment formed a DNA–protein complex with DNA substrate that either did not enter the gel or migrated a small distance into the gel (Figure 5, lanes 5–7). WRN1072–1150 formed smaller amounts of a similar complex (Figure 5, lanes 8–10). In contrast, WRN1151–1432 did not bind the ssDNA substrate (Figure 5, lanes 2–4). These results show that WRN1151–1432 is deficient in both ssDNA binding and ssDNA annealing activity (Figure 4A, middle panel). WRN949–1432 and WRN949–1236 (Figure 5, lanes 12–13 and lanes 14–15, respectively) bind ssDNA with high efficiency forming a protein–DNA complex that has greater electrophoretic mobility than the complexes formed by WRN1072–1432 and WRN1072–1150. This result may indicate that the WH motif of the RQC domain facilitates binding to ssDNA.</p><!><p>The results of gel mobility shift assays indicate that WRN variants may form protein–DNA complexes of different size/electrophoretic mobility. This could indicate that WRN variants have different aggregation, multimerization, or solubility characteristics. Here, the size and solubility of WRN variants were evaluated using gel filtration chromatography in buffer similar to the buffer for the ssDNA annealing assay. The C-terminal fragment (WRN1072–1432), which has a predicted molecular weight of 66 kDa, eluted in three peaks with molecular weights of 388, 195, and 64 kDa, corresponding to a putative hexamer, trimer, and monomer, respectively. The results also showed that the trimer was the major species formed by the fragment (Figure 6A, upper panel) and that all three peaks contained the WRN1072–1432 polypeptide (Figure 6A, lower panel). WRN1072–1150 had a similar gel filtration profile (data not shown), but WRN1151–1432, which lacks ssDNA annealing activity, eluted primarily as a trimer (Figure 6B). These results and results of the gel mobility shift assays suggest that ssDNA annealing activity may correlate with the oligomerization state of WRN.</p><!><p>Previous studies mapped WRN enzymatic functions and protein–protein interactions to distinct WRN protein domains. In these studies, WRN regions responsible for exonuclease and helicase activities were identified, and the functional properties of the acidic, RQC, and HRDC domains of WRN were characterized. The present study maps the ssDNA annealing activity of WRN to a 79-amino acid region of WRN between residues 1072 and 1150. This region, which lies between the RQC and HRDC domains, is sufficient for ssDNA annealing activity in vitro, and deletion of this region strongly inhibits, but does not completely eliminate, the ssDNA annealing activity of the WRN C-terminal region.</p><p>The C-terminal domains of BLM and RecQ5 are also required for ssDNA annealing activities, and the ssDNA annealing activity of BLM was mapped to aa1290–1350 (13). Nevertheless, the C-terminal regions of WRN, BLM, and RecQ5 show little sequence similarity (13, 14). Thus, ssDNA annealing activity does not appear to be an evolutionarily conserved function of RecQ helicases. Although there is weak sequence homology between WRN orthologues in this region, amino acids 1072 to 1150 of WRN are unique, and protein sequences closely related to this region are not found in the protein database. Furthermore, the predicted secondary and tertiary structures of this region lack similarity to any known protein structure.</p><p>WRN fragments that retain ssDNA annealing activity form a characteristic protein–DNA complex during gel mobility shift analysis (Figure 5), which correlates with the formation of putative WRN hexamers and trimers during gel filtration (Figure 6). In contrast, WRN1151–1432, which lacks ssDNA annealing activity, exists primarily as a trimer during gel filtration. The oligomerization status of the WRN C-terminal fragment appears to be independent of DNA binding (data not shown), as reported for other proteins with ssDNA annealing activity (28). In addition, previous studies showed that the C-terminal domain of WRN modulates its oligomerization (29) and that the active form of WRN 3′-5′ exonuclease may be a trimer (4). More recently, it was suggested that higher-order protein–DNA complexes of BLM may be active in ssDNA annealing (13). Although BLM also forms a hexamer in solution, the enzymatic properties of the BLM hexamer have not yet been characterized (30).</p><p>C-terminal fragments of WRN that include the WH motif of the RQC domain (i.e., WRN949–1432) appear to be more active in ssDNA binding. The WH motif of the RQC domain mediates protein–DNA interaction through the DNA recognition helix of the helix core consisting of three α-helices (20). The recognition helix interacts with the major groove of DNA as in the DNA–protein co-crystal structures of a variety of proteins involved in DNA metabolism (31–33). Therefore, the presence of the WH motif in the WRN RQC domain may offer some explanation as to why the WRN RQC fragments are the strongest DNA binding region followed by the HRDC domain and the exonuclease domain (19). The RQC domain forms a higher oligomer, but the DNA binding does not depend on higher oligomer formation; the ability of higher oligomer formation might correlate with ssDNA activity as suggested for other proteins with ssDNA activity such as Rad52 (28).</p><p>RecQ1, BLM, WRN, and RecQ5 catalyze both ssDNA annealing and DNA unwinding in vitro (6, 13, 14, 17). It has been suggested that the balance between RecQ unwinding and annealing functions may be regulated by protein conformation and/or oligomerization (34). In the case of RecQ1, oligomerization is required for ssDNA annealing, and ATP may trigger the switch from ssDNA annealing to DNA unwinding (34). Here, we showed that oligomerization is also required for the ssDNA annealing activity of WRN. The mechanism that regulates WRN ssDNA annealing is not yet known; however, it was previously shown that ATP inhibits ssDNA annealing by WRN, BLM, and RecQ5 (6, 13, 14). It is possible that ATP binding might be one of the factors for the regulation of WRN's dual function. In addition, WRN oligomerization and its ssDNA annealing activity might be regulated by protein conformation, protein concentration, presence or absence of interacting protein partners, or other as yet unidentified factors.</p><p>In future studies, it will be important to determine the in vivo biological significance of the WRN ssDNA annealing activity. This activity may play a role in dsDNA break repair (35), telomere maintenance, resolution of double Holliday junctions, or rescue of stalled or collapsed replication forks, as suggested for BLM (36, 37). Recently, it was shown that yeast and human Dna2 protein, such as WRN and BLM, has ssDNA annealing and DNA strand exchange activities (38) and that it stimulates Fen1 cleavage (39–41). Thus, ssDNA annealing and exchange activities of Dna2, WRN, and BLM may play roles in the processing of Okazaki fragments (38, 42). In support of this hypothesis, we previously reported that the C-terminal domain of WRN (WRN949–1432) interacts with and stimulates Fen1, while WRN ATPase, helicase, and exonuclease activities are not required for the stimulation of Fen1 (21).</p><p>In summary, this study clearly demonstrates that the WRN C-terminal domain (aa1072 to 1150) encodes an instrinsic ssDNA annealing activity. This poorly conserved region of WRN may play a role in ssDNA binding and protein oligomerization. Additional studies are needed to understand the in vivo biological role of WRN ssDNA annealing activity.</p>

PubMed Author Manuscript

Synthesis of Amido-Spiro[2.2]Pentanes via Simmons-Smith Cyclopropanation of Allenamides.\xe2\x80\xa0

A detailed account on Simmons-Smith cyclopropanations of allenamides en route to amido-spiro[2.2]pentanes is described here. While the diastereoselectivity was low when using unsubstituted allenamides, the reaction is overall efficient and general, representing the most direct synthesis of both chemically and biologically interesting amido-spiro[2.2]pentane systems. With \xce\xb1-substituted allenamides, while the diastereoselectivity could be improved significantly based on a series of conformational analysis, both mono- and bis-cyclopropanation products were observed. Consequently, several structurally intriguing amido-methylene cyclopropanes could also be prepared.

synthesis_of_amido-spiro[2.2]pentanes_via_simmons-smith_cyclopropanation_of_allenamides.\xe2\x80\xa0

Introduction<!>1. Cyclopropanations of \xce\xb1-Unsubstituted Allenamides<!>2. A Comparison with Chiral Enamides<!>3. Cyclopropanations of \xce\xb1-Substituted Allenamides<!>Conclusion<!>

<p>Spiro[2.2]pentanes,1 the smallest member of the triangulane or oligo-spirocyclopropane family, represent a unique structural topology with both rigidity and orthogonality that have found applications in a number of biological contexts.2 In particular, simple α-spiropentyl acetic acid [Figure 1] has been shown to mimic α-(methylenecyclopropyl) acetic acid, a well-known inhibitor against acyl-CoA dehydrogenase that is critical in the fatty acid oxidation pathway. In addition, α-(methylenecyclopropyl) acetic acid itself has also been identified as a toxic metabolite of the natural amino acid hypoglycine A found in the fruits of Jamaican ackee trees.3–7 Consequently, it is a key cause of vomiting sickness when ingesting the Jamaican ackee fruit due to the resulting deficiency it causes in the acyl-CoA dehydrogenase activity.8,9 Moreover, amino-spiro[2.2]pentanes have received much attention recently for an array of other purposes ranging from constructing deoxyribonucleotide analogs10 to exploring the chemistry and biology of spiro[2.2]pentane amino acid derivatives.11,12</p><p>Despite these biological interests, and despite a number of elegant approaches toward spiro[2.2]pentanes in literature, the overall synthetic effort toward amino-spiro[2.2]pentanes has been limited.2,13 Few involve direct bis-cyclopropanations of allenes14 with many adopting mono-cyclopropanations of methylene cyclopropanes prepared through other means.15 To the best of our knowledge, preparation of amino-spiro[2.2]pentanes directly through bis-cyclopropanations of 1-amino-allenes is not known.2,10–13,16 Our recent interest17,18 in cyclopropanations of enamides13,19,20 en route to optically enriched amino-cyclopropanes21,22 coupled with our decade long efforts in developing the chemistry of allenamides23–26 allowed us to envision the possibility of developing a direct construction of amido-spiro[2.2]pentanes via Simmons-Smith cyclopropanation of allenamides 1 [Scheme 1]. Based on our previous work on a number of different stereoselective cycloaddition manifolds employing allenamides,27 we anticipated that this cyclopropanation could proceed stereoselectively in which the zinc carbenoid can approach the bottom π-face of the more favored conformer 1a [Scheme 1]. This would lead to methylene cyclopropane 2, and while 2 is useful in its own right,28 an ensuing second cyclopropanation would provide 3a as the major amino-spiro[2.2]pentane isomer with 3b being derived from cyclopropanation of the minor conformer 1b. We report here details of these investigations.</p><!><p>The feasibility for Simmons-Smith cyclopropanations of allenamides was quickly established as shown in Scheme 2. By using 5.0 equiv Et2Zn and 10.0 equiv ICH2-X, cyclopropanation of achiral allenamide 4 proceeded in excellent yields to give amido-spiro[2.2]pentane 629 with no difference between using ICH2-I and ICH2-Cl respectively in CH2Cl2 and ClCH2CH2Cl [DCE]. We did not observe any mono-cyclopropanation product 5 after 12 h, suggesting that the second cyclopropanation via 5 took place rapidly in these reactions with Zn(CH2X)2 serving as the highly reactive cyclopropanating species.30</p><p>We turned our attention to chiral allenamides using specifically 7 [Table 1], and while the cyclopropanation was equally effective in providing de novo amido-spiro[2.2]pentane 9, the diastereomeric ratio was not desirable. We explored a range of conditions13a including different temperatures [entries 1–4] and solvents [entries 5–7], while featuring Zn(CH2Cl)2 as the cyclopropanating species. In addition, we examined the nature of cyclopropanating species such as Zn(CH2I)2 either without [entry 8] or with the addition of a chelating solvent such as DME [rendering the zinc cyclopropanating reagent more nucleophilic] [entry 9]. Finally, we explored Furukawa type reagents [entries 10–12]31,32 as well as Yamamoto's AlMe3 activation of ICH2-I [entry 13].33</p><p>We did not continue to pursue other cyclopropanating species such as Molander's Sm-Hg activation of ICH2-Cl,34 as we recognized that we were not going to improve the diastereomeric ratio of amido-spiro[2.2]pentane 9 via bis-cyclopropanation of 7 by only using different cyclopropanating species. It is noteworthy that this effort allowed us to observe by 1H NMR the mono-cyclopropanation product 8, although not isolated [entries 3 and 9]. This implies that the 2nd cyclopropanation is slower for chiral allenamides. Stereochemically, both the major and minor diastereomers of 9 were unambiguously assigned using single-crystal X-ray structures as shown in Figure 2. The ability to access both diastereomers of these structurally very interesting and novel amido-spiro[2.2]pentanes renders this none-stereoselective aspect of this reaction an opportunity and less of a limitation.</p><p>Subsequently, a number of chiral auxiliaries on the allenamide were probed in an attempt to improve the diastereoselectivity. As shown in Scheme 3, amido-spiro[2.2]pentanes such as 10, 11, 14, and 15 could be attained in good yields through bis-cyclopropanations of their respective allenamides. However, the diastereomeric ratio remained low, and when employing the more bulky Sibi's auxiliary35 or Seebach's auxiliary,36 the reaction appeared to be shut down, as only trace amounts of amido-spiro[2.2]pentanes 12 and 16 could be found. Close's auxiliary37 gave the best ratio in 17 but with a lower yield. There is essentially no difference in the level of stereoselectivity between using auxiliaries containing just the mono-substitution alpha to the amido-nitrogen atom [see 10–12] and those with vicinal substitutions on the oxazolidinone ring [see 14–17].</p><!><p>While the lack of diastereoselectivity was frustrating, it intrigued us mechanistically. We had previously examined Simmons-Smith cyclopropanations of chiral enamides and achieved a much greater success in stereochemical control.17 As shown in Scheme 4, chiral E-enamides such as 18 and E-19 gave amido-cyclopropanes 20 and trans-21 with diastereomeric ratios of 95:5 and 83:17, respectively, while chiral Z-enamides such as 22 and Z-19 led to even higher diastereomeric ratios of ≥95:5 in each case. These results are in direct contrast to our current cyclopropanation work</p><p>To rationalize the above stereochemical outcome, we examined conformations of these enamides through both X-ray structures [see structures of E-19 and Z-19 in Figure 3] and PM3 calculations via Spartan Model.™ Both the X-ray structure [see E-19] and computation model revealed that E-enamides [R = alkyl or aryl] assume the more favorable conformation E1 [Scheme 5], which was what we had speculated earlier in some epoxidation work.38,39 The other locally minimized conformation is E2 but it is less favored than E1 by 1.17 kcal mol−1. In both conformers, the olefin is approaching co-planarity with the oxazolidinone ring, allowing delocalization of the nitrogen lone pair into the olefin. Being devoid of actual transition state calculations, we will make an assumption here that these cyclopropnations proceed through the major enamide [or allenamide] conformer with the awareness that Curtin-Hamette could very well be in play here, and that we are only attempting to identif a model with some consistent rationale and predictative power at this juncture.</p><p>Based on this assumption, if the cyclopropanation proceeds through the more favored conformation E1, the necessary π-facial differentiation in E1 would provide the excellent stereochemical outcome with E2 being a possible source for the minor diastereomer. On the other hand, in both the X-ray structure [see Z-19 in Figure 3] and computation model of Z-enamides, there is a distinct shift from a coplanar motif in conformation Z1 to the more favored Z2 [ΔE = −1.03 to −1.28 kcal mol−1]. This is likely due to the oxazolidinone ring rotating along the C-N bond toward the direction so that the Ph substituent could be shifted away from the R group to alleviate the allylic strain. Despite such conformational change relative to E-enamides, the bottom π-face in the more favored conformation Z2 remains sterically more accessible, thereby providing the same sense of facial selectivity in the cyclopropanation as for the E-enamide.</p><p>Although we have not examined this in detail, the greater diastereoselectivity attained for Z-enamides relative to those of E-enamides could be a result of a greater shielding of the top face by the phenyl ring, and/or a possible chelation of the oxazolidinone carbonyl oxygen with the zinc reagent in a directed cyclopropanation manner.</p><p>In contrast, while chiral allenamides assume a similar set of conformations40 as shown in Scheme 6, calculations [PM3 calculations via Spartan Model™] suggest that the energetic difference between conformers 24a and 24b [see ΔE = −0.21 kcal mol−1 for R = H] appears to be relatively much smaller than those from enamides. In addition, we also find that the first cyclopropanation is very facile relative to the cyclopropanation of enamides. In general, the starting allenamides are consumed within 1–2 h at 0 °C [or rt] based on monitoring by NMR, leading to 25-Ma and 25-Mb, whereas cyclopropanation of enamides in most cases required 24–72 h.17 A mixture of mono- and bis-cyclopropanation products with a ratio of 1:1.5 was usually seen after 3 h at 0 °C, and the long reaction time is associated with the second cyclopropanation, leading to 25-Ba and 25-Bb.</p><p>Consequently, again, based on the assumption that these cyclopropnations also proceed through the major allenamide conformer, the lack of diastereoselectivity observed in Simmons-Smith cyclopropanations of allenamides could be due to a facile cyclopropanation through an almost equal distribution of unsubstituted allenamide conformers 24a and 24b [for R = H]. While this proposed model is based on ground state energetic difference, if valid, α-substituted allenamides [for R ≠ H] would then lead to an improved selectivity because 24a is now favored by 1.05 kcal mol−1 [for R = Me] over 24b due to its the enhanced allylic strain.</p><!><p>Based on the above conformational model, we prepared α-substituted achiral allenamides 26–28 [Scheme 7] through α-alkylation of the respective unsubstituted allenamides.41 Cyclopropanations of allenamides 26–28 were not only feasible, but also led to the observation and isolation of a substantial amount of mono-cyclopropanation products 29-M through 31-M. In the case of allenamide 28, we isolated 60% of mono-cyclopropane 31-M. These results suggest that α-substituted allenamides further impede the second cyclopropanation compared to unsubstituted chiral allenamides. The unique structural motif of the amido-methylene cyclopropane 31-M is displayed in Figure 4 through its single-crystal X-ray structure.</p><p>We proceeded to examine α-substituted chiral allenamides 32–35 as shown in Table 2. In all cases, we isolated both mono-[36-M through 39-M] and bis-cyclopropanation products [36-B through 39-B]. A longer reaction time usually resulted in more of the respective bis-cyclopropanation product. In accord with our conformational analysis, the diastereomeric ratio was indeed improved with a dependence on the size of the R groups. The stereochemistry of 37-B was unambiguously assigned using X-ray structural analysis [Figure 4].</p><p>To ensure that major isomers of mono- and bis-scyclopropanation product 37-M and 37-B in fact possess the same stereochemistry at the carbon bearing the amido group, amido-methylene cyclopropane 37-M was subjected to the same cyclopropanation conditions [Scheme 8]. While the reaction was slow and incomplete, we found a 43% yield of 37-B [as a single isomer], thereby confirming that the major isomer of bis-cyclopropanation products indeed comes from a second cyclopropanation of the major isomer of the respective mono-cyclopropanation products. This assessment would then translate the individual ratios of mono- and bis-cyclopropanation into an excellent overall or combined diastereoselectivity for the first cyclopropanation [see numbers in red] that correlates well overall with increasing in the size of the R group, and provide a solid support for the conformational model proposed above.</p><p>Lastly, the rate of the second cyclopropanation appears to be directly correlated with the degree of steric crowding of either π-face of the methylene cyclopropane intermediate. As shown in Scheme 9, in the case of unsubstituted allenamides, both p-faces of the olefin in achiral amido-methylene cyclopropane 5 are open for the second cyclopropanation, whereas chiral amido-methylene cyclopropane ent-8 is blocked on the bottom p-face with the top still available. Thus, we did not observe amido-methylene cyclopropane 5 but saw ent-8 in 12 h under the same reaction conditions. For α-substituted allenamides, both π-faces of amido-methylene cyclopropanes such as 29–31 and 36-M through 39-M are now sterically more encumbered. Consequently, the second cyclopropanation of 29–31 and 36-M through 39-M should be slower relative to those of 5 and ent-8, leading to the observation and/or isolation of methylene cyclopropanes for α-substituted allenamides.</p><!><p>We have described here Simmons-Smith cyclopropanations of allenamides in the synthesis of amido-spiro[2.2]pentanes. While the diastereoselectivity was low when using unsubstituted allenamides, the reaction is overall efficient and general, leading to an array of amido-spiro[2.2]pentanes. With α-substituted allenamides, while the diastereoselectivity could be improved significantly based on a conformational analysis, both mono- and bis-cyclopropanations were observed in these cases. Consequently, several structurally intriguing amido-methylene cyclopropanes could also be prepared. With allenamides being readily accessible, these efforts have yielded the most straightforward protocol in constructing chemically and biologically intriguing amido-spiro[2.2]pentane systems.</p><!><p>Spiro[2.2]Pentanes.</p><p>X-Ray Structures of 9a [left] and 9b [Right].</p><p>X-Ray Structures of Enamides E-19 [Left] and Z-19 [Right].</p><p>X-Ray Structures of 31-M [Left] and 37-B [Right].</p><p>Simmons-Smith Cyclopropanations of Allenamides.</p><p>Cyclopropanation of Achiral Allenamide 4.</p><p>Effect of Chiral Auxiliaries on Stereoselectivity.</p><p>Cyclopropanations of E- and Z-Enamides.</p><p>A Model for the Enamide Cyclopropanation.</p><p>A Comparison with the Enamide Cyclopropanation.</p><p>Mono- Versus Bis-Cyclopropanation of Allenamides.</p><p>Assignment of Mono-Cyclopropane 37-M.</p><p>Rate Comparisons for the Second Cyclopropanation.</p><p>Cyclopropanations of Chiral Allenamide 7.</p><p>DCE: 1,2-Dichloroethane. DME: 1,2-Dimethoxyethane.</p><p>NMR yields for 8 and Isolated yields for 9. The ratio in brackets denotes a:b with a being the major isomer as shown in the scheme, and was assigned via NMR.</p><p>Employing 5.0 equiv Et2Zn and 10.0 equiv ICH2-Cl.</p><p>Recovering 20–65% of the starting allenamide 7.</p><p>conc. = 0.05 M.</p><p>Employing 5.0 equiv Et2Zn and 10.0 equiv ICH2-I. For entry 9, 5.0 equiv of DME was added.</p><p>Employing 5.0 equiv each of Et2Zn, ICH2-I, and CF3CO2H.</p><p>Employing 5.0 equiv Et2Zn and 5.0 equiv ICH2-I. For entry 12, 5.0 equiv of DME was added.</p><p>Employing 5.0 equiv Me3Al and 5.0 equiv ICH2-I.</p><p>Cyclopropanations of Chiral α-Substituted Allenamides.</p><p>Isolated yields. Dr ratios are in the bracket with the respective major diastereomer being shown in the scheme and all ratios were assigned using crude 1H NMR.</p><p>Overall dr ratios represent the combined dr for the first cyclopropanation.</p><p>NMR yield.</p><p>See reference 42.</p>

PubMed Author Manuscript

Semiconductor-driven “turn-off” surface-enhanced Raman scattering spectroscopy: application in selective determination of chromium(<scp>vi</scp>) in water

Semiconductor materials have been successfully used as surface-enhanced Raman scattering (SERS)-active substrates, providing SERS technology with a high flexibility for application in a diverse range of fields. Here, we employ a dye-sensitized semiconductor system combined with semiconductor-enhanced Raman spectroscopy to detect metal ions, using an approach based on the "turn-off" SERS strategy that takes advantage of the intrinsic capacity of the semiconductor to catalyze the degradation of a Raman probe.Alizarin red S (ARS)-sensitized colloidal TiO 2 nanoparticles (NPs) were selected as an example to show how semiconductor-enhanced Raman spectroscopy enables the determination of Cr(VI) in water. Firstly, we explored the SERS mechanism of ARS-TiO 2 complexes and found that the strong electronic coupling between ARS and colloidal TiO 2 NPs gives rise to the formation of a ligand-to-metal chargetransfer (LMCT) transition, providing a new electronic transition pathway for the Raman process.Secondly, colloidal TiO 2 nanoparticles were used as active sites to induce the self-degradation of the Raman probe adsorbed on their surfaces in the presence of Cr(VI). Our data demonstrate the potential of ARS-TiO 2 complexes as a SERS-active sensing platform for Cr(VI) in an aqueous solution. Remarkably, the method proposed in this contribution is relatively simple, without requiring complex pretreatment and complicated instruments, but provides high sensitivity and excellent selectivity in a high-throughput fashion. Finally, the ARS-TiO 2 complexes are successfully applied to the detection of Cr(VI) in environmental samples. Thus, the present work provides a facile method for the detection of Cr(VI) in aqueous solutions and a viable application for semiconductor-enhanced Raman spectroscopy based on the chemical enhancement they contribute.

semiconductor-driven_“turn-off”_surface-enhanced_raman_scattering_spectroscopy:_application_in_selec

Introduction<!>Materials<!>Preparation of colloidal TiO 2 nanoparticles<!>SERS measurement<!>Instrument<!>Synthesis and characterization<!>Application<!>Conclusions

<p>In recent years, an increasing interest in the studies of surfaceenhanced Raman scattering (SERS) on semiconducting materials (that is semiconductor-enhanced Raman spectroscopy) has emerged owing to its potential application in biological and photoelectronic analyses. [1][2][3][4][5][6] Several semiconductor materials including TiO 2 , ZnO, graphene, Si, and Ge have been developed as SERS substrates. [7][8][9] However, the application of these materials in SERS-based quantitative measurements is still matter of debate, because of their specic dependence on molecular electronic structure and relatively weak enhancement of SERS signals. Most of the improvement in semiconductor-enhanced Raman spectroscopy is mainly induced by a charge-transfer process, leading to an enhancement of approximately 10 2 to 10 4 , because the surface plasmon resonance of semiconductor NPs typically lies in the infrared region, which does not usually coincide with the optical laser frequency. [2][3][4][7][8][9][10][11][12][13] To date, studies have largely focused on the discovery and interpretation of the SERS phenomena with different semiconductor materials. This represents the most signicant bottleneck in the application of such a technique for practical analysis and detection.</p><p>The advantage of semiconductor-enhanced Raman spectroscopy is the performance of the semiconductor, which possesses controllable photoelectric properties, good biocompatibility, and environmental stability. In order to exploit these advantages, metal-semiconductor composites were introduced into SERS-based assays with some ingenious designs. [14][15][16][17][18][19] However, the complicated process needed to prepare such composites has strongly limited their application. In addition, semiconductor materials do not respond to the Raman enhancement in these systems. [16][17][18][19] Considering the photocatalytic properties of semiconductors, quantitative analysis by semiconductor-enhanced Raman spectroscopy would be achieved through the detection of signal degradation of a labeled probe on a semiconductor. Here, we report that semiconductor-enhanced Raman spectroscopy can also be developed into a sensing platform for the detection of metal ions, even without the assistance of a noble metal. In particular, we present a novel "turn-off" SERS strategy and demonstrate its use in a SERS-based assay for the determination of Cr(VI) in water.</p><p>Cr has been extensively used in various industrial processes and has become one of the major environmental hazards. 20 The toxicological and biological properties of Cr are entirely dependent on its electric charge. 21 For instance, Cr(VI) is highly toxic; it generally exists as an oxyanion (CrO 4 2À ) in aqueous systems, and is known to be a strong carcinogen. 22 In contrast, Cr(III) is relatively non-toxic and is regarded as an essential trace element associated with the metabolism of carbohydrates and lipids. 23 Therefore, the reduction of Cr(VI) to Cr(III) is a key process for the detoxication of Cr(VI)-contaminated water and wastewater. In drinking water, the Maximum Contaminant Level (MCL) for Cr(VI) has been identied as 1 mM. However, because no efficient testing method is available for only Cr(VI), the estimated MCL by the World Health Organization (WHO) includes the total amount of Cr. 24 Evidently, this denition is not conducive to encouraging the intake of Cr(III) from a daily diet and has pushed up the cost of industrial wastewater treatment. So far, several methods, including atomic spectrometric, [25][26][27] luminescent, 28,29 electrometric, [30][31][32][33] colorimetric, 34 and X-ray uorometric techniques, 35 have been developed for the selective determination of Cr(VI). Nevertheless, none of these techniques exhibited the desired sensitivity together with easy manipulation.</p><p>Herein, charge-transfer complexes, alizarin red S (ARS)sensitized colloidal TiO 2 NPs, with a facile synthetic route are used to demonstrate how semiconductor-enhanced Raman spectroscopy enables the determination of Cr(VI) in water. We explored the SERS mechanism of ARS-TiO 2 complexes and found that the molecular polarizability tensor can be enhanced by a ligand-to-metal charge-transfer (LMCT) transition. Interestingly, the SERS intensities of the ARS-TiO 2 complexes have been found to be sensitive to the Cr(VI) concentration due to co-catalysis, indicating their potential for use in the determination of Cr(VI). Several inuencing factors such as response time, laser power, pH of the sensing system, and the loading amount of ARS on the colloidal TiO 2 NPs were taken into account to optimize the determination conditions. Our experimental results revealed that the ARS-TiO 2 complexes exhibit high sensitivity and selectivity toward Cr(VI). The practicality of this proposed method was further validated through the detection of Cr(VI) in real water samples. The method proposed here can be used for the determination of Cr(VI) in aqueous solutions for the accurate assessment of pollution levels. Thus, this work provides a clear proof of concept for extending the applications of semiconductorenhanced Raman spectroscopy.</p><!><p>Alizarin red S and titanium(IV) butoxide were acquired from Sigma-Aldrich Co. Ltd. and used without further purication. All other chemicals, obtained from Wako Co. Ltd, were analytical grade and employed without further purication. Ultrapure water (18 MU cm) was used throughout the study. The tap water and pond water were collected from the Gakuen district of Sanda and a pond near Kwansei Gakuin University, respectively. All the water samples were ltered through 0.2 mm membranes prior to use.</p><!><p>The colloidal TiO 2 NPs were synthesized according to a method described in previous reports. 36,37 Briey, a solution of titanium(IV) butoxide (5 mL) dissolved in 2-propanol (95 mL) was added dropwise (1 mL min À1 ) to an aqueous HNO 3 solution (500 mL, pH 1.5) maintained at 1 C. The solution was continuously stirred for 10-12 hours until a transparent colloid was formed.</p><!><p>A stock solution of ARS (0.1 M) was prepared in water. ARS solutions with various concentrations were obtained by serial dilution of the stock solution with sodium acetate buffer solution (0.01 M, pH 3.0). The ARS solutions with different concentrations were mixed with the colloidal TiO 2 NPs at the same volume and shaken thoroughly. For the detection of metal ions, 10 mL of each sample mixed with 10 mL of ARS-TiO 2 was dripped into an aluminum pan (0219-0062, Perkin-Elmer), and the mixture was exposed to a laser beam for 30 s before each SERS measurement. The typical exposure time for each Raman/ SERS measurement in this study was 30 s with two accumulations. The error bars represent standard deviations based on three independent measurements.</p><!><p>The image of the sample was measured on a Tecnai G2 transmission electron microscope operating at 200 kV. The UV-vis spectra were recorded on a Shimadzu UV-3600 spectrophotometer. A RS-2100 Raman spectrophotometer (Photon Design, Inc.) equipped with a CCD (Princeton Instruments) was used. Radiation with a wavelength of 514.5 nm from an Ar ion laser (Spectra Physics) was employed for the Raman excitation, with a power of 5 mW at the sample. The Raman band of a silicon wafer at 520.7 cm À1 was used to calibrate the spectrometer.</p><!><p>Colloidal TiO 2 NPs with an average diameter of 3 nm were prepared by a low-temperature acid hydrolysis route, as described previously (see Fig. 1). 36,37 The absorption spectra of the colloidal TiO 2 NPs before and aer modication with ARS are shown in Fig. 1b, together with that of ARS for the sake of comparison. In contrast to ARS, the ARS-TiO 2 complexes exhibit a more intense absorption band in the longer wavelength region with a peak maximum centered at 489 nm. This absorption band has been assigned to the LMCT transition, which arises from the strong electronic coupling between ARS and the colloidal TiO 2 NPs. 38 Based on the Benesi-Hildebrand analysis for ARS-TiO 2 complexes (Fig. S1 †), the association constant (K ass ) of the complex was determined to be 3.9 Â 10 3 M À1 , which indicates the relatively strong binding of ARS on the surface of the TiO 2 NPs. An ARS molecule contains many functional groups; the FTIR data of the ARS-TiO 2 complexes unambiguously shows that the mode of graing is bidentate chelation, which involves two hydroxyl groups (Fig. S2 †). These observations suggest that the ARS-TiO 2 composite material can be used for the development of a semiconductor-supported SERS sensing platform due to the clear charge-transfer transition process.</p><p>Mechanism for SERS of the ARS-TiO 2 system Fig. 2a compares a Raman spectrum of 0.1 M ARS in aqueous solution and a SERS spectrum of ARS-TiO 2 complexes with 514.5 nm excitation. The vibrational mode assignments listed in Table S1 † are based primarily on earlier IR and Raman studies of related alizarin dyes. [39][40][41][42] The SERS spectrum is characterized by a signicant enhancement in the 1200-1500 cm À1 region, where the bands are typically assigned to the C]C and C-O-R stretching modes. This observation conrms the presence of strong coupling between the electronic transitions and the C]C/C-O-R bond stretching modes in ARS ligands, which is consistent with the conclusions obtained from the absorption and IR spectra of the ARS-TiO 2 complexes. Such coupling has also been observed on other semiconductor NPs in colloidal suspensions such as CeO 2 , Fe 2 O 3 , ZrO 2 , etc. 43 Furthermore, the concentration-dependent SERS experiments displayed in Fig. 2c clearly demonstrate that a concentration as low as 5 Â 10 À7 M ARS can be detected. 44 The intensities of the Raman signals can be tted with the BET model well and represent a saturation effect (Fig. 2d). In addition, a linear correlation was found between the intensity at 1260 cm À1 and the ARS concentration in the range of 5 Â 10 À7 to 2 Â 10 À4 M.</p><p>Of note is that the enhancement arises from the strong coupling interaction between the dye molecules and the colloidal TiO 2 NPs and, more importantly, the formation of charge-transfer complexes opens up a new electronic transition pathway for the Raman process. 45,46 In this case, the groundstate electrons of the ARS-TiO 2 complexes are initially excited from the highest occupied molecular orbital (HOMO) level to the conduction band (CB) of the TiO 2 NPs by the incident light (Fig. 2b). Then, the excited electrons immediately transfer back to the vibrational energy level of the ARS molecule and subsequently release a Raman photon with the ARS molecule at some vibrational state. The molecular polarizability tensor can be enhanced by such a charge-transfer process due to the vibronic coupling of the conduction band states of the semiconductor with the excited states of the probe molecule through a Herzberg-Teller coupling term. 47 Therefore, unlike in resonance Raman spectroscopy, where the molecule itself should reach a resonant state with excitation by the incident light, the enhancement can be considered in this case as a SERS phenomenon, which arises from the chemical enhancement mechanism via the Herzberg-Teller contribution. 2,45,48 Mechanism for responding to Cr(VI)</p><p>In general, organic ligands are susceptible to decomposition on bulk TiO 2 , owing to their adsorption through physisorption or weak chemisorption. Compared with bulk TiO 2 , colloidal TiO 2 NPs possess abundant under-coordinated Ti defect sites, which provide plenty of coordination sites for ARS to bind via bidentate chelation. This chelation mode is favorable to the recombination between ARS + and an electron, thereby stabilizing the ARS molecule. Consequently, ARS adsorbed on the surface of colloidal TiO 2 NPs cannot be easily oxidized by visible light irradiation and preserves the SERS properties even aer exposure to high laser power (Fig. S4 †). However, the SERS intensities are decreased with the addition of Cr(VI), indicating the decomposition of ARS adsorbed on the colloidal TiO 2 NP surfaces (Fig. 3 and S5 †). As shown in Fig. 4 and S6, † the ARS-TiO 2 complexes exhibit a remarkably high selectivity and lower interference in the determination of Cr(VI), particularly in the presence of Cr(III). This specicity originates from the favorable redox potential of the couple Cr(VI)/Cr(V) (+0.55 V) for the reduction promoted by those electrons trapped in inter-bandgap states, together with the strong interaction between Cr(VI) and Ti(IV) atoms with unlled valence orbitals at the TiO 2 surface. This reduction can result in the formation of Cr(V) and decomposition of ARS-TiO 2 complexes (Fig. S5 †), thus leading to a decrease in the SERS intensities. The redox potential of the Fe(III)/Fe(II) couple (+0.77) is close to that of the Cr(VI)/Cr(V) couple. However, the relatively weak interaction between Fe(III) and the positively charged colloidal TiO 2 NPs only causes minor disturbance. To minimize the interference from Fe(III), 0.2 mM EDTA was added to the sodium acetate buffer as a masking agent. As expected, the interference from Fe(III) was found to be negligible in the presence of EDTA. Based on these results, it was inferred that the SERS intensities of ARS are sensitive to the Optimization of the sensing system</p><p>Prior to the application of such a SERS sensing platform to the detection of Cr(VI), several inuencing factors, such as response . Moreover, an aggregation-sedimentation phenomenon was observed at a pH larger than 5.0, providing us with a simple method to recycle the TiO 2 NPs from the analyte. Finally, the response mechanism was based on a co-catalysis scheme, in which both Cr(VI) and ARS are activated by the available Ti coordination sites on the surface of the colloidal TiO 2 NPs. Thus, the catalytic efficiency with different loading amounts of ARS on the colloidal TiO 2 NPs was also investigated to determine the optimum ARS-sensitized concentration. It was clearly found that increasing the loading amount of ARS results in an increase in the SERS intensity, but the best performance was obtained at 50 mM (Fig. S9 †).</p><!><p>Based on the optimized conditions, the sensitivity and linearity of this sensing system were evaluated with different concentrations of Cr(VI) (Fig. 5). A good inverse proportionality was observed between the SERS intensity and the amount of Cr(VI) in the concentration range of 0.6-10 mM. The lowest concentration at which Cr(VI) could be detected is 0.6 mM. This concentration is lower than the maximum level of Cr(VI) in drinking water allowed by the WHO. In addition, the water samples spiked with different concentrations of Cr(VI) were also measured by employing our sensing system (Fig. 6). The measurements, which accurately reported the concentrations of the added standard Cr(VI) with good recoveries (Table S2 †), conrmed that the sensing system proposed in this work has great potential for the quantitative analysis of Cr(VI) in environmental samples.</p><!><p>In this work, we showed that semiconductor-enhanced Raman spectroscopy can be used as a sensing platform for the detection of metal ions. Firstly, the possibility of utilizing the dye-sensitized TiO 2 system to promote SERS is discussed. It is found that the strong coupling interaction between the dye molecules and the colloidal TiO 2 NPs leads to the formation of charge-transfer complexes and thus opens up a new electronic transition pathway for the charge-transfer process. The molecular polarizability tensor can be enhanced by such a charge-transfer process due to the vibronic coupling of the conduction band states of the semiconductor with the excited states of the probe molecule through a Herzberg-Teller coupling term. Secondly, a novel "turn-off" SERS strategy has been proposed and its use in a SERS-based assay for Cr(VI) has been demonstrated. Colloidal TiO 2 NPs can be employed not only as an effective substrate to elicit the SERS signals of an ARS molecule, but also as a catalytic center to induce the self-degradation of the ARS response to Cr(VI). The "turn off" SERS signal upon laser irradiation allows the development of a facile assay to measure Cr(VI). Of note is that this method does not require complex pretreatment and complicated instruments, but provides a high sensitivity in a high-throughput fashion and excellent selectivity toward Cr(VI) over other common anions. Furthermore, the experiments using solutions spiked with Cr(VI) revealed that our method is effective in monitoring the Cr(VI) in real water samples. Based on this "turn-off" SERS strategy, other metal ions can also be detected by utilizing different semiconductor enhancement systems in which the energy level of the semiconductor is matched with the redox potential of the determined metal ion. Thus, we believe that the data described in this contribution clearly demonstrate that semiconductor-enhanced Raman spectroscopy integrated with the catalysis of semiconductor materials can be used as a reliable detection method for metal ions in practical applications.</p>

Royal Society of Chemistry (RSC)

Hierarchical Defect Engineering for LiCoO2 through Low-Solubility Trace Element Doping

Cation doping is a widely utilized method for modifying LiCoO 2 cathodes in an effort to improve the energy density for Li-ion batteries. However, an in-depth understanding of the underlying mechanisms remains elusive. We quantitatively characterized and thoroughly analyzed the segregation of trace-doped Ti in the LiCoO 2 cathode to reveal the hierarchical structural defects, which promote cycling stability by suppressing the undesired phase transformation at a deeply charged state.

hierarchical_defect_engineering_for_licoo2_through_low-solubility_trace_element_doping

INTRODUCTION<!>SoC Heterogeneity Effect of the Grain Boundary<!>Segregation of Ti in TLCO<!>Formation and Characterization of the Lattice Defects<!>Suppression of O 3 to H 1À3 Phase Transition<!>DISCUSSION<!>EXPERIMENTAL PROCEDURES

<p>The lithium-ion battery (LIB) is a groundbreaking invention that has enormous economic and social impacts. [1][2][3] The operation of LIBs requires the cooperation of multiple battery components, involving thermodynamically uphill and downhill reactions during charging and discharging, respectively. Although there are ongoing research efforts looking into all of the LIB components, the cathode material is currently the most significant limiting factor for further improvement of the energy density, and it is a major research focus in this field. 4,5 The structural and chemical complexity in the composite cathode electrodes is indispensable for the desired functionality, and it therefore requires a delicate control for optimal device performance. While the porous carbon-binder domain (CBD) is responsible for providing continuous diffusion pathways for the transportation of Li ions and electrons, the active materials are ultimately the energy reservoir in which Li ions are stored and released upon electrochemical cycling, and this process is accompanied by redox reactions in the host material.</p><p>Depending on the targeted applications, different active cathode materials could be chosen due to their respective pros and cons in different performance attributes. Although LCO has been utilized as the cathode material in LIBs for several decades, it is still very competitive and dominates the portable electronics market. [6][7][8][9] Stabilization of LCO's layered lattice at a deeply delithiated state is a frontier challenge that has attracted worldwide research interest. [10][11][12][13][14][15] This is because charging the LCO cathode to high voltage is of immediate and significant commercial incentives</p><p>The Bigger Picture Cation doping has long been regarded as an effective method of modifying LiCoO 2 for withstanding a higher cutoff voltage. In addition, the microstructure and chemical complexity of the cathode active particles could critically affect the overall battery performance. The underlying fundamental mechanisms, however, are often rather complicated and unclear. Herein, we present a simple yet effective multiscale defectengineering approach via lowsolubility trace element doping and demonstrate improved cycling stability. Advanced synchrotron characterization techniques reveal the multiscale segregation of Ti in the LiCoO 2 particle, formulating a hierarchical structural complexity that involves modified particle surface, grain boundaries, and lattice distortion defects. These structural defects play an important role in stabilizing the LiCoO 2 structure at a highly charged state. Moreover, this hierarchical defectengineering strategy is broadly applicable to the other material systems.</p><p>through increasing the energy density, one of the most critical battery-performance specifications. [16][17][18] Upon deep charging, there is a clear tendency for the LCO particles to build up mechanical strain, 19,20 to release lattice oxygen, 21,22 and to undergo structure reconstruction. 11,23 These negative effects are intertwined over different time and length scales and are harmful to stable battery operation at high voltage. To make it most effective, a material modification procedure must mitigate these negative effects in a systematic manner. In our previous work, we demonstrate that trace co-doping of Al, Mg, and Ti (~0.1 wt %) can greatly enhance the cycling performance of LiCoO 2 at 4.6V and reveal that each dopant contributes through different mechanisms for such performance enhancement. 24 Although such a synergistic effect formulated by the three co-existing dopants leads to overall optimal performance, it complicates the system and makes it difficult to single out the fundamental role of each dopant. While both Mg and Al have reasonable solubility in the LCO lattice, the most profound complexity lies with Ti. On the one hand, the majority of Ti segregates at the surface and interface, forming a 3D network. On the other hand, residual Ti can be found inhomogeneously in the host LCO matrix. According to the X-ray diffraction (XRD) patterns and the corresponding refinement results reported in our previous work, Ti has a major role to play in the distortion of the host lattice compared with Al and Mg. Based on these considerations, we choose to focus on Ti in the current study. Besides, because of the chemical complexities induced by different dopants-particularly for Ti that is nonuniformly distributed within particles, including Co-free cathode materials 25 -their impacts on the lattice structure across the entire particle and thus the overall structural stability have yet to be demonstrated. 26,27 Herein, we employ cutting-edge synchrotron characterization techniques, e.g., nanoresolution X-ray microscopy with composition, valence, and lattice defect sensitivities, to elucidate the spontaneous and multiscale dopant segregation in the Ti/ Mg/Al co-doped LCO particles. Our results suggest that, in addition to modifying the LCO particles' bulk and surface and interface properties as a previous study revealed, the inhomogeneous incorporation of Ti into the LCO lattice leads to a significant degree of lattice deformation (e.g., bending, twisting, and d-spacing heterogeneity). All the preexisting hierarchical defects positively contribute to the robustness of the LCO lattice at a deeply charged state as evidenced by the suppression of the LCO's phase transformation (from the O 3 phase to the H 1À3 and O 1 phases) that occurs at above 4.5 V. We clarify here that, by hierarchical defects, we are referring to the multiscale structural complexities in the material ranging from the secondary-particle level down to the atomic scale. The particle-level microscopic characterization is further supported by bulk-averaged in situ X-ray powder-diffraction data, which cover millions of particles and ensure the statistical representativeness of our conclusions. Our findings highlight a hierarchical selfassemble effect, which is originated from the low solubility of dopant in the host lattice. Such an effect could potentially be used to formulate an unconventional multiscale defect-engineering strategy that is broadly applicable.</p><!><p>The morphology and the state of charge (SoC) of a randomly selected bare LCO particle at the charge state are shown in Figure 1. A clear grain boundary with a microcrack at its edge can be observed in Figure 1A, and the grain boundary divides the particle into two domains. Further 3D Co valence distribution through peak-energy mapping using hard X-ray spectro-microscopy suggests that these two domains are in different SoCs. 28,29 Specifically, domain 2 has a higher Co valence state and exhibits a brighter color. The Co K-edge spectroscopic fingerprints of these two domains, together with that of a pristine bare LCO particle, are shown in Figure 1C. From the zoomed-in spectra in Figure 1D, it is clear that the peak energies of the pristine particle, domain 1, and domain 2 are gradually increasing, corresponding to their respective Co valence states. These results suggest that the grain boundary within a bare LCO particle not only is the mechanical weak point but also affects the local chemical states.</p><!><p>The above visualized grain boundary in a bare LCO particle appears to be chemically inactive and, consequently, prevents Li diffusion across this barrier. It is, therefore, of practical importance to modify the properties of such buried interfaces. Herein, we tackle this problem by applying trace doping of Ti, which is of a low solubility in the hosting LCO lattice. The morphology of an arbitrarily selected secondary particle of Ti/Mg/Al co-doped LCO (denoted as TLCO since we focus on the heterogeneity of Ti) is shown in Figure 2A. Its compositional heterogeneity is revealed in Figure 2B, which displays the distribution of the dopant (Ti) over a virtual slice through the Article center of the particle. The low solubility of Ti leads to its segregation at the particle surface and the buried interfaces, highlighting the separation of several primary grains as labeled in Figure 2B. Such an effect allows us to further extract and analyze individual primary grain(s). Figure 2C shows the elemental distribution results of an isolated primary TLCO particle. The depth profiles of elemental concentrations of Co and Ti as well as their relative ratios are calculated and plotted in Figure 2D. Our data suggest that the Ti concentration rapidly decreases from the surface to the particle center, while Co is relatively homogeneously distributed. These fine and quantitative analyses clearly confirm that trace doping of low-solubility dopant is an effective interface-engineering approach.</p><!><p>In addition to the modified interfaces, the lattice defects in the TLCO material could also play a significant role in the electrochemical performance. While the Bragg coherent diffraction imaging (BCDI) has been proven a powerful technique for characterizing the topological defects and local elastic properties in cathode crystalline particles, the limited coherence length of a typical third-generation synchrotron source limits BCDI's application to nanosized crystals. 30 To look into the lattice defects within an individual micron-sized primary grain of TLCO, we utilized the scanning hard X-ray nanoprobe technique (Figure 3A). 31 In a hard X-ray nanoprobe setup, the X-rays are focused to a spot of ~30 nm for illuminating the sample, which diffracts the beam in directions where the Bragg condition is satisfied. The diffraction patterns around a selected Bragg peak (101) are recorded. The crystal was rocked over a 2-degree angular range in the vicinity of the (101) Bragg peak, while a 2D raster scan was conducted at each rocking angle. Figure 3B is a typical diffraction pattern, in which we illustrate the orthogonal directions of the Bragg peak shift, relevant to the lattice defects and distortions. For example, the information collected along and perpendicular to the powder-ring direction are corresponding to the Zbending and strain, respectively. In the diffraction contrast images of LCO and TLCO particles, we observe two kinds of features that can be attributed to two different types of lattice defects (see Figure S1). The first type is worm-shaped curves (annotated with red arrows in Figure S1A), which are very similar to the edge-dislocation lines visualized in a Li-rich layered oxide particle using the BCDI method. 32 The line density is significantly higher in the TLCO particle. The second type is annotated with green lines in Figure S1B; these separate regions with different intensities in the diffraction contrast images, indicating different scattering power in different orientations. The second type of feature can therefore be attributed to the crystal twin boundaries. By tracking the diffraction pattern recorded on the pixel-area detector (see Figure 3B for the geometry) and the offsetting of the rocking curve (see Figures 3C and 3D for two representative pixels that show distinct rocking curve), the local lattice distortions can be mapped out. 33,34 To highlight Ti's role in inducing the observed lattice defects, we compare the nanodiffraction data of particles of bare LCO and TLCO in Figure 4. Figures 4A and 4B are the maps of different types of lattice distortions of the bare LCO and TLCO, respectively. The inhomogeneity in Z-bending, Y-twisting, and d-spacing can be reconstructed by conducting this calculation pixel by pixel over the entire scanned TLCO primary grain, as shown in Figure 4B. Visual assessment of these maps suggests that the bare LCO exhibits a higher degree of uniformity. This is confirmed by the histogram plots in Figure 4C, in which the peak broadening is observed in the TLCO data. Such comparison confirms that the Ti doping has induced LCO lattice deformation, which becomes a preexisting condition before the TLCO material is subjected to Article electrochemical cycling. To ensure the statistical representativeness of our particlelevel evaluation, the bulk-averaged laboratory XRD characterization is carried out. Our bulk XRD results of the pristine bare LCO and TLCO samples confirm the observations from nano X-ray diffraction-imaging experiments. Pawley refinements were performed with fundamental parameters (FPs) and pseudo-Voigt peak functions, respectively, for analysis of the lattice strain. It can be seen that TLCO exhibits much larger lattice strain than the bare LCO, indicating a larger amount of lattice defects in TLCO. More discussion on the XRD results can be found in the next section.</p><!><p>For a better understanding of how such heterogeneously distributed lattice defects affect the phase transition of LCO over the charge-discharge process, we performed in situ laboratory XRD experiments to study the phase-evolution behavior of the bare LCO and the TLCO (Figure 5). Figure 5A shows the initial charging curve of TLCO under 0.1C condition with a highlighted voltage window of 4.40-4.60 V, in which the O 3 to H 1À3 phase transition occurs. Figure 5B focuses on the (003) diffraction peak shifts of the bare LCO and the TLCO in the charging voltage range of 4.40-4.60 V. A clear sign of the two-phase reaction can be observed at the end of this charging voltage range for the bare LCO, while the TLCO displays an overall solid-solution reaction. The split of the (003) peak is a typical characteristic of the O 3 to H 1À3 phase transition. Such structural transformation involves gliding of the TMO 2 slabs, which could cause particle fracturing and, consequently, bring in a series of detrimental effects, leading to the deterioration of the cycle performance. For further quantitative analysis, the evolution of cell-lattice parameters and cell volume were obtained through Pawley XRD refinement (see Tables S1-S3). As shown in Fig- ure 5C, the changes of both the cell-lattice parameters a and c and the cell volume of TLCO are smaller than those of the bare LCO, which strongly demonstrates the significant suppression of the phase transition in TLCO. In connection with the aforementioned analysis of the diffraction images, it can be hinted that the multimodular defects induced by the low solubility of Ti, in addition to homogeneous Al/Mg lattice doping, 24,35 can well suppress the collective O 3 to H 1À3 phase transition occurring in the bare LCO. Such an effect can partially contribute to the greatly enhanced cycle performances of TLCO at 4.6 V. The underlying mechanism can be understood as shown in Figure 5D, which schematically compares the lattice defect density (top) and the microstrain distribution (bottom) in primary grains of LCO and TLCO crystal. The suppression of the O 3 to H 1À3 phase transition in TLCO could be attributed to its abundant lattice defects and, subsequently, its highly disordered microstrain distribution. It is well known that strain has a significant impact on the phase-evolution behavior of battery-electrode materials during electrochemical reactions. 30 A recent density functional theory (DFT) calculation study has also demonstrated that the anisotropic microstrain in LCO lattice increases the energy barriers of structural transition during delithiation at high voltage. 36 Another important aspect is the size effect. As the inhomogeneous Ti distribution and the resultant hierarchical lattice defects separate the sample particle into small grains, the size effect may contribute to the energy that can affect the phase transition. The effect of defect energy and size on modification of the phase-transition behavior has been demonstrated in previous literature reports. For example, Wagemaker and co-workers performed very careful studies and revealed that nanosized rutile TiO 2 has a wider Li-solubility range that can accommodate more Li deintercalation and intercalation than a micro-sized one because of the size-induced extra surface and interfacial energy. 37 We also performed DFT calculations to elucidate how size affects the O 3 to H 1À3 phase transition in LCO. The detailed discussions are expanded in the Supplemental Information. In summary, our experimental results suggest that the intergrain (e.g., grain boundary) and intragrain (e.g., lattice doping) defects synergistically inhibit the O 3 to H 1À3 phase transition at high voltages. Therefore, hierarchical defect engineering is essential for the design of high-capacity cathodes that can overcome the giant strain due to the large amount of Li deintercalation and intercalation and thus achieve high structural stability. It is worth noting that both bare LCO and TLCO show very similar structural evolution behavior in low voltage range (OCV-4.3 V). In general, the order-disorder phase transition that occurred at around 4.2 V (Li x CoO 2 , x = 0.5) is very sensitive to the element doping and doping content. This indicates that the relatively low doping content of Ti (~0.1 wt %) in TLCO does not affect the bulk structure transition at low voltages. Therefore, the Ti-induced hierarchical doping is effective at promoting the stability at highly delithiated state without affecting the LCO's behavior very much at lower SoC.</p><!><p>In this study, we show that trace Ti doping can modify the microstructure and thus the intra-and intergrain defect properties within individual particles as a result of the low solubility of Ti in LCO lattice. Quantitative analysis of the nanoresolution diffraction images on single particles and XRD patterns covering structural information of millions of particles reveal a significant amount of defects that are heterogeneously distributed in TLCO. The construction of such hierarchical defects demonstrates the effectiveness in suppressing the undesired O 3 to H 1À3 phase transition of LCO at a deeply delithiated state, contributing to the greatly improved cycle performances of TLCO at high voltages. Given that high-energy-density cathode materials require the capability of stable deintercalation and intercalation of a large numbers of alkali ions in their lattices, which will inevitably introduce a high level of strain within the particles, a hierarchical defect-engineering strategy could enable highly disordered dispersion of lattice strain across both primary and secondary particles. Such an approach could be essential for constructing cathode materials with high electrochemical stability over long-termcycling. We would like to remind in the end that the lattice defects not only affect the structural robustness but could also modulate the cation and/or anion redox reactions and rearrange the Li-ion diffusion pathways. The interplay among lattice defects, cation and anion redox, and charge heterogeneity is a frontier research topic that is worth systematic follow-up efforts.</p><!><p>Resource Availability Lead Contact Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Y. Liu (liuyijin@slac.stanford.edu).</p>

Chem Cell

Estradiol promotes rapid degradation of HER3 in ER-positive breast cancer cell line MCF-7

HER3, a member of the receptor tyrosine kinase super family, is overexpressed in a number of cancers, and is associated with malignant phenotypes. Control of the protein stability of the membrane, as well as nuclear receptors, has been known to be an important process affecting tumor cells; however, their relationships have yet to be elucidated. In this study, we demonstrate that estradiol promotes rapid degradation of HER3 via the proteasome pathway in ER-positive breast cancer, MCF-7. ER prevented HER3 degradation, and knockdown of ER expression by si-RNA promoted rapid degradation of HER3. Breakdown of HER3 and ER were regulated by a ubiquitin ligase Nedd4-1 in the presence of estradiol stimulation. We speculate that estradiol quickly degrades ER, making HER3 accessible by Nedd4-1, and leads to the rapid degradation of HER3. In addition, knockdown of ubiquitin ligase Nedd4-1 enhances estradiol induced cell proliferation. These results indicate that HER3 and Nedd4-1 in ER-positive breast cancers might be an important therapeutic target.

estradiol_promotes_rapid_degradation_of_her3_in_er-positive_breast_cancer_cell_line_mcf-7

<!>Introduction<!>Cell culture<!>Reagents and antibodies<!>siRNA and shRNA mediated knockdown<!>Cycloheximide chase assay<!>Western blotting<!>Cell proliferation assay<!>Statistical analysis<!><!>HER3 is rapidly degraded in the presence of estradiol via proteasome pathway<!><!>Nedd4-1 regulates HER3 and ER degradation in the presence of estradiol<!><!>Depletion of ER promotes the rapid degradation of HER3<!><!>Discussion<!>

<p>First time we found that estradiol promotes rapid degradation of HER3 in ER-positive breast cancer MCF-7 cells.</p><p>Knockdown of ubiquitin ligase Nedd4-1 decreased the degradation efficiency of HER3 in the presence of estradiol.</p><p>Knockdown of Nedd4-1 enhanced estradiol induced tumor cell proliferation.</p><!><p>HER3 is a member of the receptor tyrosine kinase family (RTK) and lacks intrinsic tyrosine kinase activity in the C-terminal tail. It is activated by Heregulin-1 (HRG-1) stimulation, and plays a regulatory role in cell proliferation and migration [1], [2], [3]. Overexpression of HER3 has been reported in breast, ovarian, pancreatic and gastric cancers, and is significantly associated with cancer malignancy [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. However, the mechanisms of HER3 overexpression are still not well understood. On the other hand, previous studies have reported that HER3 is regulated by ubiquitination and degradation with HRG-1 stimulation [14], [15], [16], [17].</p><p>Ubiquitination is controlled by regulatory proteins in the ubiquitin-conjugation system, and occurs through the three sequential classes of enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) [18], [19], [20]. Ubiquitin ligases interact physically with the substrate to determine the protein's fate by leading to degradation. Previous studies have reported that the HER family receptors are degraded with a help of their specific ubiquitin ligases [21], [22]. For example, HER1 is degraded by ubiquitin ligase c-Cbl [23], HER2 is mediated by c-Cbl [24], [25], [26] or chaperon-interacting protein (CHIP) [27], [28]. HER4 is ubiquitinated by WWP1 [32] or Itch [33]. In the case of HER3, HRG-1 stimulation leads to proteasome-mediated degradation of HER3, and at least three ubiquitin ligases, including neuregulin receptor degradation protein-1 (Nrdp1) [14], [15], [16], [17], [29], neural precursor cell expressed developmentally down-regulated 4 (Nedd4-1) [30] and Itchy (Itch) [31], have been identified in the degradation process. Nevertheless, the functional relationship between HER3 ubiquitination and hormones remains unknown.</p><p>In our attempt to investigate breast cancer, we have been exploring the biological role of estradiol in estrogen receptor (ER) positive breast cancer. In this line, we found that estradiol promotes rapid degradation of HER3 via the proteasome pathway, and an ubiquitin ligase Nedd4-1 controls this process. Furthermore, Nedd4-1 affects proliferation of MCF-7 cells through its dual action on HER3 and ER.</p><!><p>Human embryonic kidney cells 293 T and human breast cancer cell lines MCF-7 and MDA-MB231 were purchased from ATCC, and human breast cancer cell lines SKBR3 and BT474 were gifted by Dr. S. Hayashi (Tohoku University, Miyagi, Japan). The cells were cultured in DMEM (Wako) or RPMI 1640 (Wako) supplemented with 10% heat-inactivated FBS (Biowest), 100 units/ml penicillin G and 100 µg/ml streptomycin. For experiments evaluating the effect of 17β-estradiol (estradiol, Sigma-Aldrich), the MCF-7 cells were cultured for two days in phenol red-free DMEM (PRF-DMEM, Wako) containing 10% heat-inactivated FBS stripped of steroids by absorption to dextran-coated charcoal (DCC-FBS, Biological Industries). The cells were then cultured in a humidified 5% CO2 incubator at 37 ℃.</p><!><p>The reagents used were as follows: epoxomicin (Peptide); ethanol, cycloheximide and chloroquine diphosphate (Wako); dimethyl sulfoxide and Fulvestrant (Sigma-Aldrich). The antibodies used were as follows: anti-HER3, anti-NEDD4-1 and corresponding secondary antibodies (Cell Signaling Technology); anti-Itch and anti-Nrdp1 (Santa Cruz Biothechonology); anti-ER (Thermo Scientific): anti- β actin (Sigma-Aldrich).</p><!><p>Knockdown of human ER was performed using si-ER (Ambion, catalog# 4392420), along with a non-silencing control si-RNA (catalog# 4390843). MCF-7 cells were transiently transfected with 10 µM of the si-RNAs using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) according to the manufacturer's protocol, and were further cultured for 48 h before assays.</p><p>Human sh-Nedd4-1 expressing lentivirus vectors were constructed in pRSI12-U6-sh-HTS4-UbiC-TagRFP-2A-Puro plasmid (Cellecta). The pRSI12-U6-sh-Nedd4-1-HTS4-UbiC-TagRFP-2A-Puro or control pRSI12-U6-sh-HTS4-UbiC-TagRFP-2A-Puroplasmid was transfected into 293 T cells together with two packaging plasmids, pCMV-VSV-G/RSV-Rev and pCAG-HIVgp (RIKEN Bio-resource Center), using FuGENE HD (Promega) according to the manufacturer's protocol. At 48 h post-transfection, the supernatants were collected and filtrated through a 0.45 µm syringe filter. The lentiviral particles encoding the shRNA that targeted Nedd4-1 or a scramble control were incubated with MCF-7 cells for 48 h. Transduced cells were selected for additional incubation for 72 h in the presence of 1 µg/ml puromycin (Wako). Cells were used immediately after selection.</p><!><p>MCF-7 cells were plated in 6-well culture plates at a density of 4 × 105 cells/well with PRF-DMEM containing 10% DCC-FBS. After overnight incubation, the medium was replaced with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 5 μM epoxomicin (Epx), or 1 μM chloroquine (CQ) with 50 µg/ml cycloheximide (CHX) for 30 min, and chased for different time periods in the presence or absence of estradiol with CHX.</p><p>For CHX chase assay using Fulvestrant (Ful), MCF-7 cells were cultured in PRF-DMEM containing 10% DCC-FBS for 2 days. The cells were then plated in 6-well culture plates at a density of 4 × 105 cells/well, with PRF-DMEM containing 10% DCC-FBS added to DMSO as control or 0.1 μM Fulvestrant. After overnight incubation, the medium was replaced with serum-starved PRF-DMEM for 1.5 h in the presence of DMSO or Fulvestrant. The cells were then treated with 50 µg/ml CHX for 30 min and chased for different time periods in the presence or absence of estradiol with CHX.</p><p>The cells were collected at each time point and processed for immunoblotting by anti-HER3, anti-Nedd4-1, anti-ER and anti-β actin antibodies.</p><!><p>Cells were grown in PRF-DMEM containing 10% DCC-FBS in 6-well culture plates. The cultured cells were then washed twice with ice-cold PBS before they were lysed in RIPA buffer (40 mM Tris-HCl, pH7.5, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 2 mM Na3VO4, 50 mM NaF) containing protease inhibitor cocktail (Roche). Lysates were scraped, transferred into microtubes, and centrifuged at 13,000 g for 20 min at 4 ℃. The supernatants were used as cell extracts. Total protein concentrations were determined using a Quick Start Bradford protein assay (Bio-Rad) using bovine serum albumin as a standard. Immunoblotting was subjected to 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad), followed by transference to 0.45 µm pore size polyvinylidene difluoride membranes (Millipore), and blotting with primary and secondary antibodies. Quantification was performed using ImageJ software.</p><!><p>Cell proliferation was detected with a Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer's protocol. The sh-control or sh-Nedd4-1 knockdown MCF-7 cells were seeded in 96-well culture plates (7 × 103 cells/well) in PRF-DMEM containing 10% DCC-FBS. After overnight incubation, the cells were replaced in a medium containing ethanol (EtOH) or 1 nM estradiol reagent. At 0, 24, 48, and 72 h of incubation, 10 µl of CCK-8 solution was added to the cells. After incubating the cells for 2 h at 37 ℃, absorbance at 450 nm was measured using a plate reader (Thermo Scientific).</p><!><p>All data are expressed as mean ± SD, as indicated in the figure legends. Statistical analysis was performed using the Student t-test. Significance is denoted as * , P < 0.05; * *, P < 0.01. All experiments were replicated at least three times.</p><!><p>Expression of HER3, ER and ubiquitin ligases in human breast cancer cell lines. (A) Endogenous HER3, ER and (B) ubiquitin ligase (Nedd4-1, Itch and Nrdp1) expression in the four subtypes of human breast cancer cells (MCF7; Luminal A, BT474; Luminal B, SKBR3; HER2, MDA-MB231; triple negative) were analyzed by immunoblotting with anti-HER3, anti-ER, anti-Nedd4-1, anti-Itch, anti-Nrdp1 and anti-β actin antibodies. (C) Densitometry analysis of immunoblots. The quantification of the Nedd4-1 protein levels was done using ImageJ software. The protein levels were normalized to β actin levels. The results shown are from three independent experiments. * *P < 0.01 versus β actin. Error bars represent mean ± SD.</p><!><p>To evaluate the degradation speed of HER3 in the presence or absence of estradiol, we performed the CHX chase assay, which monitors the amount that proteins decrease under the de novo protein biosynthesis inhibition with CHX. Ethanol (EtOH) was solvent of estradiol and was used as control stimulation.</p><!><p>Estradiol induces rapid degradation of HER3 via proteasome pathway. (A) MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml cycloheximide (CHX) for 30 min, followed by treatment with indicated concentrations of estradiol. The cells were lysed at indicated time points and subjected to immunoblotting for anti-HER3, anti-ER and anti-β actin antibodies. (B) The quantification of the HER3 protein levels was done using ImageJ software. The protein levels were normalized to β actin levels. The results are shown as means ± SD of three independent experiments. *P < 0.05 versus EtOH. (C) Half-life of HER3 was calculated based on the data in Fig. 2B. (D) The MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were treated with 5 μM epoxomicin (Epx), 1 μM chloroquine (CQ), or DMSO with 50 µg/ml CHX for 30 min, followed by treatment with 1 nM estradiol or EtOH in the presence of CHX. The cells were then lysed at indicated time points and subjected to immunoblotting for anti-HER3 and anti-β actin antibodies. (E, F) Quantification of the HER3 protein levels was done using ImageJ software. Protein levels were normalized to β actin levels. All values are shown as means ± SD of three independent experiments. *P < 0.05 versus DMSO.</p><p>Nedd4-1 regulates HER3 and ER degradation in the presence of estradiol. (A) sh-control MCF-7 cells and sh-Nedd4-1 knockdown MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml CHX for 30 min, followed by treatment with EtOH or 1 nM estradiol in the presence of CHX. All protein levels were assessed by immunoblotting at indicated time points. Quantification of the HER3 (B, C) and ER (D, E) protein levels were done using ImageJ software. The protein levels were normalized to β actin levels. All values are shown as means ± SD of three independent experiments. *P < 0.05 versus sh-control.</p><!><p>To investigate the possible involvement of the same degradation process for ER, we used the CHX chase assay of ER proteins in the sh-control MCF-7 cells and sh-Nedd4-1 MCF-7 cells. After 4 h of estrogen stimulation, ER degradation in the Nedd4-1 knockdown MCF-7 cells (Fig. 3A and E, dotted line) was more suppressed than in the sh-control MCF-7 cells (Fig. 3A and E, full line). In the absence of estradiol, ER was similarly decreased in both the sh-control MCF-7 (Fig. 3A and D, full line) and sh-Nedd4-1 MCF-7 cells (Fig. 3A and D, dotted line). These results suggest that in the estradiol-stimulated condition, Nedd4-1 regulates the HER3 and ER degradation processes at a specific time point.</p><!><p>Depletion of ER promotes the rapid degradation of HER3. si-control MCF-7 cells and si-ER knockdown MCF-7 cells were incubated with serum-starved PRF-DMEM for 1.5 h. The cells were then treated with 50 µg/ml CHX for 30 min, followed by treatment with EtOH or 1 nM estradiol in the presence of CHX. All protein levels were assessed using immunoblotting at indicated time points. (B, C) Quantification of the HER3 protein levels was done using ImageJ software. All data from the three experiments were normalized to β actin. Mean values ± SD were plotted. *P < 0.05 versus si-control. (D) MCF-7 cells cultured in PRF-DMEM containing 10% DCC-FBS for 2 days. Cells were prepared with PRF-DMEM containing 10% DCC-FBS added to DMSO or 0.1 μM Fulvestrant. The medium was replaced with serum-starved PRF-DMEM for 1.5 h in the presence of DMSO or Fulvestrant. The cells were then treated with 50 µg/ml CHX for 30 min and chased for different time periods in the presence or absence of estradiol with CHX. The cells were collected at each time point and processed for immunoblotting by anti-HER3, anti-ER and anti-β actin antibodies. (E, F) Quantification of the HER3 protein levels was done using ImageJ software. The protein levels were normalized to β actin levels. All values are shown as means ± SD of three independent experiments. *P < 0.05 versus DMSO.*.</p><!><p>To confirm our findings in the ER-knockdown study, we performed the CHX chase assay using Fulvestrant, a selective ER down-regulator, which degrades ER and acts as a complete antagonist to ER function. In the estradiol-stimulated condition, degradation of HER3 was remarkably enhanced for 2–4 h by pretreatment with Fulvestrant (Fig. 4D and F, dotted line). In the control condition (EtOH), pretreatment with Fulvestrant also enhanced HER3 degradation, but this difference was small and not significant. (Fig. 4. D and E, full and dotted lines).</p><p>From these results, we speculated that estradiol induces HER3 degradation, which then liberates HER3 from its inhibition by ER, eventually leading to the degradation of HER3.</p><!><p>Knockdown of Nedd4-1 enhances proliferation of MCF-7 cells. (A, B) sh-control and sh-Nedd4-1 knockdown MCF-7 cells were incubated for the indicated time at 37 ℃ with EtOH or 1 nM estradiol. Cell proliferation was measured using CCK-8 assays. These data are representative of three independent experiments (n = 3). * *P < 0.01 versus sh-control. Error bars represent mean ± SD. (C) Summary of findings for the effect of HER3 degradation on ER-positive breast cancer cell line MCF-7.</p><!><p>We showed here that estradiol promotes rapid degradation of HER3 in ER-positive breast cancer MCF-7 cells.</p><p>HER3 is a member of the HER family, which plays multiple roles in oncogenesis [34]. Overexpression of HER3 in several types of primary tumors or culture cells, such as that in breast, ovarian pancreatic and gastric cancers, has been reported [4], [5], [6], [7], [8], [9], [10], [11], [12], [13].</p><p>In breast cancer, HER3 contributes to tumor cell survival and proliferation, and previous reports have shown that HER3 in breast cancer cases is associated with poor prognostic factors, in terms of grade, lymph node metastasis and tumor size [5], [6]. Therefore, an underlying mechanism for HER3 overexpression might be a target for drug development for breast cancer. To this end, we used ER-positive breast cancer MCF-7 cells, which had remarkably positive HER3 expression compared to the cell lines that we evaluated (Fig. 1A).</p><p>In our HER3 CHX chase assay, we found that HER3 was degraded more rapidly in the presence of estradiol than in its absence (Fig. 2A and B). As is well-known, estradiol is a ligand of ER, and estradiol stimulation causes rapid ER degradation as a result of ligand-receptor interaction [35], [36], [37]. In the current study, the half-life of both ER and HER3 were affected by estradiol stimulation (Fig. 2A and C), leading to the suspicion that the same degradation mechanism was involved in both receptors. It is known that HER3 is quickly degraded by the proteasome pathway upon Heregulin-1 (HRG-1) stimulation and, interestingly, this is also true for estradiol stimulation, as shown in our proteasome pathway inhibitor experiments (Fig. 2D-F). Moreover, ubiquitination of HER3 was observed in the estradiol-stimulated condition (data not shown).</p><p>Degradation of HER3 under HRG-1 stimulation has been associated with three ubiquitin ligases, Nedd4-1, Itch and Nrdp1 [14], [15], [16], [17], [30], [31]. Nedd4-1 is the only ubiquitin ligase which was endogenously expressed in the MCF-7 cells, and it specifically contributed to the estradiol induced rapid degradation process of HER3 and ER. We suspect that Nedd4-1 is involved in the estradiol induced degradation of HER3 in a time dependent manner. In general, the degradation process is a time-dependent multi-step process, and involves various factors. In our study, we observed that the timing of Nedd4-1's contribution is different in ER (at 4 h) and HER3 degradation (at 2 h). This might be due to differences in interaction factors of Nedd4-1 in each degradation process (Fig. 3). Interestingly, depletion of ER enhanced HER3 degradation irrespective of estradiol stimulation (Fig. 4), indicating that ER might possess a function that prevents HER3 degradation through direct interaction. Collins et al. reported that HER3 forms a complex with ER in the presence and absence of HRG-1 [38], [39]. In the current study, we were unable to prove direct interaction between ER and HER3; however, we speculate that the formation of the ER/HER3 complex could prevent HER3 degradation. Together, our current hypothetical schema is shown in Fig. 5C. ER prevents HER3 degradation through its interaction under an estradiol-negative condition. Upon estradiol stimulation, ER is quickly degraded, and HER3, which is now free from ER, is led to prompt degradation. Richard et al. reported that growth factor receptors such as EGFR or HER2 cross-talk with ER and tend to acquire resistance to endocrine therapy [39]. Since HER3 and EGFR belong to same receptor family, we hypothesize that HER3 cross-communicates with ER. This ER-HER3 crosstalk would shed light on a previously unknown aspect of breast cancer research.</p><p>Finally, Nedd4-1 knockdown showed remarkable proliferative properties, both in the presence and absence of estradiol (Fig. 5A and B). Our observation that the enhanced effect of Nedd4-1 knockdown in HRG-1 stimulated proliferation is consistent with the results of previous report [30]. Nedd4-1 contributes to estradiol-induced proliferation of MCF-7 cells through interaction with HER3, although further investigation is needed to confirm this.</p><p>A limitation of our study is that we were unable to show the difference in the ubiquitination of HER3 between in the presence or absence of estradiol. The ubiquitination of HER3 seems to have occurred for a short time and we need to establish a more precise assay system. Although we performed several experiments for evaluating the PI3K/Akt and MAPK signaling pathways of HER3 to explain the biological impact of ER/HER3 interaction through a degradation process, we could not obtain significant results. This may be due to the experiment settings or incorrect target signaling.</p><p>In summary, our findings showed a model of estradiol-induced HER3 degradation in ER-positive breast cancer MCF-7 cells. HER3 was degraded rapidly by the proteasome pathway under estradiol stimulation, and ubiquitin ligase Nedd4-1 contributed to both HER3 degradation and tumor cell growth. The impact of Nedd4-1 on breast cancer biology should continue to be studied in future research.</p><!><p>Supplementary material</p>

PubMed Open Access

Accurate cancer cell identification and microRNA silencing induced therapy using tailored DNA tetrahedron nanostructures

Accurate cancer cell identification and efficient therapy are extremely desirable and challenging in clinics.Here, we reported the first example of DNA tetrahedron nanostructures (DTNSs) to real-time monitor and image three intracellular miRNAs based on the fluorescence "OFF" to "ON" mode, as well as to realize cancer therapy induced by miRNA silencing. DTNSs were self-assembled by seven customized singlestranded nucleic acid chains containing three recognition sequences for target miRNAs. In the three vertexes of DTNSs, fluorophores and quenchers were brought into close proximity, inducing fluorescence quenching. In the presence of target miRNAs, fluorophores and quenchers would be separated, resulting in fluorescence recovery. Owing to the unique tetrahedron-like spatial structure, DTNSs displayed improved resistance to enzymatic digestion and high cellular uptake efficiency, and exhibited the ability to simultaneously monitor three intracellular miRNAs. DTNSs not only effectively distinguished tumor cells from normal cells, but also identified cancer cell subtypes, which avoided false-positive signals and significantly improved the accuracy of cancer diagnosis. Moreover, the DTNSs could also act as an anti-cancer drug; antagomir-21 (one recognition sequence) was detached from DTNSs to silence endogenous miRNA-21 inside cells, which would suppress cancer cell migration and invasion, and finally induce cancer cell apoptosis; the result was demonstrated by experiments in vitro and in vivo. It is anticipated that the development of smart nanoplatforms will open a door for cancer diagnosis and treatment in clinical systems.

accurate_cancer_cell_identification_and_microrna_silencing_induced_therapy_using_tailored_dna_tetrah

Introduction<!>Preparation and characterization of DNA tetrahedron nanostructures (DTNSs)<!>Fluorescence quenching and recovery capability of the prepared DTNSs<!>Detection and imaging of intracellular miRNAs with DTNSs<!>In vivo cancer imaging and therapy with DTNSs<!>Conclusions<!>Ethical statement<!>Conflicts of interest

<p>Accurate identication of cancer at an early stage plays an important role in cancer diagnosis and treatment. MicroRNAs (miRNAs) are a kind of cancer biomarker; their aberrant expression levels are closely related to the initiation and progression of cancers, [1][2][3][4] and therefore, sensitive detection of tumor related miRNAs holds great promise for cancer diagnostics and prognostics. [5][6][7][8] Furthermore, simultaneous detection of multiple tumor related miRNAs can avoid false-positive signals and enhance the accuracy of cancer diagnosis. With the development of nanomaterials, a variety of nanoprobes have been reported for the detection of multiple miRNAs. [9][10][11][12] For example, the Tang group developed multicolor uorescent nanoprobes based on gold nanoparticles (GNPs) for evaluating cellular migration and invasion by simultaneously imaging miRNA-221, PTEN mRNA and MMP9 in living cells. 13 The Zhu group assembled multicomponent nucleic acid enzymes onto the surface of mesoporous silica-coated gold nanorods as multifunctional nanodevices for intracellular miRNA-21 and miRNA-145 in situ imaging. 14 However, the preparation processes of these nanoprobes are oen complicated and timeconsuming, and their stability, biocompatibility and cell permeation ability are also not satisfactory for in vivo diagnosis, which has driven researchers to nd other substitutes. DNA tetrahedron nanostructures have attracted enormous interest owing to their unique advantages, such as ease of self-assembly, excellent biocompatibility, high nuclease stability, remarkable transmembrane capability through a caveolin-dependent pathway and availability for multiple modications. [15][16][17][18] To signicantly improve the survival rate of cancer patients, besides accurate cancer identication, an efficient treatment strategy is another crucial step. Gene silencing as a kind of gene therapy has now been considered as one of the most promising options to overcome the limitations of traditional cancer therapy. [19][20][21] It can induce sequence-specic inhibition of oncogene expression or translation through the delivery of antagomirs to cancer cells, which makes it possess advantages of high specicity, improved safety, high efficacy and unrestricted choice of targets. 22,23 For example, leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a novel gastric cancer marker, and silencing its expression with antagomirs could efficiently inhibit cancer angiogenesis. 24 miR-10b was overexpressed in metastatic breast tumor patients, and silencing of miR-10b with antagomirs could signicantly decrease miR-10b levels and suppress breast cancer metastasis. 25 miRNA-21 as a key oncogenic miRNA was widely overexpressed in various tumors and participated in tumor occurrence and development. Inhibition of endogenous miRNA-21 with antagomirs could suppress cancer cell proliferation, migration and invasion, and tumor growth. 26,27 In biomedical science, developing nanomaterials that integrating both ultrasensitive diagnosis and highly efficient therapy functions remains attractive and challenging. 28,29 In this work, we reported the rst example of vertebral-shaped DNA tetrahedron nanostructures (DTNSs) for accurate cancer iden-tication and miRNA silencing induced therapy. Based on the uorescence "OFF" to "ON" mode, three intracellular miRNAs (miRNA-21, miRNA-122 and miRNA-194) were simultaneously monitored and imaged, which not only effectively distinguished tumor cells from normal cells, but also identied cancer cell subtypes, and thus the accuracy of cancer diagnosis was signicantly improved. In miRNA-21 overexpressed cancer cells, antagomir-21 (one recognition sequence) was detached from DTNSs to silence endogenous miRNA-21 inside cells, which would suppress cancer cell migration and invasion, and nally induce cancer cell apoptosis. The prepared DTNSs displayed improved resistance to enzymatic digestion and high cellular uptake efficiency, and exhibited accurate cancer identication and efficient cancer therapy ability.</p><!><p>The DTNSs were prepared with seven customized singlestranded nucleic acid chains (P1-P7) through a simple thermal annealing method (Fig. 1). Four chains (P1-P4), partially complementing each other, would spontaneously and respectively fold into triangles and then assemble into a rigid tetrahedron (named TDN). Three sequences linked with quenchers (BHQ1 for FAM, BHQ2 for TAMRA and Cy5) were distributed in the vertexes of the tetrahedron, respectively, and were complementary to the recognition sequence in P5-P7 chains. Fluorescein FAM labeled P5, TAMRA labeled P6 and Cy5 labeled P7 were introduced into the above tetrahedron based on the principles of Watson-Crick base pairing to form DTNSs. The formation of DTNSs was identied by agarose gel electrophoresis analysis (inset in Fig. 1). For lanes 1 and 2, only a single band was observed, indicating that uorescence or quenching group modied nucleic acid chains maintained good purity. With the step-by-step addition of chains from lane 3 to lane 8, there clearly appeared a gradual reduction of electrophoretic mobility, which could be ascribed to the increasing molecular mass and more complicated spatial construction of assemblies. The AFM image further veried the successful formation of the DTNSs, as shown in Fig. S1, † and the prepared DTNSs were vertebral-shaped nanoparticles with a diameter of $3 nm.</p><!><p>The uorophores in the vertexes of DTNSs were quenched by the adjacent quenchers, and the uorescence quenching capability depended on the ratio of TDN to nucleic acid chains (P5/P6/P7) during DTNSs preparation, which was determined by ((F control À F DTNSs )/F control ) Â 100%, where F DTNSs was the uorescence of DTNSs and F control was the uorescence of P5/ P6/P7 chains. The uorescence of FAM/TAMRA/Cy5 was gradually quenched by increasing the ratio of TDN to P5/P6/P7 and reached a constant value at ratios of 1.5, 1.25 and 1.25, respectively, and the quenching efficiency was up to 90% (Fig. S2 and Table S1 †). In the presence of target miRNAs (miRNA-21, miRNA-122 and miRNA-194), P5, P6 and P7 would release from DTNSs, and then hybridize with the targets to form much more stable double strands. In consequence, the corresponding uorescence signals (F target ) were recovered. The degree of uorescence recovery (F target /F DTNSs ) was gradually enhanced with the increased ratio of tetrahedron to P5/P6/P7 and reached a platform at ratios of 1.5, 1.5 and 1.5, respectively (Fig. S3 †). Combining the quenching and recovery efficiency, a ratio of 1.5 was selected for TDN to P5/P6/P7. Under the optimized conditions, the uorescence intensities of FAM, TAMRA, and Cy5 increased by about 6.7-fold, 5.4-fold and 11.0-fold in the presence of the three miRNA targets, respectively.</p><p>Ability of DTNSs to detect miRNAs in a homogeneous solution Fig. 2A-C show that the uorescence intensities of FAM, TAMRA and Cy5 all increased linearly with the concentration of miRNA targets from 0.15-37.5 nM. The detection limit was calculated to be 0.13 nM for miRNA-21, 0.64 nM for miRNA-122, and 0.68 nM for miRNA-194 (Fig. 2D-F), which were signicantly lower than the previous reports (Table S2 †). 15,[30][31][32][33] We next assessed the sequence specicity and the multiple detection ability for the three miRNA targets. DTNSs were transferred into 21 wells (3 Â 7) and different mixed solutions of various combinations of the three target miRNAs were respectively added. The uorescence emission spectra of FAM, TAMRA or Cy5 were obtained only in response to miRNA-21, miRNA-122 or miRNA-194 targets and had no notable cross-reactivity with each other (Fig. 2G). And when the three targets co-existed, the uorescence signals of FAM, TAMRA or Cy5 all continuously increased with the corresponding target addition (Fig. S4 †). In contrast, treatment of DTNSs with miRNAs with a scrambled sequence (shown in Table S3 †) showed no signicant changes in uorescence intensity (Fig. S5 †). The above results indicated that the prepared DTNSs allowed high-throughput monitoring of the three miRNA targets with high sensitivity, no notable crossreactivity and good specicity.</p><p>To verify the nuclease resistance of the DTNSs, 10% fetal calf serum (FBS, v/v) was used for the preparation of DTNSs solution to closely mimic physiological conditions. As shown in Fig. S6A-C, † no detectable uorescence changes were observed with time for DTNSs in both FBS and PBS. The uorescence recovery had no signicant difference aer the addition of target miRNAs for the two groups. Moreover, the bands of DTNSs treated with PBS or FBS solution for 1-4 h in agarose gel electrophoresis still maintained the same position, demonstrating that DTNSs were undecomposed when treated with FBS for 4 h (Fig. S4D †). All the above results conrmed that the DTNSs had good nuclease stability due to their tetrahedron-like spatial structure and were suitable for in vitro and in vivo studies.</p><p>Cytotoxicity was another vital factor for living cell studies. The cytotoxicity of DTNSs was tested by the MTT assay in normal cells (HEK293). Aer incubation with DTNSs at a high concentration of 100 nM for 48 h, the HEK293 cells still maintained more than 90% of the viability, revealing the low cytotoxicity of the DTNSs to normal cells (Fig. S7 †).</p><!><p>For this study, the cell permeability of DTNSs was investigated by using non-rigid DNA tetrahedron-based nanoprobes (n-DTNSs) and DNA line nanostructures (DLNSs) as controls. Aer 4 h incubation, the uorescence in HepG2 cells incubated with DTNSs was obviously higher than that of HepG2 cells incubated with n-DTNSs or DLNSs (Fig. S8 †), indicating that the rigid DTNSs undoubtedly improved self-delivery capability without the help of transfection agents owing to their rapid internalization through a caveolin-dependent pathway, 34,35 while n-DTNSs and DLNSs had poor permeability and low biostability.</p><p>The time-dependent response of DTNSs incubated with cells was then studied by monitoring the uorescence changes with different incubation periods. Huh7 cells with high expression levels of the three target miRNAs were chosen as the model. 36 The uorescence intensities of green, orange and red all increased before 2 h (Fig. S9 †), and then stayed at the same level for another 2 h due to the complete entry of DTNSs, and thus, 2 h was used for the incubation of cells and DTNSs in the following experiments.</p><p>We next investigated whether DTNSs could monitor miRNAs in living cells. Three different cell lines, Huh7, HepG2 and HEK293 (human normal cells) were chosen, miRNA-194 was used as a model target. Both confocal images (Fig. 3A) and ow cytometric analysis (Fig. 3B) showed that the uorescence intensities of Cy5 were obviously different in each cell line, indicating different miRNA-194 expression levels in the three cell lines. In agreement with previous reports, [36][37][38] Huh7 had a higher miRNA-194 expression level than HepG2, while HEK293 exhibited a quite low expression level, and quantitative reverse transcription PCR (qRT-PCR) was further used to conrm the relative miRNA-194 expression levels (Fig. S10 †). The expression levels of miRNAs in cancer cells indicated the stage of tumorigenesis, the effect of therapy and prognosis, and dynamic monitoring of miRNA expression levels inside cells was signicant. To this end, HepG2 cells were treated with a miRNA194 mimic and inhibitor, and non-treated HepG2 cells were used as a control. An miRNA-194 inhibitor with 2 0 -Omethyl modication was chosen to transfect HepG2 cells to downregulate the expression level of miRNA-194, 39 while the miRNA-194 mimic was selected to upregulate it. 40 As shown in Fig. 3C, compared with the control group, a higher Cy5 uorescence signal was observed in the mimic treated group while a lower one was displayed in the inhibitor treated group. Additionally, the ow cytometry assay further conrmed the results of confocal images (Fig. 3D). These results demonstrated that DTNSs had the capability of real-time monitoring miRNAs inside cells.</p><p>Detection of one kind of miRNA to identify cancers will produce high false positive signals, and thus simultaneous detection of multiple miRNAs inside cells would benet the diagnostic precision of related cancers. Here we simultaneously detected three miRNAs (miRNA-21, miRNA-122 and miRNA-194) in three cell lines (two cancer cells: Huh7 and HepG2, one normal cell line: HEK293). Confocal images (Fig. 4A) and corresponding grayscale values (Fig. 4B) exhibited three uorescent signals inside cells with different intensities and spatial distributions. For Huh7 cells, the three target miRNAs were all overexpressed; for HepG2 cells, miRNA-21 was high, and miRNA-122 and miRNA-194 were low; for HEK293, the three target miRNAs were quite low, and the expression levels were in agreement with literature reports. 36,38,[41][42][43] qRT-PCR was used to quantitatively measure relative miRNA expression levels in the three cell lines (Fig. 4C), and the results were consistent with confocal images and ow cytometry. The ndings suggested that DTNSs could be used not only to effectively distinguish cancer cells from normal cells, but also to identify cancer cell subtypes.</p><p>In vitro cancer therapy induced by miRNA-21 silencing with DTNSs</p><p>Previous reports have demonstrated that silencing intracellular miRNA-21 with antagomir-21 could efficiently suppress cancer cell migration and invasion, and nally induce cancer apoptosis. 26,44 In this system, DTNSs were used as a smart carrier to deliver antagomir-21 into cells through a caveolindependent pathway. Aer cell uptake, endogenous miRNA-21 inside cancer cells served as an initiator to cause the release of antagomir-21 from DTNSs, and then suppress cancer cell migration and invasion, and trigger cell apoptosis. The woundhealing assay was employed to assess the inhibition of cancer cell migration and invasion. As shown in Fig. S11, † aer Huh7 cells being treated with DTNSs (500 nM) for a period, the cell migration and invasion were obviously inhibited, while for HEK293 normal cells, the cell migration and invasion were almost unaffected, which was ascribed to the endogenous miRNA-21 silencing effect of DTNSs.</p><p>To investigate the therapeutic effect of DTNSs in vitro, cytotoxicity assessment was accomplished by the MTT assay (Fig. 5A and B); even at an antagomir-21 dose of 2 mM in DTNSs, the HEK293 cell viability still remained over 90%. However, the cell viability of Huh7 cells was down to 58% aer treatment with 2 mM DTNSs, which was obviously lower than that of the DTNS-C treated group ($90%). Here, DTNS-C was prepared by replacing antagomir-21 (P5) with normal strand P8, which didn't have a gene silencing function. Therefore, our prepared DTNSs could efficiently induce cancer cell apoptosis and had no side effects to normal cells. And the apoptosis was further conrmed by ow cytometry analysis with the dual uorescence of Annexin V-FITC/PI (Fig. 5C). To avoid the uorescence interference of FAM/Cy5 in DTNS-C or DTNSs, DTNS-C or DTNSs without uorophore modication was synthesized for ow cytometry analysis. The Huh7 and HEK293 cells were respectively treated with DTNS-C and DTNSs without uorophore modication (2 mM) for 48 h; the Huh7 cells underwent nearly 50% apoptosis aer treatment with DTNSs but no obvious apoptosis was observed in the DTNS-C group. In contrast, no prominent apoptosis was observed in both DTNS-C and DTNSs treated HEK293 cells, and the result was in accordance with the MTT assay.</p><!><p>Before the in vivo study of DTNSs for the liver cancer therapeutic effect, the clinical signicance of the selected miRNA-21, miRNA-122 and miRNA-194 biomarkers was rstly investigated in clinical patients (Fig. S12 †). It was shown that the expression of miRNA-21 in clinical patients' liver cancer tumors (T) was signicantly higher than that in non-tumor liver tissues (NT); miRNA-122 showed decreased expression in T compared with that in NT, but there was no distinct difference for the expression of miRNA-194-1/2 in T and NT, which indicated that miRNA-21 and miRNA-122 targets were valuable biomarkers for liver cancer diagnosis while miRNA-194 mainly played a role in the discrimination of hepatocellular carcinoma cell subtypes. The normalized relative expression of miRNA-21, miRNA-122 and miRNA-194 in Huh7 cells compared with that in different tissues of mice was also studied. the tumor region and size in mice were reected by a luminescence signal, which originated from the D-luciferin catalyzed by luciferase. 45,46 Aer two weeks, mice injected with DTNS-C showed strong FAM (Green) and TAMRA (Orange) signals in the liver tumor and liver tissues, suggesting the high expression of miRNA-21 and miRNA-122 in the liver tumor and liver tissues. However, a signicantly weaker FAM (Green) signal and obvious smaller tumor size were observed in the DTNSs injected group. From the result of quantitative analysis of bioluminescence signals, we could see that the tumor growth rate of the DTNSs treatment group was obviously lower than that of the DTNS-C group (Fig. 6B). In addition, the qRT-PCR analysis result of mice liver cancer tumor models also showed that only miRNA-21 was down-regulated in the DTNSs treated group (Fig. 6C), demonstrating that antagomir-21 loaded in DTNSs silenced the expression of miRNA-21 in liver tumors and suppressed the tumor growth. All the above results indicated that the prepared DTNSs could serve as a diagnostic probe and anticancer drug to treat cancers.</p><!><p>In summary, we have successfully developed a smart nanosystem, DNA tetrahedron nanostructures (DTNSs), which can realize simultaneous monitoring of three intracellular miRNAs accompanied by efficient cancer therapy for the rst time. DTNSs exhibited high biostability and cellular uptake efficiency owing to their unique tetrahedron-like spatial structure. Based on the uorescence "OFF" to "ON" mode, three intracellular miRNA (miRNA-21, miRNA-122 and miRNA-194) expression levels could be quantitatively detected with high sensitivity, specicity and no notable cross-reactivity, and their dynamic changes could be real-time monitored. This system not only effectively distinguished tumor cells from normal cells, but also identied cancer cell subtypes, which avoided false-positive signals and signicantly enhanced the accuracy of cancer diagnosis. Moreover, DTNSs could also act as a kind of cancer drug to treat cancer efficiently through endogenous miRNA-21 silencing, which was demonstrated by experiments in vitro and in vivo. Therefore, we anticipate that our strategy will have potential application in clinical diagnosis and treatment of cancer in future.</p><!><p>All procedures involving animals were conducted in accordance with the guidelines for Care and Use of Laboratory Animals of Nanjing Medical University and experiments were approved by the Animal Ethics Committee of Nanjing Medical University.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

TMEDA in Iron-Catalyzed Hydromagnesiation: Formation of Iron(II)-Alkyl Species for Controlled Reduction to Alkene-Stabilized Iron(0)

N,N,N\xe2\x80\x99,N\xe2\x80\x99-Tetramethylethylenediamine (TMEDA) has been one of the most prevalent and successful additives used in iron catalysis, finding application in reactions as diverse as cross-coupling, C\xe2\x80\x94H activation, and borylation. However, the role that TMEDA plays in these reactions remains largely undefined. Herein, studying the iron-catalyzed hydromagnesiation of styrene derivatives using TMEDA has provided molecular-level insight into the role of TMEDA in achieving effective catalysis. The key is the initial formation of TMEDA\xe2\x80\x93iron(II)\xe2\x80\x93alkyl species which undergo a controlled reduction to selectively form catalytically active styrene-stabilized iron(0)-alkyl complexes. While TMEDA is not bound to the catalytically active species, these active iron(0) complexes cannot be accessed in the absence of TMEDA. This mode of action, allowing for controlled reduction and access to iron(0) species, represents a new paradigm for the role of this important reaction additive in iron catalysis.

tmeda_in_iron-catalyzed_hydromagnesiation:_formation_of_iron(ii)-alkyl_species_for_controlled_reduct

Introduction<!>Effect of TMEDA Loading on Catalytic Performance<!>In Situ Iron Speciation on Reaction with Grignard Reagent<!>In Situ Evolution of Iron Speciation in the Presence of Styrene<!>Iron Speciation During Catalysis<!>Conclusion

<p>Iron catalysis has seen significant development due to the increasing need for sustainable chemical synthesis.[1–12] As one of the most prevalent additives used in iron catalysis, N,N,N',N'-tetramethylethylenediamine (TMEDA) has enabled a wide variety of reactions to be achieved with high conversion and selectivity. Iron-catalyzed cross-coupling has been an area where TMEDA has been applied with particular success. Cross-coupling of a range of organometallic reagents with halides and pseudo-halides has been applied to achieve C(sp2)—C(sp3) and even C(sp3)—C(sp3) bond formation (Scheme 1 A).[13–22] Notable examples include the reductive coupling of aryl bromides with alkyl or alkenyl halides;[17,18] difluoroalkylation;[22] and release-capture ethylene coupling.[21] Additionally, the use of TMEDA as an additive extends well beyond cross-coupling, having proved essential for reactions including Miyaura-type borylation of alkyl electrophiles, C—H alkylation and the hydromagnesiation of styrene derivatives (Scheme 1 B–D).[23–27]</p><p>Despite being a crucial component of many reactions, the role that TMEDA plays in achieving effective catalysis within such reactions is largely undefined. Attempts to ascertain its function within iron-catalyzed cross-coupling reactions have been a particular focus, with Bedford and co-workers having carried out perhaps the most extensive investigations.[28–31] However, a definitive role has yet to be established. Early work by Nagashima and co-workers suggested that TMEDA coordinates the iron center, with productive reactivity taking place from the bis-transmetallated complex.[28] Bedford and co-workers later showed that this TMEDA-bound species did not react at a rate consistent with the catalytic reaction. They proposed that the tris-mesityl iron(II) ate complex was responsible for catalysis, with TMEDA acting as a "chaperone" to stabilize off-cycle species prior to their re-entry into the primary catalytic cycle.[29,31]</p><p>Despite the widespread use of TMEDA outside the realm of cross-coupling, its effects have yet to be investigated in other reactions, such as the iron-catalyzed hydromagnesiation of styrene derivatives. As a modular platform for generating molecular complexity, iron-catalyzed hydromagnesiation can generate a wealth of formal hydrofunctionalization products in high yield and with control of regio- and stereoselectivity.[26,27,32] In this reaction, TMEDA can be used in loadings as low as 1 equivalent with respect to iron, contrary to several other reactions where TMEDA is used as an additive. However, its presence is still imperative for reactivity, with trace product formation in its absence with iron salts alone. Additionally, the iron-catalyzed hydromagnesiation of styrene derivatives was initially reported using the electronically non-innocent bis(imino)pyridine (BIP) ligand (Scheme 1D).[32] Our subsequent mechanistic study of this catalytic system revealed a formal iron(0) complex, [BIPFe-(Et)(CH2=CH2)]_, as the catalyst resting state.[33] This raises the question of how TMEDA allows access to the same reactivity as an electronically non-innocent ligand. Altogether, this made the iron-catalyzed hydromagnesiation of styrene derivatives, with TMEDA, an ideal platform to examine how this versatile additive exerts its vital influence. Through the use of time-resolved, freeze-trapped 57Fe Mossbauer spectroscopy and independent synthesis and characterization of key intermediates, molecular-level definition of the role of TMEDA in achieving effective catalysis is reported herein.</p><!><p>As the loading of TMEDA used in reactions varies significantly, from catalytic to stoichiometric quantities, our initial studies examined the effect of changing the loading of TMEDA on the iron-catalyzed hydromagnesiation of 2-methoxystyrene (Figure 1).</p><p>In the absence of TMEDA, using FeCl2 alone (1 mol%), negligible reactivity was observed (< 5% yield after 1 hour). Addition of TMEDA enhanced reactivity, with increases in the loading from 0.5–2 mol% proving beneficial to both the rate and sustained catalytic activity. However, a large excess of TMEDA (10 mol%) resulted in a significantly longer induction period alongside the sustained catalytic performance. The absence of a clear stoichiometric relationship between FeCl2 and TMEDA suggests that it may not be bound to the iron center within the principal catalytic cycle. However, paramount to understanding the origin of these effects was determining the in situ iron speciation by direct spectroscopic means.</p><!><p>In order to build up a picture of iron species accessible in this reaction, it was first important to assess stoichiometric reactions with EtMgBr to identify what species may initially be formed. These reactions were carried out at reduced temperatures to stabilize any species accessed. The speciation resulting from reaction of 57FeCl2 with EtMgBr, in the presence of TMEDA (2 equiv) was examined by freeze-quenching samples in liquid nitrogen for Mössbauer spectroscopy Reaction with 1 equivalent of EtMgBr at −25 °C for 1 or 5 minutes resulted in a major species 1, constituting 89–93% of iron in solution, with Mössbauer parameters δ = 0.68 mms−1 and ∆EQ = 1.89 mms−1 (Figure 2 A, blue). The secondary species 2, accounting for the remaining iron in solution, has Mössbauer parameters δ = 0.49 mms−1 and ∆EQ = 0.74 mms−1 (Figure 2A, red). Carrying out the analogous reaction with 2 equivalents of EtMgBr resulted in species 2 being the only iron species observed (Figure 2B).</p><p>While the Mössbauer parameters and stoichiometric relationship suggested that 1 and 2 are the result of sequential transmetallation of the iron(II)-center, crystallization attempts were undertaken to confirm their identity. The relevant quantity of EtMgBr was added to a solution of FeCl2 and 2 equivalents of TMEDA at −25°C, followed by immediate cooling to −74°C. Addition of hexane and leaving to stand at −80°C for five days resulted in the formation of extremely air-, moisture-, and temperature-sensitive colorless crystals suitable for single-crystal X-ray diffraction. These were identified as the mono- and bis-ethyl TMEDA-iron(II) complexes 1 and 2, respectively. Both display distorted tetrahedral geometries with N—Fe—N angles of 82.24(12)° and 80.43(8)°, for 1 and 2 respectively (Figure 2A and B, respectively). The iron-carbon bond lengths are 2.046(4) Å for 1 and 2.0821(15) Å for 2; and the iron-nitrogen bond lengths 2.195(3) Å and 2.185(3) for complex 1, and 2.2437-(15) Å for complex 2. Additionally, 1 displays a halogen disorder with 70% bromide and 30% chloride occupancies. 57Fe Mössbauer spectroscopy of crystalline material confirmed these to be the species generated in situ.</p><p>Addition of excess EtMgBr, including during catalytic reactions, did not result in the immediate displacement of TMEDA, with bis-ethyl complex 2 still observed as the major species (see below). This contrasts observations with mesitlymagnesium bromide by Bedford and co-workers, where addition of three or more equivalents of Grignard reagent resulted in displacement of TMEDA, even when present in excess.[29] As the alkyl moiety is expected to be a stronger σ-donor, it is likely that this difference in observed reactivity is dictated by steric factors.</p><p>Alkyl–iron(II) complexes bearing β-hydrogens such as these are extremely rare, owing to their instability and propensity to undergo β-hydride elimination. To circumvent this, alkyl-iron(II) complexes have historically been prepared with these positions blocked. The most relevant example of this is the analogous high spin (S = 2) TMEDA-iron(II) bisneopentyl complex, reported by Wolczanski and co-workers.[34] An alternative strategy has been to use strongly-stabilizing ligands which result in complexes resistant to β-hydride elimination.[35–41] Chelation of the alkyl ligand has also been demonstrated to make complexes resistant to β-hydride elimination. This was observed for N-heterocyclic carbene iron(II) complexes bearing (1,3-dioxan-2-yl)ethyl groups, with chelation through the carbon and one oxygen of the acetal group.[42] Other than complexes 1 and 2, there are only two other examples of β-hydrogen-containing iron(II)–alkyl species without such stabilizing effects that have been isolated and characterized. These are a bisphosphine-iron(II) cyclohexyl complex reported by Fürstner and co-workers, with the bis-cyclohexyl analogue not isolated owing to its instability to further reaction,[35] and the partially reduced cluster [Fe8Et12][MgX(THF)5]2 reported by our group.[43] The scarcity of such β-hydrogen-containing alkyl-iron(II) complexes likely stems from their being short-lived intermediates preceding reduction to low oxidation-state iron species.[33,35,44,45] These include the formation of the formal iron(0) complex, [BIPFe(Et)(CH2=CH2)]−, as well as bisphosphine iron(0) ethene complexes, all of which result from reaction of the relevant ligand- supported iron(II) complex with EtMgBr.[33,35,46]</p><!><p>In order to assess the potential further reaction of these alkyl-iron(II) complexes, and speciation under catalytically relevant conditions, freeze-trapped 57Fe Mössbauer spectroscopy was carried out on similar reactions in the presence of styrene. A moderate excess of both EtMgBr and styrene (5 equiv), as well as the temperature of −10°C, were chosen in order to inhibit catalytic turnover and allow evaluation of the evolution of speciation. Five minutes after adding EtMgBr, TMEDA–iron(II) bis-alkyl complex 2 represented the major iron species in solution (Figure 3A, red), together with two minor components 3 and 4 (Figure 3, grey and purple, respectively). With Mössbauer parameters δ = 0.19 mms−1 and ΔEQ = 1.53 mms−1, 3 initially represents 5% of iron in solution. The remaining 2% of iron in solution is 4, with Mössbauer parameters δ = 0.36 mms−1 and ΔEQ = 1.35 mms−1. After 10 minutes only minor evolution in the speciation had taken place (see Supporting Information). The concentration of 3 remained constant, while 4 increased to 7% of iron in solution and 2 decreased correspondingly. However, after 30 and 60 minutes the speciation evolved more significantly. Although 2 remained the major species, it continued to decrease in concentration, eventually representing 40% of the iron in solution (Figure 3B & C, red). Whereas 3 is no longer observed, the concentration of 4 increased, representing 23% of iron in solution after 30 minutes and 33% after 60 minutes (Figure 3B & C, purple). After 30 minutes, a new species 5 was observed, with Mössbauer parameters δ = 0.44 mms−1 and ΔEQ = 0.88 mms−1. Initially representing 16% of iron in solution, 5 increased further to 27% after 60 minutes (Figure 3 B & C, orange). The initial presence and subsequent disappearance of 3 suggests that it reacts to form species 4 and/or 5. This is consistent with subsequent experiments during catalysis, which indicate that 3 is an intermediate on the reduction pathway to the catalytically active species (see below).</p><p>Crystallization attempts were carried out adapting the above-described reaction. A solution of FeCl2, 2 equivalents of TMEDA, and 5 equivalents of styrene was cooled to −25°C before addition of 5 equivalents of EtMgBr. The solution was then cooled to −74°C after which hexane was added, and the reaction left to stand overnight at −80°C. This resulted in the formation of dark orange plates suitable for single-crystal X-ray diffraction[50]. These highly air-, moisture-, and temperature-sensitive crystals were identified as the tris-styrene-stabilized iron(0)-ethyl complex [Fe(η2-styrene)3-(Et)][MgX(THF)5] (Figure 4). 57Fe Mössbauer spectroscopic parameters of isolated crystalline material were identical to the in situ formed 4, confirming its identity. Reduction to form this styrene-stabilized iron(0)-ethyl complex 4, in the presence of TMEDA, is in contrast to previous observations with bis-phosphine ligands by Furstner and co-workers.[35] They observed that reaction of bis(diisopropylphosphino)-propane-iron(II) dichloride with EtMgBr, in the presence of excess ethene, resulted in the iron(0) bis-ethene complex with the bis-phosphine ligand still bound. This may suggest that bis-phosphine ligands have a greater binding affinity to iron than TMEDA and are better able to stabilize the iron(0) center, avoiding dissociation in favor of styrene coordination. However, a higher binding affinity of styrene compared with ethene, due to greater π-backbonding, as well as increased sterics cannot be excluded as having influence on the species formed.[47]</p><p>Having confirmed the identity of 4, we next sought to identify the related species 5. The reactivity studies above showed that 5 was present only once 4 had increased to significant concentrations. Along with their seemingly analogous rate of formation thereafter, this suggested that 5 was somehow derived from 4. The Mössbauer parameters of 5 also correspond precisely to those of the analogous tris-styrene ligated α-aryl- iron(0) complex [Fe(η2-styrene)3(K2-CH-(CH3)Ph)][MgX(THF)5].[33] Further confirming this assignment was its crystallization from the reaction of Ph-(CH2)2MgCl with FeCl2 in the presence of 2 equivalents of TMEDA and 5 equivalents of styrene at −25°C (Scheme 2). 57Fe Mössbauer and 1H-NMR spectroscopies of the dark red-black plates were also identical to those previously obtained. While the bond metrics are consistent with 4, one notable difference is the longer iron-carbon bond of 2.150(4) Å, compared to 2.069(7) Å for 4.</p><p>The previous identification of complex 5 was as a minor component in the bis(imino)pyridine iron-catalyzed hydromagnesiation reaction, resulting from displacement of the tridentate ligand.[33] While able to be isolated, 5 had to be prepared from the starting bis(imino)pyridine iron(II) dichloride complex as starting from FeCl2 resulted in a complex mixture by 57Fe Mössbauer spectroscopy. This demonstrates the significance of TMEDA in allowing controlled reduction to the styrene-stabilized iron(0) complexes through initial formation of a TMEDA-iron(II) bis-alkyl species such as 2. Carrying out the analogous experiment to monitor the reduction of 2 in the presence of 5 equivalents of TMEDA, compared with 2 equivalents used previously, resulted in only TMEDA-iron(II) bis-alkyl complex 2 being observed even after 60 minutes (see Supporting Information). This observation, that excess TMEDA inhibits the subsequent reduction of 2 to form 4 and 5, suggests that this process proceeds by dissociation of TMEDA.</p><p>With the identities of both 4 and 5 established, a potential pathway for their associated formation becomes apparent. Net hydride transfer from the ethyl ligand to a bound styrene in complex 4, followed by exchange of the resulting ethene for another equivalent of styrene would give 5. As 5 effectively has an equivalent of product bound, turnover could be envisioned to take place by exchange with an equivalent of EtMgBr. This would release the product of hydromagnesiation, the α-aryl Grignard reagent, and regenerate complex 4. Overall, this now provides a more complete picture of how these iron(0) species are catalytically competent for the hydromagnesiation of styrene derivatives. Owing to 5 previously being only a minor component, and having to be synthesized from the bis(imino)pyridine iron(II) complex, little had been established other than its effectiveness as a pre-catalyst.[33]</p><!><p>Despite the competence of 5 as a pre-catalyst, as well as a reasonable reaction pathway involving 4 and 5 being evident, it was essential to establish whether these species are relevant and present in the catalytic reaction. The speciation during catalysis was assessed by freeze-trapped 57Fe Mössbauer spectroscopy, carried out at various time-points during the hydromagnesiation of styrene using 3 mol% 57FeCl2. In the presence of 1 equivalent of TMEDA, with respect to iron, 1 minute after adding EtMgBr (1.5 equiv) catalytic turnover has already begun, with 12% product detected. At this time 77% of the iron in solution has already been reduced to the styrene-stabilized iron(0) complexes 4 and 5 (25 % and 52%, respectively, see Supporting Information). Only a small portion (5%) of TMEDA-iron(II) bisethyl complex 2 was present, with the remaining iron consisting of the previously observed and transient species 3 (δ = 0.19 mms−1 and ∆EQ = 1.53 mms−1). Further into the reaction (5 minutes, 33% yield) 2 and 3 are no longer observed, while the combined concentrations of 4 and 5 have increased, with iron(0)-ethyl complex 4 now the major species at 64%. These results demonstrate that complexes 4 and 5 are indeed the catalytically active and major species present during catalytic hydromagnesiation using TMEDA (Table 1). Furthermore, no significant EPR-active species were present in situ during catalysis (see Supporting Information).</p><p>In order to assess whether the observed stabilization of TMEDA-iron(II) bis-alkyl complex 2 in the presence of excess TMEDA would manifest under catalytic conditions, analogous freeze-trapped 57Fe Mössbauer spectroscopy was carried out on the hydromagnesiation of styrene, this time with 5 equivalents of TMEDA with respect to iron. One minute after adding EtMgBr (1.5 equiv), starkly different iron speciation is observed compared with the reaction using 1 equivalent of TMEDA. TMEDA-iron(II) bis-ethyl complex 2 was now observed to be the major iron species present in solution, at 88% (Figure 5A, red). The remaining 12% of iron in solution is once again the transient species 3 (Figure 5 A, grey). Significantly, at this stage of the reaction no detectable product was observed. This indicates, perhaps unsurprisingly, that neither 2 or 3 are catalytically active and that the styrene-stabilized iron(0) complexes 4 and 5 are the catalytically relevant species. Further corroborating this is that after 5 minutes, catalytic turnover is occurring (21% yield) with 2 and 3 no longer present, while 4 and 5 together represent the majority of iron in solution at 86% (Figure 5 B, purple and orange, respectively). The remaining 14% of iron in solution is a previously unobserved species 6, with Mössbauer parameters δ = 0.32 mms−1 and ∆EQ = 1.87 mms−1 (Figure 5B, green). As 6 was observed during catalytic turnover, the byproduct of which is ethene, it is possible that 6 is a related iron(0) complex differing by substitution of one or more equivalents of styrene for ethene. This is consistent with reports of the reaction protocol on a large scale requiring sparging with nitrogen, indicating that reversible binding of ethene takes place.[27]</p><p>This, once again, demonstrates the degree to which excess TMEDA stabilizes 2. The consequence of this stabilization is a significantly slower reduction of 2 to catalytically active 4 and 5, as was also observed in the stoichiometric reactions. Slowing the rate of formation of the catalytically active species would in turn account for the significantly longer induction period observed when using larger excesses of TMEDA. This is further highlighted when compared to the catalytic reaction with 2 equivalents of TMEDA. In this case, after 1 minute, 4 and 5 are already the major iron species observed and catalysis occurring (see above). The more sustained catalytic activity also observed suggests TMEDA prevents catalyst deactivation by aggregation of the iron(0) species, which is consistent with the beneficial effects of TMEDA despite not being bound to the catalytically active species, 4 and 5. Further supporting this is that iron nanoparticles, demonstrated to be active for hydrogenation, proved ineffective for the reaction.[48,49]</p><p>The transient species 3, also observed during the induction period with excess TMEDA, displayed similar Mössbauer parameters (δ = 0.19 mms−1 and ∆EQ = 1.53 mms−1) to a previously identified iron(II) tri-ethyl ferrate complex (δ = 0.19 mms−1 and ∆EQ = 1.43 mms−1).[43] As the tri-ethyl ferrate species was demonstrated to have a distinctive near-infrared magnetic circular dichroism (NIR MCD) spectrum, this was used to probe whether this was the identity of 3. Carrying out the analogous catalytic reaction in 1:1.5 THF/2-MeTHF, for glassing purposes, MCD samples were frozen in liquid nitrogen after 1 minute. It should be noted that 57Fe Mössbauer spectroscopy showed that the iron speciation was not affected by this solvent mixture. The 5 K NIR MCD spectrum showed two intense transitions at circa 5940 cm−1 and circa 6900 cm−1, corresponding to TMEDA–iron(II) bisethyl complex 2 (see Supporting Information). Transitions corresponding to the tri-ethyl ferrate complex (ca. 9200 cm−1 and ca. 10200 cm−1) were not observed, although an additional transition was observed at ca. 12300 cm−1. While this rules out the presence of the tri-ethyl ferrate complex, a similar spectral feature resulting from decomposition was observed as a minor component.[43] This potentially arises from a similar reduction process through β-hydride elimination, forming a reduced species related to the transient intermediate 3. Consistent with these species being related is the fact that the stoichiometric reactivity indicates that reduction of 2 is dissociative with respect to TMEDA, after which a species similar to the tri-ethyl ferrate could form. The resulting species 3, in the case of hydromagnesiation reaction conditions, subsequently reacts further in the presence of styrene to form the styrene-stabilized iron(0) complexes observed. However, due to it being observed in only relatively small amounts along with its transient nature, the unambiguous assignment of 3 remains elusive.</p><!><p>The use of 57Fe Mössbauer spectroscopy to monitor in situ iron speciation and guide the isolation of key reactive intermediates in the iron-catalyzed hydromagnesiation of styrene derivatives with TMEDA. This study also revealed the multifaceted nature of the influence TMEDA exerts on both speciation and reactivity. Initial coordination of TMEDA to the iron center results in formation of iron(II) mono- and bis-ethyl complexes. Formation of the TMEDA–iron(II) bis-alkyl complex is key, as it undergoes selective reduction to form the catalytically active styrene-stabilized iron(0) complexes. Despite not being coordinated to the catalytically active species, TMEDA is essential to its selective formation. Additionally, the quantity of TMEDA present influences the rate of this reduction process. Beyond this, TMEDA also aids in preventing catalyst deactivation, which likely occurs through aggregation of the active iron(0) species. This manifests in more sustained catalytic performance.</p><p>The role of TMEDA in allowing controlled reduction represents a new paradigm in iron catalysis for this long-established and versatile additive, as well as demonstrating multiple ways in which it can exert beneficial effects even in a single catalytic reaction. As TMEDA is a crucial additive in various iron-catalyzed reactions, the implications of this study extend beyond hydromagnesiation. For example, despite not being on-cycle active species for the hydromagnesiation reaction, the TMEDA-iron(II) alkyl complexes represent potential intermediates in the cross-coupling of alkyl Grignard reagents with aryl chlorides and will be the subject of future work.[20]</p>

PubMed Author Manuscript

Bis-Aryl Urea Derivatives as Potent and Selective LIM Kinase (Limk) Inhibitors

The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity, and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM), excellent selectivity against ROCK and JNK kinases (> 400-fold), potent inhibition of cofilin phosphorylation in A7r5,PC-3, and CEM-SS T cells (IC50 < 1 \xce\xbcM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 \xce\xbcM inhibited only Limk1 and STK16 with \xe2\x89\xa5 80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors.

bis-aryl_urea_derivatives_as_potent_and_selective_lim_kinase_(limk)_inhibitors

Introduction<!>Chemistry<!>Results and discussion<!>Conclusion<!>Experimental Section<!>General synthetic procedures<!>3-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)ureido)-N-isopropylbenzamide (3)<!>3-(3-(4-(1H-pyrazol-4-yl)phenyl)ureido)-N-isopropylbenzamide (7a)<!>N-Isopropyl-3-(3-(4-(pyridin-4-yl)phenyl)ureido)benzamide (7b)<!>3-(3-(4-(2-Aminopyrimidin-4-yl)phenyl)ureido)-N-isopropylbenzamide (7c)<!>N-Isopropyl-4-[3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-phenyl]-ureido]-benzamide (7d)<!>4-[3-[4-(7-Ethyl-8-oxo-8,9-dihydro-7H-purin-6-yl)-phenyl]-ureido]-N-isopropyl-benzamide (7e)<!>N-Isopropyl-4-[3-[4-(9H-purin-6-yl)-phenyl]-ureido]-benzamide (7f)<!>N-Isopropyl-3-(3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)ureido)benzamide (7g)<!>N-Isopropyl-3-(3-(4-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)ureido)benzamide (7h)<!>3-(3-(4-(5,6-Dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)ureido)-N-isopropylbenzamide (7i)<!>3-(3-(2-(2-(Dimethylamino)ethoxy)-4-(5-methyl-7H-pyrrolo[2,3-d ]pyrimidin-4-yl)phenyl)ureido)-N-isopropylbenzamide (7l)<!>N-Isopropyl-3-(3-(4-(5-methyl-7H-pyrrolo[2,3-d ]pyrimidin-4-yl)-2-(trifluoromethyl)phenyl)ureido)-benzamide (7j)<!>3-(3-(2-Fluoro-4-(5-methyl-7H-pyrrolo[2,3-d ]pyrimidin-4-yl)phenyl)ureido)-N-isopropylbenzamide (7k)<!>1-(4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-3-phenylurea (10a)<!>1-(3-Fluorophenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (10b)<!>1-(2-Methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (10c)<!>1-(3-Methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (10d)<!>1-(4-Methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (10e)<!>1-[4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-phenyl]-3-thiazol-2-yl-urea (10f)<!>1-[4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-phenyl]-3-pyridin-2-yl-urea (10g)<!>3-(4-Methoxyphenyl)-1-methyl-1-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (14a)<!>3-(4-Methoxyphenyl)-1-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-(2-(pyrrolidin-1-yl)ethyl)urea (14b)<!>3-(4-Methoxyphenyl)-1-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-(2-(piperidin-1-yl)ethyl)urea (14c)<!>3-(4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-phenyl-1-(2-(pyrrolidin-1-yl)ethyl)urea (18a)<!>1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-phenylurea (18b)<!>1-(2-Fluorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18c)<!>1-(3-Fluorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18d)<!>1-(4-Fluorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18e)<!>1-(2-Chlorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18f)<!>1-(3-Chlorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18g)<!>1-(4-Chlorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18h)<!>1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-o-tolylurea (18i)<!>1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-m-tolylurea (18j)<!>1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-p-tolylurea (18k)<!>1-(2-Hydroxyethyl)-1-(2-methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18l)<!>1-(2-Hydroxyethyl)-1-(3-methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18m)<!>1-(2-hydroxyethyl)-1-(4-methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18n)<!>1-(2-Aminoethyl)-1-(4-methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18o)<!>1-(2-Aminoethyl)-1-(4-chlorophenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18p)<!>1-(2-(Dimethylamino)ethyl)-1-(4-methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18q)<!>1-(4-Chlorophenyl)-1-(2-(dimethylamino)ethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18r)<!>1-(4-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2-fluorophenyl)-1-(2-hydroxyethyl)urea (18s)<!>1-(2-Amino-ethyl)-1-(4-chloro-phenyl)-3-[4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2-fluorophenyl]-urea (18t)<!>1-(4-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2-fluorophenyl)-1-(2-(dimethylamino)ethyl)urea (18w)<!>1-(4-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2-fluorophenyl)-1-(2-methoxyethyl)urea (18x)<!>Docking of Limk inhibitors into a crystal structure of Limk1<!>Limk1 biochemical assays and kinase profiling<!>In-Cell Western assay in A7r5 cells<!>Cofilin phosphorylation cell assay in PC-3 cell lines<!>Cofilin phosphorylation cell assay in CEM-SS T cell lines<!>In vitro invasion assay in PC-3 cells<!>In vitro migration assay in PC-3 cells<!>In vitro and in vivo DMPK assays

<p>LIM-kinase (Limk) is a serine-threonine protein kinase. Two isoforms were identified as LIM kinase 1 (Limk1) and LIM kinase 2 (Limk2).1–4 Limk1 and Limk2 are highly homologous and share 50% overall identity. Both isoforms consist of two amino-terminal LIM domains, adjacent PDZ and proline/serine-rich regions, followed by a carboxyl-terminal protein kinase domain.5 Limk1 was found to be expressed widely in embryonic and adult tissues, with notably high expression in the brain, kidney, lung, stomach and testis.6 Limk2 was found to be expressed in almost all embryonic and adult tissues examined with the exceptions of glial cell, the testis, and kidney glomeruli.7 Upon activation by upstream signals, Limk phosphorylates its substrate cofilin at the Ser-3 residue, thereby inactivating it, and leading to dynamic regulation of actin cytoskeleton.8–12 Accumulated evidences suggest that Limk activity is associated with a variety of diseases including Williams Syndrome,13 Alzheimer Disease (AD),14, 15 psoriatic epidermal lesions,16 primary pulmonary hypertension (PPH),17, 18 intracranial aneurysms (IA),19 ocular hypertension/glaucoma,20 HIV and other viral infections,21–24 and cancers and cancer cell migration/invasion.25–31</p><p>Recent molecular biology studies reported that Limk1 was over-expressed in cancerous prostate cells and tissues,26 reduced expression of Limk1 retarded PC3ASL cells' proliferation by arresting cells at G2/M phase,26 altered expression of Limk1 changed cell morphology and organization of actin cytoskeleton in PC3 cells,26 increased expression of Limk1 was associated with accumulation of chromosomal abnormalities and development of cell cycle defects in cells that naturally express lower concentrations of Limk1,27 reduced expression of Limk1 abolished the invasive behavior of prostate cancer cells,27 and expression of Limk1 is higher in prostate tumors with higher Gleason Scores and incidence of metastasis.27 All these observations suggest the possibility of up-regulated Limk1 as a cellular oncogene, and inhibition of Limk1 activity in cancerous prostate cells and tissues could lead to reduction of phosphorylated cofilin and decrease of the cells' motility and thus the invasiveness of tumor cells and their evolution to metastasis. Therefore, small molecule inhibitors of Limk1 could be potential therapeutic agents for prostate cancers. Recent studies also suggest that use of Limk inhibitors may provide a novel way to target the invasive machinery in GBM (glioblastoma multiforme).32–34</p><p>HIV-1 binding and entry into host cells are strongly impaired by the inhibition of actin polymerization.24, 35 Wu et al. demonstrated that HIV-mediated Limk activation is through gp120-triggered transient activation of the Rac-PAK-Limk pathway, and that knockdown of Limk through siRNA decreased filamentous actin, increased CXCR4 trafficking, and diminished viral DNA synthesis.23 Wen et al. showed that LIM kinases modulate retrovirus particle release and cell-cell transmission events.24 This research suggest that HIV hijacks Limk to control the cortical actin dynamics for the onset of viral infection of CD4 T cells. Therefore, Limk inhibitors are supposed to have high potentials as therapeutics in anti-HIV infection applications.23</p><p>To the best of our knowledge, few small molecule Limk inhibitors have been reported in the literature.28 Bristol-Myers Squibb pharmaceuticals (BMS) disclosed potent Limk1 inhibitors based on an aminothiazole scaffold.36, 37 Tel-Aviv University recently published an oxazole based Limk1/2 inhibitor (T56-Limki) from computer-aided drug design, which was found to be effective against cancer metastasis for treatment of neurofibromatosis.34 A group of scientists from Australia reported 4-aminobenzothieno[3,2-d] pyrimidine based Limk1 inhibitors from high-through-put screen (HTS) showing activity in the micromolar range.38, 39 Recently, a Japanese group also reported a Limk inhibitor (Damnacanthal or Dam, natural product based) from HTS campaigns, and this compound (Dam) has a Limk1 inhibition IC50 of ~ 800 nM.31 Lexicon pharmaceuticals revealed a class of Limk inhibitors based on a piperidine urea or guanidine scaffold for the treatment of ocular hypertension and associated glaucoma.20 More recently, the same group of Lexicon scientists reported a novel class of Type-III binding Limk2 inhibitors that are based on a sulfonamide scaffold.40</p><p>Our group reported a novel pyrazole-phenyl urea scaffold 1 (Figure 1) as potent and selective Rho kinase (ROCK) inhibitors and their significant intraocular pressure (IOP) lowing effects on rat eyes.41, 42 Compound 1 had low Limk inhibition in counter-screen studies (IC50 > 10 μM). However, SAR investigation revealed that replacement of the hinge-binding moiety pyrazole in 1 with a 4-yl-pyrrolopyrimidine (compound 2) significantly decreased its ROCK-II affinity (ROCK-II IC50 = 188 nM of 2 vs. 2 nM of 1). On the other hand, compound 2 gained a modest Limk1 inhibition (Limk1 IC50 = 642 nM vs. > 10 μM for 1), revealing an interesting hinge-binder dependent kinase selectivity profile for this phenyl urea based scaffold. Further modification of compound 2 on its urea terminal side led to compound 3 (Figure 1) which had an even weaker ROCK-II affinity (IC50 = 1365 nM) but improved Limk1 biochemical potency (IC50 = 201 nM). Interestingly, the 4-yl-pyrrolopyrimidine moiety in 2 and 3 is also present in Lexicon's piperidine urea/guanidine based Limk inhibitors, and is believed to be involved in hinge-binding interactions.20</p><p>Encouraged by the selectivity bias of compound 3 against Limk1 and ROCK-II, we carried out further optimization for this bis-aryl urea scaffold (starting from 3), in the hope to discover highly potent, selective, and proprietary Limk inhibitors for various applications. Herein, we report the synthesis and structure-activity relationship (SAR) studies for this series of bis-aryl urea based Limk inhibitors.</p><!><p>Inhibitors 3 and 7 were accessed through a short route as shown in Scheme 1. Coupling 3-aminobenzoic acid with propan-2-amine gave carbonyl amide 4 in the presence of HATU as coupling reagent and DIEA as base. Mixing intermediate 4 with 1-bromo-4-isocyanatobenzene derivatives in dichloromethane (DCM) produced bromides 5. Finally, targeted inhibitors 3 and 7 were synthesized through a Suzuki coupling with an appropriate aryl boronic acid pinacol ester or alternatively via a two-step palladium catalyzed borylation/Suzuki coupling sequence with an aryl halide. Final targeted Limk inhibitors were all purified by the high pressure reverse-phase liquid chromatograph (HPLC) methodology to give a purity of ≥ 95% based on UV absorption (254 nm).</p><p>Pyrrolopyrimidines 10 were synthesized through the reaction of substituted anilines 8 with isocyanatobenzene derivatives in DCM at room temperature, followed by Pd-catalyzed borylation/Suzuki coupling reaction with 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (Scheme 2).</p><p>The synthesis of N-substituted (on the urea NH attached to the central phenyl ring) compounds 14 is described in Scheme 3. To make 12b and 12c (12a is commercially available, where a methyl group is attached to the alinine), N-(4-bromophenyl)oxazolidin-2-one 11 was first prepared by reacting 4-bromo-aniline with 2-chloroethyl carbonochloridate in the presence of K2CO3 in CH3CN. Intermediate 11 and a secondary amine pyrrolidine or piperidine were then dissolved in DMSO and heated at 110 °C for 1 h in a microwave reactor to give N-substituted 4-bromo-aniline 12b (from pyrrolidine) and 12c (from piperidine).43 Mixing 12 and 1-isocyanato-4-methoxybenzene in DCM with stirring gave urea 13. Finally, bromide 13 underwent a Pd-catalyzed borylation/Suzuki coupling reaction sequence to produce 14.</p><p>The preparation of N-substituted (on the urea NH attached to the terminal phenyl ring) compound 18 is shown in Scheme 4. Addition of 2-chloroethyl carbonochloridate to a mixture of anilines and pyridine in DCM gave N-phenyl-oxazolidin-2-one derivative 15.44 Then, refluxing 15 and KOH in EtOH produced N-hydroxylethyl aniline 16. Heating a mixture of iodobenzene, an N′,N′-disubstituted ethanamine, Pd(dba)2, BINAP, and Cs2CO3 in dioxane gave secondary aniline 17.45 Finally, inhibitors 18 were synthesized from 16 or 17 by following the synthetic procedures described in Scheme 3.</p><!><p>Compounds prepared were first screened in biochemical assays against Limk1 (Reaction Biology Corporation, http://www.reactionbiology.com) and ROCK-II.46, 47 Selected potent Limk inhibitors were also counterscreened against ROCK-I, PKA,42, 46, 48 and JNK3,49 as well as four selected P450 isoforms (1A1, 2C9, 2D6, and 3A4).46, 48, 50 Potent and selective Limk inhibitors were then evaluated in cell-based assays for their inhibition of cofilin phosphorylation in A7r5 cells. Due to the potential applications of Limk inhibitors for treatment of cancer and HIV-infection, selected lead inhibitors were also assessed in prostate carcinoma (PC-3) cell lines stimulated by hepatocyte growth factor (HGF),51 and in HIV related CEM-SS T cells23 using Western blot analysis. To assess the drugability of these bis-aryl urea based Limk inhibitors, a few potent, selective, and membrane permeable compounds were further evaluated in in vitro and in vivo drug metabolism and pharmacokinetics (DMPK) studies.41, 46, 50, 52–55</p><p>Since the variation of hinge-binding moieties could induce significant differences in kinase inhibition potency and selectivity, as indicated in Figure 1, we started SAR studies by varying the heteroaryl ring of 3 in order to discover the best hinge binding moiety for this bis-aryl urea scaffold of Limk inhibitors. As shown in Table 1, compounds with a simple 5- or 6-membered heteroaromatic ring as the hinge-binding moiety, such as pyrazole, pyridine, and aminopyrimidine, are all basically ROCK inhibitors (7a–7c, IC50 < 200 nM) with low Limk1 inhibition (IC50 > 10 μM). This observation was in accordance with our previous reports that pyrazole, pyridine, and aminopyrimide were suitable hinge-binding moieties for developing ROCK inhibitors.41, 46, 50, 53, 54 Application of [5,6]-fused aromatic rings, such as pyrrolopyridine (7d) and purinone (7e) still yielded compounds with good ROCK-II inhibition (IC50 = 132 and 247 nM for 7d and 7e, respectively) but low Limk1 inhibition (IC50 > 10 μM). However, the use of a 4-yl-purine moiety reversed the kinase selectivity between ROCK and Limk. Compound 7f exhibited a slightly higher potency for Limk1 inhibition (IC50 = 1.5 μM) than for ROCK-II inhibition (IC50 = 5.6 μM). Interestingly, changing hinge-binding group from the purine in 7f to a pyrrolopyrimidine ring in 3 significantly enhanced the Limk1 inhibition potency and the selectivity against ROCK. Moreover, substitution of a methyl group on the 5-position of the pyrrolopyrimidine ring (7g) further improved the inhibition potency over Limk1 (IC50 = 62 nM vs. 201 nM for 3) and the selectivity against ROCK-II (IC50 = 1608 nM). Interestingly, application of 6-methyl pyrrolopyrimidine and 5,6-dimethyl pyrrolopyrimidine rings (7h and 7i) gave slightly lower Limk1 inhibition (IC50 = 80 nM) but better selectivity over ROCK-II. Therefore, further optimizations for other parts of 3 will use 5-methyl pyrrolopyrimidine as the hinge-binding moiety. However, the 5,6-dimethyl pyrrolopyrimidine moiety will also be used in preparing drug candidate Limk inhibitors since it could lead to higher selectivity and better DMPK properties (Tables 5&6&7).</p><p>For the convenience of compound synthesis, SAR studies for the central phenyl ring were mainly based on substitutions at its ortho-position (to the urea moiety). As shown in Table 2, three substitutions were evaluated. Compared to the non-substituted inhibitor 7g, the trifluoromethyl substitution yielded a compound (7j) that had a similar Limk1 inhibition potency (IC50 = 60 nM vs. 62 nM for 7g) but lower selectivity (ROCK-II IC50 = 976 nM vs. 1608 nM for 7g). However, substitution by a small F group (with a size close to that of a proton, compound 7k) led to both enhanced Limk1 inhibition (IC50 = 18 nM) and improved selectivity against ROCK-II (based on IC50 values, the selectivity over ROCK-II is 26-fold and 43-fold for 7g and 7k, respectively, Table 2). On the other hand, substitution by a large dimethylaminoethoxy side chain (7l) significantly decreased both the Limk1 inhibition (IC50 = 710 nM vs. 62 nM for 7g) and the selectivity over ROCK-II (Table 2). Therefore, an ortho-F-substitution on the central phenyl ring is the best choice for preparing a highly potent and selective Limk inhibitor.</p><p>SAR was next investigated on the terminal phenyl ring of compound 7g, where a 5-methylpyrrolopyrimidine is used as the hinge-binding moiety and the central phenyl group is non-substituted for the convenience of organic synthesis. As shown in Table 3, removal of the 3-carbonyl amide from the terminal phenyl ring of 7g yielded a compound (10a) with a lower Limk1 inhibitory activity (IC50 = 142 nM for 10a vs. 62 nM for 7g). Interestingly, replacing the 3-carboxyl amide with a F group (10b) significantly reduced the Limk1 inhibition (IC50 = 315 nM), which is probably due to special F-bonding interactions56 between this F group and its surrounding protein residues under the p-Loop (see Figure 3 of docking studies). This special F-bonding interaction might disturb the optimal binding conformation of these urea based Limk inhibitors. The same effects were also observed in several other Limk inhibitors (see Table 4). It is important to point out that this special effect of F-bonding interactions was not observed for F-substitutions on the central phenyl ring (7k, Table 2), indicating that this effect is dependent on the position of F-substitutions. Actually, we have observed similar negative effects (reducing kinase inhibition potency) of F-bonding interactions in developing our ROCK-II inhibitors53 and JNK3 inhibitors,55 where the F-substituted aromatic moieties are all bound to an area under the p-Loop inside the ATP-binding pocket of proteins kinases.</p><p>Unlike F-substitutions, replacing the 3-carboxyl amide with a methoxy group resulted in a Limk inhibitor (10d) that had a similar Limk1 inhibitory potency (IC50 = 75 nM vs. 62 nM for 7g) and a slightly better selectivity over ROCK-II (Table 3). However, the 2-methoxy substitution (10c) significantly reduced the Limk1 inhibition activity (IC50 = 283 nM). On the other hand, the 4-methoxy substitution (10e) enhanced both Limk1 inhibition (IC50 = 35 nM) and selectivity against ROCK-II (IC50 > 10 μM, selectivity > 285-fold). Similar SAR patterns were also obtained for F-, Cl-, and methyl-substitutions on this terminal phenyl group (see Table 4), indicating that 4-substitution is the best fit for this scaffold in Limk inhibitions. Heteroaryl rings other than the benzene ring were also evaluated as the terminal aromatic moieties. For example, application of a 2-yl-thiazole (10f) resulted in lower Limk1 inhibition (compared to 10a); and the use of a 2-yl-pyridine moiety (10g) almost inactivated the compound against both Limk1 and ROCK-II.</p><p>Investigation of the substitution effects on the two urea NH groups was the next focus in our SAR studies. For the urea NH attached to the central phenyl ring, neither small nor large substitutions including pyrrolidinoethyl and piperidinoethyl could be tolerated. As shown in Figure 2, a simple methyl substitution (14a) would significantly reduce the Limk1 inhibitory potency (IC50 = 1090 nM vs. 35 nM for 10e). Larger substitutions to this NH group gave even lower Limk1 inhibitions, as evidenced by the Limk1 IC50 values of compounds 14b and 14c (Figure 2). These results demonstrated that alkylation to this urea NH disturbed the optimal binding conformations, or the NH is involved in H-bonding interactions to the protein, thus resulted in a low Limk affinity (also see docking modes in Figure 3).</p><p>In contrast to observations in Figure 2, SAR studies demonstrated that substitutions on the urea NH group attached at the terminal phenyl ring were well tolerated, and excellent Limk inhibitors could be obtained through this modification. As shown in Table 4, a pyrrolidinoethyl substitution yielded a compound (18a) with slightly lower Limk1 inhibition (IC50 = 368 nM vs. 142 nM for 10a). However, replacing the pyrrolidine ring with a hydroxyl group (18b) led to both a high Limk1 inhibitory activity (IC50 = 43 nM vs. 142 nM for 10a) and a good selectivity over ROCK-II (IC50 = 6565 nM vs. 2358 nM for 10a). Inspired by 18b, a small library of 4×3=12 analogs (of 18b), based on four functional groups (F-, Cl-, Methyl, and Methoxy) and three substitution patterns on the terminal phenyl ring (2-, 3-, 4-positions), were prepared and evaluated (compounds 18c to 18n, Table 4). Generally, the 4-substitution exhibited the highest and the 2-substitution gave the lowest Limk1 inhibitory activity. Selectivity against ROCK-II followed the same pattern with the 4-substitution being the highest and 2-substitution the lowest, no matter what was the substitution group. Among the 4-substituted Limk inhibitors, the 4-Cl analog had the best Limk1 inhibitory potency (18h, IC50 = 25 nM) and its 4-F counterpart (18e, IC50 = 86 nM) had the lowest Limk anity probably due to the special F-bonding interactions,56 while the Limk1 inhibitory activity and the selectivity against ROCK-II for the methyl and methoxy analogs (18k and 18n) were in between.</p><p>To confirm that alkylation to this urea NH group could be well tolerated, two more substitutions were explored. As shown in Table 4, aminoethyl and N′,N′-dimethylaminoethyl substitutions were applied to both 4-Cl- and 4-methoxyphenyl ureas, and the resulting 4 compounds 18o to 18r all exhibited high Limk1 inhibitions. Compounds 18p to 18r were assessed in counter-screen studies, and 18q and 18r were found to have high selectivity against ROCK-II (IC50 > 10 μM, selectivity is > 210-fold and >500-fold for 18q and 18r, respectively) while that for 18p was only ~ 21-fold. The lower Limk1 inhibition potency observed for 18a, as compared to 18q and 18r, might be due to its bulky pyrrolidine ring which might have disturbed the optimal binding conformation.</p><p>Computer modeling studies of lead compounds demonstrated that these bis-aryl urea based Limk inhibitors are all Type-I ATP-competitive kinase inhibitors. The docking mode of compound 18b in the crystal structure of Limk1 protein (PDB ID 3S95) is shown in Figure 3. Key interactions in this motif include: two H-bonds between the pyrrolopyrimidine N/NH (N1 and N7) and hinge residue I416; one plausible H-bond between N3 of pyrrolopyrimidine ring and the side chain OH group of residue T413 (not labeled in Figure 3 since this H-bonding requires rotation movement of the T413 side chain); one H-bond between the urea carbonyl moiety and the side chain amino group of K368; one H-bond between the OH group and residue D478; cation-π interactions between the terminal phenyl ring and the side chain amino group of K368; hydrophobic interactions between the terminal phenyl ring and its surrounding residues under the P-loop. It is important to point out that hydrophobic interaction between the aromatic rings of pyrrolopyrimidine/central phenyl moieties and their surrounding side chains of protein residues also contributed to the high affinity of these Limk inhibitors.</p><p>The binding motif of compound 18b supported our observed SAR. For example, both mono- and bis-methyl substituted (to the 5- and/or 6-position), or even larger group substituted (unpublished results) pyrrolopyrimidine rings were well tolerated due to the open space around this area, and these substitutions could enhance the inhibitor's Limk inhibition due to the extra interactions introduced by substitution(s). Substitution to the urea NH attached to the central phenyl ring led to inactive compounds because this substitution could disturb the orientation of the urea carbonyl group thus weakening its H-bonding to K368. On the other hand, substitutions to the urea NH adjacent to the terminal phenyl ring were well tolerated and could lead to enhanced Limk inhibition since there is enough space around this area and the substitution is directed toward the solvent. 4-Substitutions on the terminal phenyl group gave the most active Limk inhibitors (compared to the 2- and 3-substitutions) because there is a deep hydrophobic pocket around there. The H-bonding interaction between the pyrrolopyrimidine N3 and the side chain OH of T413 explained why compound 3 (Table 1) was a good Limk inhibitor while compound 7d had low Limk1 inhibition. The significant decrease of Limk1 inhibition in 7f as compared to 3 (Table 1) is probably due to the extra H-bonding interactions between N5 (of 7f) and surrounding protein residues, which might disturb the optimal binding conformation of the ligand thus reduce its affinity toward Limk1.</p><p>To summarize, our SAR analysis and docking studies for this bis-aryl urea based scaffold of Limk inhibitors showed that both 5- and 6-methyl-4-yl-pyrimidines, and the 5,6-dimethyl-4-yl-pyrrolopyrimidine could serve well as hinge-binding moieties for Limk inhibition. Among them, the 5,6-dimethylpyrrolopyrimidine was the best considering that it could render much better selectivity (against ROCK) and higher microsomal stability (see Table 6). An ortho-F-substitution on the central phenyl ring (to the urea moiety) could improve the Limk inhibitory potency while still keeping high microsomal stability (Table 6). On the other hand, an F-substitution on the terminal phenyl ring reduced inhibitory potency against Limk1 probably due to the special F-bonding interactions (under the P-loop). SAR analysis also indicated that a 4-Cl or a 4-methyl substitution on the terminal phenyl group gave overall best Limk inhibitors. Remarkably, a substitution to the urea NH attached on the terminal phenyl side could improve both biochemical and cell potency, enhance selectivity, and more importantly, increase the inhibitor's DMPK properties and bioavailability (see Table 7 below).</p><p>To take advantage of the important SAR information above, Limk inhibitors that combine the best structural elements from SAR analysis were thus prepared and evaluated. Table 5 lists the structures and biochemical potency data for four representative compounds. In compounds 18s to 18w, a 4-yl-5,6-dimethylpyrrolopyrimidine was used as the hinge binding moiety for optimal microsomal stability and better selectivity; An ortho-F-substitution on the central phenyl ring and a 4-Cl substitution on the terminal phenyl group were employed in order to achieve higher Limk1 potency; Representative substitutions on the terminal urea NH were applied to further investigate the DMPK properties (see discussion for Tables 6&7). Indeed, these compounds all had excellent Limk1 potency (IC50 ≤ 21 nM) and good selectivity against ROCK-II (IC50 > 20 μM for 18w and 18x).</p><p>In order to examine the selectivity profile of these bis-aryl urea based Limk1 inhibitors, selected lead compounds were subjected to counter screening against ROCK-I, JNK3 and four representative cytochrome P450 isoforms. As summarized in Table 6, these Limk inhibitors all exhibited low inhibitory activity over tested kinases and P450 enzymes, except that 7i showed modest inhibition against enzyme 1A2 (77%) at 10 μM. In addition to counterscreens against ROCK and JNK3, lead inhibitor 18b was also profiled against a panel of 61 kinases (Reaction Biology Corporation, http://www.reactionbiology.com/webapps/site/). Results showed that 18b at 1.0 μM inhibited only Limk1 and STK16 with ≥ 80% inhibition (~ 3% hit ratio), and hit also Aurora-a, Flt3, LRRK2, and RET with >50% inhibition (~ 10% hit ratio). Detailed profiling data for 18b is provided in Supporting Information. The profiling data demonstrated that selective Limk inhibitors can be obtained from this bis-aryl urea based scaffold.</p><p>These Limk inhibitors also had good to excellent stability in human and rat liver microsomes (Table 6) with good to excellent half-lives. It is important to point out that, compared to the mono-methyl substituted pyrrolopyrimidine based analog 7g, the 5,6-dimethyl pyrrolopyrimidine based Limk inhibitors 7i, 18s, and 18t exhibited a higher stability in both human and rat microsomes, and a higher selectivity against ROCK (see also Tables 2&5). However, when the hydroxyl or the amino group on 18s and 18t was methylated, as shown in 18w and 18x, there was a significant drop in the microsomal stability (Table 6). Apparently, the lower stability of 18w and 18x was mainly due to de-methylation on their side chain dimethylamino or methoxy groups. Other important SAR information from the selectivity profiling and stability data in Table 6 include, 1) all hydroxyethyl substituted (to the urea NH) compounds (18 series) had excellent stability in human liver microsomes with the exception of 18g (t1/2 = 22 min only), 2) F-substitution on the central phenyl ring did not reduce the microsomal stability while still keeping the excellent selectivity (7k vs. 7g), 3) F-substitution on the terminal phenyl ring not only reduced the Limk1 inhibitory potency (compared to its Cl-, methyl, and methoxy substituted counterparts) but also deteriorated the microsomal stability (18e vs. 18b, 18h, 18k, and 18n), 4) 3-substitution on the terminal phenyl ring led to significant reduction of microsomal stability, as compared to its 4-substituted counterpart (18g vs. 18h and 18m vs. 18n), to the non-substituted analog (18b), and even to its 2-substituted analog (18f).</p><p>In an effort to investigate the cell-based activity of these Limk1 inhibitors, we monitored the phosphorylation state of cofilin in several cell lines. Data in A7r5 cells (Table 6) showed that inhibitors without any substitutions on their urea NH group (7g, 7i, 7k) had a cell activity of IC50 values only in the micromolar range. On the other hand, Limk inhibitors with their urea NH group (the one attached to the terminal aryl ring) substituted by a hydroxyethyl, or an aminoethyl, or a methoxyethyl, or a dimethylaminomethyl group (18b to 18x) had IC50 values all in the sub-micromolar range, with the best one close to 100 nM (18h). In addition, SAR patterns shown in the cell-based potency were similar to those observed in biochemical potency and selectivity assays. For example, 4-Cl (18h) and 4-methyl (18k) substitutions produced compounds with better cell activity than 4-methoxy (18n) substitutions, and the 4-substitution exhibited the highest cell activity among 2-, 3-, and 4-substitutions (18f, 18g, and 18h) on the terminal phenyl ring.</p><p>Since Limk inhibitors could find wide applications, such as in glaucoma,20 cancer,27, 28, 57, 58 infection,21–24, 59 and Alzheimer's disease (AD)14, 15 etc., cofilin phosphorylation assays were also carried out for a few selected lead compounds in prostate carcinoma (PC3) cell lines stimulated by hepatocyte growth factor (HGF) and in HIV-related CEM-SS T cell lines.60 As shown in Figure 4, inhibitor 18b exhibited significant inhibition even at a concentration of only 50 nM in Western blot analysis of cofilin phosphorylation in PC-3 cells (Figure 4A). Similar cell-based potency was also observed for 18f and 18h in PC-3 cells (see Supporting Information). The phosphorylation status of p-cofilin in CEM-SS T cells for inhibitors 18p, 18r, and 18x is shown in Figure 4B. Again > 50% inhibition was seen for all these compounds at 1 μM, an inhibitory potency similar to that obtained in A7r5 cells. The results from these three tested cell lines demonstrated that the optimized Limk inhibitors had good cell permeation. Compounds 18p and 18r had almost the same biochemical Limk1 potency (IC50 values were both ~ 20 nM, Table 4). Apparently, the better cell potency observed for 18r than for 18p (Figure 4B) is due to the free NH2 group present in 18p, a structural element normally associated with deteriorated cell penetration.</p><p>In vivo pharmacokinetics (PK) studies were conducted for selected compounds during the whole optimization at various stages in order to identify structural elements that are favorable for in vivo applications, and/or to evaluate the feasibility of optimized Limk inhibitors for animal studies. PK properties of iv dosing (1 mg/kg) and the oral bioavailability (%F) for selected lead Limk inhibitors are listed in Table 7. Generally, a 2-hydroxyethyl side chain reduced the clearance (Cl) compared to the non-substituted (NH) urea derivatives (10a, 18b, 18h, 18k, 18n vs. 7g and 7k). In contrast, a side chain containing a terminal amino group increased the clearance significantly (18o, 18p, 18r vs. 18h, 18k, and 18n). Remarkably, the high clearance of compounds with an amino side chain could be reduced dramatically by introducing an F-substitution on the central phenyl ring, or by using a 5,6-dimethylpyrrolopyrimidine (instead of the 5-methylpyrrolopyrimidine) as the hinge-binding moiety, or a combination of both (18w vs. 18r). All Limk1 inhibitors listed in Table 7 had reasonable volume of distribution (Vd) values except a few which possessed an amino side chain and in which a 5-methylpyrrolopyrimidine was used as the hinge binding moiety (18o, 18p, and 18r). The much lower Cl and Vd values and higher AUC value for 18w as compared to those for 18r (and also for 18o and 18p) further demonstrated that an F-substitution on the central phenyl ring and the application of a 5,6-dimethylpyrrolopyrimidine as the hinge-binding moiety can improve the inhibitor's PK properties.</p><p>The PK data in Table 7 showed that substitution to the urea NH group could generally increase the half-lives of these urea based Limk inhibitors (the 10 and 18 series vs. the 7 series). The AUC and Cmax properties for these compounds were also excellent except for the three inhibitors (18o, 18p, and 18r) which contained an amino side chain and no F-substitutions on their central phenyl ring and in which a 5-methyl pyrrolopyrimidine was used as the hinge-binding moiety. It is important to point out that, even with an amino side chain, inhibitor 18w still exhibited good AUC and Cmax values, probably due to the presence of both an F-substitution on the central phenyl ring and a 5,6-dimethylpyrrolopyrimidine moiety in its structure. Data in Table 7 also indicated that, while the non-substituted urea compounds (7g and 7k) had no oral bioavailability (%F) at all, all inhibitors containing a hydroxyethyl side chain could exhibit reasonable oral bioavailability. However, those inhibitors containing an amino side chain (18o, 18p, and 18r) had no oral bioavailability either, probably because of the high clearance (Cl) exhibited by these compounds.</p><p>Since Limk1 expression is highly expressed in cancerous prostate cells and predominantly found in metastatic prostate tumor tissues, and is required for cancer cell migration and invasion,61, 62 Limk1 is considered as a biomarker for prostate cancer progression.63 Limk1 is involved in Rac-induced actin cytoskeleton reorganization through inactivating phosphorylation of cofilin, and also mediated with focal adhesion complexes.8, 64 Reorganization of cytoskeleton is an essential feature of motility, detachment, and invasion of cancer cells. Moreover, Limk1 expression is correlated with the aggressiveness of cancer cells, and Limk1 expression in metastatic PC-3 cells is higher than less-aggressive LNCaP and M21 cells.26 To confirm the role of Limk inhibitors on the invasion and migration of prostate cancers, we examined the effect of optimized Limk inhibitors in PC-3 cells using an in vitro invasion assay or in vitro migration assay. Thus, Transwell chambers were coated with GFR Matrigel, and PC-3 cells were seeded in the insert of the chamber as described in the Experimental Section. After incubating for 48 hours, the invasive PC-3 cells were counted and analyzed by hematoxylin staining under microscope. As shown in Figure 5 for two representative inhibitors 18b or 18f, the invasion of PC-3 cells was significantly inhibited by the treatment of 1 μM Limk inhibitors (76% for 18b, and 83% for 18f, compared to the control).</p><p>To verify the role of Limk inhibitors on migration of PC-3 cells, a wound was created by scratching in a cell monolayer as described in the Experimental Section. After incubating for 24 hours with treatment of inhibitors 18b or 18f, the closed wound area, indicating migrated cells, was analyzed by ImageJ software (Ver 1.48). As shown in Figure 6, the migrated PC-3 cells were decreased significantly even at a concentration as low as 0.1 μM, and the migration was inhibited 74% by 1 μM of inhibitor 18b (16.5% (NS) by 0.1 μM, 74.0% (p < 0.01) by 1 μM, and 77.5% (p < 0.01) by 10 μM compared to the control). Similar inhibition potency was also obtained for inhibitor 18f (13.0% (NS) by 0.1 μM, 76.1% (p < 0.01) by 1 μM, and 81.0% (p < 0.01) by 10 μM, compared to the control). These results indicated that 18b or 18f had inhibitory effects on invasion and migration of metastatic PC-3 cells. Considering that both inhibitors had low inhibition against ROCK-I and ROCK-II (Tables 4&6), the inhibition of which could also lead to suppression of cell migration/invasion,65, 66 results in Figures 5&6 also demonstrated that 18b and 18f must have played a role in Limk inhibitions in vitro.</p><p>To demonstrate the potential application of these Limk inhibitors for the treatment of glaucoma, the intraocular pressure (IOP)-lowering effect of compound 18w was monitored after applying it topically on rat eyes (Brown Norway rats, n = 6/group, housed under constant low-light conditions)67 followed a protocol described previously by our groups.41, 53 Thus, compound 18w was applied to the right eyes of an elevated IOP rat model (initial IOP was ~ 28 mmHg) using a dose of 50 μg (20 μL drop of a 0.25% solution). As shown in Figure 7, significant decreases in IOP were detected at 4 h, slightly weakened at 7 h, and IOP returning to baseline at 24 h as compared to the vehicle. It must be pointed out that the IOP drop could not be due to ROCK inhibition since 18w had a high selectivity against ROCK (Table 5).</p><!><p>Through the application of a 4-yl-pyrrolopyrimidine as the hinge-binding moiety to replace the pyrazole group in ROCK inhibitor 1, we identified compounds with high Limk1 inhibition potency. Systematic SAR studies around this bis-aryl urea scaffold (3) have led to a series of potent and selective Limk inhibitors. Docking studies demonstrated that these bis-aryl urea Limk inhibitors exhibited a typical Type-I kinase binding motif. The optimized Limk inhibitors had high biochemical potency and high selectivity over ROCK-I, ROCK-II, and JNK3. Inhibitor 18b (also coded as SR-7826) was found to hit only Limk1 and STK16 with ≥ 80% inhibition at 1 μM against a panel of 61 kinases. The lead Limk inhibitors also had good cell-based potency in cofilin phosphorylation assays and in cell-based migration/invasion assays. In addition, they had fair to excellent in vitro and in vivo DMPK properties, such as a clean inhibition profile against select CYP-450 isoforms, a high stability in human and rat liver microsomes, and favorable PK properties in iv dosing (high AUC/Cmax, low Cl, and long half-lives) and fair to good oral bioavailability (18b, 18k, 18n, and 18s) in rats. For example, compounds 18s to 18x (also coded as SR-11157) all had excellent potency against Limk1 (IC50s ≤ 21 nM), good cell-based activity against cofilin phosphorylation in A7r5 cells (IC50s ≤ 320 nM), and high selectivity over ROCK and JNK. The optimized inhibitors, such as 18b and 18f, showed excellent activities in migration/invasion cell-based assays. In addition, significant IOP drop on rat eyes (> 20%) was achieved for inhibitor 18w (also coded as SR-11124) after topical administration (at a dose of 50 μg). Applications of optimized Limk inhibitors on other indications are under investigation and will be reported in due course.</p><!><p>Commercially available reagents and anhydrous solvents were used without further purification unless otherwise specified. Thin layer chromatography (TLC) analyses were performed with precolated silica gel 60 F254. The mass spectra were recorded by LC/MS with Finnigan LCQ Advantage MAX spectrometer of Thermo Electron®. Flash chromatography was performed on prepacked columns of silica gel (230–400 Mesh, 40–63 μm) by CombiFlash® with EtOAc/hexane or MeOH/DCM as eluent. The preparative HPLC was performed on SunFire C18 OBD 10μm (30 × 250 mm) with CH3CN + 50% MeOH / H2O + 0.1% TFA as eluent to purify the targeted compounds. Analytic HPLC was performed on Agilent technologies 1200 series with CH3CN (Solvent B) / H2O + 0.9% CH3CN + 0.1% TFA (Solvent A) as eluent and the targeted products were detected by UV in the detection range of 215–310 nm. All compounds were determined to be > 95% pure by this method. NMR spectra were recorded with a Bruker® 400 MHz spectrometer at ambient temperature with the residual solvent peaks as internal standards. The line positions of multiplets were given in ppm (δ) and the coupling constants (J) were given in Hertz. The high-resolution mass spectra (HRMS, electrospray ionization) experiments were performed with Thermo Finnigan orbitrap mass analyzer. Data were acquired in the positive ion mode at resolving power of 100000 at m/z 400. Calibration was performed with an external calibration mixture immediately prior to analysis.</p><!><p>The mixture of 3-aminobenzoic acid (10 mmol), propan-2-amine (10 mmol), HATU (10 mmol), and DIEA (30 mmol) in DMF (10 mL) was stirred at room temperature until the complete conversion of the started material. Then, saturated NaHCO3 was added to quench the reaction and extracted with ethyl acetate (3 × 15 mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo to give crude aniline carboxamide 4. Aniline 4 (0.2 mmol) was then added to the solution of isocyanatobenzene derivatives (0.2 mmol) in DCM (1 mL). The mixture was stirred at room temperature for 2 h. Then, the solvent was removed in vacuo to give the crude bromide 5 for next step without further purification.</p><p>The mixture of substituted anilines 8 (0.2 mmol) and isocyanatobenzene derivatives (0.2 mmol) in DCM (1 mL) was stirred at room temperature for 2 h, then the solvent was removed in vacuo to give the crude bromides 9 for next step without further purification.</p><p>2-Chloroethyl carbonochloridate (10 mmol) was added to a mixture of 4-bromo-aniline (10 mmol) and K2CO3 (30 mmol) in CH3CN (100 mL) and the reaction was stirred for 24 h. Then, solvent was removed in vacuo and the remaining residue redissolved in water and ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, concentrated in vacuo, and purified through silica gel to give crude N-(4-bromophenyl)oxazolidin-2-one 11. Then 11 (0.2 mmol) and secondary amine (0.6 mmol) including pyrrolidine and piperidine were dissolved in DMSO (1 mL) and heated at 110 °C in microwave. After the complete conversion of 11, the mixture was diluted with water and extracted with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the intermediates 12a–12c. The mixture of 12a–12c (0.2 mmol) and 1-isocyanato-4-methoxybenzene (0.2 mmol) in DCM (1 mL) was stirred at room temperature for 2 h, the solvent was then removed in vacuo to give the crude bromide 13a–13c for next step without further purification.</p><p>Finally, the boronic acid pinacol ester (0.3 mmol) and the crude bromide 5 (0.2 mmol) were dissolved in degassed 5:1 dioxane/H2O. Pd(PPh3)4 (0.02 mmol) and 2M solution of K2CO3 (0.6 mmol) were added sequentially under Argon and the mixture was heated at 95 °C for 2 h. After cooling to room temperature, the mixture was diluted with water and extracted with ethyl acetate (3 × 5 mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was then purified by preparative HPLC to give the targeted product 7a and 7b as white solid.</p><p>In an alternative route, bis-(pinacolato)diboron (0.24 mmol), crude 5, 9, and 13 (0.2 mmol), and PdCl2(dppf) (0.02 mmol) were dissolved in degassed dioxane (5 mL). After refluxing for 2 h, the mixture was diluted with water and extracted with ethyl acetate (3 × 5 mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo to give crude boronic acid pinacol ester. Followed the synthesis procedure of 7a, 7c–7k, 10a–10f, 14a–14c were synthesized form crude boronic acid pinacol ester (0.2 mmol) and Ar-Cl (0.2 mmol).</p><p>2-Chloroethyl carbonochloridate (1 mmol) was added to a mixture of substituted anilines (1 mmol) and pyridine (3 mmol) in DCM (10 mL) and the reaction was stirred for 24 h. Then, solvent was removed in vacuo and the remaining residue redissolved in water and ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo to give crude 15. KOH (10 mmol) was added to the mixture of crude 15 (1 mmol) in EtOH (10 mL). Then the mixture was refluxed until the complete conversion of 15. The solvent was removed in vacuo and the remaining residue was redissolved in water and ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, concentrated in vacuo, and purified by silica gel to give intermediates 16. The mixture of iodobenzene (0.2 mmol), 2-(pyrrolidin-1-yl)ethanamine (0.6 mmol), Pd(dba)2 (0.01 mmol), BINAP (0.01 mmol), and Cs2CO3 (0.6 mmol) in dioxane (1 mL) was refluxing for 24 h. After cooling to room temperature, water and ethyl acetate were added. Then the organic layers were combined, dried over anhydrous Na2SO4, concentrated in vacuo, and purified by silica gel to give intermediates 17. Then 18a and 18b–18n were synthesized from 17 and 16 respectively followed the synthetic procedure of 10a–10f from 8.</p><!><p>45% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.28 (s, br, 1H), 9.06 (s, 1H), 8.96 (s, 1H), 8.81 (s, 1H), 8.18−8.16 (m, 3H), 7.87 (s, 1H), 7.71−7.64 (m, 4H), 7.46−7.44 (m, 1H), 7.38−7.36 (m, 1H), 6.95 (s, 1H), 4.14−4.07 (m, 1H), 1.18 (d, J = 5.2 Hz, 6H); 13C-NMR (DMSO-d6, 100 MHz) δ 165.50, 152.70, 152.38, 152.19, 147.81, 143.00, 139.47, 135.77, 130.01, 129.56, 128.51, 127.01, 120.81, 120.71, 118.10, 117.70, 113.78, 101.66, 40.95, 22.27; LC/MS (M+H+): 415.11; HRMS (ESI-Orbitrap) Calcd for C23H23N6O2: 415.1882 [M+H+], Found 415.1872.</p><!><p>68% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.86 (s, br, 1H), 8.82 (s, 1H), 8.69 (s, 1H), 8.18−8.16 (m, 1H), 7.96−7.93 (m, 1H), 7.84−7.79 (m, 1H), 7.78−7.74 (m, 1H), 7.72−7.68 (m, 1H), 7.65−7.60 (m, 1H), 7.53−7.51 (m, 2H), 7.45−7.41 (m, 3H), 7.36−7.32 (m, 1H), 4.08 (q, J = 6.4 Hz, 1H), 1.17 (d, J = 6.4 Hz, 6H); HRMS (ESI-Orbitrap) Calcd for C20H22N5O2: 364.1773 [M+H+], Found 364.1792.</p><!><p>65% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 9.12 (s, 1H), 9.04 (s, 1H), 8.78 (d, J=5.6 Hz, 2H), 8.19−8.17 (m, 1H), 8.12 (d, J = 5.6 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.89−7.88 (m, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.64−7.61 (m, 1H), 7.46−7.44 (m, 1H), 7.38−7.34 (m, 1H), 4.08 (q, J = 6.4 Hz, 1H), 1.17 (d, J = 6.4 Hz, 6H); 13C-NMR (DMSO-d6, 100 MHz) δ 165.46, 152.57, 152.35, 144.23, 142.99, 139.46, 135.77, 128.52, 128.49, 127.52, 121.84, 120.82, 120.68, 118.42, 117.74, 40.94, 22.28; LC/MS (M+H+): 375.14.</p><!><p>52% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 9.43 (s, br, 2H), 8.92−8.80 (m, 1H), 8.32−8.17 (m, 1H), 8.24−8.17 (m, 1H), 8.12−8.10 (m, 2H), 7.90 (s, 1H), 7.66−7.61 (m, 3H), 7.45−7.34 (m, 3H), 7.28 (s, 1H), 4.09 (q, J = 6.8 Hz, 1H), 1.16 (d, J = 6.8 Hz, 6H); 13C-NMR (DMSO-d6, 100 MHz) δ 166.79, 165.51, 159.06, 152.36, 152.00, 143.87, 139.51, 135.78, 128.67, 128.46, 128.19, 120.74, 120.66, 117.72, 117.64, 105.05, 40.93, 22.27; HRMS (ESI-Orbitrap) Calcd for C21H23N5O2: 391.1882 [M+H+], Found 391.1889.</p><!><p>40% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.26 (s, br, 1H), 9.08 (s, 1H), 8.97 (s, 1H), 8.80 (s, 1H), 8.25−8.14 (m, 3H), 8.12 (d, J = 8.8 Hz, 2H), 7.87 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.46−6.36 (m, 2H), 6.96−6.95 (m, 2H), 4.08 (m, 1H), 1.17 (d, J = 6.4 Hz, 6H); LC/MS (M+H+): 414.15.</p><!><p>45% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.08 (s, br, 1H), 9.42 (s, 1H), 8.84−8.82 (m, 1H), 8.76 (s, 1H), 8.35−8.14 (m, 3H), 8.12 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.69−7.59 (m, 1H), 4.08 (m, 1H), 3.35 (q, J = 3.2 Hz, 2H), 1.17 (d, J = 6.4 Hz, 6H), 1.07 (t, J = 3.2 Hz, 3H); LC/MS (M+H+): 460.17.</p><!><p>29% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 11.98. (s, br, 1H), 9.48 (s, 1H), 8.81−8.79 (m, 1H), 8.76 (s, 1H), 8.35−8.14 (m, 3H), 8.12 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.69−7.59 (m, 2H), 6.95 (s, 1H), 4.08 (m, 1H), 1.17 (d, J = 6.4 Hz, 6H); HRMS (ESI-Orbitrap) Calcd for C22H22N7O2: 416.1835 [M+H+], Found 416.1849.</p><!><p>40% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.63 (s, 1H), 9.21 (s, 1H), 9.10 (s, 1H), 8.92 (s, 1H), 8.19−8.18 (m, 1H), 7.89 (s, 1H), 7.74−7.68 (m, 4H), 7.65−7.61 (m, 2H), 7.46−7.44 (m, 1H), 7.39−7.35 (m, 1H), 4.10 (q, J = 6.4 Hz, 1H), 2.10 (s, 3H), 1.17 (d, J = 6.4 Hz, 6H); 13C-NMR (DMSO-d6, 100 MHz) δ165.52, 159.07, 158.74, 153.86, 152.50, 152.09, 145.81, 142.76, 139.59, 135.78, 130.90, 128.46, 125.16, 120.78, 117.71, 117.44, 114.54, 111.64, 40.94, 22.27, 12.51; HRMS (ESI-Orbitrap) Calcd for C24H25N6O2: 429.2039 [M+H+], Found 429.2029.</p><!><p>35% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.22 (s, br, 1H), 9.07 (s, 1H), 8.96 (s, 1H), 8.74 (s, 1H), 8.20−8.18 (m, 1H), 8.14−8.12 (m, 2H), 7.88 (s, 1H), 7.70−7.68 (m, 2H), 7.66−7.61 (m, 1H), 7.46−7.44 (m, 1H), 7.38−7.34 (m, 1H), 6.69 (s, 1H), 4.10 (q, J = 6.8 Hz, 1H), 1.17 (d, J = 6.8 Hz, 6H); LC/MS (M+H+): 429.17.</p><!><p>32% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 9.04 (s, 1H), 8.99 (s, 1H), 8.74−8.73 (m, 1H), 8.19−8.17 (m, 1H), 7.87 (s, 1H), 7.68−7.61 (m, 5H), 7.46-7.44 (m, 1H), 7.38−7.34 (m, 1H), 4.11 (q, J = 6.8 Hz, 1H), 1.97 (s, 3H), 1.17 (d, J = 6.8 Hz, 6H); LC/MS (M+H+): 443.16.</p><!><p>46% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.3 (s, br, 1H), 9.73 (s, 1H), 9.60 (s, 1H), 8.84 (s, 1H), 8.45 (s, 1H), 8.34−8.32 (m, 1H), 8.20−8.18 (m, 1H), 7.90 (s, 1H), 7.71−7.70 (m, 1H), 7.51 (s, 1H), 7.48−7.47 (m, 1H), 7.40−7.33 (m, 2H), 4.51 (t, J = 4.6 Hz, 2H), 4.10 (q, J = 6.4 Hz, 1H), 3.63 (t, J = 4.6 Hz, 2H), 2.94 (s, 6H), 2.12 (s, 3H), 1.17 (d, J = 6.4 Hz, 6H); 13C-NMR (DMSO-d6, 100 MHz) δ 165.44, 158.91, 158.58, 154.45, 152.33, 152.23, 146.68, 145.76, 139.52, 135.78, 131.26, 128.52, 127.95, 123.80, 120.68, 118.34, 117.72, 114.64, 113.18, 111.10, 62.96, 55.44, 42.65, 40.94, 22.26, 12.78; LC/MS (M+H+): 516.13.</p><!><p>47% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.19 (s, br, 1H), 9.71 (s, 1H), 8.83 (s, 1H), 8.33−8.26 (m, 2H), 8.21−8.19 (m, 1H), 8.01−8.00 (m, 2H), 7.85 (s, 1H), 7.70−7.68 (m, 1H), 7.48−7.46 (m,2H), 7.41−7.37 (m, 1H), 4.10 (q, J = 6.8 Hz, 1H), 2.09 (s, 3H), 1.16 (d, J = 6.8 Hz, 6H); 13C-NMR (DMSO-d6, 100 MHz) δ 165.37, 155.31, 152.57, 152.17, 149.46, 139.24, 137.50, 135.83, 134.08, 131.87, 128.70, 127.18, 126.48, 125.15, 124.24, 122.44, 120.96, 120.58, 117.53, 114.95, 108.93, 40.93, 22.27, 12.81; HRMS (ESI-Orbitrap), Calcd for C25H24F3N6O2: 497.1913 [M+H+ ], Found 497.1902.</p><!><p>42% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.20 (s, br, 1H), 9.46 (s, 1H), 8.89 (s, 1H), 8.81 (s, 1H), 8.52−8.49 (m, 1H), 8.25−8.19 (m, 1H), 7.88−7.84 (m, 1H), 7.68−7.64 (m, 2H), 7.52−7.48 (m, 3H), 7.42−7.39 (m, 1H), 4.10 (q, J = 6.8 Hz, 1H), 2.10 (s, 3H), 1.17 (d, J = 6.8 Hz, 6H); 13C-NMR (DMSO-d6, 100 MHz) δ 165.44, 154.88, 152.40, 152.10, 149.96, 148.37, 139.27, 135.83, 129.21, 128.61, 126.88, 126.43, 120.84, 120.56, 119.50, 117.45, 116.19, 115.99, 114.82, 109.82, 40.95, 22.26, 12.75; HRMS (ESI-Orbitrap), Calcd for C24H24FN6O2: 447.1945 [M+H+ ], Found 447.1934.</p><!><p>62% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.56 (s, br, 1H), 9.18 (s, 1H), 8.94−8.90 (m, 2H), 7.78−7.67 (m, 4H), 7.62−7.58 (m, 1H), 7.50−7.48 (m, 2H), 7.32−7.28 (m, 2H), 7.01−6.98 (m, 1H), 2.10 (s, 3H); HRMS, Calcd for C20H18N5O:344.1511 [M+H+ ], Found 344.1526.</p><!><p>51% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.55 (s, br, 1H), 9.17−9.12 (m, 2H), 8.88 (s, 1H), 7.69−7.68 (m, 4H), 7.60−7.50 (m, 2H), 7.36−7.31 (m, 1H), 7.18−7.16 (m, 1H), 6.84−6.79 (m, 1H), 2.10 (s, 3H); LC/MS (M+H+): 362.11.</p><!><p>60% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.49 (s, 1H), 9.66 (s, 1H), 8.88 (s, 1H), 8.35 (s, 1H), 8.16−8.14 (m, 1H), 7.69−7.68 (m, 4H), 7.56 (s, 1H), 7.06−7.03 (m, 1H), 7.00−6.98 (m, 1H), 6.96−6.89 (m, 1H), 3.90 (s, 3H), 2.10 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 154.87, 152.24, 152.18, 147.80, 146.90, 142.24, 130.81, 128.35, 127.60, 126.70, 122.12, 120.52, 118.45, 117.12, 114.61, 110.93, 110.77, 55.75, 12.66; LC/MS (M+H+): 374.09.</p><!><p>57% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.61 (s, br, 1H), 9.14 (s, 1H), 8.93−8.91 (m, 2H), 7.72−7.67 (m, 4H), 7.60 (s, 1H), 7.23−7.18 (m, 2H), 6.98−6.96 (m, 1H), 6.59−6.57 (m, 1H), 3.74 (s, 3H), 2.10 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 159.64, 153.71, 152.42, 152.07, 145.64, 142.89, 140.85, 130.91, 129.49, 128.52, 124.83, 117.37, 114.51, 111.75, 110.63, 107.32, 104.13, 54.88, 12.50; LC/MS (M+H+): 374.09.</p><!><p>45% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 11.89 (s, br, 1H), 8.86 (s, 1H), 8.71 (s, 1H), 8.60 (s, 1H), 7.61−7.60 (m, 4H), 7.38 (d, J = 8.8 Hz, 2H), 7.35 (s, 1H), 6.88 (d, J = 8.8 Hz, 2H), 3.73 (s, 3H), 2.10 (s, 3H); LC/MS (M+H+): 374.14; HRMS (ESI-Orbitrap) Calcd for C21H20N5O2: 374.1617 [M+H+], Found 374.1608.</p><!><p>40% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 11.95 (s, br, 1H), 8.76 (s, 1H), 8.71 (s, 1H), 8.60 (s, 1H), 7.71−7.68 (m, 4H), 7.53−7.48 (m, 1H), 6.99−6.97 (m, 1H), 6.58−6.56 (m, 1H), 2.10 (s, 3H); LC/MS (M+H+): 351.14.</p><!><p>48% yield in 3 steps. LC/MS (M+H+): 345.12.</p><!><p>55% yield in 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.40 (s, br, 1H), 8.87 (s, 1H), 8.40 (s, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.55−7.54 (m, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 6.8 Hz, 2H), 6.85 (d, J = 6.8 Hz, 2H), 3.38 (s, 3H), 3.17 (s, 3H), 2.11 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 159.21, 154.89, 152.28, 147.29, 146.42, 134.02, 132.78, 130.47, 127.57, 127.29, 124.54, 122.05, 114.80, 113.53, 110.66, 55.12, 37.10, 12.67; LC/MS (M+H+): 388.18; HRMS (ESI-Orbitrap) Calcd for C22H22N5O2: 388.1773 [M+H+], Found 388.1764.</p><!><p>18% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 9.59 (s, br, 1H), 8.79 (s, 1H), 8.12 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.43 (s, 1H), 7.32 (d, J = 6.8 Hz, 2H), 6.82 (d, J = 6.8 Hz, 2H), 4.08−4.04 (m, 2H), 3.70 (s, 3H), 3.65−3.64 (m, 2H), 3.37−3.32 (m, 2H), 3.12−3.06 (m, 2H), 2.14 (s, 3H), 2.04−2.02 (m, 2H), 1.90−1.86 (m, 2H); HRMS (ESI-Orbitrap), Calcd for C27H31N6O2: 471.2508 [M+H+], Found 471.2516.</p><!><p>19% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.02 (s, br, 1H), 8.77 (s, 1H), 8.15 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.41 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 4.09−4.06 (m, 2H), 3.70 (s, 3H), 3.56−3.54 (m, 2H), 3.28−3.24 (m, 2H), 2.97−2.91 (m, 2H), 2.14 (s, 3H), 1.85−1.81 (m, 2H), 1.68−1.62 (m, 4H); LC/MS (M+H+): 485.15.</p><!><p>46% yield in 4 steps. 1H NMR (DMSO-d6, 400 MHz) δ 12.04 (s, br, 1H), 9.57 (s, 1H), 8.74 (s, 1H), 8.20 (s, 1H), 7.63−7.55 (m, 4H), 7.53−7.51 (m, 2H), 7.48−7.46 (m, 1H), 7.42−7.39 (m, 2H), 4.04−4.00 (m, 2H), 3.68−3.65 (m, 2H), 3.31−3.29 (m, 2H), 3.08−3.07 (m, 2H), 2.04−2.02 (m, 5H), 1.90−1.87 (m, 2H); LC/MS (M+H+): 441.00.</p><!><p>From commecially available 2-phenylamino-ethanol, 18b was synthesized in 62% yield through 3 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 8.83−8.81 (m, 1H), 8.40 (s, 1H), 7.64−7.58 (m, 4H), 7.47−7.43 (m, 3H), 7.39−7.37 (m, 2H), 7.32−7.29 (m, 1H), 3.76 (t, J = 6.4 Hz, 2H), 3.56 (t, J = 6.4 Hz, 2H), 2.06 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 154.43, 152.14, 146.62, 142.62, 130.30, 129.37, 129.09, 127.78, 127.66, 127.59, 126.52, 119.18, 118.67, 114.56, 111.08, 58.79, 52.24, 12.61; HRMS (ESI-Orbitrap), Calcd for C22H22N5O2: 388.1773 [M+H+], Found 388.1764.</p><!><p>49% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.59 (s, br, 1H), 8.89 (s, 1H), 8.75 (s, 1H), 7.66−7.54 (m, 4H), 7.52−7.50 (m, 1H), 7.42−7.40 (m, 1H), 7.39−7.37 (m, 1H), 7.34−7.26 (m, 2H), 3.72 (t, J = 6.4 Hz, 2H), 3.57 (t, J = 6.4 Hz, 2H), 2.07 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 159.17, 158.37, 156.71, 154.39, 153.62, 152.07, 145.55, 143.00, 130.74, 130.49, 129.81, 128.64, 125.11, 119.20, 116.55, 114.51, 111.76, 58.92, 51.98, 12.47; LC/MS (M+H+): 406.07.</p><!><p>45% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.54 (s, br, 1H), 8.88 (s, 1H), 8.66 (s, 1H), 7.68−7.62 (m, 4H), 7.57 (s, 1H), 7.48−7.43 (m, 1H), 7.32−7.29 (m, 1H), 7.23−7.21 (m, 1H), 7.15−7.10 (m, 1H), 3.79 (t, J = 6.0 Hz, 2H), 3.58 (t, J = 6.0 Hz, 2H), 2.07 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 163.53, 161.11, 154.21, 152.14, 146.34, 144.40, 142.65, 130.67, 130.58, 130.36, 128.04, 123.44, 118.83, 114.75, 114.52, 112.97, 111.26, 58.81, 52.25, 12.58; HRMS (ESI-Orbitrap), Calcd for C22H21FN5O2: 406.1679 [M+H+], Found 406.1686.</p><!><p>41% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.51 (s, br, 1H), 8.86 (s, 1H), 8.37 (s, 1H), 7.66−7.62 (m, 4H), 7.60 (s, 1H), 7.44−7.41 (m, 2H), 7.29−7.25 (m, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.55 (t, J = 6.0 Hz, 2H), 2.06 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 161.76, 159.34, 154.39, 152.13, 146.35, 142.72, 138.65, 130.30, 130.14, 128.01, 118.83, 116.20, 115.98, 114.55, 111.24, 58.70, 52.33, 12.57; LC/MS (M+H+): 406.06; HRMS (ESI-Orbitrap), Calcd for C22H21FN5O2: 406.1679 [M+H+], Found 406.1670.</p><!><p>40% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.70 (s, br, 1H), 8.92 (s, 1H), 8.72 (s, 1H), 7.69−7.63 (m, 5H), 7.53−7.52 (m, 1H), 7.47−7.43 (m, 1H), 7.36−7.33 (m, 1H), 3.78 (t, J = 6.0 Hz, 2H), 3.57 (t, J = 6.0 Hz, 2H), 2.07 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 158.42, 158.07, 154.22, 152.08, 145.63, 144.25, 142.95, 133.22, 130.69, 130.46, 128.58, 127.53, 126.23, 126.19, 118.86, 114.51, 111.70, 58.82, 52.31, 12.49; HRMS (ESI-Orbitrap), Calcd for C22H21ClN5O2: 422.1384 [M+H+], Found 422.1369.</p><!><p>45% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 8.85 (s, 1H), 7.64−7.58 (m, 5H), 7.57−7.56 (m, 1H), 7.52 (s, 1H), 7.47−7.45 (m, 1H), 7.44−7.39 (m, 2H), 3.58 (t, J = 6.0 Hz, 2H), 3.42 (t, J = 6.0 Hz, 2H), 2.06 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 158.28, 157.95, 154.22, 152.12, 146.21, 142.75, 139.25, 132.59, 131.76, 130.35, 130.24, 129.21, 128.36, 128.15, 118.99, 114.53, 111.33, 58.79, 51.67, 12.58; LC/MS (M+H+): 422.06.</p><!><p>48% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.49 (s, br, 1H), 8.87 (s, 1H), 8.56 (s, 1H), 7.66−7.60 (m, 4H), 7.55 (s, 1H), 7.50−7.47 (m, 2H), 7.43−7.40 (m, 2H), 3.75 (t, J = 6.0 Hz, 2H), 3.56 (t, J = 6.0 Hz, 2H), 2.06 (s, 3H); HRMS (ESI-Orbitrap), Calcd for C22H21ClN5O2: 422.1384 [M+H+], Found 422.1375.</p><!><p>45% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.56 (s, br, 1H), 8.88 (s, 1H), 8.18 (s, 1H), 7.65−7.58 (m, 5H), 7.36−7.29 (m, 4H), 3.74 (t, J = 6.0 Hz, 2H), 3.58 (t, J = 6.0 Hz, 2H), 2.22 (s, 3H), 2.06 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 158.40, 158.06, 154.37, 153.84, 152.08, 145.75, 143.02, 136.21, 131.13, 130.43, 129.30, 128.47, 127.64, 127.09, 118.82, 114.50, 111.62, 58.82, 51.71, 17.31, 12.51; LC/MS (M+H+): 402.09.</p><!><p>43% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.70 (s, br, 1H), 8.92 (s, 1H), 8.40 (s, 1H), 7.68−7.61 (m, 5H), 7.35−7.31 (m, 1H), 7.21 (s, 1H), 7.16−7.12 (m, 2H), 3.74 (t, J = 6.4 Hz, 2H), 3.53 (t, J = 6.4 Hz, 2H), 2.35 (s, 3H), 2.07 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 158.11, 154.38, 153.97, 152.09, 145.87, 142.96, 142.34, 138.77, 130.42, 129.16, 128.37, 128.20, 127.32, 124.73, 118.69, 114.50, 111.55, 58.75, 52.20, 20.95, 12.53; LC/MS (M+H+): 402.06.</p><!><p>39% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.60 (s, br, 1H), 8.89 (s, 1H), 8.28 (s, 1H), 7.66−7.62 (m, 4H), 7.60−7.58 (m, 1H), 7.26−7.25 (m, 4H), 3.72 (t, J = 6.0 Hz, 2H), 3.54 (t, J = 6.0 Hz, 2H), 2.34 (s, 3H), 2.07 (s, 3H); LC/MS (M+H+): 402.09; HRMS (ESI-Orbitrap) Calcd for C23H24N5O2: 402.1930 [M+H+], Found 402.1920.</p><!><p>47% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 8.83 (s, 1H), 7.59−7.58 (m, 5H), 7.49−7.48 (m, 1H), 7.38−7.32 (m, 2H), 7.16−7.14 (m, 1H), 7.04−7.00 (m, 1H), 3.80 (s, 3H), 3.62−3.51 (m, 4H), 2.06 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 158.30, 155.25, 154.72, 154.23, 152.10, 146.10, 143.02, 130.49, 130.32, 130.01, 128.97, 128.16, 120.86, 118.67, 114.51, 112.74, 111.39, 58.74, 55.67, 51.22, 12.54; LC/MS (M+H+): 418.07.</p><!><p>46% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.50 (s, br, 1H), 8.86 (s, 1H), 8.39 (s, 1H), 7.66−7.60 (m, 4H), 7.55 (s, 1H), 7.36−7.32 (m, 1H), 6.98−6.97 (m, 1H), 6.93-6.88 (m, 2H), 3.78 (s, 3H), 3.75 (t, J = 6.0 Hz, 2H), 3.56 (t, J = 6.0 Hz, 2H), 2.07 (s, 3H); 13C-NMR (DMSO-d6, 100 MHz) δ 159.95, 158.32, 158.10, 154.26, 152.11, 146.22, 143.55, 142.78, 130.36, 130.05, 128.11, 119.77, 118.71, 114.53, 113.57, 112.22, 111.33, 58.75, 55.18, 52.19, 12.57; LC/MS (M+H+): 418.05.</p><!><p>48% yield in 5 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.56 (s, br, 1H), 8.88 (s, 1H), 8.13 (s, 1H), 7.66−7.59 (m, 5H), 7.30 (d, J = 6.8 Hz, 2H), 7.01 (d, J = 6.8 Hz, 2H), 3.80 (s, 3H), 3.69 (t, J = 6.4 Hz, 2H), 3.53 (t, J = 6.4 Hz, 2H), 2.06 (s, 3H); HRMS (ESI-Orbitrap), Calcd for C23H24N5O3: 418.1879 [M+H+], Found 418.1686.</p><!><p>52% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.64 (s, 1H), 8.86 (s, 1H), 8.35 (s, 1H), 7.82 (s, 2H), 7.59 (dt, J = 13.0, 6.5 Hz, 5H), 7.53 − 7.43 (m, 4H), 3.85 (t, J = 6.2 Hz, 2H), 2.93 − 2.82 (m, 2H), 2.00 (dd, J = 8.4, 2.5 Hz, 3H). LC/MS (M+H+): 415.11.</p><!><p>45% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.60 (s, 1H), 8.89 (s, 1H), 8.06 (s, 1H), 7.87 (s, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.8 Hz, 2H), 7.58 (s, 1H), 7.45 − 7.39 (m, 2H), 7.10 − 7.04 (m, 2H), 3.87 (t, J = 6.2 Hz, 2H), 3.81 (d, J = 9.0 Hz, 3H), 2.99 − 2.88 (m, 2H), 2.06 (d, J = 0.9 Hz, 3H). HRMS (ESI-Orbitrap), Calcd for C22H22ClN5O: 421.1544 [M+H+], Found 421.1563.</p><!><p>50% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.55 (s, 1H), 9.42 (s, 1H), 8.88 (s, 1H), 8.10 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.8 Hz, 2H), 7.57 (s, 1H), 7.45 − 7.38 (m, 2H), 7.11 − 7.03 (m, 2H), 3.98 (t, J = 6.2 Hz, 2H), 3.82 (s, 3H), 3.20 (d, J = 5.2 Hz, 2H), 2.88 (t, J = 8.5 Hz, 6H), 2.05 (d, J = 0.9 Hz, 3H). LC/MS (ESI-Orbitrap), Found 445.21.</p><!><p>65% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.70 (s, 1H), 9.52 (s, 1H), 8.93 (s, 1H), 8.48 (s, 0H), 7.67 (dd, J = 23.4, 8.8 Hz, 4H), 7.62 − 7.49 (m, 5H), 4.04 (t, J = 6.2 Hz, 2H), 3.21 (d, J = 4.7 Hz, 2H), 2.89 (d, J = 3.5 Hz, 6H), 2.06 (d, J = 0.7 Hz, 3H). LC/MS (ESI-Orbitrap), Found 449.21.</p><!><p>52% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.67 (s, 1H), 8.84 (s, 1H), 8.54 (s, 1H), 7.93 (t, J = 8.3 Hz, 1H), 7.58 (dd, J = 11.4, 1.9 Hz, 2H), 7.54 − 7.40 (m, 4H), 3.80 − 3.76 (m, 4H), 2.40 (d, J = 10.5 Hz, 3H), 1.93 (s, 3H). HRMS (ESI-Orbitrap) Calcd for C23H21ClFN5O2: 454.1446 [M+H+], Found 454.1434.</p><!><p>52% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.30 (s, 1H), 8.76 (s, 1H), 7.85 (d, J = 9.0 Hz, 3H), 7.80 (d, J = 8.6 Hz, 2H), 7.63 − 7.56 (m, 4H), 7.51 (dd, J = 11.4, 1.8 Hz, 1H), 7.45 (dd, J = 8.3, 1.7 Hz, 1H), 3.91 (t, J = 6.3 Hz, 2H), 3.10 (qd, J = 7.3, 4.8 Hz, 3H), 2.95 (dd, J = 11.9, 6.0 Hz, 2H), 2.37 (s, 3H), 1.92 (d, J = 4.5 Hz, 3H), 1.18 (t, J = 7.3 Hz, 4H). LC/MS (M+H+): 453.14.</p><!><p>54% yield in 4 steps. 1H-NMR (DMSO-d6, 400 MHz) δ 12.38 (s, 1H), 9.33 (s, 1H), 8.76 (s, 1H), 7.86 (s, 1H), 7.78 (t, J = 8.2 Hz, 1H), 7.66 − 7.57 (m, 2H), 7.57 − 7.42 (m, 3H), 4.02 (t, J = 6.3 Hz, 2H), 3.21 (s, 2H), 2.86 (s, 6H), 2.36 (s, 3H), 1.91 (s, 3H). HRMS (ESI-Orbitrap), Calcd for C25H26ClFN6O: 481.1919 [M+H+], Found 481.1909.</p><!><p>50% yield in 4 steps. 1H-NMR (400 MHz, DMSO) δ 12.63 (s, 1H), 8.83 (s, 1H), 8.54 (s, 1H), 7.86 (t, J = 8.2 Hz, 1H), 7.68 − 7.29 (m, 6H), 3.94 − 3.84 (m, 4H), 3.66 (s, 3H), 2.39 (s, 3H), 1.93 (s, 3H). LC/MS (ESI-Orbitrap), Found 468.14.</p><!><p>Inhibitor 18b was prepared for glide docking using LigPrep (Schrodinger, LLC, NY). The chain A of PDB ID 3S95 was prepared using protein preparation wizard in Maestro V 9.8 (Schrodinger, LLC, NY) by removing water molecules and bound ligand, and adding hydrogen atoms. The docking grid was generated around the original ligand with a box size of 18 × 18 × 18 Å3. Docking was conducted without any constraint. The top scored docking pose was merged to the protein for energy minimization using Prime (Schrodinger, LLC, NY).</p><!><p>Biochemical assays for all Limk inhibitors and kinase profiling were carried out in Reaction Biology Corporation and followed the protocols described on its website. Compounds were tested in 10-dose IC50 mode with 3-fold series dilution starting at 10 μM for IC50 measurements. Compounds were tested at 1 μM with duplicate experiments in profiling assays. Control compound Staurosporine was tested in 10-dose IC50 mode with 3-fold serial dilution starting at 10 μM. Reactions were carried out at 10 μM ATP, 1 μM substrate cofilin, and 50 nM Limk1 (final concentrations).</p><!><p>A7r5 (15,000 cells/well) were plated in a clear-bottomed Packard View black 96-well plate in 100 μl of 10% FBS DMEM:F12 medium and were allowed to attach overnight. The next day, the cells were serum starved in 1% FBS DMEM medium for 2 hr and then treated with the compounds for 1 hr. Cells were then fixed in 4% paraformaldehyde in PBS for 20 min at room temperature (RT) with no shaking. They were then washed once with 0.1 M glycine to neutralize paraformaldehyde for 5 min. Cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min at RT on orbital shaker after which they were washed once with PBS for 5 min. They were then incubated with Licor Blocking Buffer in PBS (1:1 dilution in PBS) for 1–1.5 h rocking at RT. Cells were incubated with primary antibody p-cofilin Ab (Cell Signaling # 3311) 1:100 dilution in Licor blocking buffer overnight at 4°C. Next day, they were washed twice with PBS-0.1 % Tween 20 (PBST) washing solution for 5 min each at room temperature on the orbital shaker, followed by one wash with Licor Blocking Buffer containing 0.05% Tween-20 for 5 min on the shaker at RT. The cells were then incubated with secondary antibody goat anti-rabbit IR800 (1:500 dilution) for 1 hr at RT in the dark (covered the plate with foil) in Licor blocking buffer-containing Tween-20. Following this, cells were washed twice with PBST for 5 min each at room temperature and then once with Licor blocking buffer-containing 0.05% Tween-20. The wells were then incubated with ToPro 3 stain (nucleic acid staining), diluted 1:4000 in Licor blocking buffer or Licor blocking buffer with 0.05% Tween-20 for 30 min at room temperature in the dark. Finally the plates were washed twice with PBS and analyzed using the Odyssey LICOR Infrared Scanner.</p><!><p>PC3 cells were cultured at a density of 0.5 × 106 cells/mL in 60mm culture dishes in 10% FBS RPMI1640 media. Then, the cells were treated with DMSO and the indicated concentration of Limk inhibitors. After incubating for 24 h, the cells were rinsed with ice-cold PBS twice and collected by spinning down at 4°C in 10,000 rpm for 5 min. Cellular lysates were prepared by suspending cells in SDS sample buffer, 120 mmol/L Tris, 4% SDS, 20% glycerol, 0.1 mg/mL bromophenol blue, and 100 mmol/L DTT (pH 6.8). After brief sonication, the lysates were heated at 95°C for 5 minutes. The cell lysates were separated by 12% SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corp.). Immunostaining was done using antibodies specific for phospho-Cofilin (Cell Signaling, #3313) and and β-Actin (GeneScript, #A00702) antibodies and the corresponding second antibodies for whole immunoglobulins from mouse or rabbit (Amersham Biosciences). Immunoreactive proteins were detected by chemoluminescence using the Pierce ECL Western Blotting Substrate (Thermo Scientific). We quantified the actual levels of proteins by using the Multigauge ver 3.0 software (Fujifilm). The gels were stained with Coomassie Brilliant Blue R-250 (0.25%) for 1 hour and then destained (all solutions from Bio-Rad) to check the loading amount of protein samples on the gels.</p><!><p>CEM-SS T cells (1.0 × 106) were treated with a Limk inhibitor at 10 μM and 1 μM separately at 37°C for 4 h. Cells were lysed in NuPAGE LDS Sample Buffer (Invitrogen) followed by sonication. Samples were heated at 90°C for 10 minutes, separated by SDS-PAGE, and then transferred onto nitrocellulose membranes (Invitrogen). The membranes were washed in TBST for 3 minutes and then blocked for 30 minutes at room temperature with Starting Block blocking buffer (Pierce). The blots were incubated with a rabbit anti-phospho cofilin (ser3) antibody (1:500 dilution) (Cell Signaling) diluted in 2.5% milk-TBST and rocked overnight at 4°C. The blots were washed three times for 15 minutes, then incubated with goat anti-rabbit 800cw labeled antibodies (Li-cor Biosciences) (1:5000 diluted in blocking buffer) for 1 h at 4°C. The blots were washed three times for 15 minutes and scanned with Odyssey Infrared Imager (Li-cor Biosciences). The same blots were also stripped and reprobed with antibodies against GAPDH (Abcam) as a loading control.</p><!><p>Transwell chambers coated with GFR Matrigel (BD Biosciences) were used for measurement of cell invasion. The matrigel was solidified at 37°C in a humidified incubator the day before the assay. PC3 cells (1×103 cells per well) were grown in serum-free RPMI1640 media in the upper side of the insert. The lower well was filled with RPMI 1640 supplemented with 10% FBS with a Limk inhibitor (1 μM). PC3 cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 48 h. Then transwell membrane was rinsed three times with PBS, and the cells were fixed in 2.5% EM grade glutaraldehyde for 15 min at room temperature (RT) with no shaking. After removing glutaraldehyde by aspirating, the cells were permeabilized with 0.5% Triton X-100 in PBS for 3 min at RT, and they were rinsed three times with PBS. Then, the cells were stained by Gill's hematoxylin No.1 for 15 min at RT, and they were washed by distilled water for three times. To remove any residual stain, the cells were washed by acid alcohol for 3 minutes and then rinsed by distilled water twice. The cells were exposed with 0.04% NH4OH until a blue color is observed on the membrane and then rinsed by distilled water twice. The membranes were dried overnight, and the stained cells were visualized under Leica DMI3000B microscope.</p><!><p>PC3 cells were cultured to confluence at >90% in 6 well culture dishes in 10% FBS RPMI1640 media the day before the assay. Lines were drawn with a marker on the bottom of the dishes. Using a sterile 1 mL pipet tip, the dishes were scratched three separate wounds through the cells moving perpendicular to the line drawn in the step above. The cells were rinsed with PBS twice very gently and added the RPMI1640 media, and the dishes were taken pictures using phase contrast under Leica DMI3000B microscope. Then, the cells were treated with the indicated concentration of a Limk Inhibitor and incubated for 24 h. The dishes were taken pictures to detect closed wound area as describe above, and the closed wound area was analyzed by ImageJ software (Ver 1.48).</p><!><p>All in vitro (microsomal stability, CYP-450 inhibition, etc.) and in vivo pharmacokinetics studies were carried in the DMPK Core Facility of Scripps Florida. Detailed procedures for these assays have been described in previous publications (ref. 41–42, 46, 48–50, and 52–55), and were also provided in Supporting Information.</p>

PubMed Author Manuscript

Discovery of 1,2,4-thiadiazolidine-3,5-dione analogs that exhibit unusual and selective rapid cell death kinetics against acute myelogenous leukemia cells in culture

4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) was previously identified as an antileukemic agent exhibiting no evident toxicity toward normal hematopoietic cells. An SAR study has been carried out to examine the effect of varying the C-2 and C-4-substituents on the thiadiazolidinone ring of TDZD-8 on anti-leukemic activity. These studies resulted in the identification of more druglike analogs that exhibited comparable potency to TDZD-8 in killing acute myelogenous leukemia (AML) cells in culture. Surprisingly, the cell death kinetics induced by several of these novel analogs on MV-411 cells were extremely fast, with commitment to death occurring within 30 minutes. At a concentration of 10 \xce\xbcM, 3f (LD50=3.5 \xce\xbcM) completely eradicated cell viability of MV-411 cells within 2 hours, while analog 3e (LD50=2.0 \xce\xbcM) decimated cell viability within 30 min at a concentration of 10 \xce\xbcM and effectively abolished cell viability at 5 \xce\xbcM within 1-2 hours.

discovery_of_1,2,4-thiadiazolidine-3,5-dione_analogs_that_exhibit_unusual_and_selective_rapid_cell_d

<p>Acute myelogenous leukemia (AML) is a hematologic malignancy characterized by the abnormal accumulation of immature white blood cells in the bone marrow (BM), and a poor overall survival rate; 12,330 people were diagnosed with AML in 2010.1 AML is thought to originate and be maintained from a malignant population of cells known as leukemia stem cells (LSCs). It has been shown that LSCs are not effectively eliminated by standard chemotherapy regimens and are likely to result in disease relapse.2 Thus, the development of clinically-viable small-molecule chemotherapeutics that eradicate such stem cells has become a high priority.</p><p>TDZD-8 was first identified as a highly selective inhibitor of glycogen synthase kinase 3β (GSK3β);3a-e We have recently demonstrated that 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) displays unique antileukemic properties.4 It was found that TDZD-8 possesses the ability to eradicate acute and chronic, lymphoid and myeloid forms of leukemia, including the LSC populations, at concentrations of about 20 μM with rapid kinetics (i.e. within two hours of exposure). In this concentration range, TDZD-8 possesses insignificant toxicity toward normal hematopoietic stem cells. The toxicity of TDZD-8 is also specific toward hematologic malignancies, since it was found not to be toxic toward a broad swath of other cancer cell lines in the NCI 60 anticancer screen.4 The precise antileukemic mechanism of action of TDZD-8 is not completely understood and likely involves multiple cellular targets; TDZD-8 also appears to rapidly disrupt membrane integrity and deplete free cellular thiols. The latter effect is not surprising, since we have noted in our own synthetic studies that the 1,2,4-thiadiazolidine-3,5-dione (TDZD) ring is capable of oxidizing both P(III) and Pd(0).5</p><p>The current study represents our first efforts identifying TDZD analogs which conserve or improve upon the novel selectivity and cytotoxicity of TDZD-8 toward AML cells, and that also represent more water-soluble molecules compared to the parent compound, TDZD-8. In this report, we have focused on determining the effect of varying the N-2 substituent of TDZD-8 on antileukemic activity in MV-411 cells in culture. In addition, we have investigated structural modifications that introduce a water solubilizing group into the 4-benzyl moiety of the most potent TDZD analogues in order to improve druglikeness.</p><p>The oxidative condensation of isothiocyanates with isocyanates via chlorine addition has been the subject of recent reports.3b,c Classically, 1,2,4-thiadiazolidine-3,5-dione analogs are synthesized through oxidative condensation of isothiocyanates with isocyanates in the presence of gaseous chlorine as the oxidizing agent. In our hands, we found that the introduction of chlorine as a solution in CCl4/diethyl ether was more reliable and more controllable, in terms of stoichiometry (Scheme 1, Method 1), than the treatment of a mixture of isothiocyanate and isocyanate with chlorine gas. We have also utilized SO2Cl2 in diethyl ether as an alternative reported method of preparation of 1,2,4-thiadiazolidine-3,5-diones analogs5 (Scheme 1, Method 2). Recently, we have reported that N-chlorosuccinimide is a safe and viable alternative to the use of chlorine gas in the synthesis of 1,2,4-thiadiazolidine-3,5-diones analogs (Scheme 1, Method 3).6 The TDZD analogs 3a-3f described herein were synthesized from readily available starting materials utilizing a combination of the three methodologies outlined in Scheme 1.</p><p>Despite promising in vitro antileukemic activity (Table 1), TDZD analogs 3a-3f generally had poor water-solubility. Thus, in another structural iteration, we examined the effect of introducing ionizable hydrophilic groups, such as NH2 and COOH, into the TDZD molcule to improve water-solubility. Five analogs, 10b and 10d-10g, that incorporated a protonatable aminomethyl moiety at either C4 or C3 of the phenyl ring were synthesized via the route illustrated in Scheme 2A. The appropriate Boc-protected aminomethylbenzoic acid 5 was converted to the corresponding benzylamine analog 6, utilizing standard synthetic procedures. Intermediate 6 was then converted into the corresponding benzylisothiocyanate 7 utilizing a modification of the procedure reported by Wong and Dolman7. In this procedure, a triethylammonium thiocarbamate is formed through treatment of the primary amine with 1.0 equivalent of CS2 and triethylamine in anhydrous THF, followed by addition of tosyl chloride with stirring for 3 hours. While following this protocol we experienced difficulty in obtaining a practical yield of the desired isothiocyanate. The reason was traced to the insolubility of the amine in THF, which leads to a very sluggish formation of the intermediate thiocarbamate. The yield improved dramatically when additional equivalents of CS2 were added. Consequently, we conducted the entire reaction in a 10:1 mixture of CH2Cl2/CS2 (10 mL/mmole), which resulted in instantaneous conversion of the primary amine to the thiocarbamic acid (an insoluble product). Subsequent addition of triethylamine caused the dissolution of the precipitated thiocarbamic acid. After cooling the resulting solution to 0 °C, tosyl chloride (1 equivalent) was added in one portion, and the reaction mixture worked up to afford the isothiocyanate in good yield. Reaction of 7 with the requisite isocyanate 2, utilizing Method 3 conditions (Scheme 1), afforded the corresponding Boc-protected aminomethyl TDZD analogs. The TDZD ring in the five resulting Boc-protected aminomethyl analogs was found to be stable to 4N HCl in dioxane and thus amenable to N-deprotection and HCl salt formation to afford 10b and 10d-10g.</p><p>For the synthesis of the 4-carboxylate analog 15 (Scheme 2B), the precursor tert-butyl-protected isothiocyanate 14 was synthesized from tert-butyl-4-cyanobenzoate (12)8, which was converted to tert-butyl-4-(aminomethyl)-benzoate (13), as reported by Andrew et al.9, and then subsequently converted to 14 with CS2/CH2Cl2. Reaction of 14 with isocyanate 2e utilizing Method 3 conditions (Scheme 1), followed by deprotection and sodium salt formation afforded 15 (Scheme 2B). NMR and mass spectral characterization data for all TDZD analogs are provided in the reference section10.</p><p>Cytotoxicity data (LD50) for compounds 3e-3f against cultured MV-411 cells11 are shown in Table 1 and are compared to TDZD-8 (3a). Generally, increasing the size of the N-2 substituent resulted in compounds with lower potency (3b-3d) compared to TDZD-8. However, the presence of an N-2-(2-halogenoethyl) substituent (3e; LD50 = 2.0 μM, and 3f; LD50 = 3.5 μM) afforded compounds with comparable LD50 values to TDZD-8 (3a; LD50 = 4.0 μM). Evaluation of the more water soluble analogs 10b, 10d-10g, and 15, also afforded several compounds with comparable LD50 values to TDZD-8, indicating that a water solubilizing amino moiety can be introduced into the aromatic ring of the active analogs 3e and 3f without compromising potency. Thus, the HCl salt of the N-2-(2-chloroethyl) and N-2-(2-bromoethyl) analogs 10e and 10f were found to have comparable potency (LD50 = 3.0 μM and 4.5 μM, respectively) to 3e, 3f and TDZD-8 but had improved hydrophilicity and druglikeness. Moving the aminomethyl moiety of 10e from the para-position to the meta-position on the phenyl ring to afford analog 10g had little effect on the LD50 values for these two regiospecific isomers. Unfortunately, introducing a water-solubilizing carboxylate group at the C4 position of the phenyl ring of 3e to afford 15 resulted in loss of antileukemic activity.</p><p>As with TDZD-8, none of the active TDZD analogs synthesized in this study was toxic to normal hematopoietic cells. In addition, it was previously observed that the cell death kinetics induced by TDZD-8 on MV-411 cells was extremely fast2, with commitment to death occurring within several hours. We similarly measured the kinetics of cell death caused by TDZD-8 and analogs 3d, 3e, and 3f. Figure 1 shows the time-course to determine the cell-death kinetics of these four compounds. At a concentration of 20 μM, TDZD-8 abolished MV-411 cell viability over a period of 2 hours. At a concentration of 5 μM, the activity of analog 3d was negligible, while at a concentration of 10 μM, the analog caused halving of percentage cell viability over 24 hours. However, at a concentration of 20 μM, 3d caused a 50% decrease in cell viability within 30 minutes, and effectively abolished viability over a period of 2 hours. Analog 3f caused a 50 percent decrease in cell viability within the first 30 min at a concentration of 5 μM. However, the effect was not sustained, and cell viability was retained, even at 2.5 μM after 24 hours. At a concentration of 10 μM, 3f completely eradicated cell viability within the first 2 hours. In contrast, analog 3e decimated cell viability within 30 min at a concentration of 10 μM and effectively abolished cell viability at 5 μM within 1-2 hours, and caused halving of percentage cell viability over 24 hours at a concentration of 2.5 μM.</p><p>From these results, it is clear that determination of the LD50 parameter alone provides an estimation of only the antileukemic effect of the above TDZD analogs in total populations of AML cells using MV4-11 cultures. However, the mechanism of action of each of the TDZD analogs synthesized in this study may be different. It is known that the parent compound, TDZD-8, exhibits several intracellular activities such as oxidation of cellular thiols, disruption of membrane integrity, and inhibition of protein kinases. Differential abilities of the TDZD analogs to act at different sites of action inside the leukemic cell may be responsible for the observed inconsistencies between the LD50 values and the cell death kinetics experiments. One possible mechanism for the antileukemic action of the TDZD analogs 3e, 3f, 10e, 10f, may be nonspecific chemical reactivity, such as the alkylative ability of the N-2 halogenoalkyl substituted analogs, or the aforementioned oxidative potential of the TDZD ring itself. However, our experiments show that these TDZD analogs have negligible cytotoxicity toward normal hematopoietic cells or other tumor cell types, and analog 15, which is also an N-2-(2-chloroethyl) analog, is inactive as an antileukemic agent. This suggests that alkylative or oxidative reactivity is unlikely as a mechanism of antileukemic action for these TDZD analogs, and that their mechanism of inhibition is not related to nonspecific inhibition of rapidly dividing cells.</p><p>This study has also provided useful information about the importance of the N-2-substituent in the TDZD-8 molecule for antileukemic activity. Sites for attachment of hydrophilic groups with retention of pharmacological properties have also been identified, leading the way for the the development of more druglike TDZD analogs. Future studies can now focus on a more thorough pharmacological and toxicological evaluation of these analogs. Lastly, the positive, as well as the negative SAR trends, resulting from this study will serve as a guide for further optimization of this unique series of compounds, not only with regard to potency and selectivity, but also for achieving optimal pharmacokinetics and drugability.</p>

PubMed Author Manuscript

C\xe2\x80\x93H Bonds as Ubiquitous Functionality: A General Approach to Complex Arylated Imidazoles via Regioselective Sequential Arylation of All Three C\xe2\x80\x93H Bonds and Regioselective N-Alkylation Enabled by SEM-Group Transposition

Imidazoles are an important group of the azole family of heterocycles frequently found in pharmaceuticals, drug candidates, ligands for transition metal catalysts, and other molecular functional materials. Owing to their wide application in academia and industry, new methods and strategies for the generation of functionalized imidazole derivatives are in demand. We here describe a general and comprehensive approach for the synthesis of complex aryl imidazoles, where all three C\xe2\x80\x93H bonds of the imidazole core can be arylated in a regioselective and sequential manner. We report new catalytic methods for selective C5- and C2-arylation of SEM-imidazoles and provide a mechanistic hypothesis for the observed positional selectivity based on electronic properties of C\xe2\x80\x93H bonds and the heterocyclic ring. Importantly, aryl bromides and low-cost aryl chlorides can be used as arene donors under practical laboratory conditions. To circumvent the low reactivity of the C-4 position, we developed the SEM-switch that transfers the SEM-group from N-1 to N-3 nitrogen and thus enables preparation of 4-arylimidazoles and sequential C4\xe2\x80\x93C5-arylation of the imidazole core. Furthermore, selective N3-alkylation followed by the SEM-group deprotection (trans-N-alkylation) allows for regioselective N-alkylation of complex imidazoles. The sequential C-arylation enabled by the SEM-switch allowed us to produce a variety of mono-, di-, and triarylimidazoles using diverse bromo- and chloroarenes. Using our approach, the synthesis of individual compounds or libraries of analogues can begin from either the parent imidazole or a substituted imidazole, providing rapid access to complex imidazole structures.

c\xe2\x80\x93h_bonds_as_ubiquitous_functionality:_a_general_approach_to_complex_arylated_imidazoles_

Introduction<!>Regioselectivity of the Palladium-Catalyzed Arylation of Imidazoles<!>Mechanistic Hypothesis for Positional Selectivity: Electronic Properties of C\xe2\x80\x93H Bonds and the Heteroarene Ring<!>Development of C5-Arylation of SEM-Protected Imidazoles<!>Development of New Conditions for C2-Arylation of Imidazoles<!>Synthesis of 4-Aryl-1-SEM-imidazoles and 4,5-Diarylimidazoles via the SEM-Switch<!>Synthesis of 2,4,5-Triarylimidazoles via Sequential C-Arylation and the SEM-Switch<!>Synthesis of 2-Alkyl- and 2-Dialkylamino-Substituted 4,5-Diarylimidazoles<!>Synthesis of 1-Alkyl-4-arylimidazoles from 1-SEM-5-Arylimidazoles via Trans-N-alkylation<!>Programmable Synthesis of Complex 1-Alkyl-triarylimidazoles<!>Conclusions<!>General Procedure for the C2-Arylation of SEM-Imidazoles<!>2-(3-Methoxyphenyl)-5-phenyl-1-((2-(trimethylsilyl)ethoxy)-methyl)-1H-imidazole (13)<!><!>Synthesis of 4,5-Diarylimidazolesa<!><!>Synthesis of 1-Methyl-2,4,5-triarylimidazoles via Sequential C-Arylation and N-Methylationa<!>

<p>Imidazoles are essential components of biologically active compounds, including natural products and synthetics, and display a broad range of biological activities1 (for example, antibacterial,2 anticancer,3 anti-inflammatory activity4). The imidazole ring serves as a rigid scaffold for the presentation of attached substituents in a fixed spatial orientation, creating desirable motifs for high affinity protein ligands. For example, di-, tri-, and tetra-substituted imidazoles have recently emerged as potent kinase inhibitors.5 Also, 2-arylimidazoles were found to be selective ligands for histamine receptors6 and 1,5-diarylimidazoles to display vascular disrupting activity.7 Moreover, imidazole-based N-heterocyclic carbenes have been actively pursued as transition metal ligands for the development of new catalysts,8 and imidazolium ionic liquids are used as recyclable solvents for industrial catalytic processes, affording "green" alternatives to standard organic solvents.9 The wide use of imidazoles generated a considerable interest in imidazole chemistry and revealed the need for more efficient synthetic strategies.10</p><p>There are a number of established de novo methods for the synthesis of substituted imidazoles where the imidazole ring is constructed via cyclo-condensation reactions. Although these traditional approaches have been greatly improved over the past decade, each method has its scope and efficiency limitations.11,12 Often, the condensation methods are inefficient for the assembly of series of compounds: for example, regioisomers (2,4-versus 4,5-substitution pattern) or focused analogues (different arene rings in the 4-position). In most cases, the synthesis of each analogue of the library will require the entire de novo synthetic sequence, which translates to parallel repetition of linear synthetic sequences.</p><p>To address this problem, in part of a broad program dedicated to development of new synthetic methods and strategies based on C–H bond functionalization,13,14 we have been developing catalytic arylation transformations, where multiple C–H bonds of heteroarenes are functionalized in a selective and sequential manner (topologically obvious synthesis).13,15 We have recently reported catalytic arylation of pyrazoles and a synthetic strategy based on SEM-group transposition that enables sequential arylation and preparation of complex aryl pyrazoles.15a We here report a general and comprehensive strategy for the preparation of highly functionalized aryl imidazoles through direct arylation of the imidazole core (Figure 1).16–18 All three C–H bonds of the imidazole ring can selectively and sequentially be replaced by arene rings using aryl bromides or aryl chlorides, and the amino group can be alkylated in a regioselective manner, providing rapid access to all regioisomers of mono-, di-, and triarylimidazoles.19–21</p><p>Guided by the general reactivity of imidazoles, we developed Pd-catalyzed regioselective C5- and C2-arylation protocols together with the SEM-switch and trans-N-alkylation. Figure 2 illustrates some of many possible synthetic pathways. Schematically, C5-arylation provides compound I, which can be arylated at the 2-position to give compound II, a protected 2,5-diarylimidazole. This compound could alternatively be prepared by C2-arylation, followed by C5-arylation (not shown), and depending on the specific structural context, one sequence may provide better yield than the other. The subsequent arylation of the 4-position in II is low yielding (<10%); therefore the unreactive C-4 position is converted to a reactive C-5 position via the SEM-switch, and subsequent arylation affords IV, a protected 2,4,5-triarylimidazole. This compound could be deprotected or converted to V via selective trans-N-alkylation (Figure 2A). Note that all substituents of the imidazole ring are introduced in a regioselective manner. Another possibility involves trans-N-alkylation of compound II to furnish VI, a 2,4-diaryl-1-alkylimidazole, which can be further arylated to give VII, a regioisomer of V. This latter pathway affords access to 2,4-diaryl-1-alkylimidazoles and would be the route of choice for preparation of 2,4,5-triarylimidazoles in cases where the arene ring Ar3 is not compatible with trans-N-alkylation.</p><p>We also wish to point out that the C5- and C2-arylation methods and the strategy based on the SEM-switch described in this paper are applicable to arylation and N-alkylation of advanced imidazole intermediates with various substitution patterns and substituents other than arene rings. For example, as illustrated in Figure 2B, compound VIII, a 2,4(5)-disubstituted imidazole, can be protected in a regioselective manner to give compound IX, which can be arylated in the 5-position and alkylated to furnish product XI. Both the aryl group and the alkyl group are introduced in a regioselective manner in three steps. Thus, complex aryl imidazoles can be prepared from either the parent imidazole (which is readily available and inexpensive) or substituted imidazole intermediates.</p><!><p>The general reactivity trends of imidazole are shown in Figure 3A: the C-5 position shows high reactivity toward electrophilic substitution, the C-4 position is relatively less reactive in this respect, and the C-2 position bears the most acidic C–H bond. We have previously introduced the SEM group [SEM=2-(trimethylsilyl)ethoxymethyl] as a suitable protecting group for azoles, including indoles, pyrroles, pyrazoles, and imidazoles, in the context of palladium-catalyzed C–H arylation.15a,c In this work, we developed new and practical protocols for C5- and C2-arylation of SEM-imidazoles using both aryl bromides and aryl chlorides as the arene donors (Figure 3B). With the parent SEM-imidazole, the optimized palladium protocol for C5-arylation afforded the desired product 2 in good yield along with 2,5-diphenyl-1-SEM-imidazole, in 7:1 ratio (Figure 3B). Careful examination of the reaction conditions revealed a critical role of base in the C2-arylation. Employing the stronger base sodium tert-butoxide in a nonpolar solvent, we developed a new protocol for C2-arylation of SEM-imidazoles that shows good selectivity for the 2-position (6:1, Figure 3B). To overcome the low reactivity of the 4-position, we developed the SEM-group transposition ("SEM-switch"), which leads to the rearrangement of the SEM-group from the N-1 to N-3 nitrogen. 4-Phenyl-1-SEM-imidazole can be accessed in one step by the SEM-switch (compound 2 → 5, Figure 3C). Similarly, alkylation of N-3 nitrogen and SEM-group deprotection provides 4-phenyl-1-methylimidazole ("trans-N-alkylation", compound 2 → 6, Figure 3C).</p><!><p>The C5-arylation of imidazoles may occur via electrophilic metalation-deprotonation (EMD) or concerted metalation-deprotonation (CMD), as illustrated in Figure 4A. In both mechanisms, the carboxylate or carbonate ligand is directly involved in the intramolecular deprotonation. The EMD mechanism is a step-wise metalation process similar to SEAr, while CMD is a concerted one-step process. In our previous work, we have demonstrated the importance of the carboxylate ligand both in the palladium-catalyzed15b,c,f and rhodium-catalyzed arylation15e of electron-rich heteroarenes. We have also provided kinetic evidence for the direct involvement of the pivalate ligand in the C–H activation step.15e With triarylphosphine ligands (relatively weak σ-donors), our data pointed toward the carboxylate-assisted EMD mechanism.15e,f Alternatively, other groups provided experimental and computational evidence for the CMD mechanism for the catalytic systems using strong σ-donor phosphines, where formation of the carbon–metal bond and breaking of the carbon–hydrogen bond take place simultaneously, while maintaining the aromaticity of arenes or heteroarenes.22</p><p>The C5 selectivity can readily be explained by EMD as the 5-position is more nucleophilic than the 2- and 4-positions; standard electrophilic aromatic substitutions occur preferentially at C5. To further rationalize the high selectivity for the C-5 position, we propose that the inductive effect of the N-1 nitrogen stabilizes the carbon–palladium bond at C5 (and the partially formed carbon–palladium bond in the transition state), and thus the C5-metalation is preferred considering both thermodynamic and kinetic factors (Figure 4A). In contrast, metalation of the C-2 and C-4 positions is disfavored by the electronic repulsion between the electron lone pair on the N-3 and the carbon–palladium bond, which may be particularly important in the CMD mechanism (Figure 4B).</p><p>In the presence of a strong base (NaOt-Bu), deprotonation of the C-2 position becomes efficient, presumably facilitated by complexation of the palladium complex to the N-3 nitrogen (Figure 4C). The inductive effect of two nitrogen atoms renders the C–H bond most electron-deficient and most acidic. The C2-nucleophile subsequently displaces either a halide or tert-butanol at the palladium metal to form the C2-palladium intermediate, eventually leading to the C2-arylated product.</p><p>The electronic rationale provided above explains the low reactivity of C-4; arylation of the C-4 position gives low yields even under forcing conditions when C-2 and C-5 positions are substituted (<10% yield). The C-5 to C-2 selectivity of the Pd/carbonate method is moderate, and so is the C-2 to C-5 selectivity of the Pd/NaOt-Bu system, which is attributed to relatively high reactivity of both positions (that are adjacent to the substituted nitrogen N-1) under Pd/base conditions.</p><!><p>We have previously introduced the air- and moisture-stable mixed phosphine-NHC palladium complexes, exemplified by 7 (Table 1), as a new class of catalysts for the arylation of heteroarenes and reported selective C5-arylation of 1-SEM-imidazole 1 with iodobenzene using complex 7 as the catalyst.15c We here show that a wide range of aryl donors could be employed for the arylation of SEM-imidazole (Table 1, Method A). However, this method is not compatible with chloroarene donors and the catalyst requires a multistep synthesis.</p><p>In the hope of identifying a practical and versatile catalytic procedure, we thoroughly examined the reaction parameters including metal catalyst, ligand, base, solvent, and temperature. This search found that the treatment of SEM-imidazole 1 with 5.0 mol % Pd(OAc)2, 7.5 mol %P(n-Bu)Ad2, 1.2 equiv of PhBr, and 2.0 equiv of K2CO3 in DMA (0.5 M) at 120 °C led to full conversion of the starting material, giving the desired product 2 in 72% yield along with the 2,5-diarylation product 3 in 11% yield (Table 1). Unlike our catalytic method for pyrazole C-arylation, where potassium carbonate and a substoichiometric quantity of pivalic acid (or potassium pivalate) were used,15a potassium carbonate alone proved to be an optimum base for the coupling of SEM-imidazole and bromobenzene. When we added 0.25 equiv of pivalic acid in conjunction with 3.0 equiv of potassium carbonate, a significant amount of the overarylation product 3 (21% isolated yield) was formed in addition to the monoarylation product 2 (56% yield). While the use of the carboxylic acid additive improves the performance of many palladium-catalyzed C-arylations of arenes and heteroarenes, the highly active catalytic system can decrease the regioselectivity of this reaction in cases where the substrate contains multiple C–H bonds susceptible to C–H arylation.23</p><p>Superior results to the Pd/NHC complex were achieved when the new optimized catalytic system was used (Table 1, Method B). The substituents on aryl donors did not greatly affect the coupling yields; both electron-rich and electron-poor aryl bromides coupled with SEM-imidazole, affording 5-arylimidazoles in good yields. Importantly, the optimized protocol employing the electron-rich trialkylphosphine P(n-Bu)Ad2 also enables the use of aryl chlorides as arene donors with good efficiency (Table 1).24 In addition, the reaction can be conducted on the benchtop using the air-stable phosphonium salt [P(n-Bu)Ad2H]BF4, prepared by mixing the phosphine and tetrafluoroboric acid.25 For the C5-arylation with aryl bromides and chlorides, other sterically hindered trialkylphosphines, such as tricyclohexylphosphine and 2-(dicyclohexylphosphino)biphenyl, gave comparable results to those with n-butyl-di(1-adamantyl) phosphine (see Supporting Information for experimental details).</p><!><p>Empirical studies have found that the C-5 position of imidazoles exhibited reactivity higher than that at the C-2 position for the palladium-catalyzed arylation in the presence of weak bases, as originally demonstrated by Miura and colleagues with N-methylimidazole and aryl iodides.26 It was also shown in their paper that the addition of copper(I) salts altered the bias toward the C-2 position. The latter method has subsequently been optimized by others and applied to N-methylimidazole and other azole heteroarenes.19,27 Alternatively, a rhodium-catalyzed C2-arylation of 1,3-azoles has been developed; however, only arylation of 4,5-disubstituted imidazoles has been reported.28 In sum, existing methods for C2-arylation of imidazoles are limited to aryl iodides, while aryl bromides require high temperatures (≥ 140 °C).16,28,29 Furthermore, the Pd/Cu bimetallic system gave only a trace amount of the desired product with SEM-imidazole 1 (5 mol % of Pd(OAc)2, 2 equiv of CuI, 2 equiv of bromoarene, DMF, 140 °C, 24 h).</p><p>Consequently, we carefully examined the palladium-catalyzed conditions and developed a versatile and practical method for C2-arylation of SEM-imidazoles with both aryl bromides and aryl chlorides. Inspired by the base-promoted tautomerization of imidazole ligands to N-heterocyclic carbenes,30 we envisioned that upon coordination of the palladium complex to the N-3 nitrogen of imidazole, an alkali metal alkoxide would be sufficiently basic to produce a C2-metalated intermediate. Moreover, since an electron-rich phosphine ligand should enable the activation of chloroarenes, which are not efficient donors for the previously reported procedures as discussed above, we decided to take advantage of the palladium-phosphine pair that we identified for the C5-arylation of imidazoles (and C5-arylation of pyrazoles15a).</p><p>Screening experiments with different bases and solvents led us to establish a new set of conditions, where imidazole 1 gave 2-phenylimidazole 4 in 61% yield after heating in toluene (0.5 M) in the presence of 1.5 equiv of NaOt-Bu (Scheme 1). Weaker bases were much less effective than NaOt-Bu. As expected, the formation of the double arylation product was not negligible, and it was isolated in 11–15% yield. Importantly, chlorobenzene gave a similar yield and higher C2:C5 selectivity (from 4:1 to 6:1) compared to that of bromobenzene. Not only the simple phenyl halides but also functionalized arene donors can be employed for the C2-arylation of imidazole 1 (e.g., 3-bromoanisole and 4-bromobenzonitrile afforded the desired products in 60% and 51% isolated yields, respectively; Scheme S1 in Supporting Information). This transformation represents an important advance as regioselective C2-arylation of imidazoles can be performed with aryl chlorides (and a single transition metal as the catalyst). Moreover, the simple switch from C5- to C2-arylation of SEM-imidazole by using the alkoxide base and the nonpolar solvent demonstrates the plasticity of palladium catalysis and suggests potential application of this method for regioselective arylation of N-alkyl- and N-arylimidazoles as well as other classes of heteroarenes.31 While the C2/C5 regioselectivity in the reaction of the SEM-imidazole was modest, the C2-arylation of 5-phenylimidazole 2 is efficient under the newly developed conditions. Once the 5-position of imidazoles is substituted, the overarylation is no longer a problem.</p><p>Using the alkoxide base, we were able to obtain SEM-protected 2,5-diarylimidazoles with a variety of functional groups on the C2 aryl ring in good to excellent yields (Table 2). For example, 3-chloroanisole was coupled to 5-phenylimidazole 2, giving 13 in 75% isolated yield. Naturally, as this method relies on the use of a strong base, there are limitations in the scope related to the presence of base-sensitive groups. We addressed two important limitations: (1) the use of a carboxylic acid ester and (2) the use of a nitrile. The method is compatible with tert-butyl esters of carboxylic acids as demonstrated by the efficiency of C2-arylation with tert-butyl 4-bromobenzoate (Table 2, entry 5). By using the tert-butyl ester, complications related to transesterification and hydrolysis were largely eliminated (the corresponding ethyl ester gave much lower yield). The outcome of C2-arylation with 4-bromobenzonitrile was less than satisfactory (Table 2, entry 6). However, when the air- and moisture-stable catalyst Pd(PPh3)2Cl2 was used in lieu of Pd(OAc)2 and P(n-Bu)Ad2, it gave a significantly improved result, affording product 18 in 60% yield. Finding the solution to this problem was guided by the rationale that the nitrile as an electron-withdrawing group activates the aryl halide toward oxidative addition, and therefore a less active palladium system [such as Pd(PPh3)2Cl2] would be sufficient for the aryl halide activation and at the same time less prone to inhibition by the product.32</p><!><p>The new methods for C2- and C5-arylation of SEM-protected imidazoles enable direct access to 5-aryl-1-SEM-imidazoles, 2-aryl-1-SEM-imidazoles, and 2,5-diaryl-1-SEM-imidazoles, as well as the corresponding free (NH)-imidazoles (via simple deprotection of the SEM group, vide infra). However, the C-4 position of SEM-imidazoles (and other N-substituted imidazoles) exhibits very low reactivity in the palladium-catalyzed C–H arylation described above, precluding direct C-arylation of this position. To address this problem, we developed and here describe the SEM-group switch that transposes the SEM group from the N-1 to N-3 nitrogen of the imidazole ring and in the process transforms the unreactive C-4 position to the reactive C-5 position. Analogous to the SEM-switch of pyrazoles,15a the rearrangement proceeds in the presence of a catalytic amount of SEM chloride (Scheme 2), most likely via formation of an imidazolium intermediate and subsequent loss of the SEM group through a SN1 type process, to ultimately produce the less sterically hindered 4-arylimidazole.33 The second arylation at the C-5 position of the resulting 4-arylimidazole then proceeds well to generate a 4,5-diarylimidazole.</p><p>In practice, 5-arylimidazole 19, prepared by direct arylation of SEM-imidazole 1 with 4-bromoanisole, was converted in one step to 4-arylimidazole 21 in 88% isolated yield (Scheme 2). Despite the increased steric hindrance at the "new" C-5 position, remarkably, the arylation of the C-5 position was preferred over the C-2 position, furnishing 4,5-diarylimidazole 22 in 46% isolated yield, accompanied by the starting material and the double arylation product (17% and 11% yields, respectively). The second C5-arylation becomes highly efficient with C2-substituted substrates, which is discussed in the following section.</p><!><p>Having established the C2- arylation, the C5-arylation, and the SEM-switch, we now can access 2,4,5-arylated imidazoles in short order. The three-step sequence consisting of direct C5-arylation, SEM-switch, and the second C5-arylation was extended to 2-substituted imidazoles. 2-Phenyl-1-SEM-imidazole 4, prepared by C2-arylation of imidazole 1 or by protection of commercially available 2-phenylimidazole, was used to explore the efficiency of the three-step sequence (Table 3, entries 1–3). In terms of arene donor substrate scope, several functional groups, including ester, dimethylamino, pyridyl, and pyrazyl groups, were well tolerated.34 Note that the second arylation of sterically hindered substrates is efficient, giving good to excellent yields with a variety of arene donors.</p><!><p>To further demonstrate the utility of the sequential arylation sequence, we also tested 2-alkyl- and 2-dialkylamino-imidazole derivatives. Specifically, 2-butyl-1-SEM-imidazole is an excellent substrate for the arylation and SEM-switch reactions, giving complex imidazole 34 in high yield (Table 3, entry 4). Moreover, a dialkylamino functionality on the imidazole core was compatible with not only the palladium-catalyzed arylation but also the SEM-switch; the 2-piperidylimidazole was transformed to 37 in good overall yield (Table 3, entry 5). The SEM group of the resulting arylation products can be easily removed by either acidic hydrolysis or fluoride treatment.35 For example, deprotection of complex imidazole 34 was performed in an aqueous HCl solution (1 N, 80 °C, 2 h) to give the corresponding free imidazole in quantitative yield.</p><!><p>The SEM-switch idea was extended to accomplish the trans-N-alkylation, which unlocks a short route to regioselective formation of 1-alkyl-4-arylimidazoles. Indeed, N-alkylation of 2 with benzyl bromide and subsequent acidic hydrolysis generated 1-benzyl-4-phenylimidazole 39 in excellent yield (Scheme 3).36,37 Similarly, trimethyloxonium tetrafluoroborate produced methylimidazolium salt 40, which underwent a SEM cleavage to give 1-methyl-4-phenylimidazole 6 in 91% yield over 2 steps. Given the fact that it is difficult to achieve regioselective N-alkylation of 4(5)-arylimidazoles with a small alkyl group, the procedure using SEM-imidazoles addresses this problem and enables efficient preparation of 1-alkyl-4-arylimidazoles.</p><!><p>The gathered results substantiate a general strategy for the synthesis of a broad range of mono-, di-, and triarylimidazoles from the parent imidazole as well as substituted imidazoles. For example, 2,5-diarylimidazoles can be accessed by either C5–C2 or C2–C5 sequential arylation reactions. Further, 2,4,5-triarylimidazoles can be rapidly prepared by the SEM-switch and C5-arylation of the diarylimidazoles. If the goal is to prepare free (NH)-imidazoles, SEM deprotection can be carried out under either acidic or basic (fluoride) conditions.</p><p>However, when N-alkyl-triarylimidazoles are desired, trans-N-alkylation followed by the final C5-arylation represents a good synthetic plan. This approach was implemented in a five-step synthesis of 44 (Scheme 4). In detail, the C5-arylation of imidazole 1 afforded compound 41, from which diarylimidazole 42 was derived by C2-arylation with 4-bromo-1-trifluoromethylbenzene. The N-methylation and the SEM-group deprotection provided 1-methylimidazole 43, which was subsequently subjected to the final coupling with 2-bromonaphthalene to furnish fully substituted product 44.</p><p>In this synthesis, all four substituents including the N-alkyl group were introduced to the bare imidazole core in a programmable manner. This general approach based on sequential C-arylation and N-alkylation of SEM-protected imidazoles provides rapid access to complex aryl imidazoles, and it is quite apparent how series of derivatives (with different substitutions at one or more positions) can be synthesized in short order in this manner.</p><!><p>Imidazoles are an important group of the azole family of heterocycles frequently found in pharmaceuticals, drug candidates, ligands for transition metal catalysts, and other molecular functional materials. Owing to their wide application in academia and industry, a great deal of work has been dedicated to the generation of functionalized imidazole derivatives. In contrast to conventional condensation methods, catalytic C–H bond functionalization reactions enable derivatization and elaboration of the existing imidazole rings and provide new possibilities for the synthesis of complex imidazoles.</p><p>We here described a general and comprehensive approach for the synthesis of complex aryl imidazoles, where all three C–H bonds of the imidazole core can be arylated in a regioselective and sequential manner. The synthesis of individual compounds or compound libraries can commence from either the parent imidazole or advanced imidazole derivatives (accessed from ring-forming or substituent-modifying reactions). Our approach and the new C–H arylation protocols will thus complement transition-metal-catalyzed cross-coupling reactions, including the Suzuki reaction19,38 and the catalytic N-arylation of imidazoles,39,40 as well as the C–H arylation reactions developed by others.19,20,28 The particular strength of our strategy is the flexibility with which the N-alkyl groups can be introduced in a regioselective manner at various stages of the arylation sequence. Another important advance this paper brings is that both C5- and C2-arylation reactions can be carried out with low-cost and readily available aryl chloride donors.</p><!><p>The imidazole (0.50 mmol) was weighed into a 4-mL glass vial equipped with a magnetic stir bar. Through a Teflon-lined cap, the vial was purged with argon. The aryl halide (0.75 mmol) and toluene (0.25 mL) were added to the vial under a positive pressure of argon. The reaction vial was moved to a glovebox. Pd(OAc)2 (5.6 mg, 0.025 mmol), P(n-Bu)Ad2 (13.4 mg, 0.038 mmol), and NaOt-Bu (96 mg, 1.0 mmol), which were stored under argon in the glovebox, were then added to the reaction mixture. The cap was replaced with a new Teflon-lined solid cap. The reaction vial was removed from the glovebox and then moved to a preheated reaction block(100 °C). After stirring for 24 h, the reaction mixture was cooled to room temperature and was directly loaded to a silica gel column. Purification by flash column chromatography provided the desired 2-arylimidazole. For a procedure that does not require the use of a glovebox, see Supporting Information.</p><!><p>Purification by flash column chromatography (hexanes/EtOAc = 3:1) provided 13 as a colorless oil: IR (film) 2952, 2894, 2836, 1605, 1482, 1359, 1286, 1250, 1170, 1083 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.63–7.58 (m, 2H), 7.49–7.43 (m, 4H), 7.42–7.35 (m, 2H), 7.22 (s, 1H), 7.02–6.97 (m, 1H), 5.14 (s, 2H), 3.87 (s, 3H), 3.45 (t, J=8.4 Hz, 2H), 0.97 (t, J = 8.4 Hz, 2H), 0.00 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 159.6, 149.8, 135.4, 131.8, 129.9, 129.4, 128.7, 128.6, 128.0, 127.4, 121.0, 115.3, 113.7, 73.1, 65.3, 55.2, 17.9, −1.6; HRMS (FAB) calcd for C22H29N2O2Si [M + H]+ 381.1998, found 381.1991.</p><!><p>Rapid access to complex imidazoles via direct C–H arylation.</p><p>C2- and C5-arylation methods, together with the SEM-switch, provide rapid access to complex arylimidazoles with complete control of regioselectivity. The SEM group also allows for regioselective N-alkylation (trans-N-alkylation). (A) An example of sequential elaboration of SEM-imidazole to furnish 1-alkyl-2,4-diarylimidazoles and 1-alkyl-2,4,5-triarylimidazoles. (B) Advanced intermediates (accessed for example by condensation methods) may also be arylated and alkylated in a regioselective manner via the strategy based on the SEM group.</p><p>(A) General reactivity trends of imidazole. (B) Two methods for palladium-catalyzed C–H arylation of SEM-imidazoles were developed with complementary regioselectivity (C5 and C2). The C2- and C5-arylation protocols enable the use of both aryl bromides and aryl chlorides, providing a practical method for synthesis of arylimidazoles. (C) The SEM-group transposition (SEM-switch) leads to 4-arylimidazoles and allows for subsequent arylation and preparation of complex arylimidazoles. Similarly, the SEM group enables regioselective N-alkylation (trans-N-alkylation) to afford 1-alkyl-4-arylimidazoles.</p><p>Mechanistic rationales for the observed regioselectivity of the imidazole arylation. (A) In the presence of carbonate or carboxylate base (R″ = alkyl or alkoxide), the C–H activation occurs via ligand-assisted palladation (via either EMD or CMD mechanism). The C-5 position is preferred over the C-2 and C-4 due to stabilization of the C–Pd bond by the inductive effect of N-1 nitrogen. (B) In contrast, palladation at the C-4 position is disfavored by electronic repulsion between the nitrogen e− pair and the polarized C–Pd bond. (C) In the presence of a strong base, deprotonation occurs at the C-2 position, presumably facilitated by complexation of the palladium complex to N-3. The same rationale applies to other azoles including oxazoles, thiazoles, and triazoles.</p><p>C2-Arylation of SEM-Imidazoles</p><!><p>aReaction conditions: (a) 5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.2 equiv of 4-bromoanisole, 2.0 equiv of K2CO3, DMA (0.5 M), 120 °C, 17 h, 63% yield; (b) 5.0 mol % SEM Cl, CH3CN, 80 °C, 22 h, 88% yield; (c) 5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.2 equiv of 3-bromopyridine, 2.0 equiv of K2CO3, DMA(0.5 M), 120 °C, 20 h, 46% yield. Recovered starting material and 2,5-diarylation product were isolated in 17% and 11% yields, respectively. Yields are an average of two separate isolated yields.</p><!><p>Synthesis of 1-Alkyl-4-arylimidazoles</p><!><p>aReaction conditions: (a) 5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.2 equiv of 1-bromo-3,5-dimethoxybenzene, 2.0 equiv of K2CO3, DMA(0.5 M), 120 °C, 18 h (68% yield). (b) 5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.5 equiv of 4-bromobenzotrifluoride, 2.0 equiv of NaOt-Bu, toluene (1.0 M), 100 °C, 24 h (65% yield). (c) 1.5 equiv of Me3O·BF4, CH2Cl2, rt, 1 h; 1 N HCl, H2O, 80 °C, 2 h (55% yield over 2 steps). (d) 5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.2 equiv of 2-bromonaphthalene, 2.0 equiv of K2CO3, DMA (0.5 M), 120 °C, 18 h (82% yield). Yields are an average of two separate isolated yields.</p><!><p>C5-Arylation of 1-SEM-Imidazole</p><p>Method A: 5.0 mol %7, 2.0 equiv of ArX, 2.0 equiv of CsOAc, DMA (1.0 M), 125 °C, 16–20 h. Complex 7 can be prepared according to the protocol provided in ref 15c.</p><p>Method B: 5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.2 equiv of ArX, 2.0 equiv of K2CO3, DMA(0.5 M), 120 °C, 18 h. The corresponding 2,5-diarylation product was also formed in 5–10% yield (see Supporting Information).</p><p>In the benchtop experiment, [P(n-Bu)-Ad2H]BF4 was used, and the amount of K2CO3 was increased (2.5 equiv). Yields are an average of two separate isolated yields.</p><p>C2-Arylation of 5-Phenylimidazole 2a</p><p>Reaction conditions: 5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.5 equiv of ArX, 2.0 equiv of NaOt-Bu, toluene (2.0 M), 100 °C, 24 h.</p><p>Isolated yield, average of two runs.</p><p>Pd(PPh3)2Cl2 (5 mol %) was used in place of Pd(OAc)2/P(n-Bu)Ad2. For C2-arylation of SEM-imidazole 1, see Supporting Information.</p><p>Sequential Double Arylation Enabled by SEM-Group Switch</p><p>5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.2 equiv of Ar1Br, 2.0 equiv of K2CO3, DMA (0.5 M), 120 °C, 20 h.</p><p>5.0 mol % SEMCl, CH3CN, 80 °C, 24 h.</p><p>5.0 mol % Pd(OAc)2, 7.5 mol % P(n-Bu)Ad2, 1.5 equiv of Ar2Br, 2.0 equiv of K2CO3, DMA(0.5 M), 120 °C, 20 h.</p><p>ArCl was used instead of ArBr.</p><p>In the benchtop experiment, [P(n-Bu)Ad2H]BF4 was used and the amount of K2CO3 was increased (2.5 equiv).</p>

PubMed Author Manuscript

Dual-graphite cells based on the reversible intercalation of bis(trifluoromethanesulfonyl)imide anions from an ionic liquid electrolyte

Recently, dual-ion cells based on the anion intercalation into a graphite positive electrode have been proposed as electrochemical energy storage devices. For this technology, in particular electrolytes which display a high stability vs. oxidation are required due to the very high operation potentials of the cathode, which may exceed 5 V vs. Li/Li + . In this work, we present highly promising results for the use of graphite as both the anode and cathode material in a so-called "dual-graphite" or "dual-carbon" cell. A major goal for this system is to find suitable electrolyte mixtures which exhibit not only a high oxidative stability at the cathode but also form a stable solid electrolyte interphase (SEI) at the graphite anode. As an electrolyte system, the ionic liquid-based electrolyte mixture Pyr 14 TFSI-LiTFSI is used in combination with the SEI-forming additive ethylene sulfite (ES) which allows stable and highly reversible Li + ion and TFSI À anion intercalation/de-intercalation into/from the graphite anode and cathode, respectively. By addition of ES, also the discharge capacity for the anion intercalation can be remarkably increased from 50 mA h g À1 to 97 mA h g À1 . X-ray diffraction studies of the anion intercalation into graphite are conducted in order to understand the influence of the electrolyte additive on the graphite structure and on the cell performance. Broader contextOne of the most challenging issues in the 21 st century is the preservation of a consistent energy supply that meets the world's increasing energy demands. The present energy economy based on fossil fuels is considered to be at serious risk due to several factors, such as the shortages of non-renewable resources or concerns about the environmental impact of energy consumption, and thus gives rise to the development of renewable energy sources. The need for clean and efficient storage of electrical energy will be vast, not only to meet the rising energy demand, but in particular to prevent global warming. Currently, lithium-ion batteries dominate the small format battery market for portable electronic devices and are now being widely regarded as the technology of choice for future automotive and stationary applications. The requirements for stationary batteries are signicantly different from those of power batteries in electric vehicles. For stationary batteries, high safety, low cost and long cycle life are the most important parameters. In this paper, we introduce the dual-graphite/dual-carbon battery technology as a promising option for grid applications, since it displays environmental, safety and cost benets (e.g. free of transition metals, non-ammability of the ionic liquid electrolyte, graphite as a low-cost electrode material, aqueous electrode processing possible for anodes and cathodes) over stateof-the-art lithium-ion batteries.

dual-graphite_cells_based_on_the_reversible_intercalation_of_bis(trifluoromethanesulfonyl)imide_anio

Introduction<!>Experimental<!>Results and discussion<!>Graphite anode performance in a half-cell set-up<!>Graphite cathode performance in a half cell set-up<!>In situ X-ray diffraction study of the anion intercalation into graphite<!>Dual-graphite cell performance<!>Conclusion

<p>As a redox-amphoteric material, graphite can be reduced as well as oxidized by chemical or electrochemical methods. 1 The resulting positive or negative charge can be compensated by the intercalation of a variety of certain anionic/cationic intercalation guests into the interlayer gaps of the graphite lattice. 1,2 While lithiated graphite is an established anode material in state-of-the-art lithium-ion batteries, 3 graphite intercalation compounds (GICs) intercalated by anions were suggested by Carlin et al. as possible active materials for positive electrodes in so-called "dual intercalating molten electrolyte batteries". 4 In their work, they proposed a dual-graphite battery system which uses different room temperature ionic liquids (RTILs), such as 1-ethyl-3-methylimidazolium-hexauorophosphate (EMI + PF 6 À ).</p><p>In this set-up, the electrolyte does not only act as a charge carrier but also as a source for intercalation guests, in which EMI + intercalates into the graphite anode and PF 6 À intercalates into the graphite cathode during the charge process. 4 However, the reversibility of the intercalation/de-intercalation behavior of this dual-graphite cell was relatively poor. The concept of a dualcarbon cell is also followed in industry, as recently the Japanese start-up "Power Japan Plus" announced plans to commercialize such a kind of dual-carbon battery. 5 Recently, we introduced a novel dual-intercalation battery system using a mixture of N-butyl-N-methylpyrrolidinium bis-(triuoromethanesulfonyl)imide (Pyr 14 TFSI) and lithium bis-(triuoromethanesulfonyl)imide (LiTFSI) as an electrolyte. [6][7][8][9] In this "dual-ion cell", where e.g. metallic lithium or Li 4 Ti 5 O 12 (LTO) is used as an anode material, and graphite, which is intercalated by TFSI À anions during charge, is used as a cathode material, and shows a highly reversible cycling performance with a Coulombic efficiency exceeding 99%. 6 It was demonstrated that a reversible cathode discharge capacity of more than 50 mA h g À1 at room temperature and above 110 mA h g À1 at 60 C operation temperature can be achieved. 6 X-ray diffraction investigations revealed the mechanism behind the formation of C n + TFSI À GICs. 10 The intercalation of TFSI À anions proceeds in denable and consecutive phases. The attainment of these different nphases is dependent on the operation conditions such as the cut-off potential or the temperature. A specic discharge capacity of 115 mA h g À1 is the maximum we found so far for the TFSI À uptake into graphite, which corresponds to a stage-1 GIC with a stoichiometry ranging between C 19 TFSI and C 20 TFSI. 10 In order to differentiate the energy storage mechanism of this battery system from the ion transfer mechanism which is known from lithium-ion batteries, we introduced the term "dual-ion cell". This type of cell mechanism includes all cell reactions where simultaneously anions and cations react with the electrodes. In this dual-ion battery system, which also implies the dual-graphite/dual-carbon system, the electrolyte needs to be considered as an active material as well. This is in contrast to a lithium-ion cell, in which the electrolyte "only" acts as a charge carrier between the anode and the cathode and where the thickness of the electrolyte layer needs to be minimized. According to the work of Dahn and Seel in 2000, 11 there must be a high amount of electrolyte present in the dualgraphite cell to provide enough ions which are needed during charging of the cell. In their work, they reported that an electrolyte with a high molarity is crucial to achieve a sufficiently high energy density of this system. 11 Ionic liquids (ILs), being salts of low temperature melting points, are one of the most promising classes of electrolytes providing new opportunities in electrochemistry. 12,13 ILs show several advantageous properties for electrochemical applications, such as a negligible vapor pressure, a broad liquid range and an ionic conductivity which can be in the same range of organic solvent-based electrolytes. 12,13 It should be noted that the use of ILs as solvents for electrochemical reactions is predominantly driven by their excellent oxidative and reductive stability and thus a large electrochemical stability window, which generally exceeds the ones of conventional electrolyte solvents. 14 In particular, electrolytes displaying a high stability vs. oxidation are required for the dual-ion technology, due to the very high operation potentials of the cathode, which may surpass 5 V vs. Li/Li + .</p><p>Since the compatibility of the ionic liquid Pyr 14 TFSI with graphite anodes is not sufficient, [15][16][17] so far the selection of the anode materials for the dual-ion system has been limited to metallic lithium or LTO. Due to the excellent electrochemical stability of Pyr 14 TFSI against cathodic decomposition, there is no effective solid electrolyte interphase (SEI) formation on the graphite anode. Thus, graphite undergoes exfoliation by intercalation of the relatively large organic cations (Pyr 14 + ) between the graphene layers, leading to irreversible degradation of the negative electrode. 17 This process is illustrated schematically in Fig. 1 for a dual-graphite cell.</p><p>One solution to enable the use of ILs with graphite anodes is the addition of SEI-forming electrolyte additives such as ethylene sulte (ES). [18][19][20] Sulfur-based electrolyte additives are proposed to support the SEI formation by adsorption of their reduced species onto the catalytically active functional groups of the graphite surface. 18,19,21 Unfortunately, the mechanism of the SEI formation in the presence of ES as an additive has not been well understood so far. Ota et al. proposed that an intermediate, such as SO 2 , is formed during the reductive decomposition of ES, which then reacts with various compounds such as Li 2 SO 3 or Li 2 SO 4 . 22 These compounds may contribute to more effective formation of the inorganic part of the SEI layer. Furthermore, Wrodnigg et al. suggested ES as an effective SEIforming agent in the presence of propylene carbonate (PC) as a solvent, whereby graphite is protected from rapid exfoliation caused by massive co-intercalation of PC, leading to the destruction of the graphite structure. 18 Recently, Leggesse and Jiang reported that ES can undergo a one-and two-electron reduction mechanism in PC-based electrolytes, whereas the formation of Li 2 SO 3 is more favorable in the two-electron reduction process. 23 The major reduction products which are considered to be responsible for the formation of an effective SEI layer are Li 2 SO 3 , (CH 2 OSO 2 Li) 2 , CH 3 CH(OSO 2 Li)-CH 2 OCO 2 Li and ROSO 2 Li. 23 Fig. 1 Schematic illustration of a dual-graphite cell with no effective SEI layer at the graphite anode during the charge process. The negative graphite electrode suffers from exfoliation reactions caused by cointercalation of the relatively large pyrrolidinium (Pyr 14</p><p>In this work, we present promising results concerning the use of graphite as both the anode and cathode material in a so-called "dual-graphite cell" using the ionic liquid-based electrolyte mixture Pyr 14 TFSI-LiTFSI in combination with ES as an SEI-forming additive. The energy storage mechanism of this system is based on the simultaneous TFSI À anion intercalation into the graphite positive electrode and Li + ion intercalation into the graphite negative electrode during charge. During discharge, both ions are released back into the electrolyte. In this regard, the intercalation of Pyr 14 + cations into the graphite anode needs to be prevented by the formation of a protective SEI layer, which still allows the reversible intercalation/de-intercalation of lithium ions, as depicted in Fig. 2.</p><p>Recently, Read et al. reported that a dual-graphite cell using an organic solvent-based electrolyte can be cycled reversibly. 24 However, the long-term cycling stability as well as Coulombic efficiency was relatively poor. Herein, we will demonstrate that by the use of the ionic liquid electrolyte mixture, the dualgraphite system displays a stable cycling performance with a high Coulombic efficiency, while both the discharge capacity and efficiency can be tailored by the operation conditions, such as the upper cut-off cell voltage. In addition, X-ray diffraction studies of the anion intercalation into graphite are performed in order to understand the inuence of the electrolyte additive on the graphite structure and on the cell performance.</p><!><p>The preparation of the graphite positive electrodes was carried out using a composition of 90 wt% of KS6L graphite (Imerys; D90 ¼ 8.5 mm; BET specic surface area ¼ 19.0 m 2 g À1 ), 5 wt% of conductive carbon black C-nergy Super C65 (Imerys) and 5 wt% of sodium carboxymethyl-cellulose (CMC) as a binder (Walocell CRT 2000 PPA12, Dow Wolff Cellulosics). The details of the electrode preparation have been described previously. 6 The graphite negative electrodes were prepared in the same way using 90% of commercial graphite (further abbreviated as CG; D90 ¼ 40.8 mm; BET specic surface area ¼ 3.96 m 2 g À1 ) as an active material, 5 wt% of conductive carbon black agent C-nergy™ Super C65 (Imerys), 2.5 wt% of the binder styrenebutadiene rubber (SBR; LIPATON SB 5521, Polymer Latex GmbH) and 2.5 wt% of sodium carboxymethylcellulose (CMC) as a binder (Walocel CRT 2000 PPA 12, Dow Wolff Cellulosics). The electrode paste for the graphite anode was cast on highpurity copper foil (Carl Schlenk AG ® ). The mass loading of the graphite negative electrode was ca. 2.5 mg cm À2 . 20 The ionic liquid N-butyl-N-methylpyrrolidinium bis(tri-uoromethanesulfonyl)imide (Pyr 14 TFSI, Solvionic, purity: 99.9%) used as an electrolyte solvent was dried under ultra-high vacuum (5 Â 10 À8 mbar, 110 C) for 48 hours using a turbo molecular pump TPS-compact (Varian Vacuum Technologies) before use. Under inert conditions, a mixture of dried Pyr 14 TFSI (water content less than 10 ppm, determined by Karl Fischer titration) with 1 M lithium bis(triuoromethane-sulfonyl)imide (LiTFSI, 3 M, purity: 99.95%) as conductive and electroactive salt (mole fraction of LiTFSI: 0.3) was prepared. To this electrolyte solution, 2 wt% of the SEI-forming additive ethylene sulte (ES, Sigma Aldrich, purity: 98%) were admixed. The maximum solubility of LiTFSI in the ionic liquid is strongly dependent on the temperature. While at 20 C a LiTFSI mole fraction of ca. 0.32 is possible, this can be strongly enhanced to about 0.68 at 60 C. Phase diagrams of the ionic liquid electrolyte mixtures were reported by Henderson and Passerini. 25 Electrochemical measurements were carried out either in a half-cell set-up (Subsections 3.1 and 3.2) with metallic lithium as counter and reference electrodes or in a full-cell set-up (Subsection 3.4) with the CG-based electrode as an anode, the KS6L-based electrode as a cathode and high-purity metallic lithium foil (Rockwood Lithium ® ) as a reference electrode. For that purpose a custom-made Swagelok ® type T-cell with a threeelectrode conguration was used. A borosilicate glass micro-ber (Whatman ® GF/D) drenched with 120 mL of electrolyte served as the separator.</p><p>Charge/discharge cycling was performed on a multichannel Maccor 4300 battery test system (MACCOR, INC). Aer cell assembly, the cells rested for at least 12 hours to ensure homogeneous electrolyte distribution and sufficient electrode wetting. The constant current cycling of CG-based graphite anodes in a half-cell set-up was conducted with a specic current of 37.2 mA g À1 (0.1C) between cut-off potentials of 0.02 V and 1.5 V vs. Li/Li + (see Subsection 3.1). The charge/discharge cycling of KS6L graphite cathodes in a half-cell set-up was performed with a constant current of 50 mA g À1 between cut-off potentials of 3.0 V and 4.8-5.2 V vs. Li/Li + (see Subsection 3.2). The constant current cycling of dual-graphite cells (Subsection 3.4) was carried out with a constant charge and discharge current of 10 mA g À1 for the rst three cycles in order to provide a homogeneous SEI-formation, while from the 4th cycle the current is increased in certain experiments (e.g. to 50 mA g À1 in Fig. 9 or to 500 mA g À1 in Fig. 11). For the in situ X-ray diffraction (XRD) measurements, a modied commercial CR2016 coin cell (Hohsen, Japan) with an X-ray transparent window was used. The details of this set-up have been described previously. 10 Aer assembly of the coin cell using high-purity metallic lithium (Rockwood Lithium ® ) as a counter electrode, the cell was directly placed in an X-ray diffractometer (BRUKER D8 Advance, equipped with a copper target X-ray tube). XRD measurements were conducted in a range between 2q ¼ 20 and 35 with a step size of 0.185 per second resulting in 20 minutes per in situ XRD scan at an accelerating voltage of 40 kV and a current ow of 40 mA. For in situ XRD measurements, cyclic voltammetry was performed using a VSP multichannel potentiostatic-galvanostatic system (Biologic ® Science Instrument, France) with a scan rate of 50 mV s À1 between 3.4 V and 5.2 V at 20 C with a 3 hour constant voltage step at the upper cut-off voltage.</p><!><p>A major goal for the dual-graphite system is to nd a suitable electrolyte mixture which exhibits rst a high oxidative stability, in particular with anions that are stable upon intercalation/deintercalation into the graphite cathode, and second is able to form a stable SEI layer at the graphite anode. As an electrolyte system, the ionic liquid-based electrolyte mixture Pyr 14 TFSI-1 M LiTFSI in combination with the SEI-forming additive ethylene sulte (2 wt%) is studied. At rst, lithium ion intercalation into the graphite anode and TFSI À anion intercalation into the graphite cathode are investigated independently in a half-cell set-up (Secitons 3.1 and 3.2). In Seciton 3.4, the electrochemical performance of the dual-graphite system is studied in detail.</p><!><p>As mentioned above, the compatibility of the pure ionic liquid Pyr 14 TFSI-LiTFSI with graphite anodes is relatively poor, which is related to insufficient SEI formation and the co-intercalation of Pyr 14 + cations between the graphene layers, leading to irreversible degradation/exfoliation of graphite. 15,17 Fig. 3 displays the charge/discharge cycling behavior for lithium ion intercalation/de-intercalation into/from graphite as an anode material (CG graphite) with the additive containing ionic liquid-based electrolyte (Pyr 14 TFSI, 1 M LiTFSI + 2 wt% ES). Even though the maximum discharge capacity is only about 100 mA h g À1 , which is signicantly lower than the theoretical value of fully lithiated graphite (372 mA h g À1 ), a capacity retention of 83% can be obtained aer 50 cycles at a C-rate of 0.1 (Fig. 3a). This relatively low reversible discharge capacity was also reported by others 15,26 and is most likely related to the low charge transfer kinetics for lithium intercalation/de-intercalation into/from graphite at the electrode/electrolyte interface, i.e. at the SEI. Furthermore, it can be caused by the high viscosity of the electrolyte and by the relatively low self-diffusion coefficient and transference number of Li + in the ionic liquid. 27 The reversible capacity may be further enhanced by tailoring the SEI layer by the use of certain electrolyte additives. However, these additives must not only build a stable SEI layer but also exhibit a high stability vs. oxidation for the dual-graphite cell.</p><p>The average Coulombic efficiency is about 99.0% along the cycles 10 to 50 (Fig. 3b). However, the efficiency in the rst cycle with only 49% is quite low. Fig. 3c displays a representative charge/discharge potential prole from the 50 th cycle, showing that lithium ion intercalation reversibly takes place in the potential range from 0.20 V to 0.02 V vs. Li/Li + . Overall, by addition of 2 wt% ES to the ionic liquid electrolyte, a reversible lithiation/de-lithiation of graphite is realized. In Section 3.4, we will show that this electrolyte system displays a highly reversible electrochemical performance in the dual-graphite cell, outperforming any results published so far in the literature for similar systems.</p><!><p>The electrochemical intercalation/de-intercalation behavior of TFSI À anions from the pure ionic liquid (Pyr 14 TFSI-LiTFSI) into graphite in a metallic lithium/graphite dual-ion cell was studied in detail in previous publications. 6,7,28 As the use of the additive ES is primarily intended to increase the compatibility of the ionic liquid electrolyte with the graphite anode, no negative inuence on the TFSI À anion intercalation is desirable in the presence of ES. In order to verify this assumption, electrochemical investigations of the TFSI À anion intercalation were performed with the ES-based electrolyte. It is obvious that the discharge capacity of the ES-containing system is remarkably increased to 97 mA h g À1 (50 th cycle) in comparison with the one of the pure ionic liquid, which displays a maximum value of 50 mA h g À1 (Fig. 4a and c). Apart from the rst charge/discharge cycles, the Coulombic efficiency is very similar for both systems and reaches about 99% (Fig. 4b). As we discussed previously, the low Coulombic efficiency in the rst cycles is most likely related to some type of "formation", which is presumably a kinetic hindrance due to the rst intercalation of TFSI À between the graphene sheets. 28 This "formation" may take a few cycles until all graphite particles of the porous electrode are wetted and the maximum discharge capacity is achieved. 28 Since the use of the electrolyte additive leads to an increase of the maximum discharge capacity of the graphite cathode, the inuence of a varying upper charging end potential, ranging from 4.8 V to 5.2 V vs. Li/Li + on the discharge capacity and Coulombic efficiency was investigated. Table 1 summarizes the discharge capacity and efficiency values obtained at these different potentials.</p><p>In general, the discharge capacity increases with an increasing upper charging end potential, i.e. from 49 mA h g À1 at 4.8 V vs. Li/Li + to 126 mA h g À1 at 5.1 V vs. Li/Li + , while the Coulombic efficiency slightly decreases, i.e. from 99.5% to 99.0%. If the upper cut-off potential is further enhanced to 5.2 V vs. Li/Li + , the discharge capacity drops to 116 mA h g À1 and the efficiency also deteriorates to 97.8%. Therefore, it can be assumed that potentials above 5.1 V vs. Li/Li + lead to enhanced irreversible reactions, most likely caused by electrolyte degradation and in particular by decomposition of the intercalated anion.</p><p>Fig. 5 displays the differential capacity proles of the metallic lithium/KS6L graphite dual-ion cell at different charging end potentials ranging from 4.8 V to 5.2 V vs. Li/Li + , each from the 50 th charge/discharge cycle. In Fig. 5a, the dQ/dV proles over the whole potential range are depicted, while Fig. 5b-d are magnications for a better visualization of the intercalation/deintercalation peaks. From these proles the correlation between the TFSI À intercalation and de-intercalation peaks can be realized, which are marked by Roman numerals. The correlation of these peak positions is also summarized in Table 2. When the upper cut-off potential is set to 5.2 V vs. Li/Li + , six TFSI À intercalation peaks are observable, while the main intercalation takes place in the potential range from 4.36 V to 4.65 V vs. Li/Li + (II and III) and at 4.97 V vs. Li/Li + (VI). In relation to that, also six corresponding de-intercalation peaks/potential regions can be identied (Table 2), ranging from 4.91 V to 4.64 V vs. Li/Li + (III 0 to VI 0 ) and from 4.44 V to 3.90 V vs. Li/Li + (I 0 and II 0 ). If the cut-off potential is decreased, e.g. to 5.0 V or 4.9 V vs. Li/Li + , the TFSI À intercalation peaks V and VI diminish and/or disappear, while, in the same way, the deintercalation peaks VI to III weaken or completely disappear. In contrast, the intercalation peaks I to IV and the de-intercalation peaks I 0 and II 0 nearly stay constant, independent of the upper cut-off potential from 4.8 V to 5.2 V vs. Li/Li + .</p><p>In order to gain a better understanding of the inuence of the electrolyte additive on the TFSI À anion intercalation/deintercalation behavior, the differential capacity proles from the Li/KS6L graphite dual-ion cell using the pure and the ES-containing IL-electrolyte are compared in Fig. 6. In addition, Table 2 also lists the peak positions for anion intercalation/deintercalation from the pure ionic liquid electrolyte. It is obvious that the electrolyte additive displays a strong inuence on the TFSI À uptake/release into/from graphite since the potential ranges for intercalation/de-intercalation as well as the intensities of these peaks completely change (Fig. 6). While for the pure ionic liquid ve main intercalation and de-intercalation peaks/ potential regions (I-V and I 0 -V 0 ) can be seen, the IL with ES additive displays six peaks, as described above. In particular, the onset potential for the anion uptake is shied to a lower value for the ES-containing electrolyte: 4.26 V compared to 4.37 V vs. Li/Li + (peaks I and I). In addition, the main intercalation peak for the additive containing electrolyte is below 5.0 V vs. Li/ Li + (peak VI), while for the pure ionic liquid the main intercalation takes place above 5.0 V vs. Li/Li + (peak V). Anion deintercalation from the additive-based electrolyte starts at a higher potential (4.91 V compared to 4.84 V vs. Li/Li + ) and also takes place at lower potentials (3.80 V vs. 3.98 V vs. Li/Li + , see Table 2).</p><p>Overall, it can be summarized that the addition of ES as an additive to the Pyr 14 TFSI-LiTFSI ionic liquid results in a strongly enhanced discharge capacity for the anion intercalation into graphite. This enhancement may be related to a reduced coordination of Li + ions with TFSI À anions in the presence of ethylene sulte. Deshpande et al. reported on the mobility and transport properties of ionic liquids, in particular on an enhancement of the lithium ion mobility in ionic liquid-based electrolytes which contain electrolyte additives, such as ethylene carbonate, vinylene carbonate or tetrahydrofuran. 29 They proposed that the inclusion of organic additives decreases the extent of coordination of the lithium ion with the IL-anion. 29 Furthermore, Bayley et al. reported that electrolyte additives will inuence the transport properties of cations and anions in ionic liquid electrolytes, i.e. the diffusion coefficient of the anion may be enhanced by addition of certain organic diluents. 30 These effects may also explain the differences, i.e. the increased discharge capacity and reduced onset potential for the anion intercalation process into graphite from the ES containing IL in comparison with the pure ionic liquid electrolyte. However, further investigations are necessary to conrm this assumption.</p><!><p>X-ray diffraction studies of the TFSI À anion intercalation were performed in order to examine whether the increase in discharge capacity also results in a change of the structural composition of the GIC, i.e. in a higher stage formation, for the additive containing electrolyte compared to the pure ionic Table 2 Correlation between the different peaks for TFSI À intercalation and de-intercalation into/from KS6L graphite and the corresponding peak potential positions, according to the differential capacity profiles in Fig. 6 Pyr liquid-based system. In a previous publication, we reported on the XRD investigation of the TFSI À intercalation into graphite for the pure Pyr 14 TFSI-LiTFSI electrolyte. 10 Here, we showed that the anion intercalation takes place via a stage formation process and calculated the maximum stage number, periodic repeat distance and gallery height for the C n + TFSI À GIC. 10 Fig. 7a illustrates the rst cycle of the in situ XRD pattern for TFSI À intercalation/de-intercalation from the ES containing electrolyte, which was carried out by cyclic voltammetry (CV). Fig. 7b displays the voltage and specic current proles in dependence of time from the CV experiment. The scan rate was set to 50 mV s À1 and a constant voltage step of 180 minutes was conducted at the upper cut-off voltage of 5.20 V. For a better visualization, the rst 22 XRD scans in the back of Fig. 7a are not depicted, since the (002) peak of graphite with relatively high intensity remains unaffected until the rst anion intercalation takes place at about 4.55 V (Fig. 7b). To enable a better comparison between the XRD pattern and the cyclic voltammogram, representative colored XRD scans (Fig. 7a) can be correlated with the colored dots (with the same color) in the voltage and current proles (Fig. 7b).</p><p>In addition, Table 3 summarizes the values for the most dominant stage number n, the periodic repeat distance I c , the TFSI gallery height d i , and the gallery expansion Dd, which are calculated in accordance with our previous publication. 10 For comparison reasons, also the corresponding values for the pure ionic liquid electrolyte are listed in Table 3.</p><p>The scan 23 for the pristine KS6L graphite electrode shows the (002) peak of graphite as expected at 2q ¼ 26.55 (Fig. 7a). By charging the cell to 4.55 V, the (002) graphite peak decreases in intensity and splits up into two new peaks, one more dominant peak (00n + 1) at 24.7 and the second (00n + 2) at 29.7 (scan 24). 31,32 By determining the ratio of the d (n+2) /d (n+2) peak positions and correlating these with the ratios of stage pure GICs, given in our previous publication, 10 it is possible to calculate the most dominant stage phase of the observed GIC. 31,32 Here, the rst observable intercalation stage is 4 (Table 3). When the voltage is increased to 4.86 V, the stage is raised to the next higher level of 3 (scan 29). Passing stage 2 by scan 33 at 5.10 V, the maximum intercalation stage 1 is reached during the constant voltage step at 5.2 V, exemplarily represented by scan 40 (Fig. 7 and Table 3). As the de-intercalation starts at 5.06 V (compare with peak VI 0 in Fig. 6), the intercalation stage changes to 2 at 4.40 V (scan 57) and to 4 at 4.16 V (scan 61). In contrast, for the pure ionic liquid electrolyte only a maximum stage number of 2 is observed under the same operation conditions (Table 3).</p><p>These results conrm that by addition of ES to the electrolyte, not only the discharge capacity is increased, but also the composition changes from a stage-2 GIC to a stage-1 GIC. The TFSI À gallery height of about 8 Å and thus the gallery expansion of 4.65 Å nearly stay unaffected from the type of electrolyte as well as from the stage number (Table 3).</p><!><p>Considering the investigations performed in different half-cell set-ups, it is obvious that the Pyr 14 TFSI-LiTFSI-based electrolyte in combination with the additive ES enables rst the reversible lithiation of graphite and second improves the anion intercalation into the graphite cathode. For this reason, dual-graphite full-cell investigations were conducted.</p><p>Fig. 8 and 9 illustrate the cycling behavior of the dualgraphite cells, performed with constant charge/discharge currents of 10 mA g À1 and 50 mA g À1 , respectively. In the second case, three formation cycles with a lower current were carried out (10 mA g À1 ) before increasing the current to 50 mA g À1 (Fig. 9). Assuming a theoretical capacity of 100 mA h g À1 , a specic current of 10 mA g À1 would correspond to a C-rate of 0.1. The upper cut-off voltage for these experiments was varied between 4.8 V and 5.2 V, in order to investigate the inuence on the Coulombic efficiency, discharge capacity and cycling stability. The corresponding values of the cycling data from representative cycles, including the Coulombic efficiency and discharge capacity, are given in Table 4.</p><p>In general, the discharge capacity increases with a rising upper charging end voltage. At a constant current of 10 mA g À1 , the discharge capacity changes from 60 mA h g À1 at 4.8 V to 121 mA h g À1 at 5.2 V (representative value for the 50 th cycle, see Table 4). By the increase of the specic charge/discharge current to 50 mA g À1 (Fig. 9), the discharge capacity decreases for all cutoff voltages compared to cycling at 10 mA g À1 . At 4.8 V, a discharge capacity of 32 mA h g À1 is measured, which can be enhanced to 113 mA h g À1 at 5.2 V. A detailed study on the inuence of the specic current on the electrochemical performance of the dual-graphite cell was reported in our previous work. 20 Here, we found that the charge process (¼ intercalation of lithium ions and TFSI À anions into graphite) is the rate-determining step, while a discharge capacity ranging from 100 mA h g À1 to 30 mA h g À1 is achieved for a specic current that varies from 10 mA g À1 to 500 mA g À1 . 20 Fig. 8 and 9 depict the rst 50 cycles of the charge/discharge cycling and reveal a stable cycling behavior, independent of the upper cutoff voltage.</p><p>Fig. 10 displays representative cell voltage (black curve, le yaxis) as well as the anode and cathode potential proles vs. Li/ Li + (red curves, right y-axis) for the CG/KS6L dual-graphite cell during selected cycles (1 st , 2 nd , 49 th and 50 th cycles) of the constant current charge/discharge process. The upper and lower cell voltages are 5.1 V and 3.0 V, respectively, and are given Table 3 The dominant stage index and calculated values for the in situ X-ray diffraction measurement of the TFSI À intercalation into graphite from Pyr 14 TFSI, 1M LiTFSI + 2 wt% ES electrolyte for the lithium/KS6L graphite dual-ion cell. Data correspond to Fig. 7. The comparison to the TFSI À intercalation from Pyr 14 TFSI, 1 M LiTFSI is given according to ref. by the difference of the cathode and anode potentials. 33 The charge/discharge rate for the rst three cycles was 10 mA g À1 , while the following cycles were performed at 50 mA g À1 (corresponding to Fig. 9; 5.1 V cut-off voltage).</p><p>From the cell voltage and potential proles of the anode and cathode, it is obvious that the rst cycle differs from the following cycles, which is mainly related to the SEI formation and electrolyte decomposition taking place at the graphite anode as well as to the rst intercalation of TFSI À anions into the graphite cathode. In the rst charge process, the anode potential reaches 0.115 V vs. Li/Li + , which means that the graphite anode is not fully intercalated by lithium ions, i.e. stage 2 is observed (LiC 12 ). 34 The transition to stage 1 occurs at a potential of ca. 0.09 V vs. Li/Li + and is thus about 25 mV below the potential plateau of stage 2. 34 In other words this means that the anode is oversized in terms of capacity, which is desired in order to avoid lithium metal plating and therefore safety issues at the anode. 33 According to the behavior of the anode potential, the cathode potential in the rst cycle rises to a maximum of 5.21 V vs. Li/Li + . At an increased rate (50 mA g À1 ), the anode potential drops to 0.04 V vs. Li/Li + and the cathode potential drops to the same extent to 5.14 V vs. Li/Li + , which may be related to an increased cell resistance. The potential proles for the anion intercalation have been discussed in detail in previous publications. 6,28,35 Overall, it can be observed that the intercalation of both lithium ions and TFSI À anions into the graphite anode and cathode, respectively, occurs with a high reversibility leading to a high cycling stability. Recently, we reported a capacity retention of more than 98% for this dualgraphite system aer 500 charge/discharge cycles at a rate of 50 mA g À1 and at an upper cut-off voltage of 5.0 V. 20 The Coulombic efficiency of the dual-graphite system varies in the rst charge/discharge cycle between 69.5% and 75.0% (Table 4), which is mainly related to SEI formation and electrolyte decomposition at the anode as well as the rst anion intercalation into the graphite cathode (kinetic activation), as described above. The efficiency in the rst cycle seems to be independent of the upper cut-off voltage, as no correlation can be seen. Since the electrolyte in this system can be considered as an active material, an excess of electrolyte is necessary due to the irreversible consumption of lithium for the SEI formation. In the subsequent cycles, the Coulombic efficiency increases and varies, e.g. in the 50 th cycle, between 97.2% and 98.2% at a charge/discharge rate of 10 mA g À1 (Table 4 and Fig. 8), while the highest efficiency is reached at a cut-off voltage of 5.0 V.</p><p>At an enhanced rate of 50 mA g À1 , the efficiency can be further increased and varies between 98.6% and 99.1% (Table 4 and Fig. 9). Here, the highest efficiency is obtained at 5.0 and 5.1 V. This trend can most likely be explained by a decreased decomposition of intercalated TFSI À anions as well as a decreased self-discharge for an increased charge/discharge current, which we observed in current rate investigations for the dual-ion system. Thus a higher rate results in an enhanced Coulombic efficiency. 6,28 The inuence of the cycling rate on the coulombic efficiency was studied in more detail by Smith et al. for different lithium-ion full cells based on graphite negative electrodes. 36 They reported that the time of one cycle is the dominant contributor to the irreversible capacity (coulombic inefficiency) for cells that were cycled at low rates (e.g. C/24), which indicated that parasitic reactions consuming charge proceed independent of the cycling rate. 36 Since the electrolyte oxidation and decomposition can be considered as parasitic side reactions for the dual-graphite cell, the departure of the coulombic efficiency from unity (1.0000.) will increase with cycle time and behave inversely with the C-rate. In particular at elevated operation temperatures (e.g. 60 C), the efficiency will decrease remarkably. This is also what we reported in a recent publication, showing the inuence of the operation temperature on the electrochemical performance of the dual-ion system. 28 Fig. 11 displays the constant current charge/discharge cycling behavior and efficiency curves for the dual-graphite system cycled at 500 mA g À1 (corresponding to 5C if a theoretical capacity of 100 mA h g À1 is assumed). The system exhibits a stable cycling performance over 500 cycles with a discharge capacity of about 50 mA h g À1 . In addition, the Coulombic efficiency displays an average value of 99.8%. As discussed above, one can assume that the increased charge/discharge rate results in a reduced decomposition of intercalated anions as well as reduced self-discharge reactions.</p><!><p>In this work, we demonstrated promising results for the electrochemical performance of a "dual-graphite cell" using the ionic liquid-based electrolyte mixture Pyr 14 TFSI-LiTFSI in combination with ethylene sulte (ES) as an SEI-forming additive. The main issue for this dual-graphite cell is to nd a suitable electrolyte mixture that rst enables reversible lithium ion intercalation/de-intercalation into the graphite negative electrode by formation of a stable solid electrolyte interphase (SEI) as well as second allows the reversible intercalation/deintercalation of the electrolyte salt anions (here: TFSI À anions) into the graphite positive electrode with a sufficiently high Coulombic efficiency. This in turn is dependent on a high oxidative stability of the electrolyte mixture, in particular on the stability of the intercalated anion.</p><p>By the use of the Pyr 14 TFSI-LITFSI ionic liquid electrolyte with 2 wt% of ES, it was possible to cycle the dual-graphite cell with a high reversibility, avoiding graphite exfoliation at the anode which is extensively observed for the pure ionic liquid. Furthermore, we found that the discharge capacity, which is related to the anion intercalation for a metallic lithium/graphite dual-ion cell, is nearly doubled if ES as an additive is used. At an upper cut-off potential of 5.0 V vs. Li/Li + , a discharge capacity of 97 mA h g À1 is reached, while only 50 mA h g À1 is obtained if no electrolyte additive is present. By the use of in situ XRD measurements we studied the anion intercalation into graphite with respect to the stage formation and structural composition. It was possible to determine the maximum stage number as well as to calculate the periodic repeat distance, the TFSI À gallery height and the gallery expansion. From these investigations, we found that by the use of ES as an electrolyte additive a maximum stage number of 1 is reached in the rst cycle, while for the pure ionic liquid only a stage 2 is obtained. Therefore, the addition of ethylene sulte to the electrolyte not only results in an enhanced discharge capacity for the anion uptake, but also in a different structural composition. For the pure ionic liquid, we found a maximum capacity of 115 mA h g À1 for the TFSI À uptake into graphite, which corresponds to a stage-1 GIC with a stoichiometry ranging between C 19 TFSI and C 20 TFSI. 10 However, these results were obtained at an increased operation temperature of 60 C. 10 For the Pyr 14 TFSI-LiTFSI electrolyte with ES as an additive we determined a maximum capacity of 126 mA h g À1 , which corresponds to a stage-1 GIC with a stoichiometry of ca. C 18 TFSI. The enhancement in the specic capacity and the change in the potential prole for intercalation/de-intercalation, e.g. a reduced onset potential for anion intercalation, may be related to a reduced coordination of Li + ions with TFSI À anions in the presence of ES, i.e. the diffusion coefficient of the anion may be enhanced by addition of ES. However, these assumptions need to be conrmed in future investigations.</p><p>The constant current charge/discharge cycling investigations of the dual-graphite system displayed a stable cycling performance over 50 cycles with a specic discharge capacity varying from 32 mA h g À1 to 121 mA h g À1 depending on the upper charging end voltage and the rate. In a previous publication, we showed that a stable cycling is realized even over 500 cycles for this dual-graphite system. 20 Enhanced charging end potentials and lower charge/discharge rates result in a higher specic capacity. The Coulombic efficiency can also be tailored by the charging end potential and rate, while here a contrary trend was found. With increasing rate and decreasing upper charging end potential, the Coulombic efficiency can be enhanced and may exceed 99%.</p><p>Recently, we reported that the charge process (¼ intercalation of lithium ions and TFSI À anions into graphite) is the ratedetermining step for the dual-graphite system, while a discharge capacity ranging from 100 mA h g À1 to 30 mA h g À1 can be achieved for a specic current that ranges from 10 mA g À1 to 500 mA g À1 . 20 Here, we could additionally show that a stable charge/discharge cycling performance over 500 cycles at a specic current of 500 mA g À1 (corresponds to 5C if a theoretical capacity of 100 mA h g À1 is assumed) is achieved with a specic</p>

Royal Society of Chemistry (RSC)

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes\xe2\x80\xa0

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM1-PYK). To detail similarities/differences in the allosteric function of these two homologs, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM1-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM1-PYK (i.e. the L-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs. rM1-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologs of a protein family.

effector_analogues_detect_varied_allosteric_roles_for_conserved_protein-effector_interactions_in_pyr

<!>Materials<!>Mutagenesis and Protein Expression and Purification<!>Kinetic Assays<!>Data Analysis<!>Minimum requirement for effector binding<!>Determinant of allostery<!>Summary<!>

<p>All pyruvate kinase isozymes catalyze the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP as the last step in glycolysis. However, various isozymes, including mammalian enzymes, differ in regulatory properties. The pyruvate kinase isozyme from human liver (hL-PYK) has decreased affinity for PEP when allosterically inhibited by Ala. In contrast, Ala binding to the isozyme from rabbit muscle (rM1-PYK) elicits minimal change in the affinity for PEP (1). Instead, rM1-PYK has decreased affinity for PEP when allosterically inhibited by Phe. Despite their different functional outcomes, Ala and Phe competitively bind to rM1-PYK, indicating that they bind to the same site on the protein. Thus, the differential effects of Ala on PEP binding by rM1-PYK and hL-PYK provide a means for investigating how allosteric function can vary between homologous proteins.</p><p>Previously, we identified the amino acid binding site of rM1-PYK by co-crystallization with Ala (Figure 1; PDB 2G50) (1). Ala binding in the equivalent site of hL-PYK has been confirmed by mutagenesis (e.g. H476L as shown in Supporting Information). Although, hL-PYK and rM1-PYK share 67% identity and 81% similarity overall, all of the residue side-chains in the amino acid binding site are completely conserved in these two proteins. The two non-conserved protein residues in this binding site have their backbone atoms exposed to the effector.</p><p>We report here how 45 different amino acid analogues bind to and allosterically regulate hL-PYK. This analogue series parallels that previously used in the study of amino acid inhibition of rM1-PYK (1). Using the analogue series, we distinguished the chemical moieties of the effector required for binding to hL-PYK and those that determine the magnitude of the allosteric response. A comparison of these properties in the hL-PYK and rM1-PYK (1) systems supports that effector binding, but not allosteric function, are conserved between the two homologues.</p><!><p>The potassium salts of ADP and PEP were purchased from Chem-Impex International, Inc. NADH was from Sigma. L-lactic dehydrogenase (Type III bovine heart) was purchased from Calzyme Laboratories, Inc. Other buffer components were from Fisher Scientific and Sigma. The pLC11 plasmid encoding hL-PYK was obtained as a gift from Drs. Andrea Mattevi and Giovanna Valentini (2).</p><p>Since the low solubility and low affinity for some analogues (listed by supplier) may have prevented an observed allosteric effect, the highest concentrations used to determine allosteric responses are shown in brackets, and that used in competitive binding (when completed) are in italicized brackets. The L-forms of Ala [559 mM], Arg [559 mM], Asn [50.3 mM], Asp [6.50 mM], Cys [83.80 mM], Gln [119 mM], Gly [977.5 mM], Glu [12.3 mM], His [81 mM], Ile [61.4 mM], Leu [56.4], Lys [559 mM], Met [167 mM], Phe [83.8 mM], Pro [559 mM], Ser [559 mM], Thr [265 mM], Trp [22.3 mM], Tyr [1.7 mM], and Val [111.7 mM] were purchased from Fisher Scientific. Ethanolamine [81 mM], ethylamine [40 mM] [13 mM] and isopropylamine [80 mM] [30 mM] were purchased from Sigma. (S)-(+)-2-Phenylglycine [8.4 mM], L-homophenylalanine HCl [1 mM], 2-aminoisobutyric acid [559 mM], butylamine [42 mM] [13 mM], D/L-2-aminocaprylic acid [0.15 mM], 4-nitro-L-phenylalanine [3.8 mM], and Omethyl-L-tyrosine [18.8 mM] were from by Aldrich. L-Homoserine [95 mM], L-(+)-2,3-diaminopropionic acid [56 mM], L-(+)-2-aminobutyric acid [559 mM], propionic acid [20.1 mM] [7.7 mM], and L-alanine methyl ester HCl [100 mM] [29 mM], L-norvaline [69.3 mM], L-norleucine [0.44 mM], N-methyl-L-alanine [559 mM], D-alanine [559 mM], 3-cyclohexyl-L-alanine [12.7 mM], D/L-2-aminoheptanoic acid [10.5 mM], and L-alaninol [160 mM] [40 mM] were obtained from Fluka. N-Formyl-L-alanine [15.3 mM] [10 mM], and N-acetyl-L-alanine [20.5 mM] [10 mM] were purchased from MP Biomedicals. D-phenylalanine [67 mM] and (S)-(+)-2-amino-2-methyl-3-phenylpropanoic acid [38.50 mM] were purchased from Acros Organics.</p><!><p>Mutagenesis of the hL-PYK gene to create the H476L gene was with Quikchange (Stratagene). Wild type and H476L were expressed in the FF50 strain of Escherichia coli (3). Cell lysis, ammonium sulfate fractionation and DEAE-cellulose column purification were carried out as previously reported (3). Purified proteins were used in all studies with amino acid analogs.</p><!><p>Activity measurements were carried out at 30 °C using a lactate dehydrogenase coupled assay (4). Reactions were in 350 µL bicine buffer containing 50 mM bicine/KOH, 5 mM MgCl2, 0.1 mM EDTA, 0.18 mM NADH, 19.6 U/mL lactate dehydrogenase and 5 mM ADP at pH 7.5. PEP and effector ligand concentrations were varied as indicated. PEP and effector ligand stock solutions were adjusted to pH 7.5 with KOH before addition, and dilutions were in KCl to maintain constant total K+ concentration of 150 mM in all assays (4). The enzymatic reaction was initiated with PEP and monitored at 340 nm over time. Data were collected in a 96-well plate using a Molecular Devices Spectramax Plus384 spectrometer. Initial rates were collected from the linear portion of the progress curve.</p><!><p>Throughout this work, "K-type" heterotropic allostery is defined to occur "when one ligand binds to a protein differently in the absence, versus the presence, of a second ligand," given that the two ligands bind at different locations on the protein (5). As previously discussed (5–7), this linked-equilibrium view of allostery defines allosteric coupling (Qax) as a ratio of binding constants: (1)Qax=(KiaKia/x)=(KixKix/a), where Kia = the dissociation constant for the first ligand, A, binding to the protein in the absence of the second ligand, X; Kia/x = the dissociation constant for A binding to the protein with X prebound, Kix = the dissociation constant for X binding to the protein in the absence of A; and Kix/a = the dissociation constant for molecule X binding to the protein with A pre-bound. Qax = 1 means no allosteric response, Qax > 1 defines allosteric activation, and Qax < 1 defines allosteric inhibition. Since Qax is a ratio, the magnitude of this allosteric coupling is independent of the magnitude of any one ligand dissociation constant. Therefore, individual atom-atom interactions between atoms from protein residues and atoms from chemical moieties of the ligand may contribute uniquely to ligand binding vs. allostery.</p><p>Data fitting was with the nonlinear least-squares analysis of Kaleidagraph (Synergy) software. Fits of PEP titrations of initial rates (ν) used to obtain Kapp-PEP are as previously described (3, 4). Although the potassium concentration used throughout ligand concentration range was kept constant, a control experiment was included to ensure that other counter ions and/or non-specific effects of the various Ala analogues were not contributing to the observed regulation. For this control, the impact of the analogues on the affinity of H476L for PEP was monitored. H476L completely removes an Ala elicited response but maintains an affinity for PEP similar to that of wild type hL-PYK (see supporting information). Responses of wild type protein to analogues were corrected by dividing the Kapp-PEP vs. analogue response for wild type by the Kapp-PEP vs. analogue response for H476L (i.e. subtraction of the respective free energies).</p><p>The magnitude of Qax is measured by plotting the Kapp-PEP values as a function of effector concentration and fit to equation 2 (7): (2)Kapp-PEP=Ka(Kix+[Effector]Kix+Qax[Effector]) where Ka = Kapp-PEP when [Effector] = 0. In several cases, it was not possible to obtain formation of the ternary substrate-enzyme-effector complex (i.e. formation of the upper plateau when plotting Kapp-PEP as a function of effector analogue concentration) within the working concentration range of effector analogues. When sufficient data cannot be collected to define the upper plateau, Johnson and Reinhart (8) demonstrated that eq 2 simplifies to: (3)Kapp-a−Ka(1+[Analog]Kix-analog) This simplified equation is equivalent to competitive binding between A and X. Therefore, when the upper plateau could not be obtained, data were fit to eq3 as a means of evaluating effector analogue binding. Data for effector analogues fit in this manner can be used to evaluate which region of the effector contribute to effector binding, but give no insights into the region of the effector that contribute to eliciting the allosteric response. To highlight this distinction, parameters obtained from fits to eq2 and 3 are segregated in Table 1.</p><p>When an amino acid analogue failed to elicit an allosteric response, the ability of that compound to bind competitively with Ala to hL-PYK was used to test for binding. In this approach Kix-Ala was determined (as described above using fits to eq2) at varying concentrations of analogue (see supporting information). If competitive binding is observed, a Kix value for the analogue could be determined by fitting such data to the competitive equation, i.e. same form as eq3. However we did not identify any analogues that showed competitive binding with Ala that did not also elicit sufficient influence on Kapp-PEP to allow an evaluation of Kix via fitting the allosteric response to eq2 or 3. Our discussion will consider quantitative comparisons of fit parameters (Table 1).</p><p>Due to the relatively low affinity of hL-PYK for Ala, our studies used very high ligand concentrations. Two controls can be considered to gain confidence that the results represented herein are not due to non-specific effects. First, we can consider the response of wild type hL-PYK to very high concentrations of Ala (see supporting information). Once Ala concentrations are sufficiently high to saturate the effector binding site (i.e. the upper plateau is obtained at 25 mM), Kapp-PEP is not further responsive to very high (up to 500 mM) concentrations of Ala. Therefore, it does not appear that very high concentrations of Ala alter Kapp-PEP due to non-specific effects. It follows that when mutant proteins bind Ala with affinities lower than the wild type protein, Ala concentrations up to 100 mM can be used to evaluate binding and allosteric properties. Secondly, Ala analogues (and/or counter ions associated with those analogues), as opposed to Ala may promote non-specific binding effects. H476L binds PEP with a similar affinity as the wild type protein (see supporting information). However, the Kapp-PEP of H476L shows no response to Ala up to the 100 mM concentration range used. It follows that any response of this mutant protein to high concentrations of an Ala analogue must be due to non-specific effects. Therefore, allosteric responses of wild type protein to analogues smaller than Ala (i.e. compounds that represent a fractionation of the Ala molecule) were corrected by dividing the Kapp-PEP vs. analogue response for wild type by the Kapp-PEP vs. analogue response for H476L (i.e. subtraction of the respective free energies).</p><!><p>The first question to be addressed with the analogue series is which chemical moieties of the effector are the minimum required for binding to hL-PYK. Since the amino and carboxyl groups of the effector were required for binding to rM1-PYK, modification of these regions of the effector were first considered. Complete removal of the carboxyl group of the effector (ethylamine vs. Ala) or replacement of this moiety with either a methyl (isopropylamine vs. Ala) or a methyl alcohol (alaninol vs. Ala) group reduces binding sufficiently to prevent formation of the upper plateau within the working analogue concentration range. However, L-alaninol and isopropylamine bind to hL-PYK with similar affinities, indicating that the hydroxyl oxygen contributes little to binding affinity. Therefore, we can speculate that the carbonyl oxygen must contribute to effector binding. Like rM1-PYK (1), hL-PYK is also regulated by L-alanine-methyl ester, indicating that the effector (and allosteric response) does not require a charge on the carboxylate group (i.e. a charge on the carboxylate group is not required for binding or allosteric functions). Overall, this data trend follows that observed for rM1-PYK, with the exception that there is less distinction between the binding affinities of hL-PYK for ethylamine vs. isopropylamine and alaninol.</p><p>Also similar to the finding in the rM1-PYK system (1), complete removal of the amino group (propionic acid) prevents binding of the effector to hL-PYK. Although the addition of larger chemical moieties at the amino position (N-formyl-L-ala and N-acetyl-L-ala) prevented an allosteric response, the addition of a methyl and a cyclic group lead to a minimal decrease both in binding affinity and allosteric inhibition (N-methyl-L-ala and Pro vs. Ala). Both of the respective analogues contain secondary rather than primary amines, indicating that the primary amine of standard amino acids is not required for binding or allosteric functions. Of these, the regulation by N-methyl-L-ala shows the most contrast with the results obtained for rM1-PYK; this analogue did not elicit an allosteric response in the muscle protein. However, since rM1-PYK is allosterically inhibited by Pro (1, 9), differences between the two pyruvate kinase isozymes appear limited to the ability to accommodate various chemical moieties attached to the amino group, rather than one isozyme selecting against any additions to the effector amino group. However, it appears that effector amino nitrogen is required for binding to hL-PYK, consistent both with results from effector analogue studies of rM1-PYK and the predicted contribution based on how the effector coordinates to the protein (Figure 1).</p><p>Since both the amino nitrogen and the carbonyl oxygen (above) are required for binding and removal of additional moieties (Ala vs. Gly, ethanolamine, ethylamine, and butylamine) greatly reduce affinity, the L-2-aminopropanaldehyde substructure appears to be the primary requirement for effector binding to hL-PYK. This same L-2-aminopropanaldehyde substructure was found to be required for effector binding to rM1-PYK.</p><!><p>The second question to be addressed in this study is which region of the effector elicits the allosteric response. With regards to the modification introduced at the amino and carboxyl groups (discussed above), all effector analogues that bind to hL-PYK elicit an allosteric response. In addition, the effects of additions to the chiral carbon and additions of side chain atoms beyond the Cβ of Ala were considered. However due to the lack of a strong data trend, little can be concluded regarding the accommodation of additional chemical moieties at the chiral carbon of the effector. In contrast to the lack of response in rM1-PYK (1), hL-PYK shows inhibition by 2-aminoisobutyric acid and D-alanine. These analogues bind to hL-PYK with different affinities and elicit different magnitudes of allosteric coupling as compared to Ala (i.e. the L-form of alanine). The fact that these analogues bind and influence PEP affinity greatly differs from the lack of binding in the rM1-PYK isozyme. In contrast to the small Ala analogues, only the L-form of Phe (not D-phenylalanine) elicits an allosteric response. Replacement of the α-hydrogen of L-Phe with a methyl group (S(+)-2-amino-2-methyl-3-phenyl-propionic acid vs. L-Phe) also eliminates an allosteric response, which is in contrast to the minimal effect on coupling caused by the methyl group substitution to Ala (2-Aminoisobutyric acid vs. Ala). Therefore, although there is no obvious data trend for analogues with additions at the chiral carbon, the data consistently indicates that all effector analogues that bind to hL-PYK elicit an allosteric response.</p><p>Since Pro and Ala are the only two amino acid effectors that have been co-crystallized with any of the pyruvate kinase isozymes (1, 9), a full appreciation for how large effector side-chains interact with the binding site is currently lacking. Nonetheless, effector analogues can be used to describe the functional roles of the effector side-chain. Many types of chemical moieties can be added to the Cβ without preventing the allosteric responses (Ser, Cys, L-(+)-2,3-diaminopropionic acid, 2-aminobutyric acid, Pro, Val, Thr, Met, and homoserine). Like rM1-PYK, the effector site of hL-PYK is capable of accommodating amino acids much larger than Ala (e.g. O-methyl-L-tyr). These larger analogues elicit allostery, but there is a moderate trend indicating that binding affinity is reduced as the side-chain increases in hydrophobic bulk (O-methyl-L-tyr is the exception). Also, not all side-chains allow an allosteric response (Asp and Asn; No attempt was made to distinguish if these two ligands fail to bind or bind but fail to elicit a response). Ala, 2-aminobutyric acid, Cys, Pro, and Val elicit the largest magnitude of allosteric inhibition (i.e. smaller Qax values; Table 1); this maximum effect in hL-PYK is greatly reduced relative to the maximum inhibition in the rM1-PYK system (1). Of the analogues for which a full analysis of allosteric coupling was obtained, Phe elicits the smallest antagonism of PEP affinity (i.e. Qax value closest to 1). Therefore, the nature of the effector side-chain can modify the magnitude of the allosteric response, relative to Ala. However, the chemical moieties required to elicit an allosteric inhibition are included in Ala. Overall, it appears that the L-2-aminopropanaldehyde substructure of the effector that is required for binding is also the primary determinant of the allosteric regulation.</p><!><p>Several conclusions can be drawn from the use of amino acid analogues in this study. 1) Similar to rM1-PYK, the extent of allosteric regulation of hL-PYK is dependent on the effector chemistry (an inconsistency with those two-state models that assume all-or-none allostery). 2) The L-2-aminopropanaldehyde substructure of the amino acid is primarily responsible for effector binding to both rM1-PYK and hL-PYK (Figure 2). 3) Although the length of the hydrophobic side-chain determines the magnitude of the allosteric coupling in rM1-PYK, the primary allosteric determinants in the hL-PYK system are the amino and carboxyl groups of the effector. Consequently, there is a sharp contrast between the two isozyme systems: Although the determinants of effector affinity and the magnitude of the allosteric response were separate moieties of the effector in the rM1-PYK, this separation of function was not apparent for hL-PYK inhibition.</p><p>The contrasting effector moieties that determine the magnitude of the allosteric coupling in hL-PYK vs. rM1-PYK has important implications for the common assumption of conserved allosteric function within protein families. Sequence based evolution/co-evolution of allostery within a family of homologues intrinsically assumes that this regulatory property is conserved within that family. There is growing concern that this assumption is not valid (5, 10–15). The observation that different regions of the amino acid inhibitor elicit the allosteric response in hL-PYK vs. rM1-PYK strengthens this concern.</p><!><p>This work was supported by NIH grant DK78076.</p><p> Supporting Information </p><p>Supporting information includes a competitive binding example, a high ligand concentration control, and a table including all analogue structures. This material is available free of charge via the Internet at http://pubs.acs.org.</p><p>pyruvate kinase</p><p>the pyruvate kinase isozyme found in rabbit brain and muscle</p><p>the pyruvate kinase isozyme expressed in human liver</p><p>phosphoenolpyruvate</p>

PubMed Author Manuscript

Venom Alkaloid and Cuticular Hydrocarbon Profiles Are Associated with Social Organization, Queen Fertility Status, and Queen Genotype in the Fire Ant Solenopsis invicta

Queens in social insect colonies advertise their presence in the colony to: a) attract workers\xe2\x80\x99 attention and care; b) gain acceptance by workers as replacement or supplemental reproductives; c) prevent reproductive development in nestmates. We analyzed the chemical content of whole body surface extracts of adult queens of different developmental and reproductive stages, and of adult workers from monogyne (single colony queen) and polygyne (multiple colony queens) forms of the fire ant Solenopsis invicta. We found that the composition of the most abundant components, venom alkaloids, differed between queens and workers, as well as between reproductive and non-reproductive queens. Additionally, workers of the two forms could be distinguished by alkaloid composition. Finally, sexually mature, non-reproductive queens from polygyne colonies differed in their proportions of cis-piperidine alkaloids, depending on their Gp-9 genotype, although the difference disappeared once they became functional reproductives. Among the unsaturated cuticular hydrocarbons characteristic of queens, there were differences in amounts of alkenes/alkadienes between non-reproductive polygyne queens of different Gp-9 genotypes, between non-reproductive and reproductive queens, and between polygyne and monogyne reproductive queens, with the amounts increasing at a relatively higher rate through reproductive ontogeny in queens bearing the Gp-9 b allele. Given that the genotype-specific piperidine differences reflect differences in rates of reproductive maturation between queens, we speculate that these abundant and unique compounds have been co-opted to serve in fertility signaling, while the cuticular hydrocarbons now play a complementary role in regulation of social organization by signaling queen Gp-9 genotype.

venom_alkaloid_and_cuticular_hydrocarbon_profiles_are_associated_with_social_organization,_queen_fer

Introduction<!>Insects<!>Chemical Extraction<!>Chemical Analysis<!>Chemical Identification<!>Quantitative Analysis<!>Statistical Analysis<!>Differences in Venom Alkaloid Composition Among Females of Different Classes<!>Differences in Venom Alkaloid Composition Associated with Reproductive Status of Queens<!>Differences in Venom Alkaloid Composition Between Workers from Monogyne and Polygyne Colonies<!>Differences in Cuticular Hydrocarbon Composition Associated with Gp-9 Genotype and Reproductive Status of Queens<!>Discussion

<p>Communication plays a central role in the organization of ant colonies. It is especially important for queens to communicate their reproductive potential and status to nestmates. As they mature sexually, queens must avoid aggression by nestmate workers and gain acceptance by workers as new reproductives, if the colony is recruiting such individuals. Once making the transition to reproductive status, queens must attract the attention and care of workers (Ortius and Heinze, 1999; Hannonen et al., 2002). At the same time, reproductive queens must produce signals that regulate reproductive development and behavior of other colony members (Keller and Nonacs, 1993). Elucidation of the mechanisms that underlie the communication of individual reproductive potential and status is critical for understanding the organization of ant societies.</p><p>A key question regarding these mechanisms is what compounds are used to mediate the interactions between queens and workers? Studies suggest that cuticular hydrocarbons play a central role in nestmate discrimination, queen attraction, and regulation of reproduction in many ants, bees, wasps, and termites (Monnin, 2006; Le Conte and Hefetz, 2008; Liebig et al., 2009; Peeters and Liebig, 2009; Weil et al., 2009; van Zweden and D'Ettorre, 2010). The hydrocarbon characteristic of reproductive queens in the ant Pachycondyla inversa, for example, elicits a strong antennal response in electro-antennograms of workers, thus suggesting a sensory bias to this compound (D'Ettorre et al., 2004). In the ant Aphaenogaster cockerelli, workers with highly active ovaries produce cuticular hydrocarbons identical to those of reproductive queens (Smith et al., 2008); such workers are attacked by nestmates, presumably preventing them from reproducing. These attacks can be triggered in laboratory assays by the alkane n-pentacosane, the main component of the hydrocarbon profile of reproductive queens (Smith et al., 2008). Other direct evidence for an important role for hydrocarbon profiles in the regulation of ant reproduction comes from egg discrimination experiments in Camponotus floridanus. Reproductive queens produce a hydrocarbon profile that contains roughly twice the number of components as the profiles of non-reproductive queens (Endler et al., 2006). These reproductive queen hydrocarbon profiles also are present on queen-laid eggs, but not on worker-laid eggs, allowing workers to recognize and eliminate the latter (Endler et al., 2004). Finally, a primer effect of a major hydrocarbon compound produced by fertile queens of the ant Lasius niger has been demonstrated through reduced ovarian development of workers exposed to this compound compared to controls (Holman et al., 2010).</p><p>Given the widespread association between cuticular hydrocarbon profiles and reproductive status in eusocial insects, and the direct evidence for these compounds as fertility signals, it is especially interesting to find and explain possible exceptions to this pattern. In the red imported fire ant, Solenopsis invicta, the role of cuticular hydrocarbons has been studied only in the context of nestmate discrimination, with experimental transfer of hydrocarbons having no effect, thus suggesting that these compounds are not involved in this behavior (Anderson and Vander-Meer, 2001). Because cuticular hydrocarbons are involved in nestmate discrimination in many other social insect species (van Zweden and D'Ettorre, 2010), their lack of use in nestmate discrimination for S. invicta suggests that they also may not be involved in fertility signaling. In fact, hydrocarbon patterns for the postpharyngeal glands of queens did not change over the course of 10 days following mating and initiation of oogenesis (Vander Meer et al., 1982), a period during which such signaling to potential reproductive rivals or worker nestmates should be important.</p><p>Previous studies have shown that fully reproductive S. invicta queens produce a pheromone that inhibits winged, virgin queens from shedding wings (dealating) and developing ovaries (Fletcher and Blum, 1981; Vargo, 1998); workers are not affected in this way, because they have rudimentary ovaries and cannot lay eggs. The inhibitory pheromone(s) apparently originates from the venom sac and the postpharyngeal glands (Vargo and Hulsey, 2000), but the components are yet to be identified. The venom sac also has been shown to be the source of large amounts of venom alkaloids (Brand et al., 1972, 1973) and of pyranones. The latter are attractive to workers (Rocca et al., 1983a,b) but do not inhibit the reproductive development of winged queens (Glancey et al., 1984). In workers, the most abundant components of the alkaloids are trans-2-methyl-6-(cis-4′-n-tridecenyl) piperidine (trans-C13:1), trans-2-methyl-6-n-tridecylpiperidine (trans-C13), trans-2-methyl-6-(cis-6′-n-pentadecenyl) piperidine (trans-C15:1), and trans-2-methyl-6-n-pentadecylpiperidine (C15). In alate queens, the venom constituents are different, with the most abundant component being cis-2-methyl-6-n-undecylpiperidine (cis-C11) (Brand et al., 1973). While workers inject venom directly into other animals for defense and predation, or spray it throughout the nest environment, presumably for protection against microbial pathogens, queens apply it over eggs as they are laid. It has been suggested that this behavior protects eggs from entomopathogenic fungi (Vander-Meer and Morel, 1995; Tschinkel, 2006). However, this behavior also raises the possibility that venom alkaloids on the surface of eggs may play a role in advertising the presence and fertility status of queens, as do hydrocarbons in other social insects.</p><p>An added element of complexity in the pheromonal regulation of reproduction and social organization in S. invicta comes from the occurrence of two social forms: monogyne, in which colonies contain a single queen, and polygyne, in which colonies contain multiple queens. The two social forms differ in several ways. For example, monogyne colonies are initiated by a single queen who raises her first brood by using body reserves, while polygyne colonies are initiated by budding, a process in which queens and workers leave the colony to initiate a new colony nearby (see Ross and Keller, 1995). Polymorphism in social organization is associated with allelic variation at the gene Gp-9; in the USA, monogyne colonies contain only inhabitants bearing the B allele, while polygyne colonies also include inhabitants bearing the b allele (Gotzek and Ross, 2007). Monogyne workers (all of which are BB homozygotes) accept a single BB replacement reproductive queen if their colony has been queenless for several days, but they do not tolerate queens bearing the b allele. Alternatively, polygyne workers (a mix of individuals with and without the b allele) accept multiple reproductive queens bearing the b allele, but do not tolerate BB queens (Ross and Keller, 1998, 2002). This genotype-specific pattern of aggression or tolerance of queens by workers extends to nestmate non-reproductive queens in polygyne colonies; worker aggression toward young BB queens escalates as these queens approach two weeks of adult age, coincident with attainment of sexual maturity and reproductive competence (Keller and Ross, 1993a, 1999; Brent and Vargo, 2003). Ultimately, all sexually mature BB queens in polygyne colonies are executed or leave the nest on mating flights (Keller and Ross, 1999). Importantly, worker aggression toward BB queens in polygyne colonies is induced by a transferable queen signal on the cuticle (Keller and Ross, 1998). While production of this signal is related to weight and tendency to initiate egg-laying in non-reproductive queens, it is not related to fecundity in reproductive queens (Keller and Ross, 1993a, 1999; Ross and Keller, 1998). This has led to the suggestion that at least two distinct queen-produced pheromones are involved in regulation of colony queen number and identity in S. invicta: one communicating Gp-9 genotype and the other communicating actual or potential reproductive status (Keller and Ross, 1999, Gotzek and Ross, 2007).</p><p>Here, we compared venom alkaloid and cuticular hydrocarbon profiles of queens of different stages of sexual maturity, reproductive status, and Gp-9 genotype, as well as workers, originating from monogyne and polygyne S. invicta colonies. If cuticular hydrocarbons do not encode any of the above-mentioned types of information, then the abundant piperidines, unique to the venom of this group of ants, may fill this role instead. Our goal was to detect associations between chemical profiles and reproductive state/genotype of different classes of colony members. Such associations may indicate a role for specific compounds as releaser or primer pheromones involved in communicating Gp-9 genotype and/or fertility status to workers, roles essential to the regulation of colony social organization in fire ants.</p><!><p>Large colonies of each social form of Solenopsis invicta were collected and reared in the laboratory under standard conditions (Ross, 1988). A total of 25 polygyne and 23 monogyne colonies were collected from Athens-Clarke Co. and Oglethorpe Co., Georgia, USA, respectively. Social organization of the polygyne colonies was determined by finding multiple reproductive (wingless, egg-laying) queens in each. Social organization of the monogyne colonies was determined by finding only a single reproductive queen in each. Social organization was subsequently confirmed for all colonies by genotyping several reproductive queens (polygyne colonies) or 20 adult workers (monogyne colonies) at the gene Gp-9, using horizontal starch gel electrophoresis coupled with amido black staining (DeHeer et al., 1999); all polygyne reproductive queens possessed the Bb genotype, while all monogyne workers possessed the BB genotype, as expected (Gotzek and Ross, 2007). Colonies were fed daily by alternating a high-protein diet (tuna/dog food/peanut butter mix) with a high-carbohydrate diet (assorted vegetables/granulated sugar mix). These diets were supplemented with crickets and/or mealworms on a twice-weekly basis.</p><p>Several hundred adult workers from individual laboratory colonies were collected by placing a glass vial upright in the foraging area of each rearing unit for several minutes, and allowing workers to fall into the vial; vials were placed briefly on dry ice, then stored at −80°C until extraction. Thirteen colonies of each social form were sampled in this way for determination of piperidine alkaloid profiles, while ten colonies of each form were sampled for determination of hydrocarbon profiles (see Table 1 for sample size information).</p><p>We collected queens at three different key stages of sexual and reproductive development. First, we collected 2d-old, sexually immature and non-reproductive, queens from polygyne colonies. These were obtained by transferring 1–6 newly emerged (callow) adult queens to small rearing units with several hundred worker brood and adults from the natal colony (see Table 1 for sample sizes). Forty-eight hours after transfer, queens were collected for chemical analysis. Second, we collected 14d-old queens from polygyne colonies. By this age, queens have undergone extensive weight gain and accumulation of energy reserves (fat and glycogen), characteristic of completion of maturation in S. invicta (Keller and Ross, 1993a, 1999; Brent and Vargo, 2003). Such queens are sexually mature, but typically embark on a mating flight and are inseminated before becoming reproductively active. Two to six newly emerged queens from each polygyne source colony were held individually in small rearing units, with worker brood and adults from the natal colony, for 14 d before being collected for analysis (see Table 1 for sample sizes). These queens were held individually, rather than in groups, because workers execute queens with a Gp-9 BB genotype when they attain sexual maturity in colonies that also contain queens with a Bb genotype (see Keller and Ross, 1998, 1999). This procedure also ensured that the reproductive development of young queens was not inhibited by the presence of other queens in the same unit. In the rearing units, some of the 14d-old queens dealated, a behavior associated with onset of oogenesis (Fletcher and Blum, 1981; Brent and Vargo, 2003); the identities of these queens were recorded. No 2d-old queens dealated. The third category of queens was reproductive. We collected one or two of these queens from polygyne laboratory colonies and the sole queen heading monogyne colonies (see Table 1). The mating status of the polygyne reproductive queens was unknown [approximately one-third of such queens remain unmated in invasive populations in the USA (Ross and Keller, 1995)], whereas all monogyne reproductive queens were assumed to have mated. All reproductive queens were collected after the colonies had been maintained in the laboratory for 1 month.</p><p>All collected queens were held in glass vials at −80°C, for several days to several weeks, pending chemical extraction. The Gp-9 genotypes of all 14d-old queens were determined by starch gel electrophoresis following chemical extraction; 14d-old queens of both genotypes (BB, Bb) were included in the study (Table 1). Previous work has shown that reproductive queens from established polygyne colonies always possess genotype Bb, while reproductive queens heading monogyne colonies invariably possess genotype BB (Ross and Keller, 1998; Gotzek and Ross, 2007).</p><!><p>For piperidine analyses, queens were extracted individually in 80 μl of hexane (Fluka, Buchs, Switzerland) for 2 min., and then extracted in 80 μl dichloromethane (Riedel-deHaen, Seelze, Germany) for 11 min. Adult workers were extracted as groups of 200–500 individuals in 1 ml hexane for 2 min., and then in 1 ml dichloromethane for 11 min. Solvent from the extracts was evaporated under N2, and the residue reconstituted with a known amount of hexane or dichloromethane.</p><p>We extracted individual queens and groups of workers from a second set of samples for hydrocarbon analysis. Queens were extracted in 80 μl hexane for 8 min. The solvent was evaporated under N2, and the solute was re-dissolved in 2 μl hexane. Residual solvent on 14d-old queens was allowed to evaporate for 10 min., after which queens were stored at −80°C until determination of Gp-9 genotype.</p><p>For hydrocarbon extraction of workers, subsets of 40 individuals were taken randomly from frozen colony samples and extracted in 100 μl hexane for 8 min. Solvent was removed from the extract by a N2 stream, and the residue re-dissolved in 20 μl of hexane.</p><!><p>For piperidine analyses, extracts were first analyzed qualitatively on an Agilent (Santa Clara, CA, USA) 6890 gas chromatograph (GC), coupled with an Agilent 5975 mass selective (MS) detector operated in electron impact ionization mode. Peaks in extracts then were quantified by flame ionization detection (FID). GCs were operated in splitless injection mode, and were fitted with a DB-1MS column (30 m×0.25 mm×0.25 μm; Agilent). Column ovens were programmed from 60°–200°C at 40°C min−1, after an initial delay of 2 min, then to 320°C at 5°C min−1, and held at 320°C for 5 min. The injector was set at 260°C, the MS quad at 150°C, MS source at 230°C, and transfer line at 300°C. FID temperature was set at 320°C.</p><p>For hydrocarbon analysis, the MS was used exclusively, in order to identify some alkenes that differed with queen Gp-9 genotype.</p><!><p>Venom alkaloids and hydrocarbons were identified based on retention indices, mass spectra, and fragmentation patterns, with these data compared with previously published data (Brand et al., 1972, 1973; Nelson et al., 1980; Chen and Fadamiro, 2009; Dall'Aglio-Holvorcem et al., 2009). Double bond positions in unsaturated alkyl chains were determined by GC-MS fragmentation patterns of dimethyl disulfide adducts, as described in Liebig et al. (2009). See Table 1 for details of diagnostic ions.</p><!><p>Peaks (Table 1) were aligned across samples, with only piperidines and unsaturated hydrocarbons used in the quantitative and statistical analyses. FID peak areas from both hexane and dichloromethane extracts were added, and the percentage of each peak out of total peak area calculated.</p><p>In the second set of samples, only hydrocarbon peaks were analyzed. We detected 49 compounds that were presumed to be hydrocarbons; this number was reduced to 26 for subsequent analysis. The peak area of all 49 presumptive hydrocarbons was summed and the relative proportion of each compound calculated. Then, the mean relative peak area of each compound was calculated for all queen and worker classes. For further analysis, we selected only compounds that had a mean of at least 1% of total peak area across all classes. The total amount of these 26 most abundant compounds was used as a reference for calculation of the relative proportions of each major unsaturated alkane. Peak areas of both data sets were calculated using MSD ChemStation D.02.00.275 data analysis software (Agilent Technologies).</p><!><p>For piperidines, Euclidean distances were used in non-metric multi-dimensional scaling (NMDS) analyses (Kruskal, 1964), using the program PRIMER 6.0. The first two dimensions of the model always resulted in stress values below 0.05, which is considered an excellent representation of data structure (Clarke and Warwick, 2001). The program STATISTICA 7.1 (StatSoft) was used to test for differences between monogyne and polygyne workers, and between monogyne and polygyne reproductive queens, in selected constituents of the chemical profiles, using the t-test for independent samples (these data are normally distributed; Kolmogorov-Smirnov test, P>0.2).</p><p>Data for ratios of cis-C11/(cis+trans)-C11 piperidines and peak areas of alkenes/alkadienes in queens were analyzed by means of resampling procedures coupled with binomial tests; resampling procedures were necessary because the use of multiple non-reproductive and reproductive queens from many of the polygyne source colonies meant that all replicates were not independent, violating a basic assumption of most standard statistical tests. Each class of queen was compared pairwise, with respect to ratios/peak areas, to each of the other classes, in the following way. For each iteration, a single replicate of each paired queen class was randomly selected and the difference in ratios/areas recorded; only paired replicates from different colonies were accepted. A third replicate of either of the two focal classes then was randomly selected from a different source colony than used for the first two replicates, and the difference between the two same-class replicate ratios/peak areas recorded. This procedure was repeated 1000 times (with replacement), and the paired between-class and within-class differences analyzed with binomial tests. The sample sizes, used for calculating the binomial probabilities following such resampling procedures, potentially range from the number of independent source colonies (most conservative) to the number of replicates (most liberal) used in the comparison; we chose to use the number of colonies in our calculations.</p><!><p>Qualitative differences were apparent in the venom alkaloid profiles of the different classes of S. invicta females (Fig. 1, Table 1). The 2d-old (sexually immature, non-reproductive) queens contained the lowest amounts of alkaloids relative to hydrocarbons, with the most abundant alkaloids being the two forms of C11 piperidine and, to a lesser extent, the trans configuration of C13 piperidines. The 14d-old (sexually mature, non-reproductive) queens also contained mostly C11 piperidines, but had greater proportions of both cis and trans C13 piperidines than younger queens. Reproductive queens lacked many of the trans piperidines. In contrast to queens, workers contained almost no C11 piperidines, but they had large amounts of longer-chain piperidines, especially C13 and C15 trans forms.</p><p>NMDS analysis confirmed these differences by showing clustering of piperidine profiles of the different classes (Fig. 2). Workers were separated completely from all types of queens, and workers originating from monogyne colonies were separated from those from polygyne colonies without overlap (2D stress value: 0.02). With the exception of a single individual (highlighted as an outlier in Fig. 3), reproductive queens also were completely separated from all types of non-reproductive queens. There was, however, no consistent difference between the reproductive BB (monogyne) queens and reproductive Bb (polygyne) queens. Similarly, there were no evident differences in piperidine profiles of the young non-reproductive queens of the different maturation stages and Gp-9 genotypes.</p><!><p>The proportion of cis piperidines (cis-C11/totalC11) was not different between 2-d- and 14d-old non-reproductive queens (Figs. 1, 3A). However, reproductive queens had higher proportions of cis piperidines than did either 2d- or 14d-old queens (Fig. 3A). There also was an effect of Gp-9 genotype on proportion of cis piperidines in 14d-old queens, with BB queens having a higher proportion of cis-C11 than Bb queens (Fig. 3b). In this age class, the proportion of cis piperidines also was different between queens that had dealated and those that had not (Fig. 3b), in agreement with previous observations showing that Gp-9 BB queens dealate more readily than those with genotype Bb (Keller and Ross, 1999). Indeed, in the present study, there was also an association between Gp-9 genotype and dealation, with 92.9% (26/28) of BB queens having dealated, while only 15.7% (8/51) of Bb queens dealated (Fisher's exact test, P<0.001).</p><!><p>Adult workers from monogyne colonies had a higher proportion of saturated piperidines than workers from polygyne colonies (Fig. 4). This difference stemmed mainly from differences in saturated/total C13 piperidine ratios.</p><!><p>All castes possessed the typical hydrocarbons found in previous studies (e.g., Cabrera et al. 2004). However, we also found additional unsaturated compounds not previously reported (Fig. 5; Table 1). The following compounds met our criterion for detailed analysis: heptacosene, nonacosene, nonacosadiene, hentriacontene, and hentriacosadiene. As for the piperidine profiles, these compounds gave profiles of reproductive queens that were distinct from those of other classes of females. In particular, these compounds were completely lacking in workers (Table 1); worker hydrocarbon profiles differed from those of all queen classes (resampling procedures coupled with binomial tests, all P<0.05). Importantly, the total relative amounts of the focal unsaturated hydrocarbons in queens varied according to Gp-9 genotype and reproductive status, most dramatically between reproductive queens of the alternate (social forms) genotypes (Fig. 6). Reproductive Bb queens from polygyne colonies had a median of 17.7% of unsaturated hydrocarbons in their profile, while reproductive BB queens from monogyne colonies had a median of only 0.8% of unsaturated hydrocarbons, with one of the latter queens having no detectable amounts of unsaturated hydrocarbons.</p><p>While most (19/21) of 2d-old queens lacked any detectable amounts of unsaturated alkanes, the genotype-specific pattern of alkenes and alkadienes observed in reproductive queens had already begun to develop in queens over a 14-d period of maturation following eclosion (Fig. 6). Significantly, most 14d-old-Bb queens showed pronounced peaks of unsaturated hydrocarbons (median peak amount of 1.7%), while 14d-old-BB queens either lacked detectable amounts or had peak amounts of <0.1%. Thus, this difference in amount of unsaturated hydrocarbons, characteristic of reproductive queens of alternate genotypes, is paralleled by smaller differences in young, non-reproductive queens of alternate genotypes.</p><!><p>This study shows that both venom alkaloid and hydrocarbon profiles in the fire ant S. invicta are associated with caste, queen reproductive status, queen Gp-9 genotype, and colony social organization (Electronic supplementary material Fig. 7). Venom alkaloid composition changes dramatically, both qualitatively and quantitatively, as queens become reproductively active, and also differs between workers from monogyne and polygyne colonies. Cuticular hydrocarbon profiles also show pronounced differences among different classes of queens, with the qualitative and quantitative differences observed based mainly on the amount of alkenes and alkadienes. Differences in amounts of these unsaturated hydrocarbons are related not only to queen reproductive status, but also to Gp-9 genotype of queens, with this latter difference already noticeable at 14 days post-eclosion, when queens are sexually mature but not yet reproducing.</p><p>Previous studies showed that the venom sac of reproductive fire ant queens contains mostly C11 alkaloids, which are barely present in the sac of workers (Brand et al., 1973). We found that the proportion of cis piperidines (the less abundant geometric configuration in workers), gradually increases in the venom of queens as they become fully reproductive. Moreover, in 14d-old (mature, but non-reproductive) queens, the proportion is higher in individuals of the Gp-9 BB genotype than in those of the Bb genotype. Similarly, alkenes/alkadienes are found in significant amounts only in the queen caste, and only polygyne reproductive queens contain copious amounts. It is important to note that the difference in amounts in 14d-old queens that we detected between Bb and BB queens is attributable to variation at the Gp-9 gene (or variation at linked genes), rather than to effects of social environment, because all 14d-old queens in our study originated from polygyne source colonies. This initial difference, attributable to genotype, becomes more pronounced as queens age, accelerate oogenesis, and become fully competent reproductives.</p><p>That profiles of cis piperidines and unsaturated hydrocarbons differ between sexually mature BB and Bb queens suggests that these suites of chemicals constitute signals that workers use to recognize and discriminate between queens of the two genotypes. Importantly, such behavioral discrimination by workers only becomes apparent once queens become sexually mature (Ross and Keller, 1998; Gotzek and Ross, 2007), suggesting an association between ontogeny of these presumptive queen chemical signals and discrimination behavior directed toward them.</p><p>Previous studies showed that 14d-old-BB queens accumulate greater energy reserves, dealate more readily, and lay more eggs immediately upon dealation than 14d-old-Bb queens; i.e., sexually mature BB queens are at a more advanced state of reproductive development (Keller and Ross, 1993b; Ross and Keller, 1998; DeHeer, 2002). Thus, changes in alkaloid and hydrocarbon compositions are closely tied to physiological changes that accompany sexual maturation and the onset of reproduction that, in turn, are related to Gp-9 genotype. Specifically, proportions of cis-C11 piperidines increase more rapidly during maturation in BB than Bb queens before attaining high proportions in reproductives of both genotypes, while amounts of alkenes/alkadienes increase more rapidly in maturing Bb than BB queens before reaching extremely high levels in Bb reproductives only. In line with this, 14d-old queens that dealated (i.e., those in a more advanced reproductive state, most of which lacked allele b) also had a higher proportion of cis piperidines than queens of the same age that had not dealated (most of which possessed allele b). Our results support tenets of the model of regulation of queen number proposed by Gotzek and Ross (2007), which invokes two distinct signaling components, one (piperidines) communicating actual or potential fertility status, and the other (alkenes/alkadienes) communicating Gp-9 genotype. We note, however, that our data are not fully congruent with the model, which predicts that workers bearing the b allele should display the same chemical profile as queens bearing this allele; workers lacked the unsaturated hydrocarbons that were differentially abundant in queens of alternate Gp-9 genotypes. There are several possible explanations for this incongruence, including the two classes of chemicals acting synergistically, rather than as distinct components, hydrocarbon expression depending jointly on genotype and fertility status, or other classes of chemicals on the cuticle surface, including perhaps the GP-9 protein itself (unpublished data), being involved in signaling Gp-9 genotype in S. invicta.</p><p>To date, fertility signaling has been assigned to cuticular and egg surface hydrocarbons in most social insects in which this effect has been investigated (Monnin, 2006; Le Conte and Hefetz, 2008; Peeters and Liebig, 2009; Liebig, 2010). In S. invicta, it appears that both hydrocarbons and piperidines may play roles in providing information relevant to regulation of colony queen number, as described above. Like hydrocarbons, piperidines are derived from acetate (Leclercq et al., 1996), raising the possibility that similar metabolic processes regulate the synthesis of hydrocarbon chains and piperidines.</p><p>Previously, piperidines were found not to be attractive in S. invicta (Vander Meer et al., 1980). The attractiveness of the queen poison gland secretion is attributed to two pyranones identified by Rocca et al. (1983a,b). Nevertheless, these pyranones do not delay the onset of reproduction in young queens, a primer effect that the poison sac was found to produce (Fletcher and Blum, 1981; Glancey et al., 1984; Vargo, 1998). Piperidines, with their large amounts dominating poison sac content, could be potential players in this phenomenon.</p><p>The ratio of saturated piperidines in adult worker cuticular extracts was higher in S. invicta workers from monogyne colonies than in workers from polygyne colonies (Fig. 4). This is in agreement with previous findings of differences in alkaloid composition between workers of the two social forms (Lai et al., 2008; Lin et al., 2010). Quantitative differences in piperidine composition have also been found between minor and major workers, as well as among workers of different ages (Deslippe and Guo, 2000), suggesting that piperidines also may be used for caste and possibly nestmate recognition.</p><p>Many ants use secretions from the metapleural gland as antiseptics for their nest and body surface. Fire ants have fatty acids with antimicrobial properties in the metapleural glands that may be used for that purpose (Cabrera et al., 2004), although venom alkaloids also may take part in defense against microbial pathogens. Workers of S. invicta display gaster-flagging behavior, whereby they spray the contents of the poison gland onto the nest and their body (Obin and Vander-Meer, 1985). Indeed, venom alkaloids dominate in solid phase microextraction (SPME) extracts of Solenopsis xyloni, even when the gaster cuticle is excluded from extraction (unpublished data). As high molecular weight compounds, these alkaloids are nonvolatile (MacConnell et al., 1971) and potentially can remain on the cuticle for long periods. Considering the above, it is not surprising that piperidines also are found in abundance throughout the nest (Chen, 2007). The large quantities of venom alkaloids produced for a possible role in protecting ants from microbial pathogens may have facilitated their co-option to serve as fertility signals within colonies, especially if a queen's venom becomes distributed on her cuticle. Thus, the present study suggests that venom alkaloids of fire ants may have secondarily evolved roles in communication that typically are assigned to cuticular hydrocarbons in many other social insects (Monnin, 2006; Le Conte and Hefetz, 2008; Peeters and Liebig, 2009; Liebig, 2010). Despite the large amounts of alkaloids, hydrocarbons still may play a role in regulating social structure by signaling queen Gp-9 genotype. Future manipulative behavioral studies should directly examine whether, and if so how, these and other compounds found on the queen cuticle, including GP-9 protein, function in maintaining the structure of fire ant societies.</p>

PubMed Author Manuscript

A Free Aluminylene with Diverse σ-Donating and Doubly σ/π-Accepting Ligand Features for Transition Metals

We report herein the synthesis, characterization, and coordination chemistry of a free N-aluminylene, namely a carbazolylaluminylene 2b. This species is prepared via a reduction reaction of the corresponding carbazolyl aluminium diiodide. The coordination behavior of 2b towards transition metal centers (W, Cr) is shown to afford a series of novel aluminylene complexes 3-6 with diverse coordination modes. We demonstrate that the Al center in 2b can behave as: 1. a σ-donating and doubly π-accepting ligand; 2. a σ-donating, σ-accepting and π-accepting ligand; and 3. a σ-donating and doubly σ-accepting ligand. Additionally, we show ligand exchange at the aluminylene center providing access to the modulation of electronic properties of transition metals without changing the coordinated atoms. Investigations of 2b with IDippCuCl (IDipp = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) show an unprecedented aluminylene-alumanyl transformation leading to a rare terminal Cu-alumanyl complex 8. The electronic structures of such complexes and the mechanism of the aluminylene-alumanyl transformation are investigated through density functional theory (DFT) calculations.

a_free_aluminylene_with_diverse_σ-donating_and_doubly_σ/π-accepting_ligand_features_for_transition_m

Introduction<!>Results and Discussion<!>Conclusion

<p>Ancillary ligands play essential roles in modern synthetic chemistry and materials science. [1] It is well-known that L-type ligands can not only donate electron density to transition metal centers (σ-donating) but also accept d-electrons from the metal centers via π-backdonation (π-accepting). [1][2] Such ligands in the coordination sphere of transition metals can also exhibit the σaccepting ability to act as a Lewis acid for external ligands. [3] According to the coordination modes of terminal L-type ligands (Figure 1a), they can be classified into four broadly defined categories, namely σ-donating/π-accepting type I, σ-donating and doubly π-accepting type II, σ-donating and doubly σ-accepting type III, and σ-donating, σand π-accepting type IV.</p><p>Ligands based on Al have attracted considerable attention due to the fundamental significance of the structural and electronic properties as well as their applications in synthetic chemistry. [4] The electropositive nature of aluminium (χ = 1.61) makes such ligands highly electron-releasing, thereby exhibiting unusual bonding and reactivities. [5] In the case of the terminal L-type Al ligands, representative examples include transition metal complexes A [6] and B [3g-j] derived from Schnöckel's (Cp*Al)4 [7] and Roesky's HC[(CMe)(NDipp)]2Al, [8] respectively (Figure 1b). It was independently demonstrated by the Power group [3g] and Crimmin group [3h] that unprecedented low-valent molecular complexes HC[(CMe)(NDipp)]2AlCu[(NMes)(CR)]2CH (R= Me, CF3) feature an unsupported dispersion-enhanced Al−Cu bond. Furthermore, in the late 1990s, the aluminylene complexes C of type III were disclosed by Fischer,Frenking et al. [9] In 2014, Tokitoh and coworkers described the synthesis of terminal Pt-aluminylene complexes D bearing a di-coordinate Al atom via the reaction of a dialumene-benzene adduct with Pt(PCy3)2. [10] The Al ligand in D reveals donor-acceptor interactions with Pt akin to the bonding mode of type II. Additionally, a few of aluminium-transtion metal hydride complexes have been shown to feature aluminylene character. [4k, 11] For transition metal-alumanyl complexes with a terminal X-type Al ligand, in two recent examples, Aldridge, Goicoechea et al. isolated an Au-alumanyl complex t Bu3PAuAl(NON) (NON = 4,5-bis(2,6-diisopropylanilido)-2,7-ditert-butyl-9,9-dimethylxanthene) containing an unprecedented nucleophilic Au center, [12] while Hill, McMullin et al. reported the syntheses of two Cu-alumanyl complexes LCuAl(SiN Dipp ) (L = N,N'-diisopropyl-4,5-dimethyl-2-ylidene and (1-(2,6diisopropylphenyl)-3,3,5,5-tetramethyl-pyrrolidin-2-ylidene, SiN Dipp = (CH2SiMe2NDipp)2) with ambiphilic Cu−Al bonding. [13] Taking advantage of sterically demanding terphenyl ligands, [14] Power, Tuononen et al. very recently disclosed the first and sole example of a room-temperature-stable monomeric aluminylene (alanediyl) : 1b) via a reduction reaction of AlI2Ar iPr8 with 5% w/w Na/NaCl. [15] This breakthrough allowed further explorations into unusual/unprecedented patterns of reactivity of E toward hydrogen [15] and organic azides, [16] in which the latter led to the first stable iminoalane with an Al≡N triple bond. In the present work, we report the synthesis, characterization and coordination chemistry of a free one-coordinate N-aluminylene (Figure 1c). Of note, this aluminylene functions as a σ-donating and doubly σ/πaccepting ligand for transition metals, leading to a series of unprecedented aluminylene and alumanyl complexes with diverse coordination modes via a simple one-step process.</p><!><p>Synthesis, Characterization and Bonding Analysis of N-Aluminylene. The installation of Al with bulky π-donor substituents, such as amino, [17] phosphino [18] or carbazolyl, [19] should enhance the stabilization of the inherent electron deficiency of free aluminylenes due to the possible π-donation of a N/P lone pair into an accessible vacant p orbital at Al. We thus chose the carbazolyl-substituted aluminium diiodides 1 as the precursors (Scheme 1). These species were readily accessible from a salt metathesis reaction of the respective potassium carbazolide with AlI3, and their structures were confirmed by single crystal X-ray diffraction analysis (Figure S30). [20] While all attempts of reducing 1a afforded an unidentified mixture, stirring a toluene solution of the more sterically encumbering 1b with excess 5% w/w K/KI (4 equivalents) from −15 to 13 o C for 2 days gave rise to the free aluminylene 2b as a white powder in 67% yield (Scheme 1).</p><p>Scheme 1. Synthesis of 2b. Single crystals of 2b suitable for X-ray diffraction were obtained from slow evaporation of a concentrated n-hexane solution at room temperature within 12 h. The X-ray diffraction study revealed the N(1) atom adopts a planar environment (sum of angles: 359.3 o ) (Figure 2). The Al(1)−N(1) bond length (1.913(9) Å) is slightly shorter than the Pyykkö standard value for an Al−N single bond (1.970 Å) [21] whereas much longer than those of typical Al=N double bonds (1.705(2)−1.725(1) Å) in terminal aluminum imides, [22] indicative of the presence of a weak N-to-Al π-donation. The Al(1) atom is located nearly symmetrically between the two flanking 3,5-di-tert-butylphenyl rings of the carbazolyl substituent. There is no strong secondary bonding interaction between Al and the two arenes in the solid state (the shortest Al−C distance: 3.015(3) Å), which is similar to that observed for Power's :AlAr iPr8 . [15] Infrared spectroscopic studies of 2b show no evidences for Al−H stretching frequencies (Figure S1).</p><p>Crystalline 2b can be stored at room temperature under an inert atmosphere for over a month. A benzene solution of 2b was heated up to 80 o C for 10 h without noticeable decomposition. However, it is extremely sensitive to moisture and oxygen, leading to the complete scission of the Al−N bond affording the corresponding carbazole and unidentified Al-containing species (Figure S29).</p><p>The ambiphilic nature of 2b is unambiguously demonstrated by its frontier molecular orbitals (M06-2X/def2-SVP) (Figure 3). The LUMO+6 and LUMO are mainly the in-plane and out-of-plane Al 3p orbitals, respectively (Figures 3a and 3b). The HOMO is composed of the lone pairs at both Al and N atoms as well as some π-bonding orbitals over the carbazolyl substituent, while the HOMO-1 predominantly involves the Al nonbonding lone pair (Figures 3c and 3d). These observations are different from those calculated for :AlAr iPr8 , [15] illustrating that the N-substitution at Al dramatically affects the electronic structure of aluminylenes. Moreover, the natural population analysis (NPA) shows that the Al atom is positively charged (0.79 a.u.) and the N atom carries a negative charge (-0.96 a.u.). The Wiberg bond index (WBI) of the Al-N bond is 0.28 which can be explained by its substantial ionic nature. The second-order perturbation theory of the natural bond orbital (NBO) method reveals that the donor-acceptor interaction from a N lone pair into a vacant p orbital at Al has a small stabilization energy of 16.5 kcal mol -1 due to the electropositive nature of Al (χ = 1.61) (Figure S32). For comparison, the calculated stabilization energies arising from a N-to-Al π-donation in t Bu2AlNMes2 [23] (Mes = mesityl) and (Mes*AlNPh)2 [24] (Mes* = 2,4,6-( t Bu)3C6H2) are 4.4 and 21.3 kcal mol -1 , respectively (Figure S33).</p><p>Compound 2b shows two absorption maxima in the UV/Vis spectrum in toluene at 346 and 356 nm (Figure S2), which are blue-shifted relative to those of :AlAr iPr8 (351 and 467 nm). [15] These absorptions are attributed to the HOMO-LUMO and HOMO-1-LUMO transitions according to TD-DFT calculations (Figure S35). Isolation of Aluminylene Complexes. We thus speculated that 2b should be an interesting ligand featuring σ-donor and σ/πacceptor properties for transition metals if the Al atom is kinetically accessible. 2b is completely inert upon stirring its benzene solution with an equal molar portion of W(CO)6 at room temperature for 12 h. However, UV lamp (254 nm) exposure is known to facilitate the removal of CO in metal carbonyls, [25] so the solution was irradiated for 24 h which cleanly furnished a new species 3 (Scheme 2). After workup, 3 was isolated as a yellow solid in 85%. The 1 H NMR spectrum of 3 shows two singlets for the t Bu groups of 3,5-di-tert-butylphenyl substitutes at 1.35 and 1.43 ppm, indicating the asymmetric nature with respect to the carbazolyl plane. Two singlet carbonyl resonances at 197.5 and 198.8 ppm are observed via a 13 C NMR spectroscopic study.</p><p>Slow evaporation of a concentrated hexane solution of 3 at room temperature resulted in X-ray quality yellow crystals after 5 h. The solid-state structure of 3 was determined by X-ray diffraction (Figure 4a). In contrast to 2b, the N(1) atom in 3 is slightly pyramidalized (sum of angles: 351.8 o ), and the Al(1)−N(1) bond (1.841(3) Å) is bent out of the carbazolyl plane, which consequently reduces the effective steric bulk of the substituent drastically. It is observed that the Al(1)−W(1) bond length (2.5363(11) Å) in 3 is much shorter compared to those of (TMEDA)Al(Et)W(CO)5 (2.670(1) Å) (TMEDA = N,N,N',N'tetramethylethylenediamine) and (TMPDA)Al(Cl)W(CO)5 (2.645(2) Å) (TMPDA = N,N,N',N'-tetramethylpropanediamine), [9b] indicating the stronger π-backdonation from W to Al in our case. Although the only known examples of terminal base-free aluminylene complexes D (Figure 1b) reveal an almost linear geometry at Al (R = H, 179.2(2) o ; R = t Bu, 174.0(1) o ), [10] the bond angle of N(1)−Al(1)−W(1) (147.31(10) o ) in 3 appears to be bent, likely due to the steric hindrance arising from two 3,5-di-tertbutylphenyl substituents. The aluminylene ligand in 3 acts as σdonor and double π-acceptor (vide infra). Species 3 represents the first example of an early transition metal-aluminylene complex with a di-coordinated Al atom. [10] As the aluminylene ligand in 3 formally contains two vacant p orbitals, 3 should be susceptible to Lewis base coordination. Indeed, 3 rapidly converted to a new product 4 quantitatively in THF (Scheme 2). Alternatively, treatment of 2b with W(CO)6 in THF at room temperature yielded 4 as well in 60% yield. In an analogous fashion, the reaction of 2b with Cr(CO)6 in THF led to a species 5 as a white solid in 62% yield. The NMR spectroscopic features of 4 and 5 are very comparable. The 1 H NMR spectra of Scheme 2. Synthesis of 3-6.</p><p>both cases display two diagnostic broad singlets (4: 0.96 and 3.30 ppm; 5: 0.96 and 3.32 ppm), integrating to four protons each. This suggests the presence of a coordinated THF molecule.</p><p>Colorless single crystals of 4 and 5 were obtained via slow evaporation of their concentrated benzene solutions. In the solid state, species 4 and 5 appear to be a W-aluminylene and a Craluminylene complexes, respectively (Figures 4b and S34). The structural parameters of the carbazolyl aluminylene parts in 4 and 5 are similar. The N(1) atoms in both cases are clearly pyramidalized (sum of angles: 311.9 o (4), 316.9 o (5)), and the Al(1)−N(1) bonds (1.898(7) (4), 1.883(3) Å ( 5)) are bent out of the carbazolyl plane. This allow the coordination of a THF molecule to Al with a Al(1)−O(1) bond length of 1.862(5) (4) or 1.853(3) Å (5), thereby compensating the electron deficiency of Al. The bond lengths of Al(1)−W(1) (2.601(2) Å) and Al(1)−Cr(1) (2.4087(12) Å) are shorter than those seen for (TMEDA)Al(Et)W(CO)5 (2.670(1) Å) and (TMPDA)Al(Cl)Cr(CO)5 (2.482(1) Å), respectively. [9b] These imply the presence of the π-backdonation from W/Cr to Al. 123.63(5) o ). [9b] Of note, the aluminylene ligand in 4 and 5 behaves as σ-donor, σ-acceptor and π-acceptor (vide infra), and such bonding modes are extremely rare for coordination chemistry. 3c, 7 The σ-acceptor property of ligands has been invoked in mechanistic studies using aluminylene ligands [4k, 5n] and related gallylene systems. [26] Importantly, Crimmin et al. disclosed that such property of aluminylenes is crucial to catalytic processes. [4k, 5n-p] In addition, the coordination behavior of free one-coordinate aluminylenes toward transition metals is hitherto unknown. [3g-j, 4a-j, 5a, 5d, 15-16] The formation of 3-5 demonstrates the facile access to metal-aluminylene complexes through this straightforward process.</p><p>DFT modelling reveals that dissociation of the THF from 4 to produce 3 is only unfavorable by the free energy of 4.5 kcal mol -1 , indicative of the labile nature of the THF. We thus envisioned the possibility for ligand exchange reactions at Al. To this end, 4dimethylaminopyridine (DMAP) was employed (Scheme 2). Addition of 2 equivalents of DMAP to a toluene solution of 4 at room temperature immediately yielded a sole product 6, which was isolated as a yellow powder in 90%. A C6D6 solution of 6 displays a characteristic singlet at 2.13 ppm integrating for twelve protons corresponding to the methyl groups of DMAP in the 1 H NMR spectrum, and there is no evidence for the presence of THF. This suggests that the coordinated THF in 4 is completely replaced by two DMAP molecules. Indeed, in the solid state, 6 bears a tetracoordinate Al(1) center with the tetrahedron geometry (Figure 4c). The bond length of Al(1)−N(1) (1.9549( 17 of 3 (2.5363(11) Å) and 4 (2.601(2) Å), and slightly longer than that in (TMEDA)Al(Et)W(CO)5 (2.670(1) Å). [9b] The formation of 6 undergoes a formal ligand exchange reaction at an aluminylene, reminiscent of scarce examples of ligand exchanges at low-valent main group centers, such as borylene, [27] phosphinidene, [28] carbene, [29] and vinylidene. [30] Moreover, is a rare example of complexes containing a group 13 ligand with the coordination type III (Figure 1a). [3h, 9b, 31] It is interesting to note that the presence of weak semi-bridging carbonyl interactions is observed with the asymmetry parameter (α) [32] taking values of 0.50, 0.55, 0.56 and 0.55 for these complexes 3-6, respectively. Such values are slightly larger than those of HC[(CMe)(NDipp)]2AlFe(CO)3L (L = CO, 0.47; L = Cy3P, 0.49) reported by Crimmin and Kong. [3h] The electronic properties of the aluminylene ligands in 3, 4 and 6 were next established from the carbonyl stretching frequencies (νCO). [33] With respect to the number of ligands at Al in the series N-Al(L)nW(CO)5 (n = 0-2), which can consecutively suppress Wto-Al π-backdonation while enhance Al-to-W σ-donation, there is significant decrease of the frequencies. 3 exhibits distinctly high frequencies (νCO 2060(νCO , 1974(νCO and 1922 cm -1 cm -1 ) indicative of reduced electron releasing ability of the Al ligand in 3 compared to those in 4 (νCO 2046, 1958 1897 cm -1 ) and 6 (νCO 2015, 1916 and 1854 cm -1 ). These modifications at the ligand site (i.e. coordination of THF or DMAP) drastically influence the electronic properties of the transition metal without changing the coordinated Al ligand.</p><p>Analyses. For a better understanding bonding scenarios of 3, 4 and 6, density functional theory (DFT) calculations, coupled with energy decomposition analyses with natural orbitals for chemical valence (EDA-NOCV) [34] calculations and intrinsic bond orbital (IBO) [35] investigations were carried out. The IBO method is proven to give an exact representation of any Kohn−Sham DFT wave function. [35] Inspections of IBOs of 3 demonstrate that the Al center forms two σ-bonds (Al−N and Al−W σ-bonds) (Figures 5a and 5b). It is observed that two formally vacant 3p orbitals of Al accept electron density from symmetrically accessible filled 5d orbitals of W, forming two apparent π-back-bonding (Figures 5c and 5d). This accounts for the relatively short Al(1)−W(1) bond length (vide supra). In contrast, the Al center of 4 is coordinated with a THF molecule and thus three σ-bonds (Al−N, Al−O and Al−W σ-bonds) at Al are observed (Figures 5e-5g), along with a W-to-Al π-back-bonding (Figure 5h). For 6, the coordination of two DMAP molecules prevents forming π-back-bonding (Figure S36), thereby giving four σ-bonds at Al (Al−W and three Al−N σ-bonds) (Figures 5i-5l). Additionally, EDA-NOCV calculations demonstrate that, in all cases, the orbital interactions ΔEorb are dominant between Al and W with the magnitude of -68.1, -71.1 and -92.3 kcal mol -1 for 3, 4 and 6, respectively (Figures S37-S39). Examinations of the deformation density plots allow visualization of this donoracceptor interaction (Figure S40). In all cases, the Al-to-W σdonation (3: -49.5 kcal mol -1 ; 4: -54.5 kcal mol -1 ; 6: -75.2 kcal mol -1 ) comprises the most significant contribution to ΔEorb, whereas the W-to-Al π-backdonation of 3 and 4 plays a minor role in contributions to ΔEorb (3: -12.8 kcal mol -1 ; 4: -6.0 kcal mol -1 ). Isolation of an Alumanyl Complex. Further reactivity explorations reveal that 2b is highly reducing and can readily react with (THT)AuCl (THT = tetrahydrothiophene) to afford the carbazolyl-substituted aluminium dichloride 7 as well as Au mirror (Figure S31). Repeated crystallization attempts of 7 yielded crystals of poor quality, nonetheless preliminary X-ray studies confirmed its formulation (Figure S31). In a similar vein, upon mixing 2b with IDippCuCl (IDipp = 1,3-bis(2,6diisopropylphenyl)imidazol-2-ylidene) in toluene at ambient temperature, a white solid of the Cu-alumanyl complex 8 was isolated in 71% (Scheme 3).</p><p>The solid-state structure of 8 exhibits a planar Al(1) center with the sum of angles at 359.9 o (Figure 6). The Al(1)−Cu(1) bond length is 2.3448(13) Å, which is comparable to that seen for LCuAl(SiN Dipp ) (L = N,N'-diisopropyl-4,5-dimethyl-2-ylidene, 2.3450(6) Å) [13] whereas slightly longer with respect to that of HC[(CMe)(NDipp)]2AlCu[(NMes)(CMe)]2CH (2.3011(7) Å). [3g] To date, the solid-state structural authentication of terminal Cualumanyl complexes is limited to LCuAl(SiN Dipp ) (L = N,N'diisopropyl-4,5-dimethyl-2-ylidene and (1-(2,6diisopropylphenyl)-3,3,5,5-tetramethyl-pyrrolidin-2-ylidene) [13] and K[Cu[Al(NON)]2]. [36] These species were formed by a salt metathesis reaction of the corresponding potassium aluminyl compound with a ligand-stabilized copper halide. It is important to note that the facile synthesis of 8 showcases a new avenue to terminal alumanyl complexes that are extremely rare and otherwise difficult to prepare. [4c, 4d, 12-13, 36-37] Scheme 3. Synthesis of 8. Mechanistic Investigations. The mechanism of the formation of 8 was probed via DFT calculations (SMD-M06-2X/def2-TZVP//M06-2X/def2-SVP) (Figure 7). The reaction begins with the approach of the aluminylene 2b toward the Cu atom of IDippCuCl. This prompts the slight pyramidalization of N and the formation of an Al−Cu dative bond to generate an intermediate IN1 (free energy of 11.3 kcal mol -1 ) in a barrier-less process (Figure S41). Subsequent oxidative addition of the Cu−Cl bond to Al proceeds via TS1, with the energy barrier of 13.4 kcal mol -1 (2b→TS1), to yield the stable product 8 (-4.8 kcal mol -1 ). Concurrent with this is the increase of the formal oxidation state of Al from +1 to +3.</p><!><p>In summary, a room-temperature-stable N-substituted free aluminylene 2b has been isolated and characterized by spectroscopic, crystallographic and computational techniques. While the planarization of the N atom coupled with two flanking 3,5-di-tert-butylphenyl rings of the carbazolyl substituent in 2b results in the thermodynamic and kinetic stabilization at Al, the facile pyramidalization of the same N atom making the Al kinetically accessible can occur upon treating 2b with a variety of transition metal complexes (i.e. W, Cr). This allows the isolation of a series of unprecedented aluminylene complexes 3-6. Interestingly, this Al ligand showcases σ-donor and σ/π-acceptor properties in diverse manners for transition metals. For 3, the Al is a σ-donating and doubly π-accepting ligand. For 4 and 5, the Al serves as a σ-donating, σ-accepting and π-accepting ligand. Significant to note is that 6 is prepared via an intriguing Alcentered ligand exchange reaction of 4 with DMAP and the Al functions as a σ-donating and doubly σ-accepting ligand. Infrared spectroscopic investigations show that such modifications of ligands at the Al (i.e. coordination of THF or DMAP) significantly affect the electronic properties of transition metals without changing the coordinated atoms. Finally, the first example of aluminylene-alumanyl conversion has been demonstrated, generating a Cu-alumanyl complex 8. Considering DFT calculations, the mechanism leading to 8 involves an initial Al−Cu coordination followed by an oxidative addition of a Cu−Cl bond at Al. We anticipate that these discoveries can pave a way for other unknown metal-alumanyl complexes. The utility of 2b in the production of other intriguing species, the subsequent chemistry of these new complexes and the extension of this Al ambiphilicity to catalysis are the subjects of ongoing work.</p>

ChemRxiv

Mechanosynthesis of Sydnone-containing Coordination Complexes

N-Phenyl-4-(2-pyridinyl) sydnone was shown to act as a fourelectron donor N,O-ligand in unprecedented coordination complexes featuring three different metallic centers (Co, Cu, Zn). Starting from various anilines, the use of ball-mill enabled efficiently the synthesis of N-arylglycines, subsequent nitrosylation and cyclization into sydnone, and further metalation.Sydnones, mesoionic heterocyclic compounds, are versatile building blocks for heterocyclic chemistry, for example in the formation of pyrazoles through copper-catalyzed 1,3-dipolar cycloaddition.1 They also display interesting biological activities, e.g., as antibacterial, antineoplastic and antiinflammatory agents.2 Sydnone synthesis from α-amino acids involves a nitrosylation step, using sodium nitrite in highly acidic media or isoamyl nitrite, 3 and subsequent cyclization using acetic or trifluoroacetic anhydride (TFAA). 4 In 2014, Shih et al. showed that palladium complexes featuring a sydnone-4carbaldehyde N(4)-phenylthiosemicarbazone ligand exhibited higher anticancer activities than 5-fluorouracil (Figure 1). 5 To date, this is the only reported example in which the exocyclic oxygen of the sydnone is shown to coordinate a metal. Indeed, in the literature, sydnones have been modified as phosphine ligands for palladium complexation (Figure 1), 6 directly metalated at C 4 , 7 through deprotonation or carbon-halogen insertion, or modified at N 3 with a 3-pyridine for iron coordination. 8 In this context, we wondered if the presence of a 2-pyridine at C 4 would enable the access to unprecedented coordination structures featuring sydnones as N,O-ligands.

mechanosynthesis_of_sydnone-containing_coordination_complexes

<!>This work<!>• Variety of transition metals<!>15, 7f<!>Conflicts of interest

<p>Given our expertise in mechanochemistry for organic and organometallic synthesis, 10 we envisioned a solvent-free mechanochemical approach 11 for an efficient access to sydnones and to unprecedented coordination complexes. During our study, we realized that accessing sydnone precursors could also benefit from mechanochemistry in terms of reaction time and yield. N-arylglycines were thus prepared via solvent-free mechanochemistry, using a 15 mL Teflon jar (filled with one 1 cm diameter stainless steel ball) agitated at 25 -30 Hz in a vibratory ball-mill (Scheme 1), through either alkylation of substituted anilines with ethylbromoacetate and subsequent saponification (one-pot two-step, Method A) or reductive amination involving glyoxylic acid and sodium cyanoborohydride (Method B). N-arylglycines featuring in para position electron-donating groups such as methyl (1b) or methoxy (1c) could be isolated in 95% and 80% yield over the two steps, respectively, using method A. When a bromine atom was either in meta or para position, method A was also found efficient, giving corresponding glycines in 85% (1h) and 76% (1d) yield, respectively. Mesitylamine, even though extremely sterically hindered reacted using method A to furnish the expected glycine 1i in 37% yield. Even if modest, this yield is four-fold higher than the 10% yield reported in literature. 12 When using anilines bearing electron-withdrawing groups such as NO 2 , CF 3 or CN, method B was found more</p><!><p>• Novel structures • Reagent-less mechanosynthesis</p><!><p>Known structures Scheme 1. Mechanosynthesis of N-arylglycines 1a-i efficient, giving corresponding products in 53-74% yield (1e-g). In all the cases, yields obtained using the mechanochemical approaches were similar if not better compared to literature conditions in solution. 13 In addition, reaction time was limited to 2 -3 h, and a simple extraction/precipitation procedure was required to isolate pure compounds.</p><p>Using N-phenylglycine 1a as model substrate, the synthesis of corresponding sydnone in a mechanochemical one-pot twostep approach was next investigated. Recently, the Taran group developed a one-pot procedure using tert-butyl nitrite and TFAA under solvent-free conditions and magnetic stirring, but sydnones were not isolated and directly engaged in a cycloaddition step. 14 Under mechanochemical activation, tBuONO was found to be efficient and nitrosylation was complete within 5 min, as confirmed by HPLC analysis (Table 1, entry 1). Addition of trifluoroacetic anhydride (2.5 equiv.) to the reaction mixture in the same jar furnished sydnone 2a in 51% yield. Even though this method is sodium nitrite and acid free, tBuONO has to be prepared from tert-butanol using these reagents. We reasoned that sodium nitrite, without the addition of concentrated HCl, could furnish the nitrosylated intermediate. Indeed, the two hydrogen atoms necessary for water release in the nitrosylation mechanism would in this a Total mass of the reagents was calculated to obtain a milling load (ML) of 20 mg.mL -1 . Reactions were performed in a vbm at 25 Hz.</p><p>case come from the amine and acid functions of 1a. Gratifyingly, milling 1a and NaNO 2 in stoichiometric quantity for 5 min resulted in full conversion of 1a into N-nitroso-Nphenylglycinate. Subsequent addition of TFAA to the crude mixture and milling for 30 min yielded sydnone 2a in 54% yield (Table 1, entry 2). Increasing the milling time of the second step to 1 h enabled an excellent 95% yield (Table 1, entry 3). Notably, changing the stoichiometry of TFAA did not improve the final yield (Table 1, entries 4 and 5).</p><p>Milling conditions were then applied to N-arylglycines 1a-i (Scheme 2). Reaction time and milling frequency were modulated to obtain in each case fast and full conversion, which enabled the avoidance of chromatography on silica gel. In all cases, total reaction time did not exceed 120 min with yields comparable to those obtained in solution. 13 Sydnones featuring methyl (2b) and methoxy (2c) groups in para position of the phenyl moiety were produced in 91% and 54% yield, respectively. With a bromine atom either in para or meta position, corresponding sydnones 2d and 2h could be obtained in 91% and 81% yield, respectively. Sydnones 2e-g, featuring NO 2 , CN and CF 3 electron-withdrawing groups were isolated in lower yields of 34 -46%. Even though full conversion was reached, a recrystallization step (for 2e-f) or a chromatography on silica gel (only for 2g) that reduced the final yield was required to furnish pure compounds. The same remark applies to sydnone 2i, featuring sterically hindered mesityl group, which was yielded in 53%. To the best of our knowledge, it is the first time that sydnone 2i is synthesized and fully characterized.</p><!><p>The introduction of a pyridine at C 4 of the sydnone was next performed to obtain a putative bidendate ligand. N-Phenylsydnone 2a was reacted with 2-bromopyridine in the presence of Pd(PPh 3 ) 4 or Pd(OAc) 2 /XPhos as catalytic systems according to literature methods. 16 However, yields obtained were not reproducible and separation of XPhos ligand revealed complicated. Although other palladium-catalysed crosscouplings were successful in the ball-mill, 17 attempts to perform the coupling reaction with 2a under mechanochemical conditions failed. A quick study showed that this coupling could be performed using Pd(OAc) 2 /PPh 3 (1:2) in DMC (dimethylcarbonate), an environmentally friendly solvent, giving corresponding 3-phenyl-4-(2-pyridine)sydnone 3a in 55% yield, with a facilitated purification (Scheme 3). Bidentate compound 3a was then reacted, using a vibratory ball-mill, with various metallic salts (CoCl 2 , CuCl 2 , Cu(OTf) 2 and ZnCl 2 ) under solvent-free conditions. We were pleased to observe that after 1h of milling corresponding complexes were obtained in yields of 66-74%. Conversion was followed in the solid state using IR spectroscopy, as the C=O stretching band in 3a (ν = 1757 cm -1</p><p>) shifts to lower wave numbers upon coordination (ν = 1660 -1726 cm -1</p><p>). The structure of the complexes [Co(µ-Cl)(3a) 2 ] 2 .COCl 4 , [CuCl 2 (3a) 2 ], [Cu(OTf) 2 (3a) 2 ] and [ZnCl 2 (3a)] were confirmed by single crystal X-ray diffraction after crystallisation (Figure 2). 13 Interestingly, cobalt complex possesses a dimeric structure with two sydnone ligands on each metallic center, and bridging chloride atoms. This kind of arrangement was rarely observed in literature with bidentate N,O ligands. 18 For copper containing species, an octahedral geometry with two sydnone ligands and with a trans-arrangement between the two chloride (or triflate) ligands was observed. On the other hand, reaction with zinc(II) chloride provided the complex with only one sydnone ligand and a tetrahedral geometry. In all the X-ray structures, the pyridine and sydnone rings were not found in the same plane and a twist of 22 -35° was observed in the complexes. This non-planarity was also witnessed in the X-ray structure of 3a. 13 Such observation tends to indicate that pyridine and sydnone rings may not be conjugated. Additionally, the exocyclic C-O bond was measured at 1.21 -1.24 Å, which is consistent with a double bond character. Bidentate sydnone 3a may thus act as a four-electron rather than a three-electron donor ligand.</p><p>In conclusion, the use of ball mills enabled the efficient reagent-less preparation of a series of N-aryl glycines and of corresponding sydnones. N-Phenyl-4-(2-pyridinyl) sydnone 3a was used for the first time as ligand to coordinate metals, leading to unprecedented compounds via a mechanochemical solvent-free approach. Such results pave the way for the development of novel families of coordination complexes.</p><!><p>There are no conflicts to declare.</p>

ChemRxiv

Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of the hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman) and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid phase extraction coupled with high performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3\xe2\x80\x930.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101\xe2\x80\x93105%) and high precision (5\xe2\x80\x938%) for the detection of these five nerve agent hydrolysis products in serum.

quantitation_of_five_organophosphorus_nerve_agent_metabolites_in_serum_using_hydrophilic_interaction

Introduction<!>Materials<!>Sample preparation<!>Instrumental analysis<!>Safety precautions<!>Sample Preparation<!>Separation and Detection<!>Method Characterization<!>Limits of Detection<!>Sample Analysis<!>Limitations<!>Conclusions<!>

<p>Organophosphorus nerve agents are highly toxic compounds that were originally developed as potent pesticides in the 1930s. Since that initial discovery more nerve agents have been synthesized and stockpiled for warfare purposes. Although use of these compounds has been limited in recent years [1], concerns remain that nerve agents will be used for non-sanctioned warfare or terrorist activities. Stockpiles throughout the world are gradually being decommissioned [1] and laboratory research to improve treatments for exposed persons is being pursued [2, 3]. These activities may result in accidental human contact with nerve agents; therefore, the improved and expanded ability to assess human exposure to nerve agents is needed.</p><p>Following entry into the human body, nerve agents are either hydrolyzed or bound to acetylcholinesterase, butyrylcholinesterase, and other non-specific proteins. The binding to the acetylcholinesterase is the action that results in symptoms including miosis, seizures, and potentially death [1]. The hydrolysis products, also known as nerve agent metabolites, are readily excreted in the urine, but have also been detected in blood [4–6]. More specifically, human exposure to VX was identified in serum using gas chromatography/mass spectrometry (GC/MS) to measure the hydrolysis product, ethyl methylphosphonic acid. In this exposure case which resulted in death, the amount of metabolite from VX was determined to be 125 μg/mL [5]. Additionally, the hydrolysis products were detected in minipig blood using liquid chromatography-tandem mass spectrometry (LC/MS/MS) [4] following exposures to sarin and cyclosarin.</p><p>These hydrolysis compounds have been extensively quantitated in urine through various analytical methods. Gas chromatography coupled with mass spectrometry or tandem mass spectrometry has been used with success, although a derivatization step is required [7–9]. To avoid the additional derivatization, liquid chromatography coupled with mass spectrometry or tandem mass spectrometry has been applied, achieving sensitivities on the order of pg/mL in urine [10–13].</p><p>In most cases, sample preparation, including liquid-liquid extraction (LLE) and solid phase extraction (SPE), was necessary to achieve these low levels of detection. Solid phase extraction was cited more often than LLE, most likely due to the ease of use and potential for automation. The different solid phase extraction methods used for these compounds, included aqueous normal phase [10, 11], reversed phase [4], anion exchange [12, 14], and zirconia [14, 15]. However, for the extraction of nerve agent metabolites from serum, only LLE and reversed phase SPE were used [4–6].</p><p>An analysis method which includes the other nerve agent metabolites resulting from soman and VR exposure is needed, since only three nerve agent metabolites, EMPA, IMPA and CMPA, were previously quantitated in serum [4–6]. Although the amount of metabolite present in biological matrices has not been related to dose in humans, a quantitative value is still desired to obtain as much information regarding the relative amount of exposure as possible to differentiate between exposed and unexposed persons. Given this focus, an assay with high sensitivity is desired above a quantitative method designed to capture all exposure levels. Additionally, this method should detect exposure to all five nerve agents within a single, high-throughput, and automated analysis. This study documents the development of a SPE-LC/MS/MS analysis for five nerve agent hydrolysis products in human serum which are indicative of exposure to sarin (GB), soman (GD), cyclosarin (GF), VX and VR.</p><!><p>The analytes evaluated for this method were the following: EMPA (ethyl methylphosphonic acid, CAS 1832-53-7), the metabolite of VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate, CAS 50782-69-9); IMPA (isopropyl methylphosphonic acid, CAS 1832-54-8), the metabolite of GB (isopropyl methylphosphonofluoridate CAS 107-44-8); PMPA (pinacolyl methylphosphonic acid, CAS 616-52-4), the metabolite of GD (pinacolyl methylphosphonofluoridate, CAS 96-64-0); CMPA (cyclohexyl methylphosphonic acid, CAS 1932-60-1), the metabolite of GF (cyclohexyl methylphosphonofluoridate, CAS 329-99-7); MMPA (2-(methyl)propyl methylphosphonic acid, CAS 1604-38-2), the metabolite of VR (O-2-(methyl)propyl S-2-(diethylaminoethyl) methylphosphonothioate. CAS 159939-87-4).</p><p>The internal standards were isotopically labeled as follows: EMPA, ethyl-D5; IMPA, isopropyl-13C3; PMPA, trimethylpropyl-13C6; CMPA, cyclohexyl-13C6; IMPA, methylphosphonyl-13C. The structures of the organophosphorus metabolites and internal standards are shown in Figure 1.</p><p>The analytical calibrators were prepared at concentrations of 0.5, 1.0, 2.0, 5.0, 10, 25, 50, 100 ng/mL of each analyte in serum; the quality control samples were prepared at 4.0 and 40 ng/mL of each analyte in serum. A primary stock (20 μg/mL of each analyte in methanol) purchased from Cerilliant Corporation (Round Rock, TX) was used to make stock solution I (4.0 μg/mL) and stock solution II (0.2 μg/mL). Stock solution I was prepared by aliquoting 400 μL 20 μg/mL stock to a glass vial containing 1600 μL of serum. The second stock solution was prepared by pipetting 100 μL of stock solution I to a glass vial containing 1900 μL of serum. Calibrators 0.5, 1.0, 2.0, and 5.0 ng/mL were made by aliquoting 25, 50, 100, 250 μL, respectively, of stock solution II to 10.0 mL volumetric flasks. Calibrators 10, 25, 50, and 100 ng/mL were prepared by delivering 25, 63, 125, and 250 μL, respectively, of stock solution I to their appropriate 10.0 mL volumetric flask. Quality control low (4.0 ng/mL) and quality control high (40 ng/mL) were prepared by adding 200 μL of stock solution II and 100 μL of stock solution I, respectively, to 10.0 mL volumetric flasks. An additional solution was prepared at 0.25 ng/mL in serum to evaluate the limits of detection for the assay, by delivering 17 μL of stock solution II to a 10.0 mL volumetric flask. Spiked samples, 0.75 and 3.0 ng/mL, were prepared by aliquoting 37.5, and 150 μL of stock solution II to their respective 10.0 mL volumetric flask. Spiked samples, 15, 70, and 90 ng/mL were prepared by delivering 37.5, 175, and 225 μL of stock solution I into their respective 10.0 mL volumetric flasks. All volumetric flasks were filled to 10.0 mL using serum purchased from Tennessee Blood Services Corporation (Memphis, TN). The serum was pooled from five donors, shipped at 4°C, received two days later, and subsequently stored at −20°C until use. Once prepared, all fortified samples, calibrators, and quality control samples were maintained at −20°C.</p><p>Internal standard was prepared by dilution of a 500 ng/mL aqueous mixture (Cerilliant Corporation, Round Rock, TX) to a concentration of 23.8 ng/mL in DI water.</p><p>Organic-free 18.2 MΩ Type I water from a purifier purchased from Aqua Solutions, Inc. (Jasper, GA) was used in these studies. Pelletized 98% ammonium fluoride was purchased from Alfa Aesar (Ward Hill, MA) and molecular biology grade DEPC-treated 5M ammonium acetate from CalBiochem (La Jolla, CA). HPLC-grade acetonitrile and methanol were purchased from Tedia (Fairfield, Ohio). All human serum was purchased from Tennessee Blood Services Corporation (Memphis, TN).</p><!><p>To prepare samples for extraction, 25 μL of 23.8 ng/mL internal standard was aliquoted into each well of a 2 ml 96-well NUNC plate. Fifty microliters of well-mixed serum was added to the internal standard, followed by 1000 μl of acetonitrile. The plate was sealed using a sheet of Thermo Scientific Adhesive PCR Sealing Foil (Hudson, NH) and vortexed for 5 minutes on a ThermoLab Systems Wellmix (Hudson, NH). The NUNC plate was then centrifuged for 5 minutes at 3000 rpm.</p><p>The solid phase extraction was automated by using the Life Science Zephyr (Hopkinton, MA). The Phenomenex Strata Si-1 SPE 96-well plate (55 μM, 70 Å, 100 mg) was pretreated with 1000 μl of 25% water in acetonitrile, followed by 1000 μl of acetonitrile. The entire sample mixture with precipitate was then loaded to the SPE 96-well plate, and rinsed by a two-step process of 1000 μl of acetonitrile, followed by 1000 μl of 7% water in acetonitrile. The solutions were pulled through the SPE 96-well plate using vacuum. The analytes were then eluted with 1000 μl of 28% water in acetonitrile and collected in a clean 96-well NUNC plate.</p><p>The eluent was heated at 70 °C under 25 psi of nitrogen in a TurboVap for 30 minutes and then evaporated to dryness after increasing flow rate to 70 psi. The lower initial flow rate eliminated solvent splashing. The sample was then reconstituted in 100 μl of 5% water in acetonitrile, mixed by pipetting twice, and transferred to a 150 μl 96-well PCR plate and sealed.</p><!><p>The HPLC separation was performed using an Agilent 1200 HPLC with a well-plate autosampler (Santa Clara, CA). A Waters Atlantis® HILIC 2.1-mm × 50-mm with 3-μm particles (70% porosity)HPLC column was used (Milford, MA). This hydrophilic interaction chromatography (HILIC) column consists of high purity, non-bonded silica particles. The injector was programmed to draw 20 μL of sample and wash the injector needle for 10 s in the wash port using 50% water in acetonitrile. The sample was then injected onto the column using an isocratic mobile phase consisting of 92% acetonitrile and 8% 1.0 mM ammonium fluoride with an initial flow rate of 500 μL/min. Following elution of the analytes, the flow rate was increased to 1000 μL/min at 3.01 min to remove any late eluting impurities. The flow rate returned to 500 μL/min at 5.01 min to ensure stable pressure for the following injection. This method allows a 5-min injection-to-injection cycle time.</p><p>The mass spectrometric analysis was performed using an API 4000 triple quadrupole QTrap mass spectrometer from Applied Biosystems (Foster City, CA) controlled by Analyst software. The mass spectrometer was operated in multiple-reaction-monitoring (MRM) mode using negative electrospray ionization with assisted heating. The specific operating conditions are listed in Table 1 with the proposed fragment ions.</p><p>The specific settings used were curtain gas (CUR), 10 psi; nebulizer gas (GS1), 40 psi; turbo gas (GS2), 40 psi; GS2 temperature (TEM), 550 °C; collision gas, nitrogen; collision gas (CAD), Medium, producing a gas pressure reading of 3.3 × 10−5 Torr; ionspray potential (IS), −4500 V; entrance potential (EP), −10; interface heater (IHE), on.</p><p>The data were analyzed using Analyst 1.5.2, which was provided with the instrument. This software is used to review the chromatograms for retention times, baselines and possible interferences. Quantitative analysis of the data by automated and manual integrations, linear regression, and calculation of accuracies and correlation coefficients was also performed with this software package. The chromatographic data were smoothed three times prior to integration with a bunching factor between 1 and 3, and fitted by linear regression using 1/x weighting.</p><!><p>The techniques and materials in this method do not pose any special hazards. General considerations include exercising universal precautions, such as wearing appropriate personal protective equipment, when handling chemicals and serum samples. The high voltage employed in electrospray ionization should also be considered a hazard, and the safety interlocks provided by the instrument manufacturer should not be altered. For instrument-specific safety concerns, please consult the manufacturer.</p><!><p>Initial evaluation of a protein precipitation using acetonitrile or acetone for the sample preparation of these compounds from serum resulted in lower sensitivities than desired. As exposures may be small and/or a sample may be collected many hours to days following exposure, it is important to have the highest sensitivity possible. Additionally, the presence of interfering matrix components was detected in select transitions, which would negatively impact the specificity of the analysis. To accomplish the goals of sensitivity and specificity, additional sample preparation was necessary.</p><p>Solid phase extraction was selected for sample preparation due to automation and high throughput potentials. The initial solvents and volumes used for the extraction were selected from Swaim, et al [10] from urine sample extraction. The sorbent was conditioned with 1000 μL of 25% water in acetonitrile, followed by 1000 μL of acetonitrile. After the sample mixture, comprised of 100 μL of serum, 25 μL of internal standard solution and 1000 μL of acetonitrile) was loaded, the sorbent was rinsed with 1000 μL of acetonitrile, followed by 1000 μL of 10% water in acetonitrile. Finally, the compounds were eluted using 1000 μL of 25% water in acetonitrile and collected in a 2 mL 96-well NUNC plate. Using a serum matrix fortified at 10 ng/mL, each step within the solid phase extraction protocol was then evaluated in triplicate for optimal recovery. To evaluate the loading step, 50 μL of serum was diluted with acetonitrile, with additional deionized water as needed, to yield an aqueous solution of 7, 12 and 17%. The seven percent aqueous loading solution resulted in the highest responses with no detected breakthrough. Next, the second wash step was assessed at acetonitrile content ranging from 93 – 83%. The wash step of 93% acetonitrile yielded the highest recoveries with minimal losses detected. No adjustments were made to the elution composition as complete elution was achieved in one step with the 25% water/75% acetonitrile mixture. Final extraction recoveries, determined at 10.0 ng/mL and calculated by the ratio of the measured concentration to the spiked concentration, were as follows: EMPA, 88% (standard deviation of 17), IMPA, 76%(13) MMPA , 92% (9.6)), PMPA, 94% (10), and CMPA , 95% (7.7). With the optimized solid phase extraction parameters, no analyte was detected within the two captured wash steps. Following the elution, a second elution step was evaluated to remove the analytes still remaining on the SPE. This second elution showed minimal (<1%) amounts of PMPA, CMPA and MMPA, approximately 5% of EMPA and no detectable IMPA remained on the SPE following the initial elution.</p><!><p>Previous studies have reported successful separation of these compounds with HILIC chromatography using an isocratic ammonium acetate buffer coupled with acetonitrile (14% 20 mM ammonium acetate: 86% acetonitrile) as the mobile phase [10, 11]. This mobile phase composition was used as a starting point for the separation of these compounds and is shown in Figure 2b. Since the mass spectrometric intensity of these compounds has been augmented with the addition of various solvents [16] post column, alternative buffers were investigated to determine if increases in response could be achieved. Ammonium fluoride has been reported to result in the ionization of additional compounds and may in fact enhance ionization when using negative electrospray [17, 18]. With this information the HILIC chromatographic separation was evaluated replacing the 20 mM ammonium acetate buffer with 1 mM ammonium fluoride buffer. This change resulted in an increased signal for all compounds (Table 2), with an average 7.7-fold improvement in peak area response, with astandard deviation of the peak area response between the compounds was found to be 0.77. Using the 1 mM ammonium fluoride increased retention; therefore, to minimize runtime, the isocratic mobile phase composition was adjusted to 8% 1 mM ammonium fluoride and 92% acetonitrile to achieve retention factors of 4.6–6.1 (Figure 2a). This change also resulted in further 2-fold signal increase from the 14% ammonium fluoride mobile phase, which is equivalent to at least a 10-fold signal increase (Table 2), indicating the impact of higher acetonitrile content on the ionization efficiency of these compounds.</p><!><p>Twenty sets of calibrators with QC samples were extracted and analyzed by LC-MS/MS to determine the precision and bias of this method over time. Multiple analysts prepared no more than two standard sets with QC samples per day over a period of 36 days. Quality control characterization data are presented in Table 3. The inter-day relative standard deviations for both quality control samples for all five compounds were below 8% and the accuracy was between 101–105% for all compounds. These results confirm this method provides acceptable precision and accuracy for these compounds according to the FDA Bioanalytical Method Development Guidelines [19].</p><!><p>The limits of detection (LOD) for the five compounds were estimated using low level spiked serum with concentrations from 0.25 – 1.0 ng/mL. Each low level spiked sample was analyzed 20 times within a two-week period, with no more than five replicates within one day. The absolute values of the standard deviations were then plotted versus concentration with the intercept of the least-squares fit of this line equal to S0 with 3S0 being the LOD [20]. The LODs for this method were determined and reported in Table 3. These estimates, ranging from 0.25–0.50 ng/mL, exceed other methods developed for serum which reported limits of detection from 3.9 – 30 ng/mL for EMPA[5, 6] and 16 ng/mL for PMPA and CMPA [7].</p><!><p>One hundred individual serum samples were extracted and analyzed to evaluate the presence of nerve agent metabolites in the serum of persons with no known exposure. A representative sample is shown in Figure 3. No peaks were observed above the lowest calibration point; therefore, results above the detection limit would indicate that an exposure to these compounds has occurred</p><p>To evaluate accuracy and precision across the calibration range, serum samples were prepared at the following concentrations: 0.75, 3, 15, 70, and 90 ng/mL. These samples were extracted and analyzed five times to generate the statistical data presented in Table 4. The relative standard deviations for all five levels were less than 10%, with the exception of the 0.75 ng/mL spike of IMPA which resulted in a variance of 18%. Similarly, the four highest levels resulted in accuracy between 96% and 112%. However, for the lowest spiked sample at a concentration of 0.75 ng/mL the accuracies were between 78–93%. This drop in accuracy and increase in variability at the low concentration may be due to the closeness of the value to the limit of detection.</p><p>To widen application of this method to samples with a metabolite concentration greater than 100 ng/mL, a 4.0 μg/mL fortified sample was prepared in serum. This sample was diluted to 4.0 and 40.0 ng/mL using blank serum; the diluted samples were then extracted and analyzed in triplicate. The average quantitated values ranged from 3.91–4.27 ng/mL and 39.5–41.8 ng/mL for the two levels. When the quantitated values were calculated with the dilution factor, the accuracies ranged from 97.8–106%.</p><p>The stability of these compounds in serum stored for 6 months at −20°C was evaluated at 4.0 ng/mL and 40.0 ng/mL. The stored samples were analyzed in triplicate and quantitated relative to freshly prepared calibrators. The concentrations of the two levels were within the 10.0% of the original spiked concentration for all five analytes. Given the variability of the method, these results indicated that the metabolites were stable for at least 6 months stored frozen.</p><!><p>Due to a high background response resulting from the ammonium fluoride buffer, the initial transition selected for CMPA (177>79 m/z) did not result in a discernible peak within the reportable range. An alternative transition of 177>63 m/z was evaluated; however, this new transition was only detectable to about 1 ng/mL due to limited signal. Similarly, the confirmatory ion for PMPA was not consistently identified below 2 ng/mL due to a high background response. Previous publications have indicated that this background may be attributed to the impurities within the ammonium fluoride used for the buffer and potentially the interaction of this buffer with the stationary phase [18]. It should be noted that these issues were only detected in the confirmatory ions and do not affect the reported limits of detection for these compounds</p><!><p>A method to detect exposure to five nerve agents, including VX, VR, soman (GD), sarin (GB), and cyclosarin (GF) in serum has been developed and evaluated. The novel use of ammonium fluoride as the mobile phase buffer increased the ionization of the compounds up to 10-fold over the use of ammonium acetate, which substantially increased the sensitivity of this assay. Both the resulting precision and accuracy, 8% and 100–105% respectively, were within FDA recommendations. Since no peaks were detected within 100 individual serum samples with no known exposure to nerve agents, a response above the limit of detection is indicative of exposure to nerve agents. Given the potential for blood to clot and lyse, often due to poor handling or storage, which would impact the quality of the serum, future studies are planned to apply this assay to quantitate nerve agent exposure in non-ideal blood matrices.</p><!><p> Disclaimer </p><p>The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the US Department of Health and Human Services.</p><p>Structures of nerve agent metabolites and corresponding internal standards, * = 13C</p><p>Chromatograms of 100 ng/mL calibrator in serum containing five nerve agent metabolites in different mobile phase compositions: a) 8% ammonium fluoride (1 mM)/92% acetonitrile, and b) 14% ammonium acetate (20 mM)/86% acetonitrile</p><p>Chromatograms of 0.5 ng/mL serum calibrator containing five nerve agent metabolites (a) and blank serum (b)</p><p>Mass spectrometric transitions and parameters</p><p>Percent of peak area response relative to the buffer composition of 14% ammonium acetate (20 mM) with 86% acetonitrile</p><p>Mean, precision (relative standard deviation), accuracy, and limit of detection (LOD) values for five nerve agent metabolites in serum (n=20)</p><p>Mean, precision, and accuracy of spiked serum samples (n=5)</p>

PubMed Author Manuscript

A Rapid, Efficient and Economical Method for Generating Leishmanial Gene Targeting Constructs

Targeted gene replacement is a powerful tool in Leishmania genetics that can be time-consuming to implement. One tedious aspect that delays progress is the multi-step construction of gene targeting vectors. To accelerate this process, we developed a streamlined method that allows the assembly of a complete targeting vector from all its constituent parts in a single-step multi-fragment ligation. The individual components to be assembled are flanked by sites for the restriction endonuclease SfiI that generates nonidentical, non-palindromic three base 3\xe2\x80\x99-overhangs designed to allow annealing and ligation of the parts only in the proper order. The method was optimized by generating constructs for targeting the Leishmania donovani inosine monophosphate dehydrogenase gene (LdIMPDH) encoding six different drug resistance markers, and was found to be rapid and efficient. These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species.

a_rapid,_efficient_and_economical_method_for_generating_leishmanial_gene_targeting_constructs

<p>Targeted gene replacement via homologous recombination has been an invaluable tool for the genetic dissection of important metabolic and virulence pathways in Leishmania species [1], as well as for many other protozoan parasites [2]. The general experimental approach for the genetic manipulation of model organisms is essentially the same: DNA sequences of sufficient length to direct homologous recombination flanking the gene to be targeted - referred to herein as 5'- and 3'-targeting sequences (TS) - are independently isolated and joined to an alternative gene (i.e., drug resistance gene) that allows selection of cells in which the appropriate integration event has occurred. The most common method for generating leishmanial and other parasite gene targeting constructs involves the sequential cloning of 5'-TS and 3'-TS DNAs into a vector encoding a drug resistance cassette flanked by restriction sites [3]. This multi-step process can be time consuming and is complicated by the fact that commonly used vectors have limited restriction sites for TS insertion, exchange of drug resistance markers, and excision of the targeting cassette from the vector backbone prior to transfection. The ability to assemble the complete gene targeting construct in a single step would greatly enhance the throughput of gene replacement studies. Numerous techniques have been described for the simultaneous assembly of multiple DNA fragments, including variations on ligation-independent cloning [4–6], overlap extension polymerase chain reaction (PCR) [7], site-specific recombination [8], and recombination in Escherichia coli, i.e., "recombineering"[9]. Some of these methods have been used to construct gene targeting constructs for various trypanosomatids [10,11]. However, these techniques can involve tedious, complicated, or unfamiliar technology [5,6,8,9,12], require expensive kits or reagents [4,8], and are complicated by inefficiency [13].</p><p>To overcome these many impediments to gene replacement in Leishmania, we have developed a simple, cost-effective, and efficient method (see Fig. 1) for construction of leishmanial targeting constructs that is based on the properties of the SfiI restriction endonuclease and requires only readily available and familiar technologies, (i.e., PCR, restriction digestion, and DNA ligation). The SfiI restriction endonuclease was chosen as the basis of this multi-fragment ligation strategy for several reasons. First, the recognition sequence for SfiI, GGCCNNNNNGGCC, is an interrupted palindrome that generates an asymmetric three base 3'-overhang (underlined and in boldface type in the sequence) upon DNA cleavage that can only ligate to complementary overhangs but not to itself nor to overhangs from non-identical SfiI sites. SfiI sites designed to produce non-identical 3'-overhangs incorporated at the ends of the individual targeting vector components allows the directional and ordered assembly of the targeting vector in a single ligation reaction (see Fig. 1C). Second, the eight base pair recognition sequence of SfiI should only rarely occur within any given TS, rendering this strategy applicable to the generation of targeting constructs for virtually any leishmanial gene. Indeed, examination of one megabase of L. infantum genome sequence identified SfiI sites with a frequency of approximately one in every 38 kb. If a TS encompasses an SfiI site, alternative restriction enzymes with six base pair recognition sequences that are also interrupted palindromes are potentially available for generating compatible 3'-overhangs (see Supplementary Protocol). Third, SfiI has been utilized by other groups for the ordered assembly of multiple DNA fragments and the method has proven to be quite efficient [13,14]. While the use of SfiI in other strategies for producing gene targeting constructs has been described [12,15], none of these schemes involve the simultaneous assembly of a complete targeting vector by single-step ligation of all of the constituent components.</p><p>Standard targeting vectors are normally composed of four parts: a minimal plasmid backbone to permit selection and propagation in Escherichia coli, a drug resistance cassette to allow selection in Leishmania, and a 5'-TS and a 3'-TS to facilitate gene replacement via homologous recombination. In the multi-fragment ligation method, all four parts are digested with SfiI, gel purified, and combined in a single ligation reaction to generate the complete targeting construct. As depicted in Fig. 1A, the minimal plasmid backbone is donated by the pBB plasmid that contains two incompatible SfiI sites (SfiI-A and SfiI-D) flanking a stuffer fragment that allows the plasmid backbone fragment to be readily distinguished from uncut vector during gel purification. An expression cassette encoding one of six possible drug resistance genes currently available for Leishmania transfection [1,16–18] is donated by the corresponding pCR-DRC plasmid (Fig. 1A) and is also flanked by incompatible SfiI sites (SfiI-B and SfiI-C). Importantly, the SfiI overhangs encoded by the plasmid backbone fragment and the drug resistance cassettes cannot ligate to each other, but, rather, require the complementary SfiI overhangs provided by the 5'- and 3'-TS PCR fragments (Fig. 1B) to serve as a bridge between them (Fig. 1C). The complete targeting vector (Fig. 1D) encodes ampicillin resistance and the fact that the pCR-DRC plasmids are kanamycin-resistant eliminates the possible contribution of uncut pCR-DRC plasmid to the occurrence of background colonies following transformation of the ligation reaction. All the drug resistance cassettes are flanked by restriction sites for the rare cutting enzymes SbfI and AscI to facilitate the ready exchange of drug resistance cassettes between targeting constructs (i.e. pTRG in Fig. 1D) and the pCR-DRC plasmids (Fig. 1A). The targeting cassette can be conveniently excised from the plasmid backbone using either PacI or PmeI endonucleases, whose eight bp recognition sequences occur rarely.</p><p>To demonstrate the efficacy of the multi-fragment ligation method for targeting vector construction, we generated constructs for targeted replacement of the L. donovani inosine monophosphate dehydrogenase (LdIMPDH) gene [19]. As a first step, the conditions for the four-way ligations were optimized by varying the molar ratio of inserts (3'- and 5'-IMPDH TS fragments and puromycin (PAC) or phleomycin (PHLEO) drug resistance cassettes) to plasmid backbone fragment in trial ligation reactions. While the transformation efficiencies were uniformly high for all insert to vector ratios tested, the percentage of clones containing plasmids with the correct structure was consistently lower at the 0.5:1 molar ratio (Table 1). SfiI digestion of plasmid DNA from twenty colonies each from the 2:1 ratio ligations of both the PAC and PHLEO LdIMPDH targeting constructs revealed that 85% and 75%, respectively, had the correct structure (data not shown) confirming the efficiency of the method at this ratio. Therefore, a 2:1 insert to vector ratio was employed for construction of LdIMPDH targeting constructs encoding neomycin (NEO), hygromycin (HYG), blasticidin (BSD), and nourseothricin (SAT) expression cassettes, since it consistently allowed smaller ligation volumes to be used and yielded a large number of colonies containing a high percentage of targeting vectors with the expected structure in each case (data not shown).</p><p>While the overall number of colonies and the percentage of colonies containing the correct construct was high, it was noted that all of the incorrect plasmids could be readily identified as contaminating pBB-GFP plasmid (data not shown). Other systems for simultaneous assembly of multiple DNA fragments reduce the occurrence of background by including a conditionally lethal gene such as ccdB or sacB in donor vectors [8,12]. To reduce the potential for background colonies derived from the plasmid backbone donor plasmid, the GFP stuffer fragment of pBB-GFP was replaced with a chloramphenicol resistance/ccdB expression cassette to generate plasmid pBB-CmR-ccdB. This plasmid cannot be propagated in standard E. coli strains used for cloning but instead requires a specialized bacterial strain expressing the antitoxin to CcdB encoded by the ccdA gene [20]. When pBB-CmR-ccdB was used as the plasmid backbone donor in a four-way ligation to generate LdIMPDH targeting constructs containing a PAC cassette, no background was observed (data not shown). In fact, in eight additional targeting constructs have been produced in our laboratory using pBB-CmR-ccdB as the plasmid backbone donor and no background has been observed (A. Fulwiler and R. Soysa, unpublished observations).</p><p>To establish the utility of constructs generated by multi-fragment ligation for targeted gene replacement, LdIMPDH targeting constructs encoding BSD-, HYG-, NEO-, PHLEO-, and PURO resistance cassettes were transfected into wild type L. donovani parasites and plated on the appropriate selective medium. Southern blot analysis indicated that each targeting construct was capable of generating IMPDH/impdh heterozygotes with the expected genomic structure (Fig. 2).</p><p>The multi-fragment ligation strategy presented here for production of leishmanial gene targeting vectors is markedly faster than traditional approaches [3] and is significantly cheaper and more straightforward than alternatives [11]. Using this technique, we routinely generate gene targeting constructs in three to four days from the time of TS fragment PCR to confirmation of vector structure by restriction analysis. The general strategy is adaptable to the generation of targeting constructs for other parasites and genetically manipulable organisms by simply producing species-specific selectable markers flanked by the appropriate SfiI sites. All of the components of this system are available by request from the authors.</p><p>Strategy for assembly of gene targeting vectors via multi-fragment ligation. This method is modular in design with each component derived independently prior to simultaneous assembly in a single ligation reaction. (A) Plasmid pBB serves as the donor of a minimal (2165 bp) plasmid backbone (denoted by the thick black line) that encodes ampicillin resistance (AMPR- open arrow) and a plasmid replication origin (small black arrow). There are two versions of pBB, pBB-GFP (Genbank accession no. HQ416901) and pBB-CmR-ccdB (Genbank accession no. HQ416902), that encode either GFP (~800 bp) or CmR-ccdB (~1600 bp) stuffer fragments, respectively, flanked by non-identical SfiI restriction sites (SfiI-A and SfiI-D). Drug resistance cassettes (thick black line) are donated by the pCR-DRC plasmids that were constructed by cloning drug resistance cassettes (~3 kb) PCR amplified with primers encoding unique SfiI restriction sites (SfiI-B and SfiI-C) into pCR®-Blunt II-TOPO®. Six pCR-DRC plasmids were constructed that contain drug resistance cassettes conferring resistance to either neomycin/G418 (pCR-NEO), hygromycin (pCR-HYG), phleomycin (pCR-PHLEO), puromycin (pCR-PAC), blasticidin (pCR-BSD), or nourseothricin (pCR-SAT). A detailed description of pBB and pCR-DRC plasmid construction is provided in the supplementary materials and methods. For all of the drug resistance cassettes, the appropriate trans-splicing and polyadenylylation of the drug resistance gene is directed by 5'- and 3'-flanking sequences, respectively, derived from the L. major DHFR-TS gene as described [1] (B) The 5'- and 3'-targeting sequences (TS) are generated by PCR amplification using primers that add SfiI restriction sites SfiI-A and SfiI-B (5'-TS) or SfiI-C and SfiI-D (3'-TS) (see Supplementary Protocol). (C) Targeting vector assembly. The 5'- and 3'-TS PCR fragments, along with the pBB and pCR-DRC donor plasmids are digested with SfiI and the appropriate DNA fragments are agarose gel purified and combined in a ligation reaction. Each DNA fragment has unique three base 3'-overhangs on the sense (indicated by A, B, C, D) and antisense (indicated by A', B', C', D') strands that can only anneal to complementary overhangs. (D) The assembled targeting vector (pTRG) contains restriction sites for exchange of drug resistance cassettes (SbfI and AscI) and release of the targeting fragment from the plasmid backbone (PacI or PmeI). Note that the plasmids are not drawn to scale.</p><p>Southern blot analysis of Δimpdh/IMPDH parasites. Total genomic DNA (~2 μg) from wild type L. donovani (lane 1), or strains in which one IMPDH allele has been replaced by BSD (lane 2), HYG (lane 3), PAC (lane 4), PHLEO (lane 5), or NEO (lane 6) cassettes was digested with XhoI, fractionated on a 1% agarose gel, and blotted onto a nylon membrane. The blot was hybridized under high stringency conditions with a probe to the 3'-IMPDH TS. Bands above the IMPDH band correspond to the targeted IMPDH allele and vary in size in accordance with the sizes of the drug resistance cassettes (labeled DRC). Targeted replacement of LdIMPDH using a targeting construct containing a SAT cassette (pTRG-IMPDH-SAT) was not attempted.</p><p>Optimization of multi-fragment ligation conditions</p><p>The molar ratio of inserts (5'- and 3'- IMPDH TS fragments and the drug resistance cassette) to plasmid backbone was varied with respect to 10 ng of the plasmid backbone. The Inserts: Plasmid Backbone Ratio denotes the molar ratio of each insert fragment relative to the plasmid backbone. The number of colonies from plating 100 μl (21% of total transformation volume) of E.coli transformed with 2 μl ligation reaction (representing 0.67 ng plasmid backbone fragment) is presented. Transformation efficiencies are given as colony forming units per μg of DNA and are calculated based on 0.67 ng of plasmid backbone fragment per transformation. Plasmid pUC19 (0.02 ng) was included to assess the transformation efficiency of the lot of commercially prepared competent E. coli used in this experiment. Transformation of a ligation reaction performed using only 10 ng of the purified plasmid backbone fragment (originating from the same batch used in inserts:vector ratio optimization) served to indicate the contribution of pBB-GFP to background. Further assessment of transformants derived from ligation reactions using 2:1 inserts to plasmid backbone ratio showed that the percentage of clones positive for the correct PHLEO- or PAC-containing targeting vector was 75% and 85%, respectively (N=20).</p>

PubMed Author Manuscript

DID it or DIDn\xe2\x80\x99t it? Exploration of a failure to replicate binge-like alcohol-drinking in C57BL/6J mice

We previously reported that commercially-sourced C57BL/6J (B6) male mice with a history of adult-onset binge-drinking exhibit anxiety-like behavior in early withdrawal, while the negative affective state incubates during protracted withdrawal in adolescent-onset binge-drinking males. As the results of such studies are potentially confounded by age-related differences in reactivity to environmental stress, we employed a 2-bottle-choice DID procedure (20 and 40% alcohol; 20 min habituation to the drinking cage) to examine the effects of binge-drinking on negative affect in male and female, adult and adolescent, B6 mice from our university colony. Unexpectedly, the mice in the initial experiment exhibited very low alcohol intake, with little sign of withdrawal-induced negative affect. This failure to replicate prompted us to examine how the duration of drinking cage habituation, the number of alcohol concentrations presented and the animal source might influence the propensity to binge-drink. Herein, we show that both male and female adult mice from our colony will binge-drink when allowed 45 min to habituate to the drinking cages, irrespective of whether mice are offered a choice between 2, 3 or 4 alcohol concentrations. Further, when drinking under 4-bottle-choice procedures (5, 10, 20 and 40% alcohol), adult-onset binge-drinking females exhibit robust negative affect in early withdrawal akin to that reported previously for adult males; however, the negative affective state persists for at least 30 days into withdrawal. Also unlike males, adolescent-onset binge-drinking females exhibit some signs of negative affect, as well as potentiated alcohol intake, in early withdrawal, which persist into later withdrawal. These latter data suggest that the age-related differences in the temporal patterning of the negative affective state produced by alcohol withdrawal may vary as a function of sex, which may have implications for understanding sex differences in the etiology of affective disorders and alcoholism co-morbidity.

did_it_or_didn\xe2\x80\x99t_it?_exploration_of_a_failure_to_replicate_binge-like_alcohol-drinking_in

Introduction<!>MATERIALS AND METHODS<!>Experiment 1: Replication and Extension of Age-Related Differences in Withdrawal-induced Negative Affect.<!>Experiment 2: Influence of the number of alcohol concentrations available upon alcohol intake in male and female UCSB mice.<!>Experiment 3: Influence of animal source upon withdrawal-induced negative affect and subsequent binge-drinking expressed by female mice.<!>Subjects.<!>Multi-Bottle-Choice Drinking-in-the-Dark (DID) Procedures.<!>Behavioral Testing for Negative Affect.<!>Acoustic Startle.<!>Novel Object.<!>Porsolt Forced Swim Test.<!>Light-Dark Shuttle Box.<!>Marble-burying.<!>Failure to reproduce binge-like levels of alcohol consumption under 2-BC conditions in UCSB mice.<!>Low-dose alcohol-drinking does not alter acoustic startle.<!>Low-dose alcohol-drinking does not alter prepulse inhibition of acoustic startle.<!>Low-dose alcohol-drinking does not alter behavior in the novel object test.<!>Low-dose alcohol effects upon behavior under light-dark shuttle-box procedures.<!>Failure to replicate withdrawal-induced anxiety-like behavior under marble-burying procedures.<!>Some replication of withdrawal-induced negative affect under forced swim procedures in UCSB mice.<!>Experiment 2: Effect of varying the number of alcohol concentrations available upon alcohol intake by male and female UCSB mice.<!>Animal source does not significantly affect alcohol intake under 4-BC procedures.<!>Replication of age-related differences in alcohol withdrawal-induced anxiety in female B6 mice under light-dark shuttle-box procedures.<!>Partial replication of age-related differences in alcohol withdrawal-induced anxiety in female B6 mice under marble-burying procedures.<!>Partial replication of age- and withdrawal-related differences in alcohol withdrawal-induced negative affect in female B6 mice under forced swim procedures.<!>Replication of a sensitization of subsequent alcohol intake in female mice with a prior history of binge-drinking.<!>Discussion<!>\xe2\x80\x9cSub-binge\xe2\x80\x9d levels of alcohol-drinking does not elicit a negative affective state during alcohol withdrawal.<!>Habituation to the drinking environment appears to facilitate binge-drinking.<!>Extension of the interaction between binge-drinking history, age of drinking-onset and withdrawal to female mice.<!>Limitations of study.<!>Conclusion.

<p>In humans, alcohol drinking typically commences during adolescence (ages 13–14), with binge-drinking being the most common pattern of alcohol intake (e.g., SAMHSA, 2010). Binge-drinking, as well as the incidence and prevalence of alcohol use disorders (AUDs), peaks in late adolescence/early adulthood (e.g., Chen et al., 2004; Harford et al., 2005; Martin and Winters, 1998). This is concerning as this age-range constitutes a critical period for the establishment of corticofugal connectivity within the extended amygdala neural circuit regulating emotional reactivity (e.g., Gogtay et al., 2004; Sowell et al., 2001; Spear, 2000a; Steinberg et al. 2005). As such, binge-drinking is theorized to be an important neurodevelopmental insult that impedes the resolution of a hyper-emotional state upon the transition from adolescence to adulthood (e.g., Guerri and Pascual, 2010; McClure and Pine, 2007; Miller et al., 2007). In support of this theory, AUDs exhibit a very high degree of co-morbidity with all affective disorders (e.g., SAMHSA, 2010). However, it is unclear from the epidemiological data which disorder precedes the other in comorbid individuals. Complicating the issue, AUDs, affective disorders and their comorbidity are sexually dimorphic in humans (e.g., Grant et al., 2004; Hasin et al., 2007; Johnston et al., 2008; Kessler et al., 2005; Merikangas et al., 2007) and this dimorphism appears in early adolescence (Fox and Sinha, 2009; Johnston et al., 2008; Sonne et al., 2003; Witt, 2007). The gender gap in heavy drinking is closing, particularly amongst youth (e.g., Johnston et al., 2008; Keyes et al., 2010) and clinical evidence indicates accelerated AUD and neuropsychiatric disease progression in women versus men (e.g., Harford et al., 2009; Hommer et al., 2001; Keller et al., 2010; Schuckit et al., 1998; Sharrett-Field et al., 2013). Thus, there is a pressing need for more basic animal research focusing on the interactions between the age of heavy drinking-onset and sex with respect to the manifestation of negative affect (c.f., Fox and Sinha, 2009; Gueri and Pascual, 2010).</p><p>Adolescence in rats and mice is approximately 2 weeks long, and occurs between postnatal days (PNDs) 35–48 (e.g., Spear, 2000b). Thus, the comparative study of adult and adolescent binge-drinking upon emotional reactivity requires animals to voluntarily consume sufficiently high levels of alcohol during a "single sitting" to produce BECs ≥ 80 mg% (see National Institute on Alcohol Abuse and Alcoholism, 2007) and to do so consistently over the course of at least 2 weeks. However, as highlighted in several recent reviews (c.f., Becker, 2017; Spear, 2018), the majority of the rodent literature regarding age-related differences in alcohol-affect interactions employed forced alcohol exposure procedures (i.e., experimenter-administered injections, oral infusions or vapor chamber delivery) or lengthy self-administration procedures. Nevertheless, as reported in humans (e.g., Bogin et al., 1986; Brown et al., 2008), the results of such animal studies indicate that adolescents are resilient to the behavioral and physiological "hang-over" effects of early alcohol withdrawal (incl. anxiety-like behavior) that are typically manifested by alcohol-exposed adults.</p><p>Likewise, a limited number of voluntary binge-drinking studies using modified Drinking-in-the-Dark procedures (see Rhodes et al., 2005 for description of original procedure) have also reported an interaction between the age of drinking-onset and withdrawal in the expression of anxiety-like behavior in male C57BL/6J (B6) mice. More specifically, adult male B6 mice with a 2-week history of binge-drinking under 2-h-access, multi-bottle-choice procedures [e.g., 4-bottle-choice (4-BC): 5, 10, 20 and 40%; 3-bottle-choice (3-BC): 10, 20, 40% alcohol, v/v; bottles presented at 3 h into the dark phase of the circadian cycle], exhibit robust signs of anxiety-like behavior in several paradigms (light-dark shuttle box, marble-burying and forced swim test) when assayed 1 day following drinking cessation (e.g., Lee et al., 2016, 2017a,b, 2018a,b). Further, behavioral signs of anxiety-like behavior are lower or completely absent when adult-onset drinking males are tested in later withdrawal (i.e., 30 days post-drinking cessation). Such findings argued that the alcohol-induced neuroadaptations driving the negative affective state in adult-onset binge-drinkers resolve or normalize with the passage of time in withdrawal. In contrast, despite consuming more alcohol over the 2-week drinking period and despite exhibiting more signs of basal anxiety than their adult counterparts, male B6 adolescents exhibit no overt emotional anomalies when tested at 1 day withdrawal. However, when tested 30 days later, the adolescent-onset binge-drinkers manifest very robust behavioral signs of negative affect (e.g., Lee et al., 2017b, 2018a). Thus, the manifestation of negative affect incubates during protracted alcohol withdrawal in male B6 mice with a history adolescent-onset binge-drinking, supporting the hypothesis that adolescent-onset binge-drinking alters the neurodevelopmental trajectory of circuits governing emotionality.</p><p>One major drawback of our prior work is the study of commercially-sourced mice, as the results are subject to the interpretational confound of age-related differences in transportation/relocation stress. Another drawback relates to the sole focus on male subjects, as female rodents tend to binge-drink more alcohol than males (e.g., Mélon et al., 2013). This report describes a primary, large-scale, experiment designed to replicate our prior interaction between binge-drinking history, the age of drinking-onset, and withdrawal in the manifestation of negative affect in male and female mice derived from a B6 colony established in our university vivarium (Experiment 1). For this, distinct cohorts of male and female littermates were tested for signs of negative affect on withdrawal days 1 or 30, under a similar behavioral test battery as that used in our prior studies of vendor-sourced, male mice (e.g., Lee et al., 2017b). Based on evidence that both adolescent and adult male mice binge-drinking under multi-bottle-choice procedures consume the majority of their daily intake from the sipper tubes containing 20 or 40% alcohol (e.g., Lee et al., 2016, 2017b), the mice in Experiment 1 were offered only 20 and 40% alcohol to facilitate study through-put. Further, to accommodate the large number of animals required of this study, mice were group-housed with same-sex littermates and then transferred singly to drinking cages, located in a non-colony procedural room, to determine individual alcohol intake. To expedite the timing of bottle presentation, mice were allowed 20 min to habituate to the drinking cages prior to alcohol presentation. As in our prior work (e.g., Lee et al., 2017b), the alcohol was offered for 2 h/day, beginning at 3 h into the dark phase of the circadian cycle, for 14 consecutive days prior to behavioral testing.</p><p>Appropriate for inclusion in this special edition on reproducibility and replication, the results of this primary experiment failed to replicate the levels of binge-drinking observed in our published work (e.g., Lee et al., 2017b) and, not surprisingly, we detected no signs of withdrawal-induced negative affect. As these completely negative outcomes were unexpected, additional follow-up studies were then conducted to understand the basis for this replication failure. As one procedural variable that differed from prior work was the number/range of alcohol concentrations presented, the first follow-up experiment (Experiment 2) determined how the number of alcohol concentrations presented (2, 3 or 4) influenced the amount of alcohol consumed by adult, male and female, mice from our UCSB colony. As mice in our prior studies (e.g., Lee et al., 2017b) were allowed a minimum of 45 min to habituate to the drinking cages prior to bottle presentation, a >45 min habituation period was employed in Experiment 2. As all mice in Experiment 2 consumed levels of alcohol demonstrated previously to result in BACs ≥ 80 mg%. The second follow-up experiment (Experiment 3) then examined how animal source influences the expression of alcohol withdrawal-induced negative affect. As we have replicated age-related differences in the temporal manifestation of negative affect and excessive alcohol-drinking during withdrawal in male mice (e.g., Lee et al., 2016, 2017b), Experiment 3 employed females exclusively to begin to make head-way towards characterizing how withdrawal from binge-drinking influences emotionality in female subjects. Experiment 3 employed 4-bottle-choice procedures (5, 10, 20 and 40% alcohol), based on the findings of Experiment 2 indicating that this procedure engendered the highest alcohol consumption in female subjects.</p><p>Combined, the results of these three experiments provide additional insight into experiential factors that impinge upon the levels of binge-drinking in male and female B6 mice to impact the manifestation of negative affect during withdrawal.</p><!><p>The Materials and Methods section begins with an outline of the designs of, and rationales for, the three experiments summarized in this report. The first subsection also outlines the procedural time-lines of the experiments and the statistical approaches employed to analyze the data. The remaining subsections then detail the specific procedures employed in this study.</p><!><p>Experiment 1 examined for the interactions between sex, binge-drinking history (2-week DID vs. water), age of drinking-onset (adolescent vs. adult) and withdrawal (1 vs. 30 days) upon negative effect in mice bred in our UCSB colony. In Experiment 1, mice underwent a 2-BC or water-drinking procedure for 14 consecutive days and then were tested for negative affect at their predetermined withdrawal period. The average total alcohol intake (i.e., g/kg alcohol consumed in a 2-h period) over the 14-day drinking period was analyzed using a Sex X DID (water vs. 2-BC) X Age (adolescent vs. adult) ANOVA. The data from our behavior test battery were analyzed using a Sex X Drinking (DID vs. Water) X Age (adolescent vs. adult) X Withdrawal (1 and 30 days) ANOVAs. For both analyses, significant interactions were deconstructed and followed-up using corrected t-tests, when appropriate. In the cases where non-significant main effects or interactions were detected, the data were collapsed across the non-significant factor(s) and re-analyzed along the relevant factors.</p><!><p>The study of binge-drinking-induced changes in emotionality requires that our UCSB-bred mice engage consistently in binge-drinking behavior in the first place. Thus, Experiment 2 determined whether or not the low levels of alcohol intake exhibited by the mice in Experiment 1 reflected the decision to employ at a 2-BC procedure, in lieu of a 3- or 4-BC drinking procedure or the employ of a shorter habituation period prior to daily bottle presentation. As adult mice tend to binge-drink less alcohol than adolescents (e.g., Lee et al., 2017b; Mélon et al., 2013), Experiment 2 employed adult mice exclusively. A between-subjects design was employed in which different groups of adult male and female UCSB-bred mice were presented with alcohol under 2-BC, 3-BC or 4-BC procedures (see Sect. 2.2) for 14 consecutive days. The data for alcohol intake and our measures of negative affect were analyzed using a Sex X Bottle (2-BC, 3-BC, 4-BC) ANOVA and significant interactions were deconstructed prior to follow-up analyses. As conducted in Experiment 1 (see Sect. 2.4.1), the data were collapsed across the non-significant factor(s) and re-analyzed along the relevant factors.</p><!><p>The results of Experiment 2 pointed to the duration of the habituation period, rather than the number of bottles presented, as an important procedural variable regulating alcohol intake in adult UCSB mice. While the results of Experiment 2 demonstrated clearly that both male and female adult UCSB will engage in binge-drinking when allowed a minimum 45-min habituation period to the drinking-cages, it remained to be determined whether or not age-related differences in amount of binge-drinking, and its consequent effects on emotionality, might also vary with animal source. Thus, Experiment 3 compared the effects of withdrawal from binge-drinking between adult and adolescent B6 mice from The Jackson Laboratory or our UCSB colony. As the results of Experiment 1 could not inform as to how a binge-drinking history impacts emotionality in female mice, Experiment 3 employed female subjects exclusively. Based on the data from Experiment 2, Experiment 3 employed a 2week, 4-BC procedure (5, 10, 20 and 40% alcohol, v/v) to maximize alcohol intake and consequent effects upon negative affect in early and later withdrawal. The data for alcohol intake were analyzed using a Source (JAX vs. UCSB) X Day (14 days) X Withdrawal (1 vs. 30 days) X Concentration (5, 10, 20 and 40% v/v) ANOVA, with repeated measures on the Days and Concentration factors. The behavioral data were analyzed using a Source (JAX vs. UCSB) X Age (adolescent vs. adult) X DID (water vs. 4-BC) X Withdrawal (1 vs. 30 days) ANOVA. The data were collapsed across non-significant factors prior to deconstruction of significant interactions, as appropriate. All data were analyzed using SPPS v.23.</p><!><p>Male and female C57BL/6J (B6) mice were obtained from The Jackson Laboratory (Sacramento, CA). A subset of these animals (herein, referred to as JAX) were assayed for the effects of binge-drinking upon withdrawal-induced negative affect (see Sect. 2.3 and 2.4 below), while another subset was employed to establish a breeding colony of B6 mice in the Psychology Building vivarium (minimum of 20 breeding pairs) and generate research subjects (F2–5) naïve to transportation/relocation stress (herein, referred to as UCSB). As in our prior studies of male B6 mice (e.g., Lee et al., 2017b), the JAX adolescent and adult males and females arrived 1 week prior to testing to acclimate to the colony conditions and 12-h reverse cycle (lights off: 1000 h). The UCSB mice were maintained on a regular 12-h light cycle (lights on: 0700 h) until 1 week prior to testing, at which time they were relocated to the reverse cycle room such that the time allowed for acclimation to the reverse cycle was consistent with that of the JAX mice. All animals were housed in same-sex, age-matched groups of 4 per cage, with standard rat chow (Purina LabDiet) and water available ad libitum except during the 2-h alcohol-drinking period. All cages were lined with woodchip bedding. All procedures were approved by the Institutional Animal Care and Use Committee of the University of California Santa Barbara and were conducted in compliance with The Guide for the Care and Use of Laboratory Animals (2014).</p><!><p>Half of the animals from each experimental group were subjected to 14 consecutive days of binge-drinking under 1 of 3 different multi-bottle-choice DID procedures. Consistent with our prior adolescent work (e.g., Lee et al., 2017b), alcohol access was restricted to 14 days for all animals, which corresponds to the approximate duration of early-mid adolescence in mice (e.g., Spear, 2000a). Adolescent mice began drinking on PND 28–29, while adult mice began drinking on PND 56–58. Each day, animals were separated into individual drinking cages and allowed to habituate (see Section 2.1 for duration of habituation for each individual experiment). Beginning 3 h into the circadian dark cycle, binge-drinking (DID) mice were presented with sipper tubes containing different concentrations of alcohol. All DID mice in Experiment 1 were given simultaneous access to 2 bottles containing 20% and 40% alcohol (v/v; 2-BC). In Experiment 2, subsets of mice underwent the same 2-BC procedures, or were presented with either 3 bottles containing 10%, 20% and 40% alcohol (v/v; 3-BC; Lee et al., 2017a) or 4 bottles containing 5%, 10%, 20% and 40% alcohol (v/v; 4-BC) for 2 h (e.g., Lee et al., 2017b). In Experiment 3, 4-BC procedures were used exclusively. In all experiments, control animals were transported to the drinking room but received water only (Water). Daily alcohol consumption was calculated by weighing the bottles immediately before and after the 2-h drinking period and expressed as a function of the animal's body weight (g/kg), which was determined weekly. Throughout this report, "alcohol intake" refers to the amount of alcohol consumed (g/kg) in a 2-h period. In Experiment 1, submandibular blood samples were collected from a subset of alcohol-drinking animals on day 11 of drinking, immediately upon conclusion of the 2-h drinking period. Blood alcohol concentration (BAC) was determined using an Analox alcohol analyzer (model AM1, Analox Instruments USA, Lunenburg, MA).</p><!><p>Our prior studies of JAX male mice indicated that adult-onset binge-drinking augments anxiety-like behavior during early (1–2 days) alcohol withdrawal, but that this effect dissipates by 30 days withdrawal. In contrast, adolescent-onset binge-drinking produces no observable effects upon negative affect in early withdrawal, but a robust increase in both anxiety- and depressive-like behavior during protracted withdrawal (e.g., Lee et al., 2016, 2017b). To replicate and extend these initial findings in Experimental 1, behavioral testing was conducted across withdrawal days 1 and 2 (WD1) and withdrawal days 30 and 31 (WD30), using a 2-day test battery, similar to that employed in our original study of binge-drinkingl-induced negative affect (Lee et al., 2015). At both time-points and starting at lights-out, testing for affect began on Day 1 with an test for acoustic startle and pre-pulse inhibition of acoustic startle (see Sect. 2.4.1), which was followed by the novel object encounter test (see Sect. 2.4.2) and the forced swim test (see Section 2.4.3). On Day 2, testing began (again at lights-out) with the light-dark shuttle-box test (see Section 2.4.4), followed by the marble-burying test (see Section 2.4.5). All tests were conducted under standard ambient lighting and animals were tested in the aforementioned order in cohorts of 8, with assays spaced ~30 min apart, with no more than 4 cohorts run per test day.</p><!><p>Testing was conducted in sound-attenuated startle chambers (SRLAB, San Diego Instruments, San Diego, CA), each consisting of a Plexiglas cylinder (3.8 cm diameter) mounted on a Plexiglas platform, with a high frequency loudspeaker (28 cm above the cylinder) producing all acoustic stimuli. The background noise of each chamber was 70 dB. A piezoelectric accelerometer, attached to the base, detected and transduced movements within the cylinder were detected and transduced by a piezoelectric accelerometer and then digitized and stored by a PC-type computer. As conducted previously (Szumlinski et al., 2005), six different trial types were presented: startle pulse (st110, 110 dB/40milliseconds), low prepulse stimulus given alone (st74, 74 dB/20milliseconds), high prepulse stimulus given alone (st90, 90 dB/20 milliseconds), st74 or st90 given 100 milliseconds before the onset of the startle pulse (pp74 and pp90, respectively) and no acoustic stimulus (i.e. only background noise was presented; st0). The st110, st0, pp74 and pp90 trials were applied 10 times, st74 and st90 trials were applied five times, and all trials were given in random order. The average inter-trial interval was 15 seconds (10–20seconds) and the data for startle amplitude were averaged across the different stimulus trials for each mouse prior to statistical analyses. Elevated basal activity and increased startle amplitude were interpreted as reflecting anxiety-like behavior, while reduced pre-pulse inhibition of acoustic startle was interpreted as reflecting a sensorimotor gating deficit. This test was approximately 20 min in length and immediately following testing, mice were removed from the startle chambers, the chambers and cylinders were wiped down with 30% ethanol and mice were transported within the lab to a distinct testing room housing the novel object test chambers.</p><!><p>To test reactivity to a novel object as an index of neophobia-related anxiety (e.g., Misslin and Ropartz, 1981), animals were placed in a black Plexiglas activity arena, measuring 46 cm long X 42 cm wide X 40 cm high. In the center of the arena was placed a novel, inedible, object (patterned ceramic candlestick holder;measuring ~6 cm in diameter X 12 cm high). A zone was designated around the novel object and was used to monitor the animals' interaction with the novel object during the 2-min trial using AnyMazeTM tracking software (Stoelting Co., WoodDale, IL, USA).The number of contacts and total time spent in contact with the novel object, as well as the total distance traveled within the activity arena, were recorded.</p><!><p>Each animal was placed into an 11-cm diameter cylindrical container and the latency to first exhibit immobility (defined as no horizontal or vertical displacement of the animal's center of gravity for ≥5 s), total time spent immobile, and the numbers of immobile episodes were monitored throughout the entire 6-min trial period using ANY-Maze™ video-tracking. In the forced swim test, decreased swimming/increased immobility or floating is conventionally interpreted as reflecting greater depressive-like behavior or "behavioral despair" as it is sensitive to pharmacological reversal by anti-depressant drugs (e.g., Porsolt et al., 1977). However, in prior work, we have observed an age-related effect of alcohol withdrawal in this assay, with adults and adolescents showing, respectively, decreased and increased immobility during withdrawal (e.g., Lee et al., 2017b, 2018a). Interestingly, the reduced immobility exhibited by adult binge-drinking mice temporally coincides with the manifestation of anxiety-like, but not depressive-like, behavior in other paradigms (e.g., Lee et al., 2017b, 2018a) and can be reversed by systemic treatment with the anxiolytic busperione (Lee et al., 2017a). Thus, we interpret the reduced immobility expressed by adult binge-withdrawn mice as reflecting an anxiety-like state (i.e., "panic"; Lee et al., 2017a). Conversely, adolescent-onset binge-drinking increases immobility in the forced swim test, coincident with reduced sucrose preference (Lee et al., 2017b) – a behavioral sign interpreted as reflecting anhedonia (see Katz, 1982). Thus, we interpret the increased immobility exhibited by adolescent-onset binge-drinking mice as reflecting a depressive-like state. Unfortunately, the limited available colony space at the time of testing precluded our ability to assay the mice in the present series of experiments for their sucrose preference. Upon completion of forced swim testing, animals were allowed to dry and then returned to the colony room to recuperate prior to testing the next day.</p><!><p>Animals were placed into a polycarbonate box measuring 46 cm long × 24 cm high × 22 cm wide containing two distinct environments for a 15-min trial. Half of the box was white and uncovered, the other half black and covered, and these two environments were separated by a central divider with an opening. The animals were first placed on the dark side and the latency to enter the light side, number of light-side entries, and total time spent in the light-side of the shuttle box were recorded using ANY-Maze™ tracking software. The dependent measures were: the number of light-side entries, latency to first light-side entry, total time spent on the light side, and the total distance traveled. Decreased interaction with the light-side was interpreted as reflecting increased anxiety-like behavior (e.g., Crawley, 1985), while the distance traveled in the light-side provided an index of general locomotor activity. Immediately following completion of light-dark testing, the mice were transported, in their home cages, to another distinct behavioral testing room where marble-burying was assayed.</p><!><p>In our paradigm, 12 square glass pieces (2.5 cm2 ×1.25 cm tall) were placed in an empty home cage, lined with woodchip bedding, 6 at each end. The entire 20-min trial was recorded using ANY-Maze™ video-tracking and the latency to start burying the marbles, as well as the time spent burying were determined upon video playback by an observer blind to the history or sex of the mice using a stopwatch. The total number of marbles buried at the end of each trial was also recorded upon removal of the mouse from the testing cage. The dependent measures were: the number of marbles buried, latency to first begin burying, and total time spent burying. Increased burying behavior was interpreted as reflecting increased defensive anxiety-like behavior (Njung'e and Handley, 1991).</p><!><p>Initial analyses of the average total alcohol intake/2h session across the 14 days of drinking under 2-BC procedures indicated a main Sex effect [F(1,108)=16.09, p<0.0001] and a main Age effect [F(1,108)=4.60, p=0.03]. There was no difference in the alcohol intake between the mice slated to be tested at the different withdrawal time-points, as indicated by no Withdrawal effect (p=0.19) and no interactions between any of the factors (see Supplemental Table 1). As depicted in Figure 1, females consumed more alcohol than males, irrespective of their age of drinking-onset, and adolescents consumed more alcohol than adults, irrespective of their sex. BACs were significantly, albeit weakly, correlated with intakes in the subset of mice tested (r=0.34, p=0.02; N=44) and an analysis of group differences in BACs collected on Day 11 of drinking (see Table 1) failed to support any significant main effects of, or interaction between, the Sex and Age factors (see Supplemental Table 2). The average total alcohol intakes exhibited by all groups were either at, or below, those predicted from published correlational analyses to result in BACs ≥ 80 mg% (e.g., Cozzoli et al., 2014). Thus, while we replicated prior reports of sex- and age-related differences in alcohol intake by B6 mice (e.g., Mélon et al., 2013; Strong et al., 2009), Experiment 1 failed to replicate binge-levels of alcohol intake under our 2-bottle DID procedures.</p><!><p>Initial analysis of group differences in the startle amplitude failed to indicate any main effect of, or interaction with, the DID factor (see Supplemental Table 3). Thus, the data were collapsed across this factor for reanalysis. As alcohol-drinking did not influence any of the dependent variables in the study, the detailed results are presented in the Supplemental Material (Sect. S.1.1) and summarized in Supplemental Figure 1A, B.</p><!><p>Initial analysis of group differences in the inhibition of acoustic startle by the 74 and 90 dB prepulses revealed a significant DID X Age X Sex X Prepulse interaction [F(1,196)=6.97, p=0.009], but no main effect of Withdrawal (see Supplemental Tables 3 and 4 for complete results). Thus, the data were collapsed across the Withdrawal factor prior to deconstruction of the significant 4-way interaction along the Sex factor. In females, we detected a significant interaction between prepulse intensity and binge-drinking history [DID X Prepulse: F(1,104)=3.93, p=0.05], that was independent of the age of drinking-onset (no Age effects or interactions, p's>0.10). However, when the data was collapsed across the Age factor, we did not detect any significant effect of alcohol-drinking history upon the prepulse inhibition produced by either the 74 dB [t(106)=1.72, p=0.09] or the 90 dB pre-pulse (t-test, p=0.69) in female subjects (Figure 2A). In males, we detected a significant 3-way interaction between drinking history, age of drinking-onset and prepulse intensity [F(1,100)=4.09, p=0.04]. Deconstruction of the interaction along the Age factor indicated no effect of alcohol-drinking in adolescent males (Figure 2B, left; DID effect and interaction, p's>0.40). We did detect a significant interaction between drinking history and prepulse intensity in adult males [F(1,50)=6.03, p=0.02]; however, post-hoc analyses failed to indicate any effect of drinking history on the percent inhibition produced by either pre-pulse stimulus (Figure 2B, right; t-tests, df=50, p's>0.18).</p><!><p>Initial analysis of the number of contacts, and the time spent in contact, with the novel object failed to indicate any group differences for these variables (data not shown; see Supplemental Tables 5 and 6). Initial analyses of the distance traveled indicated a main Age effect [F(1,216)=6.995, p=0.009] and an Age by Withdrawal interaction [F(1,216)=4.2, p=0.04; see Supplemental Table 7 for complete results]. As no significant Sex or DID effect was detected, the data were collapsed along both factors for re-analysis and the results of this analysis are presented in the Supplemental Material (Section S.1.2; Supplemental Figure 1C).</p><!><p>Initial analysis of group differences in the latency to enter the light-side of the shuttle box failed to indicate any main effect of, or interaction with, the DID factor (see Supplemental Table 8 for complete results). Thus, the data were collapsed across this factor for re-analysis, the detailed results are presented in the Supplemental Material (Section S.1.3) and significant findings summarized in Supplemental Figure 1D.</p><p>Opposite our prediction, there was a trend for alcohol-experienced mice to spend more time in the light-side of the shuttle box [DID effect: F(1,216)=3.61, p=0.06], but this did not vary as a function of any of the other factors examined (see Supplemental Table 9 for complete results). Collapsing across all other factors, the alcohol-related difference in the time spent in the light-side reached statistical significance (Figure 2C) [t(215)=1.99, p=0.048].</p><p>Initial analysis of the number of light-side entries indicated a significant 4-way interaction [F(1,216)=8.00, p=0.005; see Supplemental Table 10 for complete results]. Thus, the interaction was deconstructed along the Withdrawal factor. On WD1 (Figure 2D, left), alcohol-drinking mice made more light-side entries than did water controls [DID effect: F(1,110)=4.63, p=0.03] and females made more entries than did males [Sex effect: F(1,110)=9.18, p=0.003]. No age-related effects nor interactions were detected for the number of light-side entries on WD1, although the 3-way interaction approached statistical significance (Figure 2D, left) [F(1,110)=3.62, p=0.06]. Given that we predicted an age-related difference in anxiety during withdrawal, we deconstructed the 3-way interaction for WD1 along the Age factor. In adult mice (Figure 2D, left, right subpanel), alcohol-drinking animals exhibited a greater number of light-side entries than water controls [DID effect: F(1,50)=8.01, p=0.007] and females entered the light-side more than males [Sex effect: F(1.50)=9.10, p=0.004]. Although inspection of Figure 2D (left) suggested that the sex difference in light-side entries was driven primarily by the alcohol-drinking females, the Sex X DID interaction was not statistically significant (p=0.14). In contrast, no group differences were observed in adolescent mice on WD1 (Figure 2D, left, left subpanel; DID X Sex ANOVA, all p's>0.16). On WD30, the 3-way interaction was statistically significant (Figure 2D, right) [DID X Sex X Age: F(1,105)=4.85, p=0.03]. Thus, we deconstructed this interaction along the Age factor and detected no group differences in adult mice on WD30 (Figure 2D, right, left subpanel; DID X Sex ANOVA, all p's>0.14). However, a significant interaction was observed for the adolescent animals (Figure 2D left, right subpanel) [F(1,51)=4.54, p=0.04], which reflected significantly more lightside entries in female alcohol-drinking mice versus their water controls [t(23)=2.29 p=0.03], but no drinking effect in males (t-test, p=0.45).</p><p>Taken together, the results of the light-dark shuttle-box test represent a failure to replicate an alcohol withdrawal-induced increase in anxiety-related behavior, as well as the age-related differences therein. While we did detect age- and/or alcohol-related differences in behavior in this assay, the direction of the observed effects were opposite those reported previously by our group (e.g., Lee et al., 2017b).</p><!><p>Initial analysis of the latency to begin burying marbles indicated significant main effects of Withdrawal [F(1,216)=4.24, p=0.04], which reflected an overall longer latency to bury in early versus later withdrawal (WD1: 100.01 ± 6.76 vs. WD30: 79.81 ± 6.87 sec). We also observed a main alcohol-drinking effect [F(1,216)=5.81, p=0.02], as well as an interaction between alcohol-drinking history and the age of drinking-onset [F(1,216)=7.20, p=0.008] (see Supplemental Table 11 for complete results). As there were no significant interactions with the Withdrawal factor and no statistically significant effect of, or interactions with, the Sex factor (see Supplemental Table 11), the data for the latency to begin marble-burying were collapsed across these factors for reanalysis of drinking-related effects. Re-analysis supported a significant DID by Age interaction [F(1,216)=6.52, p=0.01], which reflected a shorter latency to bury by adult water controls, relative to their alcohol-drinking counterparts (Figure 1E, [t(103)=3.54, p=0.001], but no alcohol-related differences in adolescent animals (Figure 1E; t-test, p=0.97).</p><p>Initial analysis of the data for the time spent burying indicated an overall alcohol-drinking effect [F(1,216)=9.36, p=0.003], which reflected a shorter time spent burying by alcohol-experienced versus water control mice [Water: 17.85 ± 0.93, n=108 vs. DID: 12.77 ± 0.75, n=109]. While sex differences were not detected for the latency to begin burying marbles (see above), there were significant or near-significant interactions with the Sex factor as indicated by the initial analysis of the data for the time spent burying (see Supplemental Table 11 for complete results). However, as initial analyses failed to indicate any age-related differences in the time spent burying (see Supplemental Table 12), the data were collapsed across this factor for re-analysis. Re-analysis indicated a significant interaction between sex and withdrawal [F(1,216)=19.20, p<0.0001], which varied, albeit modestly, with the drinking history of the mice [DID X Sex X Withdrawal: F(1,216)=3.66, p=0.06]. We also detected a near-significant interaction between drinking history and withdrawal for the time spent burying [F(1,216)=3.84, p=0.05] and we explored these interactions further by deconstructing this interaction along the Withdrawal factor. On WD1, alcohol-drinking mice spent less time burying than water controls, irrespective of sex (Figure 2F, left) [DID effect: F(1,110)=21.64, p<0.0001; Sex effect and interactions, p's>0.14]. However on WD30, the effect of drinking varied as a function of sex [DID X Sex interaction: F(1,105)=5.66, p=0.02], with female alcohol-drinking mice spending less time burying than their water controls (Figure 2F, right) [t(49)=2.55, p=0.01], but no alcohol-related differences in males (t-test, p=0.56).</p><p>Initial analysis of the total number of marbles buried revealed a complex interaction between all of our factors [Sex X Age X DID X Withdrawal: F(2,216)=6.83, p=0.01; see Supplemental Table 13 for complete results]. Thus, the data were deconstructed along the Withdrawal factor to examine for time-dependent changes in behavior. On WD1, we detected less marble-burying, overall, in alcohol-drinking mice versus their water controls (compare open vs. closed bars in Figure 2G, left) [DID effect: F(1,110)=21.26, p<0.0001] and in adolescent versus adult mice (compare right vs. left panel in Figure 2G, left) [Age effect: F(1,110)=8.48, p=0.004; Adolescents: 1.53 ± 0.24, n=60 vs. Adults: 2.55 ± 0.30, n=51]. However, no significant interactions were observed between any of the factors on WD1 (Figure 2G, leftI; DID X Age X Sex ANOVA, all p's>0.10). In contrast, a significant 3-way interaction was observed for the number of marbles buried on WD30 (Figure 2G, right) [F(1,105)=5.06, p=0.03]. Deconstructing this interaction along the Age factor failed to indicate any effects of our factors in adolescent subjects (Figure 2G, right; DID X Sex ANOVA, all p's>0.45), while a significant DID by Sex interaction was observed for adult mice (Figure 2G, right) [F(1,53)=7.02, p=0.01], which reflected significantly lower marble-burying in female alcohol-drinking mice, relative to their water controls [t(24)=2.61, p=0.02], but no alcohol-related effect in adult male mice (t-test, p=0.28).</p><p>Taken together, the results of the marble-burying test also represent a failure to replicate an alcohol withdrawal-induced increase in anxiety-related behavior, as well as the age-related differences therein. As observed for the light-dark shuttle box test (Section. 3.1.4), we did detect age- and alcohol-related differences in marble-burying behavior, however, the direction of the observed effects were opposite those reported previously by our group (e.g., Lee et al., 2017b).</p><!><p>Initial analysis of the latency to first exhibit floating behavior in the forced swim test indicated a significant 4-way interaction [F(1,210)=4.80, p=0.03; see Supplemental Table 14 for complete results]. Deconstruction of the interaction along the Sex factor failed to indicate any significant main effects or interactions in female subjects (Figure 3A; DID X Age X Withdrawal ANOVA, all p's>0.10). In contrast, a significant 3-way interaction was detected in males [F(1,104)=6.82, p=0.01]. Deconstructing this interaction along the Withdrawal factor indicated that adult males took significantly longer to float than did adolescent males on WD1 [Age effect: F(1,50)=5.22, p=0.03], but there was no main DID effect or interaction detected in early withdrawal (Figure 3B, left; p's>20). On WD30, a significant DID by Age interaction was detected [F(1,53)=8.32, p=0.006]. Relative to their water controls, this interaction reflected a shorter and longer latency to float, respectively, in adult and adolescent alcohol-drinking mice on WD30 (Figure 3B, right) [for adolescents: t(25)=2.03, p=0.053; for adults: t(25)=2.09, p=0.047]. These data constitute a replication of our prior work (e.g., Lee et al., 2017b; 2018a) and indicate that even low-dose alcohol consumption can alter behavior in the forced swim test.</p><p>While the data for the latency to float replicated our prior work in male mice (e.g., Lee et al., 2017b, 2018a), we failed to detect any effect of drinking upon the number of immobile episodes in the forced swim test (all p's>0.40; Supplemental Table S15). Thus, the data were collapsed along the DID factor for re-analysis and the data are presented in the Supplemental Materials (Sect. S1.4).</p><p>However, a modest 4-way interaction was noted for the time spent floating [F(1,215)=3.89, p=0.05; see Supplemental Table 16 for complete results]. As conducted for the latency to float, we constructed this interaction along the Sex factor and follow-up analyses of the data from females indicated a significant Age effect [adolescents > adults; F(1,107)=4.84, p=0.03], a significant Withdrawal effect [WD30 > WD1; F(1,107)=4.69 p=0.03], but no DID effect or interactions (Figure 3C). A significant DID by Age interaction was observed in males [F(1,107)=4.89, p=0.03], and while inspection of Figure 3D suggested that this interaction was driven by the data from WD1, the DID by Age interaction did not vary significantly as a function of withdrawal (3-way ANOVA, p=0.14).</p><p>Taken together, the results of the forced swim test replicate, but only partially, age-related differences in alcohol withdrawal-induced changes in negative affect.</p><!><p>Experiment 2 examined total alcohol intake under 2-BC (20 and 40% v/v), 3-BC (10, 20 and 40% v/v) or 4-BC (5, 10, 20 and 40% v/v alcohol) in adult male and female mice bred in-house at UCSB. As depicted in Figure 4A and B, the total daily alcohol intake exhibited by UCSB mice fluctuated somewhat across the 14-day drinking period [Day effect: F(13,507)=14.32, p<0.0001] and, overall, females consumed more alcohol than males [Sex effect: F(1,39)=18.10, p<0.0001; Sex X Day: p=0.81]. While intake tended to be the highest under 4-BC procedures, neither the amount nor the pattern of intake varied significantly as a function of procedure [Procedure effect: F(2,39)=3.13, p=0.06; Procedure X Day: p=0.08; Sex X Procedure X Day: p=0.33; see Supplemental Table 17 for complete results).</p><p>As expected based on the time-course data, an analysis of the average total alcohol intake exhibited by the mice over the 14-day drinking period indicated higher alcohol consumption in female versus males (Figure 4C) [Sex effect: F(1,39)=18.10, p<0.0001]. Although inspection of Figure 4C suggested that the alcohol intake of females increased linearly as a function of the number of alcohol concentrations available, the interaction between sex and procedure was shy of statistical significance (Sex X Procedure: p=0.06; see Supplemental Table 18). As the alcohol intake under 2-BC procedures exhibited by both male and female mice in Experiment 2 was above that predicted to result in BACs ≥ 80 mg%, these data argue that the low alcohol intake exhibited by the UCSB mice in Experiment 1 did not simply reflect the employ of 2-BC drinking procedures, but rather likely reflected an insufficient habituation to the drinking cages prior to bottle presentation. However, because (1) alcohol consumption exhibited by the female mice in Experiment 2 tended to increase as a function of the number of alcohol concentrations presented and (2) our original studies of age-related differences in alcohol withdrawal-induced negative affect in males were conducted under a 4-BC procedure (Lee et al., 2016, 2017b), a 4-BC procedure was employed in Experiment 3 to engender the highest possible alcohol intake in female mice prior to testing for withdrawal-induced changes in negative affect and subsequent alcohol consumption.</p><!><p>An examination of the dose-response function for the average alcohol intake exhibited by female, adult and adolescent, mice indicated a significant Age X Concentration interaction [F(3,183)=14.67, p<0.0001]. However, we failed to detect any significant effect of, or interactions with, the Source factor with respect to alcohol intake (see Supplemental Table 19 for complete results). As depicted in Figure 5A, JAX and UCSB females exhibited equivalent alcohol consumption, irrespective of their age of drinking-onset. Collapsing across Source, a comparison of alcohol intake at each concentration indicated that adolescent females consumed more 40% alcohol (v/v) than their adult counterparts [t(63)=4.26, p<0.0001], while there were no significant age-related differences at the other concentrations tested (t-tests, all p's>0.08). These findings extend to females our prior work in males, indicating that adolescent mice consume more high-dose alcohol than adult mice under our 4-BC DID procedures (e.g., Lee et al., 2017b). Furthermore, the average total alcohol consumption exhibited by the female mice in this study exceeded 4 g/kg in a 2-h period, with adolescent females consuming nearly 1 g/kg more alcohol than their adult counterparts (Figure 5B) [Age effect: F(1,64)=10.34, p=0.002; Source effect and interaction, p's>0.45; see Supplemental Table 20 for complete results]. Thus, all of the female mice in Experiment 3 were engaged in high levels of binge-drinking behavior prior to testing for behavioral signs of negative affect and subsequent alcohol consumption.</p><!><p>The influence of animal-source upon anxiety-like behavior in the light-dark shuttle box depended upon the dependent variable examined. For example, no group differences were apparent regarding the latency to enter the light-side of the shuttle-box (data not shown; see Supplemental Table 21 for complete results).</p><p>However, an age-related difference was observed for the effect of alcohol withdrawal upon the number of light-side entries exhibited by female mice in Experiment 3 [Age X DID X Withdrawal: F(1,130)=27.80, p<0.0001]. Although UCSB females made fewer light-side entries, overall, than did JAX mice (data not shown) [Source effect: F(1,130)=10.72, p=0.001], animal source did not significantly affect age-related differences in withdrawal-induced anxiety as determined by this measure [Source X Age X DID X Withdrawal, p=0.79; see Supplemental Table 22 for complete results]. As such, the data were collapsed across the Source factor for follow-up analyses and the interaction was deconstructed along the Withdrawal factor. On WD1 (Figure 6A, left), we observed a significant DID X Age interaction [F(1,65)=10.85, p=0.002], that reflected fewer light-side entries in water-drinking adolescents versus both their alcohol-drinking counterparts [t(31)=2.30, p=0.03] and water-drinking adult mice [t(31)=2.62, p=0.01]. In contrast, the number of light-side entries exhibited by alcohol-drinking adult females was lower than that of their water-drinking adult controls [t(32)=2.17, p=0.04]. On WD30 (Figure 6A, right), we also observed a significant DID X Age interaction [F(1,64)=14.16, p<0.0001], however, this interaction reflected significantly fewer light-side entries in adolescent-onset alcohol-drinking mice, relative to both their water-drinking counterparts [t(32)=5.35, p<0.0001] and mice with a history of adult-onset alcohol-drinking [t(30)=3.17, p=0.004]. No significant age-related difference was detected for the number of light-side entries between adult- and adolescent-onset water-drinking mice (t-test, p=0.07).</p><p>The pattern of group differences in the time spent in the light-side of the shuttle box (Figure 6B) was similar to that observed for the number of light-side entries provided above, although the animal source exerted a more modest overall effect upon the time spent in the light-side (UCSB<JAX; data not shown) [F(1,130)=3.88, p=0.05]. Again, we detected a significant interaction between the DID, Age and Withdrawal factors for this variable [F(1,130)=12.92, p<0.0001] that was independent of animal source (4-way interaction, p=0.11; see Supplemental Table 23 for complete results). Thus, the data were collapsed along the Source factor for follow-up analyses and the interaction deconstructed along the Withdrawal factor. On WD1 (Figure 6B, left), no significant group differences were detected in the time spent in the light-side, although adult alcohol-drinking mice spent the least amount of time of all the groups tested. However, on WD30 (Figure 6B, right), a significant Age X DID interaction was detected [Age X DID: F(1,65)=13.10, p=0.001], which reflected significantly less time spent in the light-side by adolescent-onset alcohol-drinking mice versus their water controls [t(32)=4.53, p<0.0001], with no alcohol effect observed in adult-onset females (t-test, p=0.26).</p><p>Together, these data from the light-dark shuttle-box test argue that despite behavioral indices of higher overall anxiety in UCSB-bred mice, animal source does not significantly impact the age-related difference in the temporal patterning of alcohol withdrawal-induced anxiety-like behavior in female mice. Importantly, as reported for males (e.g., Lee et al., 2016, 2017b), females with an adult-onset binge-drinking history exhibit behavioral signs of anxiety-like behavior during early withdrawal that dissipate with the passage of time. In contrast, anxiety-like behavior incubates with the passage of time during alcohol withdrawal in females with a prior history of adolescent-onset binge-drinking.</p><!><p>Analysis of the latency to marble-bury indicated an age-related difference in the effect of alcohol withdrawal (Figure 7A) [Age X DID X Withdrawal: F(1,130)=7.17, p=0.009], but no effect of, or interactions with, the Source factor (all p's>0.10; see Supplemental Table 24 for complete results). Thus, the data were collapsed across the Source factor prior to deconstruction of the 3-way interaction along with the Withdrawal factor. On WD1 (Figure 7A, left), a significant DID X Age interaction was noted [F(1,65)=11.92, p=0.001], which reflected a shorter latency to marble-bury in alcohol-drinking adults versus both their water-drinking controls [t(32)=7.10, p<0.0001] and alcohol-drinking adolescents [t(31)=4.32, p<0.0001]. In contrast, no alcohol effect observed in adolescent mice (t-test, p=0.93), and no age-related difference in the latency to marble bury was observed in water-drinking controls (t-test, p=0.53). On WD30 (Figure 7A, right), we detected a main effect of binge-drinking only [DID effect: F(1,64)=77.44, p<0.0001; Age effect and interaction, p's>0.50].</p><p>The pattern of group differences for the number of marbles buried was consistent with that observed for the latency to begin marble-burying (Figure 7B) [DID X Age X Withdrawal: F(1,130)=13.62, p<0.0001], with no effect of animal source detected for this variable (see Supplemental Table 25 for complete results). Thus, the data were collapsed across the Source factor and the 3-way interaction deconstructed along the Withdrawal factor. On WD1 (Figure 7B, left), we detected a significant DID X Age interaction [F(1,65)=8.70, p=0.004], that reflected more marbles buried by adult alcohol-drinking mice versus both their water controls [t(32)=4.34, p<0.0001], as well as alcohol-drinking adolescent mice [t(31)=5.30, p<0.0001]. No alcohol effect was detected in adolescent mice on WD1 (t-test, p=0.91) and no age-related difference was observed between the water controls (t-test, p=0.18). On WD30 (Figure 7B, right), a significant Age X DID interaction was also detected [F(64)=4.84, p=0.03], with post-hoc comparisons indicating more marble-burying in both adult-onset [t(29)=4.40, p<0.0001] and adolescent-onset [t(32)=6.90, p<0.0001] alcohol-drinking mice relative to their respective water controls. The significant interaction reflected a modest, but significant, difference in the number of marbles buried between adult- and adolescent-onset water-drinking mice [t(31)=2.07, p=0.05], which was not apparent in their alcohol-drinking counterparts (t-test, p=0.17).</p><p>Finally, the analysis of the time spent burying also indicated a significant DID X Age X Withdrawal interaction (Figure 7C) [F(1,130)=20.81, p<0.0001]. As no Source effect or interactions were observed (p's>0.30; see Supplemental Table 26 for complete results), the data were collapsed across Source prior to deconstruction of the significant interaction along the Withdrawal factor. On WD1 (Figure 7C, left), we detected a significant DID X Age interaction [F(1,65)=63.99, p<0.0001], which reflected a longer burying time in alcohol-drinking adults, relative to their water-drinking controls [t(32)=4.59, p<0.0001], as well as adolescent alcohol-drinking animals [t(31)=4.08, p<0.0001]. No alcohol-related effect was observed in adolescent mice (t-test, p=0.70). On WD30 (Figure 7C, right), we detected a main effect of drinking [F(1,64)=232.90, p<0.0001], but no Age effect or interaction (p's>0.90).</p><p>The present data from the marble-burying test provide novel evidence that certain signs of withdrawal-induced negative affect can persist into protracted withdrawal in female mice with a 2week history of binge-drinking in adulthood. Further, the data from adolescent-onset alcohol-drinking females are consistent with those observed in males, indicating that signs of negative affect in this assay incubate during protracted withdrawal. Finally, the data fail to support any role for animal source in regulating behavior in this paradigm.</p><!><p>Analysis of the latency to float during the forced swim test indicated a significant DID X Age X Withdrawal interaction (Figure 8A) [F(1,130)=7.53, p=0.007], with no indication that animal source influenced this variable (see Supplemental Table 27 for complete results). Thus, the data were collapsed across Source to deconstruct the 3-way interaction along the Withdrawal factor. On WD1 (Figure 8A, left), we detected a significant DID X Age interaction [F(1,65)=34.55, p<0.0001], which reflected longer latency to float in adult alcohol-drinking mice, relative to their water-drinking controls [t(32)=7.31, p<0.0001], but not relative to their alcohol-drinking adolescent counterparts (t-test, p=0.21). No alcohol-related effect was observed in adolescent mice on WD1 (t-test, p=0.98), although adolescent water-drinking mice exhibited a longer latency to float than did their adult counterparts [t(31)=6.11, p<0.0001]. On WD30 (Figure 7A, right), we detected a significant effect of drinking [DID effect: F(1,64)=127.98, p<0.0001], a modest effect of Age [F(1,64)=4.03, p=0.05], but no significant interaction (p=0.09). Irrespective of age of drinking-onset, alcohol-experienced mice exhibited a longer latency to float, relative to their water-drinking counterparts, with adult mice tending to have a longer float latency than the adolescent mice at this withdrawal time-point (Figure 8A, right).</p><p>A similar pattern of group differences were observed for the time spent floating as that observed for the latency to float (Figure 8B) [DID X Age X Withdrawal: F(1,130)=8.74, p=0.004], with no effect of animal source apparent from the results of the statistical analyses (see Supplemental Table 28 for complete details). As such, the data were collapsed across Source prior to deconstruction of the 3-way interaction along the Withdrawal factor. On WD1 (Figure 8B, left), we detected a significant DID X Age interaction [F(1,65)=15.07, p<0.0001]. However, follow-up analyses indicated a shorter float time in both adult and adolescent alcohol-drinking females, relative to their water-drinking controls [for adults: t(32)=3.12, p=0.004; for adolescents: t(30)=3.32, p=0.002] and no age-related differences in float time observed in either water-drinking or alcohol-drinking mice (t-tests, for water: p=0.90; for alcohol: p=0.37). On WD30 (Figure 8B, right), we detected a main DID effect only [F(1,64)=26.99, p<0.0001; other p's>0.70], which reflected a shorter float time in alcohol-experienced animals, relative to their water-drinking controls.</p><p>In contrast to the above data, we detected no effect of alcohol upon the number of floats exhibited by either adolescent- or adult-onset drinking animals (no DID, Age or Withdrawal effects or interactions, all p's>0.1; see Supplement Table 29 for complete results). Overall, JAX mice tended to exhibit more floats than UCSB mice [F(1,130)=5.69, p=0.02; JAX: 18.60 ± 0.78, n=63; UCSB: 15.69 ± 0.96, n=68], but source did not interact with any of the other independent variables examined (data not shown; see Supplemental Table 29).</p><p>The present data from the forced swim test provide novel evidence that a history of either adolescent- or adult-onset alcohol-drinking elicits signs of withdrawal-induced anxiety-like behavior early in withdrawal in female mice that persist for at least 30 days. Interestingly, the direction of the effect of alcohol withdrawal upon floating behavior observed in the female mice with a history of adolescent-onset drinking is opposite that reported previously for males with a comparable drinking history (e.g., Lee et al., 2017b), suggesting potential sex differences in the manifestation of negative affect during alcohol withdrawal.</p><!><p>Analysis of the average total alcohol intake exhibited by female mice during the week following behavioral testing indicated a significant Age X DID X Withdrawal interaction (Figure 9) [F(1,130)=5.87, p=0.02], with no significant main effect of, or interactions with the Source factor (all p's>0.10; see Supplemental Table 30 for complete results). Thus, the data were collapsed across the Source factor prior to deconstruction of the significant 3-way interaction along the Withdrawal factor. On WD1 (Figure 9, left), mice with a prior binge-drinking history consumed more alcohol than prior naïve controls, irrespective of their age of drinking-onset [DID effect: F(1,65)=35.10, p<0.0001; no Age effect or interaction, p's>0.30]. However, an analysis of drinking during protracted withdrawal indicated a significant DID X Age interaction [F(1,64)=17.92, p<0.0001], which reflected higher alcohol intake by adolescent-onset alcohol-drinking mice, relative to both their water-drinking controls [t(31)=4.96, p<0.0001] and adult-onset alcohol-drinking mice [t(30)=4.53, p<0.0001]. No alcohol-related effect was observed adult-onset alcohol-drinking females during protracted withdrawal (t-test, p=0.93). These data indicate that, regardless of the age of drinking-onset, a prior history of binge-drinking promotes or sensitizes subsequent binge-drinking behavioral in early alcohol withdrawal in female mice. Further, these data indicate that this potentiating effect persists in female mice with a prior history of binge-drinking during adolescence.</p><!><p>Binge-drinking is the most common form of alcohol abuse and alcoholism (Center for Disease Control, 2012), particularly amongst adolescents and young adults (e.g., Chen et al., 2004; Harford et al., 2005; Martin and Winters, 1998; SAMHSA, 2010). In humans, early onset heavy drinking is a strong predictor of affective disorders in later life (e.g., Agosti, 2013; Grant et al., 2004, 2005; Hasin and Grant 2004, Kessler et al., 2005; Merikangas et al., 2007, Pettinati et al., 1989). Fitting with this, recent studies by our group have demonstrated using multi-bottle DID binge-drinking procedures that behavioral signs of negative affect (i.e., an anxious- and/or depressive-like state) incubate during protracted withdrawal in male B6 mice with a history of adolescent-onset binge-drinking (Lee et al., 2017b, 2018a,b). However, clinical evidence indicates greater neuropsychiatric disturbances in female versus male adolescent-onset binge-drinkers (e.g., Caldwell et al., 2005; Harford et al., 2009; Hommer et al., 1996, 2001; Keller et al., 2010; Medina et al., 2008; Sharrett-Field et al., 2013; Schuckit et al., 1998). Thus, Experiment 1 was designed to extend our results from males to female subjects and test the hypothesis that alcohol withdrawal-induced negative affect would be more severe in female mice, particularly those with a history of binge-drinking during adolescence. To avoid interpretational confounds associated with age- and/or sex-related differences in reactivity to environmental stressors, Experiment 1 employed male and female mice bred in our UCSB colony.</p><!><p>As detailed in Section 3.1, Experiment 1 successfully replicated published work indicating higher alcohol intake in female versus male B6 mice drinking under 2-h limited-access procedures (e.g., Melón et al., 2013; Metten et al., 2011; Rhodes et al., 2005; see Figure 1). However, this initial experiment failed to replicate age-related differences in 2-h alcohol intake (e.g., Lee et al., 2016, 2017b, 2018a,b; Melón et al., 2013; Metten et al., 2011). Further, the levels of alcohol intake, as well as BACs attained during drinking, were well below those expected of an animal model of voluntary binge-drinking (see Crabbe et al., 2011 for discussion). Not surprisingly, given their low alcohol intake, the mice in Experiment 1 exhibited no evidence of alcohol withdrawal-induced negative affect. This result fits with circumstantial evidence from our prior working suggesting that both the severity and longevity of the negative affective state observed during alcohol withdrawal with the amount of alcohol consumed. As mice were tested under ambient lighting over the course of 2 days, it is possible that circadian disruption may have mitigated our ability to detect water-alcohol differences in Experiment 1, particularly on the second day of testing. However, we have replicated water-alcohol differences in anxiety-like behavior expressed by male mice under both 1- and 2-day anxiety testing procedures. Thus, we attribute the failure to detect alcohol withdrawal-induced anxiety in Experiment 1 to the low level of alcohol intake.</p><p>Not expected, however, was the observation that mice with a history of repeated low-dose alcohol-drinking exhibited behaviors more consistent with a hypo-anxious state – a finding opposite that reported previously in B6 mice with a binge-drinking history. The "hypo-anxiety" exhibited by the mice in Experiment 1 was most notable in the light-dark shuttle box (Figure 2C,D) and marble-burying assays (Figure 2E-G), in which alcohol-drinking mice exhibited less defensive behaviors than their water-drinking counterparts. While we did not expect that alcohol withdrawal would impact every dependent variable assayed, we did expect, based on our prior work, to observe some age-related differences in the temporal manifestation of withdrawal-induced affective behavior across the different assays, with adult and adolescent mice exhibiting more pronounced behavioral effects, respectively, in early versus later alcohol withdrawal. Further, given that females consumed more alcohol than males during the 2-week drinking period, we anticipated sex-related differences in affective behavior that reflected the observed sex difference in alcohol-drinking. However, the behavioral changes observed in the alcohol-drinking mice of Experiment 1 did not vary systematically as a function of sex, age of binge-drinking onset or withdrawal, either within or between paradigms. We reasoned that the discrepancies in findings could reflect a number of procedural differences that we explored in follow-up experiments.</p><!><p>Within the confines of this report, the results of Experiment 2 argue an important role for the duration of this acclimatization period for the manifestation of binge-drinking. When allowed a longer time to habituate to the drinking cages, the adult UCSB B6 mice in Experiment 2 consumed doses of alcohol at, or above, 4 g/kg in 2 h, which corresponds to BACs greater than 80 mg/dl (e.g., Lee et al., 2016, 2017b, 2018a). In fact, a comparison of the total alcohol intake of the adult female mice undergoing our 2-BC DID procedure in Experiment 1 versus 2 indicated an ~ 1 g/kg difference in intake between the two experiments (3.7 g/kg vs. 4.7 g/kg), with an even larger difference apparent in the adult males (2.7 g/kg vs. 4.4 g/kg). The alcohol intakes observed in Experiment 2 are more in line with those observed in our much earlier reports in which adult, male, B6 mice were continuously single-housed in their drinking cages (e.g., Cozzoli et al., 2014; Lee et al., 2016) than the results from Experiment 1. While requiring direct study, this collection of results, coupled with casual observations of high levels of exploratory behavior upon cage transfer (e.g., climbing on, or handing from, cage lid bars, hyper-locomotion and rearing; i.e., behaviors that are physically incompatible with drinking), argue that an acclimation period may be an important procedural factor when examining alcohol intake in a novel test cage.</p><p>As suggested from our earlier reports employing 3-BC versus 4-BC procedures, the alcohol intake of the UCSB B6 males in Experiment 2 was nearly identical under 2-, 3- or 4-BC procedures (Figure 4C). Although total alcohol intake trended upwards as a function of the number of alcohol concentrations in the B6 females of Experiment 2 (Figure 4C), the difference in intake across procedures was not statistically significant. The findings of Experiment 2 contrast with an earlier report indicating that alcohol intake by both male mice and rats increases proportionately with the number of bottles containing 10% alcohol presented under continuous-access procedures (Tordoff and Bachmanov, 2003), as well as our prior binge-drinking research using 1 vs. 4 concentrations (see Cozzoli et al., 2012). Whether or not our failure to detect a robust "bottle-dependent" increase in alcohol intake reflects the fact that we offered mice a choice of different alcohol concentrations ranging from 10–40% (v/v), the alcohol was presented under limited-access conditions and/or no water was available during the drinking period cannot be discerned from the design of the present studies. Nevertheless, from the results of Experiment 2, we conclude that, at least in adult B6 mice, no robust sex difference exists with the respect to the influence of the number of alcohol concentrations presented under limited-access upon the manifestation of binge-drinking, as operationally defined by NIAAA (NIAAA, 2007). Although, female B6 mice exhibit a tendency to consume higher amounts of alcohol when the range of alcohol concentrations available is restricted to higher concentrations, which might reflect a failure to titrate dosing. Such an interpretation would be consistent with clinical evidence that females tend to develop an AUD at a faster rate than males and exhibit greater AUD severity (e.g., Hardford et al., 2005; Keyes et al., 2010; Schuckit et al., 1998). Whether or not such sex-related patterns in alcohol consumption are apparent in other mouse strains or rodent species that exhibit lower alcohol intake than C57BL/6 mice requires further study.</p><!><p>As reported in male B6 mice from JAX, adolescent female B6 mice binge-drank more alcohol than their adult counterparts under 4-BC DID procedures in Experiment 3. While a prior alcohol-drinking study indicated that UCSB-bred male and female mice on a mixed C57BL/6J-129/SvlmJ background exhibit binge-like levels of alcohol intake under similar testing procedures as those employed herein (Quadir et al., 2017), we have never directly compared alcohol intake between commercially- versus in-sourced mice. The results of Experiment 3 indicted that the age-related difference in alcohol intake exhibited by female mice did not vary with animal source (Figure 5), nor did animal source interact in any statistically reliable manner with the other independent variables with respect to either our behavioral indices of negative effect or subsequent binge-drinking. Based on these findings and our prior work with B6-hybrid mice (Quadir et al., 2017), we suggest that factors associated with animal transportation/relocation are not major determinants in the manifestation of binge-drinking or behavioral signs of negative affect following a 2-week period of binge-drinking, at least in female B6 mice tested within the confines of our laboratory setting. Future studies are necessary to determine whether or not the same is true for male B6 mice.</p><p>Male B6 mice from JAX with a 2-week history of binge-drinking exhibit age-related differences in the temporal manifestation of negative affect and excessive alcohol-drinking during withdrawal (Lee et al., 2016, 2017b). As reported for B6 males, the adult binge-drinking females from Experiment 3 manifested signs of anxiety-like behavior in the light-dark shuttle box, the marble-burying test and the forced swim test when assayed 24 h post-drinking. As also reported for males, the female binge-drinking adolescents exhibited no signs of anxiety-like behavior during early withdrawal in both the shuttle-box (Figure 6) and marble-burying tests (Figure 7). However, different from males, binge-drinking adolescent females spent less time floating than their water controls and the magnitude of this alcohol effect was comparable to that of the adult counterparts (Figure 8). The observed alcohol effect on the float time of the adolescent binge-drinking females may simply be spurious, as these same mice exhibited no other behavioral signs of anxiety during early withdrawal. However, akin to the binge-drinking adults, the adolescent binge-drinking females also consumed more alcohol than their water-drinking controls during the days following the anxiety testing at the early withdrawal time-point (Figure 9). These latter results suggest that adolescent females may be more prone than males to manifesting an anxiety-like state during early withdrawal from binge-drinking and, while relatively mild (i.e., only observed on one measure), the anxiety-like state may be sufficient to promote heavy drinking. As this finding is in line with human data indicated greater neuropsychiatric disturbances in female versus male adolescent binge-drinkers (e.g., Bekman et al., 2013; Squeglia et al., 2009), future work will involve a direct examination for sex differences in negative affect during early alcohol withdrawal and its relation to the propensity to consume the drug.</p><p>In our prior studies of males, no anxiety-like behavior is apparent during later withdrawal in adult-onset male mice with a 2-week history of binge-drinking (i.e., 30 days withdrawal), while their adolescent-onset counterparts exhibit very robust signs of anxiety-like behavior in the light-dark shuttle box and marble-burying tests, as well as signs of depressive-like behavior in the forced swim test and these signs of hyper-emotionality are associated with increased subsequent drinking. In Experiment 3, the pattern of age-related differences in the expression of anxiety-like behavior in the light-dark shuttle-box was very much in line with those observed previously in males; only adolescent-onset binge-drinking female mice exhibited signs of an anxiety-like state at 30 days withdrawal (Figure 6). Similarly, only adolescent-onset binge-drinking females exhibited augmented alcohol intake when tested in protracted withdrawal (Figure 9). However, on both the marble-burying and forced swim tests, both adult and adolescent females exhibited signs of anxiety-like behavior during protracted withdrawal; these effects were robust and observed across the majority of measures (Figure 7, 8). Thus, a robust negative affective state (1) incubates during protracted withdrawal in female mice with a 2-week history of binge-drinking during adolescence and (2) persists in female mice with a 2-week history of binge-drinking during adulthood. We know from prior work that a persistent negative affective state can be observed in adult male B6 mice with a more extensive (30-day) binge-drinking history, which is correlated with cellular markers of hyperexcitability within the central nucleus of the amygdala and the bed nucleus of the stria terminalis (Lee et al., 2015). Thus, the possibility exists that the more enduring effect of binge-drinking exhibited by the adult B6 females in Experiment 3 merely reflects their propensity to consume more alcohol, resulting in more enduring neuroplasticity within the extended amygdala. Unfortunately, the single-sex approach of Experiment 3 precludes any firm conclusions in this regard. Nevertheless, the apparent sex-difference in the longevity of the negative affective state produced by a 2-week binge-drinking history in adult mice is very intriguing and warrants further, more directed, investigation.</p><!><p>The purpose of this article was to report a failure by our laboratory to replicate a binge-drinking phenotype in C57BL/6 mice under specific limited-access drinking procedures and to summarize our efforts aimed at understanding the potential procedural bases of this failure. Given this, all data were obtained through studies of C57BL/6 mice and we cannot know if the findings generalize to other mouse strains or rodent species or to other drinking paradigms. Similarly, we cannot know if the age-related differences in alcohol withdrawal-induced anxiety exhibited by C57BL/6 mice in this or our prior work generalize across mouse strains or rodent species. While we have reported comparable withdrawal-induced anxiety in male C57BL/6 mice under both 1-day and 2-day behavioral test batteries, we cannot know how the magnitude of our group differences would compare if negative affect was assayed only in a single test or if mice were tested under low or red lighting conditions. Further, we have reported comparable alcohol intake and withdrawal-induced anxiety in male C57BL/6 mice housed singly or in groups; however, we have never systemically tested how individual versus group-housing impacts our dependent measures. Unless otherwise stipulated, the drinking and anxiety-testing procedures employed in the experiments described herein were conducted according to standard protocols that have been established in the laboratory for nearly 5 years. This operando facilitates data interpretation across studies and facilitates cross-study comparisons and the results of the present study were interpreted and discussed almost exclusively within the context of our own binge-drinking research. Indeed, the hypothesis that high levels of behavioral reactivity to the non-colony drinking cage interferes with focused drinking behavior under limited-access conditions was derived from behavioral observations during early pilot work when developing our "alternate cage" drinking procedures, and was supported by the differential findings from Experiment 1 versus 2. However, the reader is cautioned that our hypothesis was not tested in any parametric fashion and thus, the conclusions derived herein could benefit from additional follow-up study.</p><!><p>Herein, we show that a history of low-dose alcohol consumption is insufficient to elicit behavioral signs of negative affect in mice, irrespective of sex, the age of binge drinking-onset or the duration of alcohol withdrawal. We also suggest that the time allotted for group-housed mice to habituate to a novel drinking cage is an important procedural variable regulating alcohol consumption in both male and female adult and adolescent mice and make recommendations that, when studying C57BL/6J mice, habituation periods of at least 45 min should be employed prior to alcohol presentation to minimize behavioral reactivity that is physically incompatible with drinking behavior. Further, we show that when allowed sufficient time to habituate to the drinking cage, both male and female mice will binge-drink alcohol when presented simultaneously with 20 and 40% alcohol over a 2-h period, but the level of consumption does not vary significantly with the opportunity to also consume lower alcohol concentrations. Finally, we show that prior binge-drinking history, age of drinking-onset and withdrawal are factors that can interact to influence negative affect in female mice, with evidence suggesting that the underlying processes and temporal patterning of emotional dysregulation may be different from that of males.</p>

PubMed Author Manuscript

Novel Re(I) tricarbonyl coordination compounds based on 2-pyridyl-1,2,3-triazole derivatives bearing a 4-amino-substituted benzenesulfonamide arm: synthesis, crystal structure, computational studies and inhibitory activity against carbonic anhydrase I, II, and IX isoforms†

AbstractIn this work, two bidentate 2-pyridyl-1,2,3-triazole ligands (3a and 3b) containing a 4-substituted benzenesulfonamide pharmacophore prepared by classical click chemistry procedures, as well as their corresponding rhenium complexes, 4a and 4b of general formula [ReCl(CO)3(L)] (L = 3a or 3b) were prepared and fully characterised by spectroscopic methods (IR, NMR, MS, UV-Vis), elemental analysis, X-ray diffraction, and theoretical studies using DFT and TD-DFT methods. In particular, we showed that, in the solid state, the pyridine and the triazole rings of 3b adopted an uncommon cis configuration which stems from intermolecular hydrogen bonds. Preliminary assays demonstrated a promising nanomolar inhibitory activity against carbonic anhydrase isoform IX for both ligands and complexes with a strong affinity Ki of 2.8 nM for ligand 3a. More interestingly, complex 4b exhibited a pronounced selectivity against hCA IX over the off-targets hCA I and hCA II which makes this compound a promising potential anticancer drug candidate.

novel_re(i)_tricarbonyl_coordination_compounds_based_on_2-pyridyl-1,2,3-triazole_derivatives_bearing

Introduction<!>Materials and equipment<!>4-Azidobenzenesulfonamide (2a).<!>4-(Azidomethyl)-benzenesulfonamide (2b).<!>4-(4–(2-pyridyl)-1H-1,2,3-triazol-1-yl)-benzenesulfonamide (3a)<!>4–(4-(2-pyridyl)-1H-1,2,3-triazol-1-ylmethyl)-benzenesulfonamide (3b)<!>[(3a)Re(CO)3Cl], (4a)<!>[(3b)Re(CO)3Cl], (4b)<!>Crystal structure determination<!><!>Crystal structure determination<!>Computational details<!>Carbonic anhydrase inhibition assays<!>Synthesis and structural characterisation<!><!>Synthesis and structural characterisation<!><!>Synthesis and structural characterisation<!><!>Computational study<!><!>Computational study<!>Carbonic anhydrase inhibitory activity<!>Conclusion<!><!>Disclosure statement

<p>Over recent decades, rhenium(I) tricarbonyl complexes have been intensively studied by the inorganic chemist community, due to their significant photophysical and photochemical properties. These features make them interesting tools for numerous potential practical applications, such as photo sensitisers in solar cells 1 , CO2 reduction catalysts 2 , organic light-emitting devices (OLEDs) 3 , luminescence sensors 4 , and CO-releasing moieties (CORMs) 5 . Additionally, radioactive fac-[188Re(CO)3]+ complexes have recently drawn the attention of several research groups for their use as therapeutic radiopharmaceuticals 6 .</p><p>In this context, the efficient design and synthesis of chelating ligands represent the pivotal key to the successful application of this type of complexes. Most of the reported ligands which coordinate efficiently to the rhenium(I) tricarbonyl core are based on bidentate and tridentate chelating systems including N-heteroaromatic nitrogen, oxygen and to a lesser extend sulfur or phosphorus donor atoms 7 . While tridentate ligands lead to the most stable rhenium(I) tricarbonyl complexes, bidentate ligands based on α,α'-diimines are currently very popular. Indeed, their corresponding rhenium complexes exhibit interesting photo-physical properties which can be tuned by small changes in the ligand scaffold or around the rhenium centre by substituting the chlorine bound to the metal by a ternary ligand (generally, cyanides or N-heterocycles) 8 . Among efficient bidentate α,α'-diimine chelators, 2,2'-bipyridine (bpy) has been originally used 9 . More recently, similar chelating systems based on pyridine-triazole (pyta and tapy systems 1 ) were developed as alternative ligands to 2,2′-bipyridines 10 . Obata et al. first reported that pyta moieties acted like a bipyridine mimic with strongly electron-donating substituents, the corresponding complexes of general formula fac-[Re(pyta)(CO)3Cl] exhibiting luminescence properties 11 .</p><p>The growing interest on pyta (and tapy) architectures is mainly due to their easier synthetic access and functionalization. Unlike bpy systems, it is possible by using a classical copper-catalysed alkyne-azide cycloaddition (CuAAC reaction) not only to generate a large number of this important class of heterocyclic compounds but also to allow different functionalizations of the triazole ring 12 . As an example, we previously reported the preparation of two [M(CO)3]+ complexes (M = Re and 99mTc) from a pyridyltriazole-based ligand bearing the bioactive (2-methoxyphenyl) piperazine pharmacophore, which is a central nervous system (CNS) receptors-targeted vector. Thus, we demonstrated that (i) both complexes were iso-structural, as expected, (ii) the rhenium complex exhibited fluorescence at room temperature and the radioactive 99mTc-complex presented a suitable lipophilic character for its use as CNS imaging agent 12a . Given these first results, we decided to explore similar systems as potential human carbonic anhydrase inhibitors (CAIs) by adding a heterocyclic sulfonamide moiety to a pyta scaffold.</p><p>While sulfonamides (and bioisosteres sulfamates and sulfamides) are known to possess very efficient carbonic anhydrase inhibitory properties 13 , only a few examples of benzenesulfonamide-based compounds incorporating a [M(CO)3]+ complex (M = Re and 99mTc) have been reported 14 . Among these examples, Alberto and coworkers showed that simple piano-stool-type rhenium complexes with a pendent arylsulfonamide arm inhibited hCA IX and hCA XII with nanomolar affinities 14c . Using a similar strategy, i.e. coupling a simple and compact rhenium complex to a 4-substituted benzenesulfonamide moiety, we report here the synthesis of two tricarbonyl rhenium(I) conjugates based on a pyta ligand as well as their full characterisation by spectroscopic methods, X-ray crystallography study and DFT calculations. Preliminary biological assays against cytosolic/membrane-associated carbonic anhydrase isoforms I, II and IX (hCAs I, II and IX) were performed with both ligands and complexes and first results are promising.</p><!><p>All purchased chemicals were of the highest purity commercially available and used without further purification. Analytical grade solvents were used and were not further purified unless specified. Starting materials 1a and 1b were purchased from Aldrich Chem. Co. [Re(CO)5Cl] was purchased from Acros Organics.</p><p>1H and 13 C nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Avance 300 (75.5) MHz; chemical shifts are reported in parts per million (ppm) relative to a residual solvent peak and coupling constants (J) are given in Hertz (Hz). Multiplicities were recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quadruplet) … and m (multiplet). Infra-red (IR) spectra were recorded with Perkin–Elmer FTIR 1725 spectrophotometer in the range 4000–400 cm−1. Electrospray (ESI) mass spectra were obtained on a Q TRAP Applied Biosystems spectrometer and High-Resolution Mass Spectra (HRMS) on an LCT Premier Waters spectrometer. Microanalysis was performed by the microanalytical department of the Laboratoire de Chimie de Coordination de Toulouse (LCC, Toulouse, France). Electronic spectrum was measured on a Hewlett Packard 8453 temperature-controlled spectrometer in the range 1000–200 nm in methanol solution. Melting points were measured in capillaries using Mettler Toledo and are reported as uncorrected.</p><p>Literature methods were used to prepare intermediates 2a and 2b 15 (if azido compounds are potentially explosive intermediates, in our hands, we never observed hazardous reactions for both azido intermediates).</p><!><p>Yield: 267 mg (93%). 1H NMR (300 MHz, DMSO-d6): δ/ppm = 7.29 (d, J = 8.7 Hz, 2H, HAr), 7.36 (s, 2H, NH2), 7.82 (d, J = 8.6 Hz, 2H, HAr). Spectroscopic analysis is in agreement with reported data 15 .</p><!><p>Yield: 391 mg (85%). 1H NMR (300 MHz, DMSO-d6): δ/ppm = 4.56 (s, 2H, NCH2), 7.37 (s, 2H, NH2), 7.55 (d, J = 8.7 Hz, 2H, HAr), 7.83 (d, J = 8.6 Hz, 2H, HAr). Spectroscopic analysis is in agreement with reported data 15 .</p><p>General procedure for CuAAC reaction: Azide compound (2a or 2b, 1.1 equiv.) and 2-ethynylpyridine (1 equiv.) were suspended in acetonitrile (10 ml). Copper(II) acetate monohydrate (0.2 equiv) and sodium ascorbate (0.4 equiv) were added and the mixture is stirred in the dark at 45 °C overnight (16–18 h). The solvent was then removed under reduced pressure and remaining residues purified by column chromatography on silica gel using CH2Cl2/MeOH (95:5) as eluent.</p><!><p>Two fifty milligams (1.26 mmol) of 2a and 116 μL (1.15 mmol) of 2-ethynylpyridine with 46 mg (0.23 mmol) of Cu(OAc)2.H2O and 91.4 mg (0.46 mmol) of sodium ascorbate yielded the desired compound 3a as a yellow solid. Suitable crystals of 3a for X-ray crystal structure determination were grown by slow evaporation of methanol solution.</p><p>Yield: 236 mg (69%). mp 244–248 °C. 1H NMR (300 MHz, DMSO-d6): δ/ppm = 7.43 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H, Hpyr), 7.54 (s, 2H, NH2), 7.97 (td, J = 7.7, 1.8 Hz, 1H, Hpyr), 8.04 (d, J = 8.7 Hz, 2H, HAr), 8.14 (dt, J = 7.9, 1.1 Hz, 1H, Hpyr), 8.26 (d, J = 8.7 Hz, 2H, HAr), 8.63-8.71 (m, 1H, Hpyr), 9.46 (s, 1H, Hta). 13 C NMR (75 MHz, DMSO-d6): δ/ppm = 119.9, 123.5, 137.4, 149.2 (CHpyr), 120.5, 127.5 (CHAr), 121.6 (CHta) 138.6, 144.0 (CAr), 148.5 (Cpyr), 149.8 (Cta). ESI+-MS: m/z = 302.1 [M + H]+, 324.1 [M + Na]+. HRMS calculated for C13H12N5O2S ([M + H]+) 302.0712; found 302.0716. Elemental analysis for C13H11N5O2S: calculated (found) C, 51.82 (51.64); H, 3.68 (3.63); N, 23.24 (22.26).</p><!><p>Two twenty milligrams (1.04 mmol) of 2b and 95 μL (0.94 mmol) of 2-ethynylpyridine with 38 mg (0.19 mmol) of Cu(OAc)2.H2O and 75.5 mg (0.38 mmol) of sodium ascorbate yielded the desired compound 3b as a yellow solid. Suitable crystals of 3b for X-ray crystal structure determination were grown by slow evaporation of methanol solution.</p><p>Yield: 180 mg (61%). mp 192–195 °C. 1H NMR (300 MHz, DMSO-d6): δ/ppm = 5.78 (br s, 2H, NCH2), 7.29–7.41 (m, 3H, Hpyr, NH2), 7.53 (d, J = 8.7 Hz, 2H, HAr), 7.83 (d, J = 8.7 Hz, 2H, HAr),7.90 (ddd, J = 7.9, 7.5, 1.8 Hz, 1H, Hpyr), 8.04 (dt, J = 7.9, 1.1 Hz, 1H, Hpyr), 8.60 (ddd, J = 4.8, 1.8, 1.0 Hz, 1H, Hpyr), 8.73 (s,1H, Hta). 13 C NMR (75 MHz, DMSO-d6): δ/ppm = 52.4 (CH2), 119.4, 123.7, 137.2, 149.6 (CHpyr), 123.1 (CHta), 126.2, 128.4 (CHAr), 139.7, 143.9 (CAr), 147.6 (Cpyr), 149.8 (Cta). ESI+-MS: m/z = 316.1 [M + H]+. HRMS calculated for C14H14N5O2S ([M + H]+) 316.0868, found 316.0868. Elemental analysis for C14H13N5O2S: calculated (found) C, 53.32 (53.19); H, 4.16 (3.94); N, 22.21 (21.49).</p><p>General procedure for complexation reaction: A solution of 3a/3b (1 equiv) and commercial [Re(CO)5Cl] (1.1 equiv.) in methanol (8 ml) was stirred 12 h at 65 °C. After cooling to room temperature, the solution was concentrated until 3 ml and then a precipitate was obtained. Five millilitres of methanol was added to the precipitate. The mixture was heated, then cooled and stored at 4 °C for 3 h and then a supernatant was carefully removed. This process was repeated three times before the precipitate was dried under vacuum.</p><!><p>3a (70 mg, 0.23 mmol) and [Re(CO)5Cl] (92.5 mg, 0.25 mmol) yielded the desired complex 4a as a white solid. Suitable crystals of 4a for X-ray crystal structure determination were grown by slow evaporation of methanol solution.</p><p>Yield: 95 mg (67%). mp >300 °C. 1H NMR (300 MHz, DMSO-d6): δ/ppm = 7.64 (s, 2H, NH2), 7.71 (ddd, J = 7.3, 5.5, 1.6, 1H, Hpyr), 8.14 (d, J = 8.8 Hz, 2H, HAr), 8.25 (d, J = 8.8 Hz, 2H, HAr), 8.31 (ddd, J = 7.9, 1.6, 0.8 Hz, 1H, Hpyr), 8.38 (td, J = 7.7, 1.5 Hz, 1H, Hpyr), 9.04 (dd, J = 5.6, 1.2 Hz, 1H, Hpyr), 10.08 (s, 1H, Hta). 13 C NMR (75 MHz, DMSO-d6): δ/ppm = 121.4, 127.8 (CHAr), 122.7, 126.9, 137.4, 149.2 (CHpyr), 124.6 (CHta), 141.1, 145.5 (CAr), 148.3 (Cpyr), 153.3 (Cta), 189.4, 196.5, 197.4 (CO). IR: νCO=2027, 1931, 1905 cm−1, ESI+-MS: m/z = 570.0 [M-Cl]+, 627.9 [M + Na]+. HRMS calculated for C16H11N5O5SClRe ([M + Na]+) 627.9597, found 627.9616. Elemental analysis for C16H11N5O5SClRe: calculated (found) C, 31.66 (31.37); H, 1.83 (1.44); N, 11.54 (11.10).</p><!><p>3b (75 mg, 0.238 mmol) and [Re(CO)5Cl] (95 mg, 0.26 mmol) yielded the desired complex 4b as a white solid. Suitable crystals of 4b for X-ray crystal structure determination were grown by slow evaporation of methanol solution.</p><p>Yield: 98 mg (66%). mp 268–271 °C. 1H NMR (300 MHz, DMSO-d6): δ/ppm = 6.00 (s, 2H, NCH2), 7.41 (s, 2H, NH2), 7.60–7.68 (m, 3H, 1Hpyr, 2HAr), 7.90 (d, J = 8.3 Hz, 2H, HAr), 8.19–8.37 (m, 2H, 2Hpyr), 8.96 (dd J = 5.4, 1.3 Hz, 1H, Hpyr), 9.31 (s, 1H, Hta). 13 C NMR (75 MHz, DMSO-d6): δ/ppm = 54.1 (CH2), 122.8, 126.6, 137.8, 148.6 (CHpyr), 126.2 (CHta), 126.4, 129.2 (CHAr), 140.7, 144.5 (CAr), 148.6 (Cpyr), 153.0 (Cta), 189.5, 196.7, 197.5 (CO). IR (KBr): νCO=2029, 1920, 1902 cm−1. ESI+-MS: m/z = 584.0 [M-Cl]+, 642 [M + Na]+. HRMS calculated for C17H13N5O5NaSClRe ([M + Na]+) 641.9753, found 641.9750. Elemental analysis for C17H13N5O5SClRe: calculated (found) C, 32.88 (32.79); H, 2.11 (1.84); N, 11.28 (10.97).</p><!><p>X-ray intensity data of ligands 3a, 3b and corresponding rhenium complexes 4a, 4b were collected on a Bruker D8 VENTURE diffractometer (MA, USA) and using graphite monochromated Mo Kα radiation (λ = 0.71073 Å) for 3b, 4a and 4b and Cu Kα radiation (λ = 1.54178 Å) for 3a at 193 K. The semi-empirical absorption corrections were employed [SADABS, Programme for data correction, Bruker-AXS]. Crystallographic data and refinement details are given in Table 1. The structures were solved using an intrinsic phasing method 16 , and refined by full matrix least squares procedures on F2. All non-hydrogen atoms were refined with anisotropic displacement coefficients. The hydrogen atoms bound to carbon atoms were placed in calculated positions and treated as riding on their parent atoms with d(C–H)=0.93 Å, Uiso(H)=1.2 Ueq(C) (aromatic); and d(C–H)=0.96 Å, Uiso(H)=1.5 Ueq(C) (methyl). The methyl groups were allowed to rotate about their local threefold axis. The ShelX software package 16 was used for the calculations.</p><!><p>Crystal data and structure refinement for 3a, 3b, 4a, and 4b.</p><p>Inhibition data of human CA I, II and IX isoforms with compounds 3a, 3b, 4a, and 4b in comparison with the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped-flow CO2 hydrase assay.</p><p>mean from 3 different assays, by a stopped-flow technique (errors in the range of ±5–10% of the reported value).</p><p>Italic values are the references values of AAZ (acetalozamide). These are used to compare with our results.</p><!><p>CCDC 1871452 (3a), CCDC 1871453 (3b), CCDC 1871454 (4a) and CCDC 1871455 (4b) contain the supplementary crystallographic data. These data can be obtained free of charge from http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; tel: +44 (0)1223 336408; fax: +44 (0)1223 336033; or e-mail: deposit@ccdc.cam.ac.uk.</p><!><p>The reported calculations for the studied tricarbonyl rhenium(I) complexes 4a and 4b were carried out using the GAUSSIAN 09 programme package 17 with the aid of the GaussView visualisation program 18 . Full geometries optimizations were performed in gas-phase and methanol solvent without any symmetry restrictions with the DFT method using the hybrid B3LYP (Becke, three-parameter, Lee-Yang-Parr) functional 19 . The pseudo-potential (ECP) LANL2DZ 20 was employed to describe the electrons of Re atom while the other atoms (H, C, O, N and S) were assigned by the standard 6–31 G (d) basis set. The optimised geometries were evaluated using vibrational frequencies calculations to ensure that the true local minimum was attained and all eigenvalues are positive. Natural bond orbital analysis was carried out using NBO code included in GAUSSIAN 09 21 . At the optimised structures, time-dependent density functional theory (TD-DFT) method 22 using B3LYP functional was applied to calculate the vertical excitation energies and corresponding oscillator strengths. In addition, the solvent effect (methanol) was modelled using the polarisable continuum model with the integral equation formalism (IEF–PCM) 23 . The initial geometries were taken from X-ray structures, and all calculations were based on the optimised geometries.</p><!><p>An Applied Photophysics stopped-flow instrument was used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining the constant ionic strength), following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7–17 mM for the determination of the kinetic parameters and inhibition constants. In particular, CO2 was bubbled in distilled deionised water for 30 min so that the water was saturated (the concentration at a specific temperature is known from literature). In addition, a CO2 assay kit (from Sigma) was used to measure the concentration in variously diluted solutions obtained from the saturated one (which was kept at the same temperature and a constant bubbling during the experiments). For each inhibitor at least six traces of the initial 5–10% of the reaction was used for determining the initial velocity 24 . The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water and dilutions up to 0.01 nM were done thereafter with distilled-deionised water. Inhibitor and enzyme solutions were pre-incubated together for 15 min–2 h (or longer, i.e. 4–6 h) at room temperature (at 4 °C for the incubation periods longer than 15 min) prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation 25 , 26 , as reported earlier, and represent the mean from at least three different determinations. hCA I was purchased by Sigma-Aldrich and used without further purification, whereas all the other hCA isoforms were recombinant ones obtained in-house as reported earlier 27 , 28 .</p><!><p>The synthesis of rhenium tricarbonyl complexes 4a and 4b were readily synthesised following a three-step procedure as depicted in Scheme 1. The ligands 3a and 3b were prepared by classical copper-catalysed Huisgen cycloaddition (CuAAC reaction) using the corresponding azido intermediates and commercial 2-ethynylpyridine derivatives. Both azido compounds 2a and 2b were obtained in good yields (>80%) by following a procedure reported earlier 15 . By applying classical CuAAC conditions we previously reported for pyta ligands preparation 10b (i.e. catalytic system: Cu(OAc)2.H2O/sodium ascorbate in tert-butanol/water mixture or in acetonitrile at room temperature), 3a and 3b were obtained in low yield or at trace level. In turn, the yield of the cycloaddition was noticeably improved by performing the reaction at 45 °C in acetonitrile (average yield of 65%). Pyta-based rhenium complexes 4a and 4b were finally obtained in modest yields (c.a. 66%) by the reaction of commercial rhenium pentacarbonyl chloride with chelating ligands in refluxing methanol during 12 h. All our compounds are stable in the solid state as well as in organic solvents and their stability is not affected by the presence of air or moisture. They were fully characterised by NMR, IR, MS and elemental analysis and exhibited classical spectroscopic features for pyta-based compounds (see experimental section and ESI part, Tables S1–2 for NMR data). Briefly, upon complexation with the tricarbonyl rhenium core, a significant down-field shift (∼0.6 ppm) of the singlet due to the triazole proton and very minor shifts of the hydrogens of the phenyl ring compared to those of the free ligand were observed in proton NMR for each complex, confirming the coordination of the rhenium by the triazolyl unit and the pendent nature of the 4-substituted arylsulfonamide moiety. The pattern of the CO stretching frequencies in the IR spectra (three intense bands in the 2030–1900 cm−1 region) confirms the facial arrangement of the CO ligands in the complexes. More interestingly, as reported recently by Sarkar et al., the average value of the CO stretching frequencies gave information on the overall donor strength of the chelating pyta pincer 29 . In our case, both complexes exhibited an average value of 1950 cm−1, this value being consistent with those reported for other pyta ligands 29 and indicating an overall donor strength similar to bpy ligands. Fortunately, we obtained crystals of ligands 3a/3b and complexes 4a/4b suitable for single crystal X-ray diffraction from the slow evaporation of a concentrated sample in methanol solution. Selected crystallographic parameters are listed in Table 1 and selected bond lengths and angles values are gathered in the ESI part (Tables S3–S5).</p><!><p>Synthesis of ligands 3a/3b and their corresponding benzenesulfonamide rhenium tricarbonyl complexes 4a/4b. Conditions and reagents: (i) 1a, NaNO2, NaN3, HCl/THF/DMF (v/v/v: 1:1:1), 0–25 °C, 1 night (93%) or 1b, NaN3, Tf2O, CuSO4.5H2O, K2CO3, CH2Cl2, 0–25 °C, 1 night (85%); (ii) Cu(OAc)2.H2O, sodium ascorbate, CH3CN, 45 °C, 1 night, (3a: 69%, 3b: 61%); (iii) [Re(CO)3Cl], 65 °C, MeOH, 12h, (4a: 67%, 4b: 66%).</p><!><p>Both ligands (3a and 3b) were crystallized in triclinic and monoclinic system and their crystals were solved in space groups P 1¯ and P2 1/c, respectively (see ORTEP diagrams in ESI part, Figure S1). As expected, the structural characteristics of 3a/3b are similar to those reported for related pyta ligands 10 a,b, 11 , 12 , 30 . The azo character of the triazolyl ring is confirmed by the N(2)–N(3) distance of the 1,2,3-triazole unit which is shorter than the N(3)–N(4) and N(2)–C(6) bonds (1.314(3) Å vs. 1.357(3) Å and 1.366(4) Å for 3a with similar bond length values for both molecules of the asymmetric unit, and 1.313(2) Å vs. 1.343(2) Å and 1.363(2) Å for 3b.</p><p>While 3b is essentially planar with an angle between the least-square planes of the 2-pyridyl and triazole rings of 8.1(1)°, 3a displays an exceptional deviation between the pyridyl part and the triazolyl entity. The values of the dihedral angle (33.3(1)° and 27.9(1)° for both molecules of the asymmetric unit) are the largest observed for the pyta scaffold compared to the previously reported values (range from 1.56°–15°) 10 a, 11 , 30 . In turn, one of the two molecules of 3a exhibits a singular planar geometry with respect to the triazolyl moiety and the phenyl group of the benzenesulfonamide unit (dihedral angle value of 3.2(1)° vs. 30.3(2)° for the second molecule of the asymmetric unit). Generally, in pyta scaffold bearing a phenyl group directly connected to the triazole ring, the dihedral angle between the pyridyl and triazole rings is shorter than that between the triazole and phenyl rings (dihedral angle value: c.a. 20–30°) 11 , 30a . A network of inter-molecular hydrogen interactions (see ESI part, Table S6) and π–π interactions could explain these unexpected structural features for 3a (see ESI part, Table S7).</p><p>More interestingly, while the nitrogen atoms N(1)/N(2) and N(6)/N(7) of 3a exhibits a classical anti configuration 11 , 12 , 30 with minimal electronic repulsions between both nitrogens, an unprecedented cis arrangement is clearly adopted by the nitrogen atoms of the pyridine and triazole rings of ligand 3b, as illustrated in Figure 1. Stabilisation through a network of non-covalent interactions has been invoked to explain thus unexpected arrangement. The single-crystal X-ray structure of 3b shows two types of hydrogen-bonding interactions (see ESI part, Table S6): stronger intermolecular hydrogen-bonding interactions of the type N–H···N [2.939(2) Å] between the N(5)–H(5b) proton of the sulfonamide moiety and the N(1) atom of the pyridine ring, as well as C–H···O [3.260(2) Å] between the C(7)–H(7) proton of the 1,2,3-triazolyl group and O(1) atom of the sulfonamide moiety. π–π interactions have also been detected in the crystallographic structure for 3b (see ESI part, Table S7).</p><!><p>Unprecedented cis-configuration of the 2-pyridyl-1,2,3-triazole unit of ligand 3b; a partial view of the crystal packing of 3b with N–H•••N and C–H•••O hydrogen bonds and π–π contacts shown as dashed lines. The purple spheres represent the centroids of the rings involved in π–π interactions.</p><!><p>Rhenium complexes 4a and 4b shown in Figure 2 (molecular views), crystallise as neutral molecular complexes in the orthorhombic space groups Pbcn and triclinic space groups P 1¯, respectively. As suggested by the spectroscopic data, each rhenium centre adopts a distorted octahedral coordination geometry. The rhenium atoms are coordinated to two nitrogens of the pyta pincer, three carbonyl donors in a fac-orientation and one chlorine atom. The bond lengths and angles for both complexes 4a and 4b are unexceptional and similar to those previously reported for other rhenium tricarbonyl complexes based on bidentate ligands 10–12 , 30 . As an example, the average Re-carbonyl bond length is 1.92 Å, and the OC–Re–CO angles range from 87.4(2)° to 90.3(2)° and 88.8(1)° to 90.3(1)° for 4a and 4b, respectively. The trans bond angles exhibit a moderate distortion from an idealised octahedral geometry (range of 170.3–179.0°), as expected. The Re–Cl bond lengths [2.490(1) Å for 4a, 2.485(1) Å for 4b] lie in the upper side of the range (2.454–2.505 Å) found for other rhenium tricarbonyl complexes based on pyta derivatives 10 g, 11 , 12a . For both complexes 4a and 4b, the chelate part is essentially planar with a very slight deviation of 2.5(1)° and 8.2(2)° between the mean planes through the triazolyl and pyridyl rings, respectively. As observed previously, the co-planarity was extended to the benzenesulfonamide pendent arm in 4a. The 4-substituted aromatic arm shows a dihedral angle of 2.1(1)° with the mean plane of the triazolyl ring, as a result of conjugation 10 b, 31 . In both complexes, a network of π–π interactions and inter-molecular hydrogen bonds between two adjacent complex molecules for 4a and between two adjacent complex molecules plus the lattice methanol molecule for 4b are involved in the crystal cohesion (see ESI part, Table S6, and Figure S2).</p><!><p>The molecular structures of rhenium complexes 4a and 4b. Displacement ellipsoids are drawn at 50% probability, solvent molecule (4b) and hydrogen atoms (except H atoms on nitrogen) have been omitted for clarity.</p><!><p>The density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations were performed in order to assess the impact of the methylene linker (−CH2−) between the benzenesulfonamide moiety and the chelate unit of 3b on the electronic structures of its rhenium complexes (See Tables S8–S10 and Figure S3 in ESI part for more details). While HOMOs energy levels exhibit small changes, the introduction of the methylene spacer in the complex 4b has an effect on LUMOs energy, as illustrated in Figure 3. Compared to 4a, the introduction of this methylene linker increased the energy level of LUMO (−2.29 eV) causing wider energy gap (3.22 eV for 4b v s. 3.09 eV for 4a) and a slight blue-shift of the lowest energy absorption band (331 nm for 4b vs. 333 nm for 4a). The lowest virtual orbitals of complexes are mainly concentrated on the π-anti-bonding orbitals of the chelate and CO ligands. The lowest virtual molecular orbital (LUMO) is centred on π-anti-bonding orbitals of the 2-pyridyl-1,2,3-triazole part and CO ligands. The LUMO + 1 and LUMO + 4 are mainly localised on the full pyta ligand for both complexes. The LUMO + 2 is located on the 2-pyridyl-1,2,3-triazole unit in 4a, and with a slight contribution from the phenyl ring in the case of 4b. The LUMO + 3 is wholly centred on phenyl ring for the complex 4a, whereas for 4b, it is localised on the full connected ligand.</p><!><p>Molecular orbital diagrams of 4a (Left) and 4b (Right).</p><!><p>For 4a and 4b, the low-energy absorption bands at 333 and 331 nm have mixed metal-to-ligand [MLCT] and ligand-to-ligand [LLCT] charge transfer character. Accordingly, these electronic transitions originate mainly from the mixed orbitals of the rhenium centre and chlorine to the π-antibonding orbitals of the chelating ligand and carbonyl group, which can be described as {d/π(Cl) → π*(L)/π*(CO) or π*(L)}. The band assignments were presented like those previously reported by other researchers, for related Re(CO)3-complexes with similar bidentate ligands 32 .</p><!><p>As mentioned in the introduction, the group of Alberto demonstrated that compact tricarbonyl rhenium complexes acted as excellent carbonic anhydrase IX and XII inhibitors, with nanomolar affinities 14c . More recently, Tim Storr et al., reported that tricarbonyl rhenium complexes based on a dipyridylamine (DPA) or an iminodiacetate (IDA) ligands with a pendent benzenesulfonamide pharmacophore exhibited interesting CA-IX inhibition (KI of 37 nM for the best candidate) 14d .</p><p>In our case, both ligands 3a and 3b and their corresponding complexes 4a and 4b were tested for their efficacy to inhibit physiologically relevant hCA I, II and the tumour-associated hCA IX isoforms, as a preliminary study. The synthesised compounds were screened for their inhibition potential by means of stopped-flow carbon dioxide hydration assay and compared with the clinically used reference drug acetazolamide (AAZ) (Table 2).</p><p>Both complexes 4a and 4b and the reference drug acetazolamide (AAZ) exhibited similar nanomolar affinities (ca. 25 nM) against hCA IX isozyme. Compared to reported affinity values with different tricarbonyl rhenium systems bearing an arylsulfonamide moiety, our values are better than those recently reported by the group of Storr (37–220 nM), in the lower range than those obtained by Babich et al. (3–116 nM) but higher than those obtained by Alberto et al. (3–7 nM) 14b–d . The introduction of a methylene group between the metallic part and the sulfonamide unit has a slight negative effect on the inhibition values (18.7 nM for 4a vs. 27.3 nM for 4b), as previously reported 14c . Additionally, a better inhibition is observed with ligands 3a and 3b compared to their corresponding metallic analogs. In particular, a very strong inhibition is found for ligand 3a, which displayed 7-fold higher affinity than its rhenium complex 4a (Ki(3a)=2.8 nM vs. Ki(4a)=18.7 nM). This unexpected result should be confirmed on other hCA isoforms, the non-metaled free ligands exhibiting generally weaker affinity than that of the corresponding rhenium complexes 14b . In turn, this first study showed that both rhenium complexes were highly inactive against hCA I and exhibited a low affinity against hCA II but a nanomolar affinity against hCA IX. In terms of selectivity, complex 4b was our best candidate with promising hCA I/hCA IX and hCA II/hCA IX ratios of 261.75 and 30.64, respectively.</p><!><p>In short, two new bidentate 2-pyridyl-1,2,3-triazole ligands with a pendent 4-substituted benzenesulfonamide arm and their corresponding Re(CO)3-complexes were successfully synthesised. All the prepared compounds were fully characterised by classical spectroscopic methods and X-ray, as well as theoretical studies for both complexes. While in the solid state, rhenium complexes showed classical distorted octahedral geometry, ligand 3b adopted an uncommon cis-configuration arising from a network of intermolecular hydrogen-bonding interactions. DFT calculations revealed that the presence of methylene (−CH2−) linker between the benzenesulfonamide moiety and the chelate unit has a slight effect on the absorption bands position of 4b as well as the HOMO-LUMO gap which is slightly blue-shifted compared to 4a.</p><p>Preliminary investigation on the inhibitory activity of these compounds against the cytosolic human carbonic anhydrase I, II and the membrane-associated isoforms IX (hCAs I, II and IX) revealed an activity in the nanomolar range for hCA IX isozyme. Surprisingly, better inhibition was found with ligands compared to rhenium complexes. In particular, this preliminary study showed that ligand 3a exhibited a strong affinity Ki of 2.8 nM for hCA IX. Additionally, complex 4b exhibited a pronounced selectivity against hCA IX over the off-targets isoforms hCA I and hCA II which makes this compound a promising potential anticancer drug candidate. Further in vitro studies against other hCA isoforms with both ligands and rhenium complexes and other derivatives based on the same scaffold are under progress in order to rationalise these first biological results.</p><!><p>Pyta and tapy mean 4-(2-pyridyl)-1,2,3-triazole and 1-(2-pyridyl)-1,2,3-triazole, respectively.</p><!><p>No potential conflict of interest was reported by the authors.</p>

PubMed Open Access

Dynamics of Hydration Water Plays a Key Role in Determining the Binding Thermodynamics of Protein Complexes

Interfacial waters are considered to play a crucial role in protein-protein interactions, but in what sense and why are they important? Here, using molecular dynamics simulations and statistical thermodynamic analyses, we demonstrate distinctive dynamic characteristics of the interfacial water and investigate their implications for the binding thermodynamics. We identify the presence of extraordinarily slow (~1,000 times slower than in bulk water) hydrogen-bond rearrangements in interfacial water. We rationalize the slow rearrangements by introducing the "trapping" free energies, characterizing how strongly individual hydration waters are captured by the biomolecular surface, whose magnitude is then traced back to the number of water-protein hydrogen bonds and the strong electrostatic field produced at the binding interface. We also discuss the impact of the slow interfacial waters on the binding thermodynamics. We find that, as expected from their slow dynamics, the conventional approach to the water-mediated interaction, which assumes rapid equilibration of the waters' degrees of freedom, is inadequate. We show instead that an explicit treatment of the extremely slow interfacial waters is critical. Our results shed new light on the role of water in proteinprotein interactions, highlighting the need to consider its dynamics to improve our understanding of biomolecular bindings.Water is an active and indispensable component of cells. Understanding its versatile roles in determining the structure and dynamics of biomolecules and mediating their interactions is of fundamental importance 1-3 . The versatility of water in biological contexts arises from its ability to alter its characteristics depending on its interaction with biomolecules. For example, the DNA sequence-dependent behavior of hydration water serves as a sequence-specific "hydration fingerprint" 4 ; changes in water dynamics during binding of a substrate to an enzyme play a vital role in protein-ligand recognition 5 ; and the non-bulk behavior of water inside the translocon strongly affects the partitioning of hydrophobic segments from the translocon to the membrane 6 . However, although our understanding of the behavior of hydration water around biomolecules has advanced significantly in recent years [7][8][9][10][11][12][13][14] , it remains a challenge to elucidate the extent to which water molecules located between two biomolecules are modified through concurrent interactions with the two binding surfaces and how such altered water molecules in turn affect the binding affinity.In this connection, it has been suggested that water-mediated contacts substantially complement direct protein-protein contacts, providing an additional layer of biomolecular recognition 15,16 . The necessity of an explicit treatment of interfacial water molecules to properly describe such water-mediated interactions has also been noted 17 . Indeed, recent computational studies have reported on the relevance of explicitly handling "key" interfacial waters in protein-protein interaction 18 and protein-ligand binding 19 : for example, including two, rather than all, interfacial water molecules was crucial to correctly obtaining the trends observed in mutation effects on protein-protein binding affinity 18 ; in another study, explicitly taking into account interfacial water molecules ranging in number (N wat ) from 30 to 70 significantly improved the correlation with the experimental binding affinities for four different systems, where the optimum value of N wat depended on the specific system 19 . What, however, distinguishes those key interfacial water molecules from others? Do any distinctive characteristics of the interfacial water emerge upon protein-protein complex formation?

dynamics_of_hydration_water_plays_a_key_role_in_determining_the_binding_thermodynamics_of_protein_co

<!>Methods<!>Explicit inclusion of the water molecules of interest.<!>Results and Discussion<!>Thermodynamic-dynamic relationship diagram.<!>∆<!>Conclusions

<p>In this paper, we investigate the dynamic and thermodynamic features of interfacial water in the barnasebarstar complex 15 . This is a well-studied paradigm for protein-protein interactions and is also an ideal system for analyzing the interfacial water because X-ray measurements indicate the presence of waters filling the gap between the binding surfaces 15,20 . We perform molecular dynamics simulations to explore dynamic characteristics of the interfacial water. We focus on the rearrangements of hydrogen bonds, which are the most important protein-water interaction because the protein-protein binding surfaces comprise mainly polar and charged residues 21 . We then conduct statistical thermodynamic analyses to rationalize the dynamic characteristics of the interfacial water. Finally, we discuss the impact of the interfacial water dynamics on the protein-protein binding affinity. We find that the conventional approach to the water-mediated interaction, which assumes the time-scale separation between the protein and hydration water dynamics, fails owing to the extremely slow dynamics exhibited by the interfacial waters. We show instead that an explicit treatment of those slow waters as an integral part of biomolecules is critical. Thereby, we would like to shed new light on the role of water in protein-protein interactions based on a dynamic view point.</p><!><p>Molecular dynamics simulations. We conducted explicit-water molecular dynamics simulations for the barnase-barstar complex (Fig. 1) and for the free barnase and barstar proteins. The initial complex structure was modeled based on the X-ray structure (PDB ID: 1 BRS 15 ) as detailed in ref. 22. The starting structures of the free barnase and barstar simulations were taken from their NMR structures (1 BNR 23 and 1 BTA 24 , respectively). The complex structure was solvated by 23,477 waters and neutralized by 4 counter Na + ions in a cubic box of the initial side length 95.4 Å; the free barnase (barstar) was solvated by 14,346 (8,397) waters and neutralized by 2 Cl − (6 Na + ) ions in a cubic box of the initial size 81.7 Å (69.5 Å). All the simulations were performed using the AMBER14 suite 25 with the FF99SB force field 26 for proteins and the TIP3P model 27 for water. The temperature and pressure were maintained at T = 300 K and P = 1 bar using the Berendsen's method 28 . We adopted the same simulation procedures as described in ref. 22, and three independent 1 μs production runs were performed for each system starting from different random initial velocities.</p><p>We also conducted pure-water simulations to obtain dynamical quantities for bulk water. Three independent 100 ns simulations were performed at T = 300 K and P = 1 bar with 2,539 waters.</p><p>Hydrogen-bond rearrangement dynamics. We analyzed the hydrogen-bond time-correlation function, which quantifies the extent to which hydrogen bonds found at time t = 0 survive to subsequent times t 29 , to investigate hydrogen-bond rearrangements between protein and hydration water. A hydrogen bond is considered formed when the water oxygen is located within 3.5 Å from heavy atoms in a protein. The hydration water is classified as follows (see Fig. 2a for an illustration). A water molecule forming a single hydrogen bond to a protein is referred to as single HB water. The locations of single HB waters in a simulation snapshot are indicated by cyan spheres in Fig. 2b, and their average number (±standard deviation) computed from the whole simulation trajectories for the complex is 325.7 ± 13.7 (Table 1 summarizes the number of water molecules and the number of water-protein hydrogen bonds). A water molecule making two or more hydrogen bonds to a protein is termed double HB water: the positions of double HB waters in a snapshot are shown by orange spheres in Fig. 2b. There are 133.0 ± 8.0 double HB waters in the system, and the average number of hydrogen bonds to a protein is 2.4 ± 0.1. Finally, a water molecule forming concurrent hydrogen bonds with two proteins is called bridging water. By definition, bridging waters are present only at the interface between two proteins (red spheres in Fig. 2b). We find 19.6 ± 3.0 bridging waters located at the interface, with the average number of water-protein hydrogen bonds being 2.9 ± 0.2.</p><p>We investigate the hydrogen-bond time-correlation function defined by Trapping free energy. We introduce the trapping free energies of individual hydration waters to quantify how strongly they are bound to the protein surface. The trapping free energy refers to the reversible work (i.e., the potential of mean force) for transferring a water molecule from an infinite separation to a specific position and orientation relative to the protein-protein complex. We consider here the transfer process to a fixed position and orientation relative to the solute for two reasons. First, this allows us to compute the trapping free energies of individual hydration waters solely based on the simulation snapshots such as the one presented in Fig. 2b. Second, we are interested in whether the trapping free energies so computed at time t = 0 serve as a descriptor of the degree of retardation of the subsequent (t > 0) dynamics of individual water molecules. The thermodynamic cycle shown in Fig. 3 is used to obtain this quantity; in this cycle, we consider the transfer of the i-th water molecule to a specific position and orientation around the solute u, which includes the hydration water molecules of interest (e.g., all the waters displayed in Fig. 2b). The solute from which the i-th water molecule is excluded is denoted as u′.</p><p>Process (1) in Fig. 3 is the independent solvation processes of the i-th water molecule and the solute u′; hence, the associated Gibbs free energy change is given by G G G u</p><p>(1) w ater solv solv ∆ = + ′ , which consists of the solvation free energies of a single water molecule (G water solv ) and of the solute u′ ( ′ G u solv ). In process (2), the i-th water molecule is transferred to a specific position and orientation around the solute u′ from an infinite separation in vacuum. The reversible work required for this process is given by the interaction energy ′ − E u i between the solute u′ and the i-th</p><p>. Process (3) is the solvation of the solute u(=u′ + i), and we obtain</p><p>. From the thermodynamic cycle, one obtains the trapping free energy from -ΔG (1) + ΔG (2) + ΔG (3) ; that is, Table 1. Average number of waters and water-protein hydrogen bonds a . a Average ± standard deviation.</p><p>We computed the interaction energy ′− E u i from the force field, whereas the solvation free energy G u solv is obtained using the 3D-RISM theory 30 , whose details are presented in Supplementary Methods. An efficient method for computing the contribution G G</p><p>′ which is based on the atomic decomposition of the solvation free energy 31,32 is also provided there. The trapping free energies for the hydration waters surrounding free barnase and barstar proteins can be obtained in a similar manner.</p><p>Recently, several computational methods have been developed for evaluating thermodynamic functions of individual hydration waters [33][34][35][36][37][38][39][40][41] . However, these methods typically demand performing additional distinct simulations. For example, the application of the inhomogeneous solvation theory [33][34][35] requires conducting simulations in which restrains are added on protein atoms to sample waters' positions and orientations for a given protein conformation. Further complications in analysis will arise when hydration waters exchange with bulk waters during those additional simulations. On the other hand, our computational method for the trapping free energy that employs the integral-equation theory (3D-RISM) is applicable solely based on snapshots taken from unrestrained equilibrium simulations, and it is in this sense more computationally efficient.</p><p>Standard binding free energy. Conventional approach. The statistical thermodynamic expression for the standard binding free energy is given by refs 22 and 42</p><p>Here, ΔX denotes the change in X upon complex formation from two free proteins (labeled 1 and 2),</p><p>solv comprises the gas-phase energy (E u ) and the solvation free energy (G u solv ) of the solute u (here, u refers to the complex or one of the two free proteins and excludes hydration waters); the bar denotes the average over the simulated configurations; S config is the configurational entropy associated with the solute's internal degrees of freedom; and ΔS ext is the entropy change originating from the reduction in the external (positional and orientational) degrees of freedom upon complex formation. ΔS ext carries the standard-state dependence, which is chosen here to be the one of the standard concentration (1 M).</p><p>We computed the gas-phase energy E u from the force field adopted in the simulations. (E u for free proteins represents only the intra-protein energy, but E u for the complex includes the inter-protein interaction energy as well). For the solvation free energy G u solv , we employed the 3D-RISM theory 30 (see Supplementary Methods). For the configurational entropy S config , we used an energetic approach 43,44 that expresses S config in terms of the statistical properties of f u . In particular, when the probability distribution W(f u ) of f u is Gaussian, the following holds: where k B is Boltzmann's constant, and f f f u u u δ = − . For the external entropy ΔS ext , we use the estimate TΔS ext = −6.8 ± 0.1 kcal/mol for the barnase-barstar complex, which was computed in ref. 22. by extending the energetic approach to the binding process and is close to the value reported in ref. 45.</p><!><p>A statistical thermodynamic formulation of the binding free energy which allows one to explicitly include certain solvent molecules was also derived in ref. 42. In essence and using our notation, what is required is to replace</p><p>In this expression, the solute u now explicitly includes the water molecules of interest (e.g., E u now contains interactions with and among those water molecules), n is the number of water molecules included, and G water solv is the single water molecule's solvation free energy. S config then needs to be evaluated by combining equations ( 4) and (5).</p><!><p>Hydrogen-bond rearrangement dynamics. We study the dynamic and thermodynamic features of the hydration water surrounding the barnase-barstar complex by conducting molecular dynamics simulations and statistical thermodynamic analyses. In particular, we aim to uncover the distinctive characteristics of the interfacial water between two proteins that emerge upon complex formation. This is done by contrasting the dynamics of the interfacial water with that of the hydration water surrounding free proteins; for this purpose, we also perform simulations and analyses for the free barnase and barstar proteins. We focus on the rearrangements of hydrogen bonds, which are the most important protein-water interactions because of the largely hydrophilic nature of the protein-protein binding surfaces 21 .</p><p>Figure 4 shows the hydrogen-bond time-correlation functions, which quantify the extent to which hydrogen bonds found at time t = 0 remain at subsequent times t. For water molecules making a single hydrogen bond to a protein (referred to as single HB water; see Fig. 2 and Table 1), we observe profound slowing down of the relaxation dynamics compared to those of bulk water (see Table 2 for a comparison of the average relaxation times). For water molecules making two or more hydrogen bonds to a protein (double HB water), the hydrogen-bond rearrangement is even slower. For bridging water molecules, that is, interfacial water molecules making concurrent hydrogen bonds with two proteins, the relaxation is extraordinarily slow (~1,000 times slower than the relaxation in bulk water). Furthermore, the relaxation curve is anomalous, exhibiting a logarithmic decay over three orders of magnitude in time.</p><!><p>To rationalize the slow relaxations of hydration waters, we conducted statistical thermodynamic analyses. We focused on the long-time region where the time-correlation functions decay from 0.3 to 0.1 (light yellow region in the upper panel of Fig. 5) and extracted the water molecules contributing to the relaxation there by examining the hydrogen-bond survival times (τ i ) of individual molecules. For each of those water molecules, we computed the trapping free energy (G i trap ) using the simulation snapshot at t = 0. The trapping free energy can be considered as the effective potential characterizing how strongly each water molecule is captured by the biomolecular surface: a more negative trapping free energy means that a water molecule is more stable near the protein complex than in the bulk, and hence, is more favorably "trapped" by the protein complex. The lower panel of Fig. 5 shows scatter plots of the relaxation times and trapping free energies of individual water molecules. (The average relaxation times and trapping free energies listed in Table 2 are obtained from these plots. Since the distribution of the individual waters' relaxation times τ i is well represented on the logarithmic axis as shown in the lower panel of Fig. 5, Table 2 also provides the statistics computed with log 10 τ i ). The resulting "thermodynamic-dynamic relationship diagram" clearly illustrates that the trapping free energy (G i trap ) at t = 0 serves as a good descriptor of the degree of retardation (τ i ) of the subsequent dynamics of hydration water.</p><p>Thermodynamic-dynamic relationship diagram is presented schematically in Fig. 6a. Single HB water exhibits slower dynamics than bulk water because it is more stable near the protein surface, which in turn reflects the fact that the hydrogen bond between water and protein is stronger than the one between waters. The even slower dynamics of double HB water can be understood similarly; further stabilization originates from an additional water-protein hydrogen bond. Why, then, is bridging water, which has the comparable number of hydrogen bonds with proteins as double HB water (Table 1), more strongly trapped than double HB water? We also notice here that the dynamic, as well as thermodynamic, characteristics of single and double HB water molecules are nearly the same, irrespective of whether they are placed near the isolated monomer or protein-protein complex (Supplementary Figs S1 and S2). The emergence of the "red region" for bridging water in the diagram (Fig. 6a) thus arises solely from the formation of the protein-protein interface. Is there any special factor that is effective only at the interface?</p><p>We notice in this regard the electrostatic complementarity of the barnase-barstar binding surfaces (Fig. 6b), which creates a strong electrostatic field that is exerted on the interfacial water. Indeed, we find that the magnitude of the electrostatic field is stronger and the water's dipole vector is more oriented along the electrostatic field for bridging water than for single and double HB water (Supplementary Fig. S3). Thus, whereas essentially no change in the hydration water dynamics is observed in the non-interfacial region before and after the binding (Supplementary Figs S1 and S2), the strong electrostatic field created at the binding interface produces an extra stabilizing factor for bridging water, causing it to exhibit extremely slow (nanosecond timescale) hydrogen-bond relaxations (Table 2). It would be interesting to investigate how the transition in the hydration water dynamics occurs during the binding process, but for this purpose, one needs to perform spontaneous binding simulations. The trapping free energy introduced in the present work serves as a valuable quantity not only to characterize the formation of the binding interface from the water's perspective, but also to discuss how and whether the hydration water is rearranging to go from the unbound protein to bound complex. While the barnase-barstar complex studied here is known to be a system in which the interfacial waters are particularly immobile 46 , we anticipate the emergence of the extremely slow water relaxations to be a generic feature of hydrophilic protein-protein interfaces because electrostatic complementarity of the binding surfaces has been observed in numerous protein complexes 47,48 .</p><p>"Conventional" binding thermodynamics. Among the terms that contribute to ∆G bind 0 (see equation ( 3)), the quantity</p><p>solv plays a special role for two reasons. First, it is generally difficult to compute the configurational (ΔS config ) and external (ΔS ext ) entropies for complex macromolecules such as proteins. Second, ΔS config and ΔS ext are usually negative upon complex formation and thus make unfavorable positive contributions to G bind 0</p><!><p>; hence, the driving force for binding must originate from Δf u . Indeed, Δf u is the central quantity, termed the effective binding free energy 49 , in computational approaches to biomolecular bindings such as the molecular-mechanics Poisson-Boltzmann surface area (MM-PBSA) method [50][51][52] .</p><p>We computed f u ∆ by averaging Δf u over the simulated protein conformations. (Our approach is referred to as the three-trajectory approach because we conducted separate computations for the complex and for two free proteins. Numerical values for the binding thermodynamics shall be reported with standard errors computed based on the respective independent trajectories of the complex and free proteins and on the rule of error propagation). The energetic contributions (ΔE u ) were calculated directly from the force field, and the solvation contributions ( G u solv ∆</p><p>) were computed using the 3D-RISM theory (see Supplementary Methods). We obtained f 25 7 2 6 u ∆ = + . ± . kcal/mol; this result leads to an unphysical positive value of ∆G bind 0 , which is in obvious disagreement with the experimental observation ( G 18 9</p><p>bind 0 ∆ =− . kcal/mol) 53 . Interestingly, positive effective binding free energy has also been reported based on the MM-PBSA calculations for the barnase-barstar complex: ∆ = + f 14 u kcal/mol in ref. 54 and ∆ = + . f 3 6 u kcal/mol in ref. 55. (The difference in these values may originate from the use of the one-trajectory approach in the MM-PBSA calculations, in which both the complex and monomer configurations were taken from simulations of the complex; the use of different force fields; and the use of different approximations for the solvation free energy). Basic assumption behind the conventional approach. At this point, we critically examine the basic assumption behind the expression (3) for the standard binding free energy. To simplify the discussion, we work in the canonical ensemble and ignore the external entropy, which would not alter the essential point here. We start from the configuration integral, the potential part of the partition function, for a solute-solvent system:</p><p>Here, r u and r v collectively denote the solute and solvent degrees of freedom, respectively; β = 1/(k B T) is the inverse temperature; and E u , E uv , and E v are the solute energy, solute-solvent interaction energy, and solvent-solvent interaction energy, respectively. Z tot is the principal object in free energy simulations: the change in the free energy F tot = −k B T log Z tot , e.g., upon mutation, is computed from simulations in which both the solute and solvent degrees of freedom are explicitly handled. However, equation (6) does not serve as a basis of equation (3): for example, by introducing the probability distribution</p><p>the entropy that naturally arises from equation ( 6) is</p><p>, and it is non-trivial to partition the solute and solvent terms from this total entropy. This is in contrast to equation (3) where the solute (S config ) and solvent (contained in G u solv ) entropies are separated. To arrive at equation ( 3) from equation ( 6), one has to resort to a pre-averaging of solvent degrees of freedom. For a given solute configuration r u , this pre-averaging can be performed as</p><p>in terms of the solute-configuration dependent solvation free energy G r ( )</p><p>v v is the configuration integral for the pure solvent. Now, the configuration integral after the pre-averaging of the solvent degrees of freedom is given by</p><p>, the associated entropy is given by</p><p>, which is the defining equation for the solute configurational entropy S config . It is therefore clear that equation ( 3) is based on the pre-averaging of solvent degrees of freedom (see refs 22 and 42 for a complete derivation of equation (3) from equation ( 8)). By the "conventional" approach, we mean the one that is based on this pre-averaging, and do not refer to specific methods such as PBSA and 3D-RISM.</p><p>In practical applications of the conventional approach, one takes only the protein conformations from simulation trajectories, replacing all the explicit water molecules by the equilibrium continuum model (PBSA) or molecular distribution function (3D-RISM). Such a treatment is usually justified because of the timescale separation between the typical water dynamics (picoseconds) and the protein conformational motions (nanoseconds) 16 , i.e., because of the rapid equilibration of surrounding waters. However, the extreme slowness of the bridging-water relaxation may invalidate such a naive treatment of the water at biomolecular interfaces, and we conjectured that this might the origin of the unphysical positive value of u ∆ .</p><p>Explicit inclusion of bridging water. We therefore investigated the impact of explicit inclusion of the slow bridging waters. For this purpose, we not only take the protein configurations from the simulation trajectories for the complex, but also bridging waters located at the interface: the number (n) of bridging waters depends on the simulation snapshot, and its average value is 19.6 ± 3.0 (Table 1). Now, those bridging waters are considered as a structural part of the complex, and we apply equation ( 5) to compute f u for the complex. We obtain a negative value f 34 2 2 1 u ∆ = − . ± . kcal/mol, which now serves as the driving force for binding. This result indicates the necessity of considering the dynamics of the interfacial water in the binding thermodynamics.</p><p>To support our explicit inclusion of bridging waters through a comparison with experiment, we computed the binding free energy G bind 0 ∆</p><p>. To this end, we need to estimate the configurational (ΔS config ) and external (ΔS ext ) entropy contributions. For the configurational entropy, we used an energetic approach 43,44 that expresses ΔS config in terms of the fluctuations in f u . In particular, when the probability distribution W(f u ) of f u is Gaussian, S config is simply given by the mean-squared fluctuations of f u (see equation ( 4)). Indeed, W(f u ) of the barnase-barstar complex with bridging water is well approximated by Gaussian, as well as that of the free barnase and barstar proteins (Supplementary Fig. S4), from which TΔS config = −4.5 ± 18.5 kcal/mol is obtained. For the external entropy ΔS ext , we use the estimate TΔS ext = −6.8 ± 0.1 kcal/mol, which was obtained using the method developed in ref. 22 and is close to the value reported in ref. 45. Combining all these results, we obtain ∆ =− . ± . G 22 9 18 6 bind 0 kcal/mol, which is in reasonable accord with experiment (−18.9 kcal/mol) 53 . (As can be inferred from the numerical values presented above, the large standard error for G bind 0</p><p>originates from that for TΔS config ; indeed, the protein configurational entropy is known as the most difficult thermodynamic parameter to estimate).</p><!><p>Water molecules are ubiquitously found at the interfaces between biomolecules, and it is often stated that the interfacial water must be considered as an integral part of biomolecules. The work presented here sheds new light on this statement based on the dynamic viewpoint. We demonstrate the emergence of "special" waters in the interfacial region that bridge two biomolecules through concurrent hydrogen bonds and exhibit extremely slow hydrogen-bond rearrangements. By analyzing the thermodynamic-dynamic relationship diagram, we find that the extremely slow nature of bridging water is due to not only the number of hydrogen bonds involved, but also the additional stabilization resulting from the strong electrostatic field between the binding surfaces of electrostatic complementarity. The role of such slow interfacial waters in determining the binding affinity cannot be described using the conventional approach to the water-mediated interaction, which assumes rapid equilibration of the waters' degrees of freedom. Indeed, we observe that a meaningful estimate of the binding affinity is achieved only with a unified treatment of both the biomolecules and the interfacial bridging water. Our work thus demonstrates the impact of the hydration dynamics on the protein-protein binding thermodynamics.</p>

Scientific Reports - Nature

A proton shelter inspired by the sugar coating of acidophilic archaea

The acidophilic archaeons are a group of single-celled microorganisms that flourish in hot acid springs (usually pH , 3) but maintain their internal pH near neutral. Although there is a lack of direct evidence, the abundance of sugar modifications on the cell surface has been suggested to provide the acidophiles with protection against proton invasion. In this study, a hydroxyl (OH)-rich polymer brush layer was prepared to mimic the OH-rich sugar coating. Using a novel pH-sensitive dithioacetal molecule as a probe, we studied the proton-resisting property and found that a 10-nm-thick polymer layer was able to raise the pH from 1.0 to . 5.0, indicating that the densely packed OH-rich layer is a proton shelter. As strong evidence for the role of sugar coatings as proton barriers, this biomimetic study provides insight into evolutionary biology, and the results also could be expanded for the development of biocompatible anti-acid materials. Several mechanisms have been suggested to act synergistically to maintain the pH homeostasis of acidophilic archaea, including the intracellular positive transmembrane potential that inhibits proton influx and antiporters that pump out excess protons [1][2][3][4] , with the most comprehensive evidence being reported for the extremely low proton permeability of the cell membrane 1,3,5,6 . The plasma membrane of acidophilic archaeons is unique in two aspects (Fig. 1). First, unlike the bilayer structure commonly found in other archaeal, bacterial or eukaryotic cell membranes, it is a monolayer composed of unique ''tetraether lipids'' in which two hydrophilic heads attached to the same hydrophobic tail through ether bonds and is, therefore, physiochemically more stable and less fluid 2,6-10 . Second, in addition to the tetraether core structure, the membrane lipids are also characterized by a substantially high content of glycolipids (as high as . 90% in some species), with one or more sugar units exposed at the outer surface of the cell 8,9 . This structure has been suggested to provide proton resistance because the average number of sugar units attached increases when the environmental pH decreases 11 . However, a deeper understanding of this mechanism had been hindered, mainly due to the lack of proper tools. In the present study, this biological proton shelter was studied within a novel biomimicry context by mimicking the hydroxyl (OH)rich sugar coating with OH-rich polymer brushes. ResultsDesign of the biomimicry regime. Three tools made a detailed study of the above enigma possible: (1) a newly designed acid-probing dithioacetal molecule (Compound 1, Fig. 2a), (2) the surface-initiated polymerization (SIP) to prepare the finely tuned polymer brushes 12 (Fig. 2b) and (3) the quartz crystal microbalance (QCM) that is sensitive to interfacial changes [13][14][15] (Fig. 3a).Compound 1 is a novel pH-sensitive initiator of the SIP that forms self-assembled monolayers 16 (SAMs) on a gold surface that is stable at neutral pH values (down to 5.0), whereas undergoes partial disassembly when exposed to dilute hydrochloric acid (HCl, pH 1.0, Supplementary Fig. S2). The SAM of 1 plays two roles in this study: (1) because of its acid-sensitivity, it was used as a sensing layer that could probe local pH changes, and (2) because of the bromoisobutyryloxy end it contains, it was used as a layer of initiators from which the poly(oligo(ethylene glycol) methacrylate) (hereafter abbreviated as poly(OEGMA)) brushes were grafted via the SIP (Fig. 2b). The monomer OEGMA 526 (M n 5 526 g mol 21 ) was used to prepare the OH-rich brushes to mimic the OH-rich sugar coating on acidophilic archaeons, whereas OEGMA 475 (M n 5 475 g mol 21 ) was used to prepare the OCH 3 -rich brushes as the control.

a_proton_shelter_inspired_by_the_sugar_coating_of_acidophilic_archaea

<!>Discussion

<p>The SAM and polymer brushes were prepared on the gold electrode of a QCM chip for QCM measurements under various pH solutions. QCM is an acoustic-based sensor that detects interfacial mass losses as frequency increases (Df . 0) 15,17 . The polymer-coated chip was treated with HCl (pH 1.0) to mimic the environmental pH of acidophiles (Fig. 2b). If the H 1 from the HCl reached the Au-S bonds, one would observe frequency increases due to the partial disassembly of the SAM and attached polymer chains. If the polymer layer exerted any inhibiting effect on proton permeation, the frequency response would be weakened or silenced.</p><p>Examination of the effect of the OH-rich polymer as a proton shelter. First, the chips grafted with poly(OEGMA 475 ) were challenged with HCl. As is shown by the red line in Fig. 3a, the QCM frequency remained unresponsive to the pH 5.0 HCl but increased significantly when exposed to the pH 1.0 HCl, indicating that the H 1 from HCl penetrated the poly(OEGMA 475 ) layer and reached the Au-S bonds. As a control, poly(OEGMA 475 ) was also grafted from the acid-treated 1 SAMs and thiol initiator (vmercaptoundecyl bromoisobutyrate) SAMs (Supplementary Figs S5 and S6). The resulting chips showed no acid responses, confirming that the mass loss in Fig. 3a (red line) was driven by the acid-sensitivity of the 1 SAMs. The acid-induced partial loss of surface mass was also evidenced by atomic force microscopy (Supplementary Fig. S7).</p><p>The poly(OEGMA 526 )-covered chips were also investigated. As shown in Fig. 3a (the black line), no significant Df was recorded when the pH value dropped from 5.0 to 1.0, indicating that the H 1 from HCl did not fully penetrate the poly(OEGMA 526 ) coating and that the local pH near the Au-S bonds was at least . 5.0. This is strong evidence that the replacement of -OCH 3 groups to -OH groups enabled the polymer coating to act as a proton shelter. Similar results were observed when OEGMA 300 (M n 5 300 g mol 21 , -OCH 3 terminated) and OEGMA 360 (M n 5 360 g mol 21 , -OH terminated) were used as another pair of -OCH 3 /-OH-presenting monomers in the SIP (Supplementary Figs S8 and S9).</p><p>To confirm the inhibition effect of -OH groups on H 1 penetration further, we designed the following series of experiments. First, we copolymerized OEGMA 526 and OEGMA 475 at different feed ratios to tune the relative content between the -OCH 3 and -OH ends (Supplementary Fig. S10). The result demonstrated that the pH response was completely silenced when the proportion of OEGMA 526 in the copolymer was as low as 10%; a significant proton-resistant effect was still observed even when that ratio dropped to 0.1%. Second, when the -OH ends in the 10% OEGMA 526 -containing copolymer were converted to -OCH 3 groups using MeI, the pH-responsiveness was regained, as expected (Supplementary Fig. S11). Cyclic voltammetry (CV) experiments also indicated a higher electronic resistance of poly(OEGMA 526 ) than poly(OEGMA 475 ), consistent with protonblocking property 18 (Supplementary Figs S12-14). All of these results support the notion that the OH-rich polymer brushes could act as a proton shelter.</p><p>The -OH groups must be presented in a brush form to gain the proton-shelter function, i.e., the -OH groups must be spatially confined. When a poly(OEGMA 475 )-grafted chip was treated with a pH 1.0 HCl solution containing 1 mM OEGMA 526 monomer, a Df . 0 was observed (Supplementary Fig. S15), indicating that the free form of the -OH groups in solution did not suppress the pH response. Therefore, the surface-grafted polymer brushes were necessary for the confinement and enrichment of the -OH groups, a mimic to the conditions in glycolipids.</p><p>To assess further the effect in three-dimensional compartments, the proton-shelter was lifted up from the SAM surface by block copolymerization in which a ''proton-conductive'' layer containing exclusively poly(OEGMA 475 ) was first prepared as a spacer to set the distance between the ''proton shelter layer'' and the SAM (Fig. 3b). The chips with this two-layer architecture were subjected to acid treatment, and a prominent proton-resisting function was observed, even when the distance increased to 80 nm (24 nm of dry thickness of the poly(OEGMA 475 ) layer corresponded to 80 nm of wet thickness in HCl), proving the proton resistance effect of the -OH groups in three-dimensional space.</p><!><p>In dramatic contrast to those in bacteria or eukaryotic cells, the majority of the lipids in the cell membrane of acidophilic archaeons are glycolipids or glycophospholipids that contain at least one sugar residue on the extracellular side (Fig. 1b). When the environmental pH decreased from 3.0 to 1.2, the content of glycolipids in the plasma membrane of Thermoplasma acidophilum increased and the percentage of the lipids that contained 2-4 sugar units rose from 14% to 35%, suggesting an adaptation to low pH values by extending the sugar chains on the cell surface 11 . As a systematic investigation of the effect of -OH groups on proton resistance, our study presents strong evidence for that adaptation mechanism and pinpoints the effective site as -OH groups. The layer remained resistant to proton permeation after 1,000-fold dilution of the -OH group concentration, suggesting that there a redundancy still exists with regard to the amount of -OH groups in our experiments. Thus it is reasonable to suppose that a layer of a few sugar units (1-2 nm in thickness) is capable of exerting moderate effects on acid defense (Fig. 1c).</p><p>The concentration of -OH groups inside the poly(OEGMA 526 ) brushes was estimated to be 0.5 M (see Supplementary Information for the detailed calculation), which is on the similar order of magnitude with that on the cell surfaces of acidophilic archaeons, thus justifying our simulation 19 .</p><p>Our study proves the strong proton-resistance of surface confined -OH groups and also paves the way for the investigation of the underlying mechanism. The proton-resistance properties of other groups, such as -NH 2 and epoxy groups, could be studied by changing the monomers, the direction of our ongoing experiments. The results, combined with a theoretical analysis, are expected to reveal the mechanisms.</p><p>The bioinspired poly(OEGMA 526 ) membrane possesses a strong proton-sheltering effect that has been quantitatively defined as raising the pH from 1.0 to . 5.0. Therefore, our biomimetic study may be expanded for the development of biocompatible anti-acid strategies, such as tooth protection and drug-carriers that withstand gastric acid digestion 20 . Our conclusion also provides information for the design of novel paints that offer protection to vehicles, buildings and infrastructures against acid rain corrosion, an area for which realistic solutions are still lacking. Furthermore, as the first organosulfur compound that has been found to form an acid-sensitive Au-S bond, compound 1 could be used to study the nature of Au-S bonds. The dithioacetal initiator (1) was synthesized using the following procedure:</p><p>(1) Synthesis of 4-formylphenyl -2-bromo-2-methylpropanoate 21 : Hydroxybenzaldehyde (0.61 g, 5 mmol), triethylamine (0.75 mL, 1.033 mmol), and dry dichloromethane (25 mL) were added to a 50-mL round-bottom flask with a stir bar. The mixture was cooled to 0uC, followed by the dropwise addition of ice-cold bromoisobutyryl bromide (0.67 mL, 5.39 mmol). After stirring at 0uC for 1 h, the reaction was continued for another 12-16 h at room temperature. Water (20 mL) and dichloromethane (10 mL) were added to the mixture for a two-phase extraction. The aqueous phase was further extracted with dichloromethane (2 3 30 mL), and the organic phase was concentrated by rotary evaporation to remove the dichloromethane. The resulting crude extract was dissolved in dichloromethane (40 mL), washed with a saturated sodium bicarbonate solution (3 3 40 mL), and dried over MgSO 4 . The removal of the dichloromethane resulted in a yellowish oil, which was passed through a column (silica gel, neutral, with petroleum ether:ethyl acetate 5 20:1) and then vacuum dried overnight. The final product (4-formylphenyl -2bromo-2-methylpropanoate) was a white solid, obtained in high purity and with a high yield (0.2168 g, 80% yield). 1 (2) Synthesis of dithioacetal initiator 22 : Mercaptoundecanol (0.48 g, 2 mmol): 4formylphenyl -2-bromo-2-methylpropanoate (0.271 g, 1 mmol) and toluene (10 mL) were added to a 50 mL round-bottom flask with a stir bar. The mixture was refluxed to 110uC, followed by the addition of p-toluenesulfonic acid (0.009 mL, 0.05 mmol). After stirring at 110uC for 3 h, the reaction was stopped by the addition of triethylamine (3 mL). The mixture was concentrated by rotary evaporation to remove the toluene. The removal of the toluene resulted in an orange oil, which was passed through a column (silica gel, neutral, with petroleum ether: ethyl acetate5 2:1 with 2% triethylamine as eluent) and then vacuum dried overnight. The final product was a white solid, obtained in high purity and with a high yield (1.4040 g, 93.1% yield). 1 H NMR (400 MHz, CDCl 3 ): d7.493 ppm (d, J 5 6.4, 2H), 7.111 (d, J 5 8.8, 2H), 4.886 (s, 6H), 3.642 (t, J 5 13.2, 4H), 2.542 (m, J 5 43.6, 4H), 2.073 (s, 6H), 1.554 (m, J 5 27.6, 10H), 1.322 (m, J 5 39.6, 26H). 13 SAMs. The QCM chips were incubated in 1 mM ethanol solution of 1 for 18 hours. The reaction was executed at ambient temperature, under nitrogen atmosphere protection and protected from light.</p><p>SIP. The SIP of OEGMA from the SAMs of 1 as the initiators was performed as reported previously 15 using bipyridine as a ligand and water: ethanol 5 1: 1 as a solvent, with a molar ratio of OEGMA/CuBr 2 /bipyridine/CuBr/ 5 400/1/30/10 (i.e., 10/0.025/0.75/0.25 mM). The reaction was terminated by MilliQ-water (pH 6.2), and the chips were then rinsed thoroughly with MilliQ-water and ethanol to remove any salt particles and dried under a nitrogen flow. The dry thicknesses of the surfacetethered polymer brushes were determined by Ellipsometry.</p><p>QCM measurements. All of the QCM measurements were conducted with a relative humidity controlled below 25%. The operation temperature was set at 25uC. The chips (AT cut, 5 MHz; HZDW, Hangzhou, China) were placed in a home-built QCM with control software purchased from Resonant Probes GmbH (Goslar, Germany). For the liquid-phase measurements, the QCM was operated in a flow-through mode at a speed of 80 mL min 21 . Milli-Q water (pH 5 6.2) with a resistivity of 18.2 MV cm 21 was used for the solution preparation.</p><p>AFM. The AFM images of the samples in their dry state were taken in the tapping mode (Digital Instruments, Santa Barbara). The polymer-coated QCM chips were imaged before and after washing with a dilute acid solution.</p><p>Ellipsometry. The dry film thickness was measured using an M-2000V spectroscopic ellipsometer (J. A. Woollam Co., Inc.) at angles of 65u, 70uand 75uand wavelengths from 400 nm to 800 nm. The ellipsometric data were fitted for thickness using materialspecific models (Cauchy layer model) from a vendor-supplied software, with fixed (A n , B n ) values (1.46, 0.01). Each datum was an average of three measurements.</p>

Scientific Reports - Nature

LIPID PHOSPHATE PHOSPHOHYDROLASE TYPE 1 (LPP1) DEGRADES EXTRACELLULAR LYSOPHOSPHATIDIC ACID IN VIVO

Lysophosphatidic acid (LPA) is a lipid mediator that stimulates cell proliferation and growth and is involved in physiological and pathological processes such as wound healing, platelet activation, angiogenesis and the growth of tumors. Therefore, defining the mechanisms of LPA production and degradation are of interest in understanding the regulation of these processes. Extracellular LPA synthesis is relatively well understood whereas the mechanisms of its degradation are not. One route of LPA degradation is de-phosphorylation. A candidate enzyme is the integral membrane exophosphatase lipid phosphate phosphohydrolase type 1 (LPP1). We report here the development of a mouse wherein the LPP1 gene (Ppap2a) was disrupted. The homozygous mice, which are phenotypically unremarkable, generally lack LPP1 mRNA and multiple tissues exhibit a substantial (35\xe2\x80\x9395%) reduction in LPA phosphatase activity. Compared to wild type littermates, Ppap2atr/tr animals have increased levels of plasma LPA and LPA injected intravenously is metabolized at a four-fold slower rate. Our results demonstrate that LPA is rapidly metabolized in the bloodstream and that LPP1 is an important determinant of this turnover. These results indicate that LPP1 is a catabolic enzyme for LPA in vivo.

lipid_phosphate_phosphohydrolase_type_1_(lpp1)_degrades_extracellular_lysophosphatidic_acid_in_vivo

INTRODUCTION<!>Generation of LPP1 null mice<!>Genotyping of mice<!>Real-Time Reverse Transcriptase-Polymerase Chain Reaction (Real-Time RT-PCR)<!>[32P]LPA and [32P]PA synthesis<!>Measurement of lipid phosphate phosphohydrolase activity in tissue homogenates<!>Measurement the lipid phosphate phosphohydrolase activity of tissue slices and splenocytes<!>Measurement of plasma LPA concentration<!>Preparation of plasma for clearance studies<!>Measurement of LPA clearance in vitro<!>Measurement of LPA clearance in vivo<!>Measurement of LysoPLD activity<!>Animal Care<!>Generation of Ppap2a+/+ Ppap2a+/tr and Ppap2atr/tr mice<!>LPP1 mRNA expression<!>LPA phosphatase activity<!>LPA exophosphatase activity<!>Plasma LPA levels<!>Degradation of LPA in blood<!>DISCUSSION<!>Structure and characterization of the normal and trapped Ppap2a mouse genes<!>LPP1 and LPP3 mRNA expression in selected organs of Ppap2atr/tr and Ppap2a+/+ mice<!>Lipid phosphate phosphohydrolase activity of selected organs of Ppap2atr/tr and Ppap2a+/+ mice<!>Lipid phosphate phosphohydrolase activity of intact cells and tissues from Ppap2atr/tr and Ppap2a+/+ mice<!>Plasma levels of LPA in Ppap2atr/tr and Ppap2a+/+ mice<!>Ex vivo and in vivo clearance of LPA in Ppap2atr/tr and Ppap2a+/+ mice

<p>Lysophosphatidic acid (LPA)1 (1-acyl-2-lyso-sn-glycero-3-phosphate) has long been known as an intermediate in de novo biosynthesis of all glycerophospholipids. LPA gained credence as a signaling molecule with the discovery in the early 1990's by Moolenaar and collaborators that LPA promotes cell proliferation (see [1] for a review). LPA and other biologically active lipid phosphates, notably sphingosine 1-phosphate (S1P), have since been studied extensively and found to have a role as mediators in a variety of physiologic and pathologic processes. The stimulatory effect of LPA on cell proliferation, survival and migration serves as the underlying mechanism of the major biological effects of LPA. The most conspicuous examples are the involvement of LPA in wound healing, platelet activation, vascular remodeling and the progression of some forms of cancer such as ovarian tumors (see [2] for a review).</p><p>The role of LPA and other lipid phosphates as first messengers was firmly established by the realization that many of the biological effects of these molecules are mediated by their interaction with specific, seven-transmembrane domain G protein-coupled receptors (see [3] for a review). As with any bona fide signaling molecule, specific mechanisms are expected to exist that ensure its timely destruction to prevent the detrimental effects of overstimulation. LPA catabolism is presumed to be mediated by a group of phosphatases termed lipid phosphate phosphohydrolases (LPPs) rather than phosphatidate phosphatase-1 (lipins) that are specific for phosphatidate [4,5].</p><p>LPPs (formerly known as phosphatidic acid phosphatases (PAPs) (see [6,7] for reviews) are enzymes that catalyze the hydrolysis of a variety of lipid phosphate mono-esters. The LPPs are Mg2+-independent, NEM-insensitive (LPP1, LPP2 and LPP3 formerly known PAP2A, PAP2B and PAP2C respectively; see [8] for a review). LPP1-3 are cell surface, N-glycosylated, integral membrane proteins. LPP3 in some cell types also localizes to intracellular membranes [9]. The topology of membrane LPPs is such that the active site amino acids are exofacial, thus these enzymes function as exophosphatases [10].</p><p>The hypothesis that one or more LPP isotypes may function to delimit LPA signaling has been difficult to test. On the one hand, LPP1 and LPP3 are nearly ubiquitous among mammalian cells (see [11] for a review) and forced expression of these proteins is either toxic to cells or provide information restricted to cells in culture under somewhat artificial conditions [12,13]. On the other hand, the difficulties inherent in the study of integral membrane proteins – a lack of quality antibodies and purified protein – have slowed progress in understanding these enzymes. A chemical biology approach has not been particularly informative in that selective inhibitors have yet to be developed and structure activity relationship of substrates is rather uninformative. That is, the LPP isotypes act on a wide variety of lipid mono-phosphates including LPA, PA, diacylglycerolpyrophosphate, S1P and ceramide 1-phosphate. Using in vitro assays, LPP1 exhibits a preference for glycerol-versus sphingoid base-containing lipids, while LPP3 does not differentiate as well among these substrates (see [10] for a review). These ecto-activities of LPP1 have two potentially important functions. First, they could regulate circulating concentrations of LPA or S1P and thus the activation of their respective receptors. Decreased LPP activities occur in tumors and this has been proposed as a mechanism that results in increased LPA-induced growth in ovarian cancers [14]. Secondly, the dephosphorylated product formed by the LPP is taken up by the cell and thereby it stimulates cell signaling itself or after re-phosphorylation. [5,6,15,16].</p><p>An alternative approach – genetic manipulation – has yielded several studies reporting the consequences of altering the expression of LPP genes. Disruption of the mouse gene encoding LPP3 (Ppap2b) is embryonic lethal (E9.5), apparently as a consequence of aberrant embryonic and extra-embryonic development of the vasculature [17]. By contrast, mice lacking LPP2 (Ppap2c −/−) are viable, fertile and not noticeably different from wild type littermates [18]. Ablation of the Drosophila homologs of the mammalian LPPs (wun and wun2) results in a defect in germ cell migration, suggesting that a spatial pattern of phospholipid hydrolysis is required for the migration and survival of germ cells during fly development [19]. Global forced expression of LPP1 (Ppap2a) in mice resulted in runted animals with fur abnormalities and impaired spermatogenesis, a phenotype that is not readily ascribed to increased extracellular LPA or PA degradation. Interestingly, theses LPP1 transgenic mice had normal levels of plasma LPA [20].</p><p>In this communication we report the generation and characterization of a mouse strain wherein the gene encoding LPP1 (Ppap2a) is disrupted by insertion of an exon-trapping element. Mice with one (Ppap2a+/tr) or both (Ppap2atr/tr) alleles disrupted are viable and fertile. We used these mice as an in vivo model to establish whether LPP1 plays a physiological role in controlling the degradation of circulating LPA. Our results demonstrate that LPP1 performs this function in vivo.</p><!><p>An embryonic stem (ES) cell line (RRD231) reported to harbor a gene trap vector (pGT1Lxf) in the first intron of the Ppap2a gene were obtained from BayGenomics via the Mutant Mouse Regional Resource Center (MMRRC) at Davis, CA. We verified the presence of the trapping element and further established its precise location by sequencing genomic DNA from Ppap2atr/tr mice (see Fig. 1). Chimeric founder mice were generated at the University of Virginia Transgenic Mouse facility using C57BL/6 blastocysts. The ES cells were sv129 strain, animals used in this study were of mixed genetic background (F1N1, F1N5).</p><!><p>Mice were genotyped by PCR using genomic DNA from tail biopsies, liver or brain samples and the following primers (see Fig. 1): 5′-GAGAGTGAGCGAGTGTCTGAGTTTCTGATG-3′ (forward), 5′-AGTACTGGGCATCTCACACCACAT-3′ (reverse wild type allele), 5′-CCTTCAAAGGGAAGGGGTAAAGTGGTAGGG-3′ (reverse trapped allele). Amplification in presence of 20 ng DNA and 4 mM MgCl2 was carried out as follows: at 94 °C for 3 min, followed by 35 cycles at 94 °C (30 sec), 57 °C (1 min), 72 °C (1.5 min); and a final 72 °C step for 10 min.</p><!><p>RNA was extracted from tissues and cDNA was obtained using the SuperScript First-Strand Synthesis System (Invitrogen) using random hexamers according to the manufacturer's instructions. Amplification was performed in a iCycler iQ System (Bio-Rad, Hercules, CA) at 95 °C for 4 min, followed by 40 cycles at 95 °C, 55 °C, and 72 °C (30 s each step) in 50 μl of a SYBR Green-based medium (iQ SYBR Green Supermix; Bio-Rad) using the following forward and reverse primers: 5′-TGTACTGCATGCTGTTTGTCGCAC-3′, 5′-TGACGTCACTCCAGTGGTGTTTGT-3′ for LPP1; and 5′-ATAAACGATGCTGTGCTCTGTGCG-3′, 5-TTTGCTGTCTTCTCCTCTGCACCT-3′ for LPP3. The levels of mRNA expression were normalized to the expression level of the 18s ribosomal RNA gene measured using a commercial primer kit (QuantumRNA, Classic II; Ambion, Austin, TX). Quantitative gene expression was obtained from cycle differences at appropriate thresholds and the efficiency of amplification as described by Pfaffl [21]. The size and singularity of the RT-PCR products was established by agarose gel electrophoresis and melting point analysis. For the detection of the different LPP1 isoforms, a similar protocol was followed but using the following forward and reverse primers: 5′-ATCCATTTCAGAGGGGCTTT-3′, 5′-AACCTGCCCTCCTTGACTTT-3′ for isoform 1; and 5′-TTCAAGGCATACCCCCTTC-3′, 5′-GGTGGCTATGTAGGGATTGC-3′ for isoform 2. To amplify the cDNA region containing the trap insertion point in intron 2, a similar protocol was used except for annealing at 53 °C and the use of the following primer 5′-TCTGTTCCTCCCGCCACT-3′ in conjunction with the reverse primer used for LPP1 isoform 1.</p><!><p>[32P]-labeled LPA and PA were synthesized as previously described [22] using E. coli diacylglycerol kinase (Sigma D3065), ATP (Sigma A2383), γ-[32P]ATP (MP Biochemicals 35001; 7,000 Ci/mmol), cardiolipin (Sigma C0563), and 1-monooleoyl-glycerol (Sigma M-7765) or 1,2-dioleoyl-glycerol (Avanti Polar Lipids 800811C). Final purification by thin layer chromatography (TLC) was carried out on Whatman Silica Gel plates (4865-621) using a mobile phase consisting of 1-butanol:acetic acid:H2O 3:1:1. Small aliquots of the labeled compounds dissolved in chloroform were stored in light-protected, chloroform-rinsed glass vials at −20 °C for no more than four days. No radiolysis was observed under these conditions as judged by TLC analysis.</p><!><p>Mice were anesthetized with isofluorane and sacrificed by cervical dislocation. Organs were immediately obtained, frozen in liquid nitrogen and stored at −80 °C. Brain samples consisted of tissue from the cerebral hemispheres. Skeletal muscle samples were obtained from the hind legs. Frozen tissue samples were homogenized using a motor driven Teflon pestle homogenizer in 10 volumes of 20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM PMSF, 1mM DTT, 10 μg/ml aprotinin (Sigma A6279), 10 μg/ml leupeptin (Sigma L2023). Homogenates were centrifuged at 500 × g and the supernatant fluids were aliquoted and stored at −80 °C (pellets were discarded). The protein content of these preparations was measured according to Bradford [23] using a commercial Coomassie blue solution (Bio-Rad). LPP activity was measured as the release of [32P]H3PO4 from [32P]-labeled substrates presented as mixed Triton X-100 micelles as previously described [24] with some modifications. Assays were carried out in 200 μl of a buffer consisting of 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM DTT, 3.2 mM Triton X-100, 100 μM LPA or PA and 50,000 dpm [32P]LPA or [32P]PA. Assays were carried out at 37 °C in presence of 100 μg protein for different times, 5 min to 30 min, according to the specific LPP activity of each tissue so that degradation did not exceed 5% of the total substrate. Different incubation times were preferred over incubation with different amounts of protein to preserve the ratio Triton X-100/protein content. Reactions were started by mixing 100 μl of mixed micelles with 100 μl of a solution containing the remaining components. Activity was constant with time for every tissue. No LPP activity was observed in absence of tissue. Reaction was stopped by adding 200 μl of 1 M HClO4 containing 100 μM phosphoric acid. Samples were then centrifuged for 5 min at 15,000 × g and the supernatant extracted twice with H2O-saturated 1-butanol. Orthophosphate was recovered from the extracted aqueous phase as described [25] by precipitation with 12.5 mM ammonium molybdate and extraction with isobutanol:benzene 1:1. Radionuclide in the organic phase was measured by liquid scintillation spectrometry. Specific activity of the substrates was determined by measuring the amount of 32P present in known volumes of reaction media. To measure the NEM-sensitive, Mg2+-independent activity, tissue samples containing 100 μg of protein were brought to 80 μl and supplemented with 10 μl of 50 mM NEM. After a 15 min incubation, NEM was neutralized by adding 10 μl of 22 mM DTT (1mM final DTT). This solution was then added to 100 μl of mixed micelles and processed as described above except for the omission of MgCl2 and the addition of 1 mM EGTA and 2 mM EDTA [26].</p><!><p>Ppap2a+/+ and Ppap2atr/tr mice were anesthetized with isofluorane and sacrificed by cervical dislocation. The brain hemispheres, kidney, liver and spleen were removed, placed in ice cold Buffer A (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM glucose, 20 mM Hepes-NaOH, pH 7.4) and sliced into 300 μm thick slices using a Vibratome type slicer (Dosaka DTK 1500E microslicer). Slices were trimmed to approximately 3 mm × 3 mm squares and incubated in 200 μl of Buffer A containing 0.1% Fatty Acid Free bovine serum albumin (FAF-BSA, Sigma A6003), 100 μM LPA and approximately 2 × 105 cpm [32P]LPA for variable times at 37 °C. At the end of the incubation periods, 100 μl aliquots were withdrawn and the presence of [32P]H3PO4 was assayed as described for tissue homogenates. The remaining 100 μl were discarded and the tissue dissolved in 2% SDS and the protein content measured by the bicinchonic acid method using a commercial kit (Pierce 23223). Activity was linear with time. Linearity with protein concentration was tested only for slices of the same thickness and roughly the same size and shape. In particular, we carried out incubations with half, one or two 3 mm × 3 mm slices and found that activity and protein were linearly correlated. For slices of different thickness, activity and protein were not linearly correlated, therefore and to ensure thickness homogeneity similar organs from Ppap2a+/+ and Ppap2atr/tr were mounted together and sliced simultaneously. Splenocytes were obtained by passing the spleens through a nylon cell strainer (100 μm mesh, Falcon 352360) and discarding the capsule. Cells were resuspended in 1 ml of Buffer A and centrifuged at 300 × g for 5 min. The pellet was resuspended in 1 ml of erythrocyte lysis buffer (8.29 g/l NH4Cl, 1 g/l KHCO3, 0.0372 g/l Na2EDTA, pH 7.3) and incubated on ice for 5 min. Lysis was quenched by adding 9 ml of Buffer A and washing the cell preparation twice (at 300 × g for 5 min each). Finally cells were resuspended cells in 1 ml Buffer A and counted in a hemocytometer. Viability, assessed by Trypan Blue exclusion, was > 99%. LPP activity was measured as described above and started by resuspending pellets (300 × g for 5 min) containing 5 × 105 lysed or non-lysed cells (approximately 100 and 20 μg of protein respectively) in 200 μl of reaction medium. In both cases, slices and splenocytes, incubation times were identical for similar tissues from Ppap2a+/+ and Ppap2atr/tr mice and they were adjusted for substrate degradation not to exceed 5%. No release of [32P]H3PO4 was observed in absence of slices or cells.</p><!><p>Plasma was obtained by cardiac puncture using EDTA (~ 5 mM) as anticoagulant. LPA levels were determined by liquid chromatography-tandem mass spectrometry using a hybrid ABI-4000 triple quadrupoleion trap mass spectrometer (Applied Biosystems, Foster City, CA) coupled with an Agilent 1100 liquid chromatography column and C17:0 LPA as an internal standard [27]. LPA species were separated on a Zorbax Eclipse XDB-C8 HPLC column (4.6 × 150 mm, 5 μm) using methanol/water/HCOOH, 79:20:0.5 v/v as solvent A and 99:0.5:0.5 v/v as solvent B, containing in both cases 5 mM NH4COOH. Elution was carried out in solvent A for 1 min. A gradient change to solvent B lasting 1 min was then effected and a final, isocratic elution of LPA was carried out in solvent B for 7 min. LPA species were analyzed in negative ionization mode with declustering potential and collision energy optimized for 17:0, 18:0, 18:1, 18:2 and 20:4 LPA. Multiple reaction monitoring parameters for nine other LPA molecular species were selected with the closest possible approximation to the available LPA standards. The following transitions, indicated as the m/z values and, in parentheses, chain length: number of double bonds, were monitored: 407.0/153.0 (16:1); 409.0/153.1 (16:0); 423.0/153.1 (17:0, an unnatural species used as an internal standard); 431/153.0 (18:3); 433.0/153.0 (18:2); 435.1/152.9 (18:1); 437.0/153.0 (18:0); 455.1/153.0 (20:5); 457.0/153.0 (20:4); 459.1/153.0 (20:3); 461.1/153.0 (20:2); 481.1/153.0 (22:6); 483.1/153.0 (22:5); and 485.1/153.0 (22:4).</p><!><p>A series of Ppap2a+/+ and Ppap2atr/tr mice were anesthetized and exsanguinated by cardiac puncture using EDTA (~5 mM) as anticoagulant. Plasma was prepared as previously described [28] by centrifuging the blood twice, at 1,000 and 10,000 × g for 3 min each time at 4 °C, and stored at −80 °C. Aliquots of these plasma preparations were used as a vehicle for [32P]LPA to study LPA clearance as described below. Plasma used in clearance experiments was in each case of the same genotype as the animals whose clearance was being investigated.</p><!><p>Blood samples from a series of Ppap2a+/+ and Ppap2atr/tr mice were obtained and anticoagulated with EDTA (~ 5 mM). Half of these samples were used as whole blood and the other half used as plasma (obtained as described above). Blood and plasma samples were supplemented as soon as they were obtained with [32P]LPA dissolved in either plasma (see above) or saline (0.9 % NaCl) containing 0.1% FAF-BSA. Incorporation of [32P]LPA into plasma or saline solution was carried out by first drying in a glass vial an appropriate amount of [32P]LPA dissolved in chloroform and then dissolving the dried [32P]LPA into the appropriate vehicles (plasma or saline). Two hundred μl blood samples and 100 μl plasma samples were supplemented with plasma amounting to 5% of their volume (10 and 5 μl respectively) and containing approximately 105 cpm [32P]LPA. Immediately after the addition of [32P]LPA ("time zero" sample) or after 5, 15 and 30 min incubation at 37 °C, [32P]LPA was measured as follows: plasma samples and plasma obtained from blood samples as described above were immediately acidified with 3.33 volumes of ice-cold 30 mM citric acid, 40 mM Na2HPO4, pH 4.0, and extracted twice with 8.88 volumes of H2O-saturated butanol. Butanol extractions were then combined, dried under a stream of N2, re-dissolved in chloroform and subjected to TLC as described above. Radioautographic analysis of the developed plates showed only one band that comigrates with authentic LPA (Rf = 0.5). LPA was then measured by scrapping off the LPA bands from the developed TLC plates and measuring their 32P content by liquid scintillation spectrometry. The "time zero" plasma samples were also used to assess the quantitative recovery of LPA from plasma by comparing the amount of [32P]LPA added to the plasma samples with the amount of [32P]LPA recovered from TLC plates. Similar to the previously reported 99% recovery figure for this technique [29], our recovery was not significantly different from 100%.</p><!><p>Mice (24–27 g) were anesthetized with methoxyfluorane and injected through the tail vein with approximately 108 cpm [32P]LPA (6.5 × 10−12 mole) dissolved in 150 μl of either plasma (see above) or sterile saline (0.9 % NaCl) containing 0.1% FAF-BSA. Immediately after injection, a blood sample (3 to 6 drops or approximately 50–100 μl) was taken by retro orbital bleeding using a Micro-Hematocrit capillary tube and EDTA (~5 mM) as anticoagulant. This first sample was obtained approximately 2–3 min after injection and it was considered to be the "time zero" sample. Additional samples were taken at 5 min, 15 min and 30 min. Immediately after they were obtained, blood samples were processed as described above to measure plasma [32P]LPA. Similar to the in vivo experiments, no band other than LPA was observed by radioautography.</p><!><p>Plasma LysoPLD activity was measured according to Umezu-Goto et al. [30] by mixing 10 μl of plasma with 90 μl of a buffer consisting of 100 mM Tris-HCl, pH 9.0, 500 mM NaCl, 5 mM MgCl2, 30 μM CoCl2, 0.05% Triton X-100 and 1.1 mM LPC (Avanti Polar 148870). After 4 h incubation at 37 ° C, choline in samples was measured colorimetrically at 555 nm by adding 100 μl of 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 50 U/ml horseradish peroxidase, 18 U/ml choline oxidase, 5 mM 4-aminoantipyrine, and 3 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine. Net production of choline was calculated by subtracting the amount of choline present in parallel samples that were kept frozen. Blood was obtained by retro-orbital bleeding using heparin as anticoagulant. Choline chloride (Fluka 26978) was used as standard.</p><!><p>The University of Virginia's Animal Care and Use Committee approved all experiments with animals.</p><!><p>We first established the exact location of the exon trap element by sequencing genomic DNA from Ppap2atr/tr mice. As shown in Fig. 1A, insertion occurred in intron 1 as reported by BayGenomics. Mouse LPP1 is expressed as two different isoforms as a result of alternative splicing of exon 2 [31]. However, in the present instance, both variants were expected to be trapped because insertion occurred upstream of either exon 2 (Fig. 1A). Based on the location of the trap element we designed primers to detect by PCR the normal and trapped versions of the gene and were able to identify wild type, heterozygous and homozygous animals or Ppap2a+/+, Ppap2a+/tr and Ppap2atr/tr animals (Fig. 1B).</p><p>Ppap2atr/tr and Ppap2a+/tr mice are phenotypically unremarkable, that is their anatomy, behavior, and fertility are not readily distinguished from those of wild type littermates. We examined the gross anatomy of all major organs of these animals and found no differences with wild type animals. Our examination included the male reproductive tract and the prostate in particular because LPP1 mRNA is particularly prominent in prostate and LPP1 is an androgen-induced gene [32].</p><!><p>We investigated next the levels of LPP1 mRNA expression in different organs of Ppap2atr/tr and Ppap2a+/+ mice by quantitative Real-Time RT-PCR. In these experiments, primers were anchored to exons 3 and 4 (forward primer) and 5 (reverse primer) and therefore results correspond to the combined expression of both LPP1 isoforms which, as depicted in Fig. 1A, are generated by alternative splicing of exon 2. As presented in Fig. 2, Ppap2atr/tr mice exhibited much reduced levels of LPP1 expression in relation to Ppap2a+/+ littermates in all organs studied except the brain. LPP1 mRNA expression in Ppap2atr/tr mice ranged from 1/300 (kidney) to 1/800 (muscle) the levels found in Ppap2a+/+ mice. The unaltered expression of LPP1 mRNA in the brain prompted us to investigate whether these animals lack the trapping element in this organ, but we found no evidence for a wild type allele in Ppap2atr/tr mice in brain genomic DNA (not shown). Further, we found no difference in the expression of LPP1 mRNA isoforms in comparing brains of Ppap2atr/tr and Ppap2a+/+ mice (not shown). Finally, we examined the expression of LPP3 in the same organs and found that, unlike LPP1, levels of LPP3 expression in Ppap2atr/tr and Ppap2a+/+ mice were similar (Fig. 2).</p><!><p>Tissue homogenates from Ppap2atr/tr mice exhibited reduced phosphatase activity in all organs studied except the brain, although that the extent of reduction varied among tissues (Fig. 3A). Using [32P]LPA as a substrate, we observed values ranging from 43% in heart (i.e. activity in Ppap2atr/tr mice was 57% that of Ppap2a+/+ mice) to 91% in the spleen. With [32P]PA as a substrate, values ranged from 31% in the kidney to 88% in the spleen. We found major differences in LPP activity between the brain and the other organs studied. As predicted from LPP1 mRNA quantification, LPA phosphohydrolase activity was not diminished in brain tissue from Ppap2atr/tr mice. We also found that the lipid phosphohydrolase activity from both Ppap2atr/tr and Ppap2a+/+ mice was higher with PA (vs. LPA) as a substrate in brain homogenates as compared to other tissues. In toto, these results lead us to conclude that Ppap2atr/tr mice are not entirely null for LPP1, rather these animals are LPP1 hypomorphs that exhibit severely reduced expression of LPP1 mRNA and reduced LPP activity in all organs studied except the brain.</p><p>As mentioned in the Introduction, LPPs such as LPP1 are NEM-insensitive, Mg2+-independent and act as exophosphatases, i.e. degrade lipids presented to the extracellular face of the cell membrane. A second group of lipid phosphohydrolases have opposite characteristics: they are sensitive to NEM, require Mg2+ and are located inside the cells. We carried out a series of experiments to further map to LPP1 the reduction of lipid phosphohydrolase activity observed in Ppap2atr/tr mice. To this end, we investigated first the effects of NEM alkylation and lack of Mg2+ on our assay of tissue phosphohydrolase activity (Fig. 3A), and, second, the exophosphatase activity of intact tissues (Fig. 4). As shown in Fig. 3A, only a small fraction of the total activity was NEM-sensitive. We compared the NEM-sensitive, Mg2+-dependent fractions from similar organs of Ppap2atr/tr and Ppap2a+/+ mice and found that in some cases Ppap2atr/tr mice show an increased amount of NEM-sensitive, Mg2+-dependent activity. This phenomenon is illustrated in Fig. 3B, where the Ppap2atr/tr/Ppap2a+/+ ratio of NEM-sensitive, Mg2+-dependent activities is shown for each organ. In the cases of the kidney, liver and muscle, ratios were much higher than unity: 7.6, 5.9 and 15 respectively. For the other organs studied, values ranged from 0.83 to 1.1.</p><!><p>The topology of LPP1 is such that the active site is at the cell surface, thus we measured exophosphatase activity (Fig. 4). We prepared tissue slices from different organs, incubated them with [32P]LPA and measured the release of [32P]H3PO4. Similar to our previous findings with tissue homogenates, Ppap2atr/tr mice organs, with the exception of the brain, showed a decreased level of exophosphatase activity and, once again, the spleen was the most severely affected organ. Values were 65, 74 and 94 % reduction for the kidney, liver and spleen (Figs. 2 and 3). We examined in more detail the exophosphatase activity of the spleen. Splenocytes isolated from Ppap2atr/tr mice exhibit, as expected, a greatly reduced exophosphatase activity (> 99%)</p><!><p>If LPP1 metabolizes LPA in vivo, the reduced levels of LPA phosphohydrolase activity in Ppap2atr/tr mice should result in elevated levels of plasma LPA. We tested this prediction by measuring plasma LPA levels in Ppap2atr/tr and Ppap2a+/+ mice by LC-MS-MS. We found that Ppap2atr/tr mice have higher levels of plasma LPA on average, but there was substantial variation in LPA levels among animals (Fig 5). To determine the source of this variation, we measured plasma LPA levels in a homogeneous population of pure C57BL/6j mice. We found that LPA levels in age- and gender-matched C57BL/6j mice were in agreement with previously reported values (see Discussion) and exhibited low variability (Fig. 5). This result suggests that the observed difference in plasma LPA levels between Ppap2atr/tr and Ppap2a+/+ mice is genuine and the wide range of LPA plasma values is a reflection of a diverse (genetically as well as age and gender) population. To test this idea, we backcrossed the mutant Ppap2a mice against C57BL/6j mice for four additional generations. After ascertaining that mutant and wild type the F1N5 mice retained the differences in LPP1 mRNA and activity delineated above, we measured plasma LPA levels. Although the F1N5 mice had much less variation in plasma LPA levels, the difference between Ppap2atr/tr mice and their wild type littermates remained (Fig. 5). Interestingly, the mole fractions of the LPA species measured was not different in Ppap2atr/tr and Ppap2a+/+ mice (not shown). The rank order of abundance was as follows: 18:2 > 20:4 > 22:6 > 18:0 ~ 16:0 > 18:1 > 20:3 ~ 22:5 > 16:1 ~ 22:4 ≫14:0 ~ 20:5 ~ 20:2 ≫ 22:3 ~ 22:2. This result suggests that LPP1 is not selective for any of these molecular species.</p><!><p>Next we examined the ability of Ppap2atr/tr and Ppap2a+/+ mice to metabolize LPA by measuring the rate of disappearance of [32P]LPA in blood both ex vivo and in vivo. Fig. 6A documents that the amount of LPA that can be recovered from whole blood incubated at 37°C rapidly diminished as a function of time. The rate of decay was slower for blood obtained from Ppap2atr/tr mice. Half-lives were approximately 5 and 20 min for Ppap2a+/+ and Ppap2atr/tr mice, respectively. Similar experiments measuring the decay in plasma samples revealed that [32P]LPA was stable in plasma up to 30 min (not shown). Fig. 6B shows a similar series of experiments except that in this case [32P]LPA was injected into the bloodstream of mice. In this case decay was faster and, similar to the ex vivo observations, disappearance of [32P]LPA was markedly slower in Ppap2atr/tr mice. Half lives in this case were approximately 3 and 12 min. Fig. 6 also documents that there were no differences in [32P]LPA decay when a less physiologic alternative — saline solution containing FA-free BSA — was used as a vehicle for [32P]LPA instead of plasma. Finally, we explored whether Ppap2atr/tr animals show any abnormality in the production of LPA, likely a compensatory reduction as a consequence of the elevated plasma LPA levels and the reduced rate of LPA degradation. We found no differences between Ppap2a+/+ and Ppap2atr/tr mice when we used a colorimetric method to detect choline produced by the breakdown of LPC into choline and LPA catalyzed by plasma lysophospholipase D. Values were (average ± S.D. of 5 animals): 7.53 ± 0.21 and 7.73 ± 0.23 × 10−13 moles choline/μl plasma/min.</p><!><p>In vitro studies have implicated LPP1 in the phosphohydrolysis of extracellular LPA, but the lack of specific inhibitors has made testing the physiologic relevance of LPP1 in this process difficult. With this paper we document that disruption of the LPP1 encoding gene, Ppap2a, results in mice with substantially reduced NEM-resistant LPA/PA phosphatase activity, moderately elevated plasma LPA levels and slowed clearance of exogenously administered LPA. Our data demonstrate that LPP1 contributes 43–81% of the total tissue LPA phosphohydrolase activity and 65–99% of the exophosphatase activity. We suppose that the residual LPA/PA phosphatase activity is due to other lipid phosphatases such as LPP3. Our results support the contention LPP1 is a physiologically relevant LPA exophosphatase.</p><p>Exophosphatase activity was virtually nil in Ppap2atr/tr splenocytes suggesting that LPP1 is the only functioning exo-LPP in these cells. This is a somewhat surprising observation in view of the presence of LPP3 mRNA in splenocytes. Nevertheless, the lack of LPA exophosphatase activity may be exploited to examine the role of LPP1 further.</p><p>The unaltered level of LPP1 mRNA expression and enzyme activity in the brains of Ppap2atr/tr mice was unexpected. The possibility that Ppap2atr/tr mice lack the trapping element in brain tissue, i.e. mosaicism, was ruled out by genotyping brain DNA. We reasoned that alternative splicing might be responsible for the failure of the exon trapping element in brain but we were unable to detect differential expression of known LPP1 isoforms [31]. Thus we do not have a plausible explanation as to why the pre-mRNA splicing machinery does not recognize the exon trap splicing acceptor site in brain.</p><p>Our LPP1 hypomorphic mice have elevated plasma LPA levels. The range of reported mouse plasma LPA concentrations is 170–625 nM [34, 35, 36, 37]. The lowest concentration detected in our LPP1 mice was 368 nM, while the highest was 2.45 μM (Fig. 5). We considered whether the source this large variation in LPA levels was an artifact of our methods (plasma preparation, LC-MS-MS) or a reflection of the genetic diversity of our LPP1 colony (F1N1, C57BL/6 x sv129 strains). When one of our laboratories (KRL) generated plasma from age- and gender-matched pure-bred C57BL/6j mice and blinded another of our laboratories (VN) to the source of this plasma, our methods yielded values that were similar to previously reported mouse plasma LPA concentrations and, most importantly, exhibited much less variation: 348 ± 98 nM, i.e. 28%. Additionally, the intra-assay variability was found to be slight (Fig. 5). Thus we reasoned that the disparity in plasma LPA levels measured in our F1N1 Ppap2atr/tr mice was due largely to differences in genetic background and, perhaps, in gender and age. When our LPP1 mouse colony was re-derived at F1N5 after additional crossings onto the C57Bl/6j background, the variability in plasma LPA levels was substantially reduced in age-matched mice (Fig. 5). Nonetheless, our data indicate consistently that LPP1-deficient mice exhibit plasma LPA levels significantly higher than control littermates supporting the hypothesis that LPP1 acts as an LPA exophosphatase. Further, our results document that plasma LPA levels can exceed 2 μM in mice without apparent ill-effects.</p><p>We also investigated the ability of Ppap2atr/tr mice to catabolize LPA by comparing the rate of disappearance of tracer amounts of [32P]LPA from the blood of Ppap2atr/tr and Ppap2a+/+ animals. We found that [32P]LPA disappears rapidly from the blood of Ppap2a+/+ animals when incubated ex vivo at 37 °C and that this process is even faster in vivo. As Fig. 6 documents, Ppap2atr/tr mice exhibit, both ex vivo and in vivo, a still rapid but distinctly slower rate of LPA disappearance. We have measured only how much LPA remains after injection so it is not possible to ascribe the disappearance of exogenous LPA to any catabolic process. Nevertheless, the mutant mice demonstrate that LPP1 is in part responsible for this process.</p><p>In conclusion, we developed an LPP1 hypomorph mouse that enabled us to provide a definitive demonstration that LPP1 plays a role in the extracellular catabolism of LPA in intact animals under physiological circumstances. Further, our results suggest a role of LPP1 in controlling the kinetics of extracellular LPA turnover in vivo. These results provide evidence that LPP1 is an important determinant of LPA turnover and circulating LPA concentrations, which might alter LPA signaling patterns.</p><!><p>A schematic representation of the normal and trapped Ppap2a alleles are shown in A. The wild type Ppap2a gene consist of six exons (numbered 1 to 6 in figure) located on chromosome 13. Two isoforms arise by alternative splicing of two different exons 2 (2.1 and 2.2). Mice used in this study harbor an exon trapping element ("Exon Trap" in figure) between exons 1 and 2 and therefore generate a truncated mRNA ("Trapped Ppap2a" in figure) consisting of exon 1 spliced to the coding sequence of the trapping element followed by a poly(A) sequence. Because insertion occurred upstream of exons 2, only one trapped isoform exists. The exact location of the trapping element was determined by genomic DNA sequencing and found to be some 25,600 bp downstream of the 5′ end of the gene (about 75% the length of intron 1). Sequence at the insertion point shows an intervening 5-nucleotide sequence of unknown origin ("unk") between the Ppap2a gene and the trap element sequence ("pGT1Lxf"). Genotyping was carried out by PCR amplification of genomic DNA using a forward primer complementary to a sequence upstream of the insertion point, and two reverse primers corresponding to sequences downstream in the normal and in the trapped gene. Expected products sizes were 675 bp and 931 bp for the normal and trapped genes respectively. The analysis of PCR products by agarose gel electrophoresis for homozygous (Ppap2atr/tr, "HO" in figure), heterozygous(Ppap2a+/tr,"HE" in figure) and wild type (Ppap2a+/+, "WT" in figure) animals using the indicated combinations of forward and reverse primers is shown in B. In A, the relative positions of the exons and the trap element are shown according to their actual locations within the gene, exon sizes however have been exaggerated for clarity (they constitute only about 2% of the gene).</p><!><p>The expression of LPP1 and LPP3 mRNA was measured by Real-Time RT-PCR in Ppap2atr/tr and Ppap2a+/+ mice. In the case of LPP1, appropriate primers were used to obtain the combined expression of both isoforms (see Results). mRNA expression for LPP1 and LPP3 was normalized by the expression of the 18s ribosomal RNA gene. The main figure shows the expression of mRNA for LPP1 (gray bars) and LPP3 (white bars) as a ratio (Ppap2atr/tr/Ppap2a+/+). Data correspond to the average ± S.D. of three ratios measured in three different pairs of animals (Note the split y axis). Inset (top right) shows the expression of LPP1 mRNA in the brain, liver, kidney and spleen ("B", "L", "K" and "S" in figure) of Ppap2atr/tr and Ppap2a+/+ mice. In this case a standard PCR reaction was carried out for 33 cycles using a forward primer anchored to exon 1 and a reverse primer anchored to exons 3 and 4. The expected molecular mass of the PCR product is 531 bp.</p><!><p>The lipid phosphate phosphohydrolase activity of Ppap2a+/+ and Ppap2atr/tr mice was measured as the ability of organ homogenates to release [32P]H3PO4 from 32P-labeled LPA or PA in a Triton X-100 mixed micelles assay. Main graph (A) shows the lipid phosphate phosphohydrolase activity of Ppap2a+/+ (white bars) and Ppap2atr/tr (gray bars) mice using either LPA or PA as substrates as indicated in the figure. In the case of LPA, activity was also measured under conditions that abolish the NEM-sensitive, Mg2+-dependent component of the total activity. This is shown as composite LPA bars that represent the total activity as the full height of the bar and the NEM-insensitive, Mg2+-independent activity as the height of the bottom part. Their difference, i.e. the NEM-sensitive, Mg2+-dependent portion, is represented by the blackened parts of the bars. Data were obtained from two sets of three animals (three Ppap2a+/+ and three Ppap2atr/tr). Activity is expressed as nmole of [32P]H3PO4 released per mg of protein in one minute. Values represent the average ± S.D. for each genotype (n=3). In B, the NEM-sensitive, Mg2+-dependent activity observed in organs of Ppap2atr/tr animals is expressed in relation to the activities observed in the corresponding organs of Ppap2a+/+ animals, i.e. the Ppap2atr/tr/Ppap2a+/+ ratio of NEM-sensitive, Mg2+-dependent activities. For clarity, organs are identified in this case with the initial letter or letters of the their names ("B": Brain, "H": Heart, etc).</p><!><p>The lipid phosphate phosphohydrolase activity of small tissue slices and spleen cells from Ppap2a+/+ and Ppap2atr/tr mice was measured as their ability to release [32P]H3PO4 from [32P]LPA. Slices and cells were incubated in a physiological solution containing [32P]LPA for a similar period of time for each tissue or cell type. The leftmost group of bars (indicated as "slices" in the figure) show the phosphohydrolase activity of tissue slices prepared from Ppap2a+/+ (white bars) or Ppap2atr/tr (gray bars) mice organs. The group of bars on the right indicated as "Spleen cells" in the figure show the phosphohydrolase activity of spleen cells obtained by mechanical disruption of spleens from Ppap2a+/+ (white bars) or Ppap2atr/tr (gray bars) mice. Bars labeled as "Total" represent the activity of a crude preparation of cells, i.e. all the cells that were obtained. "Splenocytes" represents the activity of cell population constituted mostly by lymphocytes that is obtained by removing the red blood cells from a crude preparation by osmotic lysis. Values represent the average ± S.D of four measurements obtained from tissues of two animals and are expressed as nmole of 32P[H3 PO4] released per mg of protein in one minute.</p><!><p>The levels of plasma LPA in Ppap2atr/tr, Ppap2a+/+ and C57BL/6j mice were measured by liquid chromatography-mass spectrometry. Each dot represents a single measurement from an individual animal, horizontal bars represent the average. Data for Ppap2atr/tr and Ppap2a+/+ animals were either F1N1 or F1N5 generations. The biologic and inter-assay variability was assessed in a homogenous C57BL/6j mouse population (4.5 month old females). Values for these animals, depicted as "B6", were 0.35 ± 0.098 μM (n =7). To assess intra-assay variability, portions of the same plasma samples were mixed and assayed repeatedly ("B6mix"). Values in this case were 0.30 ± 0.046 μM (n = 6). Values for F1N1 and F1N5 Ppap2atr/tr mice (n = 5 and n=25 respectively) were significantly higher (p < 0.05, Student's t test) than those for F1N1 and F1N5 Ppap2a+/+ mice (n = 5 and n=6 respectively) as indicated in the figure. (Note the split x axis and the magnified y axis (on right), which applies to C57BL/6j and F1N5 animals.)</p><!><p>The clearance of LPA was measured by observing the disappearance of [32P]LPA in plasma obtained from blood samples that had been supplemented with tracer amounts [32P]LPA ex vivo or in vivo. For ex vivo experiments, blood samples anticoagulated with EDTA were supplemented with [32P]LPA and incubated for 0, 5, 15 and 30 min at 37°C. Addition of [32P]LPA was accomplished by adding a small amount (5% of the volume of blood) of either [32P]LPA-containing plasma from another animal of the same genotype or a solution consisting of 0.9 % NaCl, 0.1% FAF-BSA and [32P]LPA. In vivo experiments were carried out by injecting mice through the tail vein with 150 μl of either [32P]LPA-containing plasma from another animal of the same genotype or a solution consisting of sterile 0.9 % NaCl, 0.1% FAF-BSA and [32P]LPA. After injection, a series blood samples (~ 50–100 μl) were obtained by retro-orbital bleeding. For practical reasons, the first sample could not be obtained earlier than 2–3 min after injections. This first blood sample was considered to be the "time zero" sample and therefore subsequent samples were taken 5, 15 and 30 min after the moment the first sample was obtained (rather than after the moment of injection). In both cases, ex vivo and in vivo, plasma was immediately obtained from each timed blood sample by centrifugation and then extracted with 1-butanol under acidic conditions. Butanol extracts were then analyzed by TLC. The amount of [32P]LPA present in each sample was measured by scraping off the [32P]LPA bands from the developed TLC plates and determining their 32P content by liquid scintillation spectrometry. A and B show the results of the ex vivo and in vivo experiments respectively by expressing the observed amounts [32P]LPA as a percentage of the "time zero" value. In A, the disappearance of [32P]LPA ex vivo in blood samples from Ppap2a+/+ mice (white symbols) and Ppap2atr/tr mice (gray symbols) is shown. Addition of [32P]LPA was carried out as described above by adding plasma (square symbols or "Plasma" in figure) or saline solution (triangular symbols or "Saline" in figure). Each value corresponds to the average ± S.D. of three measurements obtained in blood samples from three different animals. Values for plasma and saline were obtained in blood from the same animals. Plasma used for additions was a combined plasma preparation from two animals. B shows the in vivo experiments in which the clearance of [32P]LPA in the bloodstream of live Ppap2a+/+ (white symbols) and Ppap2atr/tr (gray symbols) mice was measured. Administration of [32P]LPA was carried out as described above by injecting plasma (square symbols or "Plasma" in figure) or saline solution (triangular and circular symbols or "Saline" in figure). Values for Ppap2atr/tr mice injected with saline (gray squares) are the average ± S.D. of three measurements obtained in three different animals. Values for Ppap2atr/tr mice injected with saline (gray triangles) are single determinations. Values for Ppap2a+/+ mice are single determinations carried out twice with saline (white circles and triangles) and once with plasma (white squares). Because the volume of the blood samples obtained in the in vivo experiments were in general not equal, the observed content of [32P]LPA of each sample was corrected by dividing this value by the volume of the "time zero" blood sample. Each injection of plasma was carried out with plasma obtained from a different animal of the same genotype.</p>

PubMed Author Manuscript

Structural Insights into the Autoregulation and Cooperativity of the Human Transcription Factor Ets-2*

Background: ETS transcription factors regulate expression of genes involved in development and cancer.Results: Crystal structure of the Ets-2 DNA binding domain reveals how closely spaced binding sites are recognized.Conclusion: A significant difference in binding regulation is observed between the closely related Ets-1 and Ets-2 transcription factors.Significance: This may explain the different physiological roles of different Ets proteins.

structural_insights_into_the_autoregulation_and_cooperativity_of_the_human_transcription_factor_ets-

Introduction<!>Cloning and Overexpression<!>Protein Purification<!>Crystallization and Structure Determination<!>Electrophoretic Mobility Shift Assays<!>Modeling of Ets-2 on Palindromic DNA Substrates<!>Structure of the Ets-2 DNA Complex<!><!>Structure of the Ets-2 DNA Complex<!>Structure of the Autoinhibited Form<!><!>Structure of the Non-autoinhibited Form<!><!>Structure of the Non-autoinhibited Form<!>Autoinhibition Properties of Ets-2<!><!>Cooperative DNA Binding by Ets-2<!><!>Cooperative DNA Binding by Ets-2<!><!>Cooperative DNA Binding by Ets-2<!><!>DISCUSSION<!>Protein Data Bank Accession Numbers<!>

<p>The Ets4 (E26 transformation-specific) family of transcription factors are conserved throughout the metazoan phyla (1) with 28 members in the human genome (2). Ets proteins share an 85-amino acid winged helix-turn-helix DNA binding domain (the Ets domain) that recognizes the conserved core DNA sequence GGA(A/T) through direct polar contacts mediated by residues in the highly conserved recognition helix (α3). Ets family proteins have been assigned into four classes based on the binding preferences of up to two residues upstream and downstream of the GGA(A/T) core (3). Class I proteins (which include Ets-2) are by far the most widely represented, comprising more than half of the human Ets proteins. Despite the similarities in the mode and sequence specificity of DNA binding, the functional roles and biological processes regulated by this family of transcription factors are diverse, being able to both activate and repress the transcription of genes involved in proliferation, differentiation, and apoptosis of various cell types. Unsurprisingly, given their importance in cellular proliferation and cell death, a number of Ets family members, (e.g. Ets-1, Pu-1, Etv-1, Etv-4, and Tel) are associated with tumorigenesis and tumor progression. Various factors influence the functional roles of Ets proteins, including tissue-specific expression, alternate splicing, subcellular localization, presence of additional domains, post translational modifications, and the modulation of DNA binding through the formation of homomeric or heteromeric interactions with protein partners.</p><p>Several of the Ets family members such as Ets-1, Ets-2, Etv-4, Etv-5, and Etv-6 are subject to autoinhibition in which additional structural elements outside of the core Ets domain inhibit DNA binding. Much of the knowledge of autoinhibition in Ets family proteins comes from studies on Ets-1, which is closely related to Ets-2 (84% sequence identity for the Ets domains and 55% across the entire length of the polypeptide) and, like Ets-2, contains 4 additional α-helices located immediately N-terminal (HI-1 and HI-2) and C-terminal (H4 and H5) to the core Ets domain. Deletion of these helices or expression of an alternately spliced isoform of Ets-1, which lacks these elements, results in a protein with increased DNA binding affinity (4–6). Structural and functional studies have established a mechanism for the autoinhibition of Ets-1 where, in the absence of DNA, the additional helices (HI-1 HI-2 and H4 H5) form a four-helix bundle that serves as an allosteric inhibitory module, packing against the opposite face from the DNA binding interface (7). Upon binding DNA the first N-terminal inhibitory helix HI-1 unfolds (8, 9), forming a mostly disordered section of random coil (7). The effect of this allosteric inhibition in Ets-1 is increased by phosphorylation of serine residues immediately N-terminal to HI-1 (10) and can be relieved by protein-protein interactions (11, 12). A further layer of complexity was added to this model when it was discovered that the presence and phosphorylation status of an intrinsically disordered serine-rich region immediately N-terminal to HI-1 was found to stabilize the inactive state of Ets-1 to a much greater extent (10–20-fold inhibition) than the structural transitions of HI-1 (2–3-fold inhibition) (13, 14). A recent study established a possible mechanism by which this inhibition proceeds, finding that the phosphorylated serine-rich region is intrinsically disordered and forms dynamic interactions with the core Ets domain, including regions of the DNA recognition interface (15).</p><p>Another mechanism of regulation that is shared by both Ets-1 and Ets-2 is the phenomenon of cooperative binding to palindromic repeats of the Ets binding site (EBS) (16–18), which is not generally observed in the wider Ets family proteins, which typically bind single EBS motifs with high affinity. Structural studies on Ets-1 bound to a palindromic EBS repeat in the core GGA(A/T) motif separated by 4 bp (a sequence naturally occurring on the stromelysin-1 promoter) have identified a small predominantly polar interface between monomers that may explain the structural basis of cooperative binding (19, 20). Although the contact area for this interface is minimal, it is formed from residues present in the N-terminal autoinhibitory regions, and the stimulation of binding to cooperative sites was found to be approximately equal to the relief of autoinhibition (11, 19). An intriguing aspect of the structural studies of Ets-1 is the propensity for the dynamic HI-1 inhibitory helix to associate with a neighboring Ets-1 molecule in a domain-swapped manner. This phenomenon has been observed for both free (PDB IDs 1MD0 (12) and 1GVJ) and DNA-bound Ets-1 structures (PDB ID 3RI4 (21), 2NNY (19), and 3MFK (20)). In one of these structures (PDB ID 2NNY (19)) HI-1 was modeled as a random coil; however, a re-examination of the electron density maps reveals clear density for a domain-swapped helix similar to that observed in PDB ID 3MFK (20). It is not clear if this propensity for domain swapping has any functional significance, yet the fact that it is present in multiple structures with different crystal forms and involves the same regions that have been shown to be important for allosteric transitions suggest that it may have a functional importance for relief from autoinhibition or cooperativity.</p><p>Despite the many similarities between Ets-1 and Ets-2 in their structural and regulatory mechanisms, the function of the two paralogues in vivo is distinctly different. It is clear that some of these differences can be explained by the fact that they have different tissue-specific expression profiles, with both proteins expressed in a wide range of cell types, but Ets-1 is much more highly expressed in thymus, lung, and Jurkat cells, with corresponding differences in the phenotypes of knock-out mice (22). Nevertheless, significant differences have been observed in their transactivation activity and the protein-protein interactions in which they participate; for example, Ets-2 but not Ets-1 is able to interact with both ERG (23) and CDK10 (24). To gain insights into the structural basis for these functional differences and to further investigate the structural basis for autoinhibition and cooperativity, we have determined the crystal structure of the Ets domain of Ets-2. The structure, determined in complex with a single EBS DNA oligonucleotide, unexpectedly contains archetypes of both the autoinhibited and non autoinhibited forms, allowing us to analyze the structural and conformation differences that accompany autoinhibition. The association and interfaces formed between molecules in the asymmetric units give insights into novel aspects of the autoinhibitory mechanism and cooperative binding.</p><!><p>Plasmid DNA templates for full-length human Ets-2 (IMAGE: 3852274) and full-length murine Ets-1 (IMAGE: 40056547) were obtained from the Mammalian Gene Collection (Source BioScience, Nottingham, UK).</p><p>The fragments of Ets-2 and Ets-1 used in this study are: Ets-2325–464 (Ets and autoinhibition domains, crystallized fragment), Ets-2308–469 (Ets domain, autoinhibition domain and N-terminal flanking sequence, used in electrophoretic mobility shift assays (EMSAs)), Ets-2325–469 (Ets and autoinhibition domains, used in EMSA), Ets-2360–464 (ETS domain, used in EMSA), Ets-1280–440 (Ets domain, autoinhibition domain, and N-terminal flanking sequence, used in EMSA), Ets-1300–440 (Ets and autoinhibition domains, used in EMSA), Ets-1331–440 (Ets domain, used in EMSA). The gene fragments were amplified by PCR and cloned into the pNIC28-Bsa4 expression vector, encompassing a tobacco etch virus protease-cleavable N-terminal His tag MHHHHHHSSGVDLGTENLYFQ↓SM), as described elsewhere (25). Plasmids were transformed into BL21(DE3)-R3-pRARE2, and cultures were grown in UltraYield baffled flasks (Thomson Instrument Co.) in Terrific Broth medium containing 50 μg/ml kanamycin at 37 °C to an A600 of 2–3, at which point the cultures were cooled to 18 °C and expression was induced by the addition of 0.1 mm isopropyl β-d-1-thiogalactopyranoside, with cells harvested 18 h after induction.</p><!><p>For purification of both the Ets-1 and Ets-2 domains, cell pellets were thawed and resuspended in buffer A (50 mm HEPES, pH 7.5, 500 mm NaCl, 5% glycerol, 10 mm imidazole, 0.5 mm Tris(2-carboxyethyl)phosphine) and disrupted by sonication. Cell debris and nucleic acids were removed by the addition of 0.15% polyethyleneimine, pH 7.5, and centrifugation at 50,000 × g for 1 h at 4 °C. The supernatants were applied to a 3-ml Ni2+-iminodiacetic acid (Ni-IDA) agarose immobilized metal ion affinity chromatography gravity flow column, washed with wash buffer (buffer A with 30 mm imidazole), and eluted with 5 column volumes of elution buffer (buffer A with 300 mm imidazole). Proteins were incubated overnight at 4 °C in the presence of tobacco etch virus protease (1:40 mass ratio) while being dialyzed using 3.5-kDa molecular weight cutoff snakeskin membrane (Thermo Fisher Scientific, Rockford, IL) into buffer B (20 mm HEPES, pH 7.5, 500 mm NaCl, 5% glycerol, 0.5 mm Tris(2-carboxyethyl)phosphine). Tobacco etch virus protease and contaminating proteins were removed by reapplication of dialyzed proteins to a Ni2+-iminodiacetic acid agarose immobilized metal ion affinity chromatography column (2-ml column volume). Proteins passing through the column were pooled and concentrated using a 10-kDa molecular weight cutoff centrifugal concentrator to 1 ml before loading onto a HiLoad 16/60 Superdex S75 gel filtration column equilibrated in buffer B. Proteins were identified by SDS-PAGE and confirmed by mass spectrometry, and concentrations were determined by absorbance measurement at 280 nm (Nanodrop) using the calculated molecular mass and extinction coefficients.</p><!><p>For crystallization of the Ets domain DNA complex, the oligonucleotides ACCGGAAGTG and CACTTCCGGT were resuspended to 900 μm in 10 mm Tris-HCl, pH 8.0, 50 mm NaCl, mixed in a 1:1 ratio, heated to 95 °C for 5 min in a heating block, and allowed to cool slowly over several hours. The Ets-2 Ets domain protein was concentrated to 300 μm (5 mg/ml) and mixed with an equal volume of double-stranded DNA (1:1.5 molar ratio) before being concentrated on a 3-kDa molecular weight cutoff centrifugal concentrator to 15 mg/ml for crystallization. Sitting drop vapor diffusion crystallization trials were set up with a Mosquito (TTP Labtech) crystallization robot. Crystals grew at 20 °C from conditions containing 0.1 m BisTris, pH 5.5, 0.25 m NaCl, and 15% PEG 3350 and were transferred to a cryoprotectant solution consisting of well solution supplemented with 25% ethylene glycol before being loop-mounted and plunged into a pool of liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I02 and processed using XDS (26), and the structure was solved by molecular replacement using the program MOLREP (27) with the structure of the FEV DNA complex (PDB ID 3ZP5) as a search model. Model building was performed using the program COOT (28) and refined using and PHENIX REFINE (29). A summary of the data collection and refinement statistics is found in Table 1.</p><!><p>The affinity and cooperativity of Ets-2 Ets domain DNA binding was measured using EMSAs. The probes consisted of the following oligonucleotide sequences annealed to the complementary strands (the core Ets binding sites are underlined): single site (ATCTCACCGGAAGTGTAGCA) and palindromes (4-bp, AGCGGAAGTACTTCCGGA; 5-bp, AGCGGAAGTGACTTCCGGA; 6-bp, AGCGGAAGTGCACTTCCGGA; 7-bp, AGCGGAAGTGACACTTCCGGA; 8-bp, AGCGGAAGTGATCACTTCCGGA).</p><p>Radiolabeled double-stranded DNA probes were prepared by incubating the forward strand oligonucleotides for 1 h at 37 °C with T4 polynucleotide kinase in the presence of [γ-32P]ATP. Complementary (non-radiolabeled) oligonucleotides were added, and the mixture was heated to 95 °C and allowed to cool slowly to room temperature. The double-stranded DNA probes were purified before use on a Bio-Rad P6 micro-biospin column. EMSAs were performed by incubating radiolabeled probe (at a concentration of 0.1 nm for Ets-2 and 0.02 nm for Ets-1) with protein titrated by serial dilution. The buffer was 50 mm Tris-HCl, pH 7.5, 25 mm NaCl, 50 mm l-arginine-HCl, pH 7.5, 0.5 mm EDTA, 0.1% Tween 20, 2 mm DTT, and 5% glycerol (inclusion of arginine in the buffer prevented the precipitation of protein-DNA complexes). Reactions were performed for 15 min at room temperature, and 5 μl of each reaction was mixed with loading dye to 0.25% and resolved by 12% native polyacrylamide gel electrophoresis in Tris borate EDTA buffer at 180 V for 1 h on ice. Gels were visualized using phosphorimaging, quantitation was performed using quantity one 1-D analysis software (Bio-Rad), and apparent dissociation constants were calculated using a sigmoidal four-parameter logistic nonlinear regression model in PRISM (GraphPad).</p><!><p>To assess potential interactions between Ets-2 molecules on the various palindromic DNA substrates, we have superposed chain A of the Ets-2 structure as a rigid body onto successive positions on the pseudo-contiguous DNA duplex (formed by chains E, F, H, and I in the Ets-2 structure) as dictated by the spacing requirements of the GGAA motifs of the Ets-2 consensus sequence using the program COOT (28). For the case of substrates with the GGAA motifs separated by 4 bp, the result of this superposition was found to be almost identical to the arrangement of the chains in the Ets-1 stromelysin-1 promoter DNA complex (20).</p><!><p>Crystals of the Ets-2 DNA complex were obtained with an Ets-2 construct spanning residues 325–464, including the N-terminal (HI-1 and HI-2) and C-terminal (H4 and H5) autoinhibitory regions and the DNA oligonucleotides 5′-ACCGGAAGTG and 5′-CACTTCCGGT. The crystals belong to a primitive monoclinic crystal system, space group P 21, and diffracted to 2.5 Å resolution with 3 copies of Ets-2 and three 10-bp DNA duplexes in the asymmetric unit. The structure was solved by molecular replacement using the structure of FEV DNA complex (PDB ID 3ZP5) as a search model. The three DNA duplexes in the asymmetric unit pack with base-stacking interactions that are extended through contacts with symmetry mates to form a pseudo-contiguous double helix spanning the length of the crystal (Fig. 1A). The electron density map is of overall high quality for both DNA and protein chains, with the final model containing 301 of a possible 417 protein residues, and was refined to a crystallographic Rfactor of 0.235 (Rfree of 0.255). A summary of the data collection and refinement statistics can be found in Table 1.</p><!><p>Structure of the Ets-2 Ets bound to DNA. A, schematic representation of the contents of the asymmetric unit of Ets-2; DNA crystals with Ets-2 molecules represented in the ribbon format. Crystallographic symmetry mates are shown in gray, and dotted lines represent stacking of blunt ends of DNA oligonucleotides, which form a pseudo-continuous DNA molecule in the crystal. B, stereo view of the overall structure of the Ets-2 Ets domain with secondary structure elements labeled. The three chains are superposed and color-coded as for A. C, stereo view of the interaction of Ets-2 with DNA with key interacting residues and DNA molecules shown in the stick format.</p><p>Data collection, phasing, and refinement statistics</p><p>Data are provided for the ETS DNA complex.</p><p>a Rmerge = ΣhklΣi|Ii − Im|/ΣhklΣiIi where Ii and Im are the observed intensity and mean intensity of related reflections, respectively.</p><p>b Rp.i.m. = Σhkl √(1/n − 1) Σin = 1|Ii − Im|/ΣhklΣiIi.</p><!><p>An examination of the three Ets-2 molecules in the asymmetric unit reveals that, although they all share the same core Ets family fold and interact with double-stranded DNA in a similar manner, the autoinhibitory regions are significantly different, being well ordered in chain A but almost completely disordered in the other two chains (D and G) (Fig. 1B). The core Ets family fold, common to all chains in the asymmetric unit, consists of a four-stranded antiparallel β-sheet, (strand order β1, β2, β4, β3) flanked on one side by three α-helices with an additional short helix immediately preceding the first β strand, which is of intermediate character (α and 310). The recognition helix (H3) inserts deep into the major groove of the DNA and forms a number of base pair-specific interactions with the DNA, with additional contacts to the DNA backbone being formed from residues in the C terminus of H2, the H2-H3 loop, β3, and the β3-β4 loop (Fig. 1C). Overall the protein DNA interface is very similar to that described in other structural investigations of Ets family proteins (12) and as such is not described in detail here.</p><!><p>In chain A the entire autoinhibitory module can be seen to form a complete folded helical structure and associates with the core of the Ets domain via an extensive interface formed by H1 and the β1-β2 loop. The contact area for the interface is ∼790 Å2 with 7 hydrogen bonds being formed between the core and autoinhibitory module, although the majority of the contacts are hydrophobic in nature, with the core Ets domain providing several aromatic residues (Trp-366, Leu-370, Leu-373, Trp-384, Trp-389, and Phe-442) that contact predominantly branched chain amino acids on the autoinhibitory module (Ile-349, Leu-354, Leu-446, and Leu-457) (Fig. 2A). The HI-1 helix, which was suggested to undergo a structural transition from ordered to disordered upon DNA binding (8, 9), is well ordered and forms a helical secondary structure that packs against the C-terminal ends of helices H4 and HI-2, burying two predominant aromatic residues (Phe-331 and Tyr-334). Overall the structure of chain A, hereinafter referred to as the autoinhibited form, is generally similar to the NMR structure of the autoinhibited Ets-1 (7) (PDB ID 1R36) (1.7 Å root mean square deviation over 123 aligned Cα atoms). The most notable difference is the HI-1–HI-2 loop (residues 338–346), which in Ets-2 contains a single amino acid insertion (Pro-341), and is in a different conformation, forming an extended solvent-exposed loop with a single salt bridge formed between Arg-338 and Glu-343. In contrast, the equivalent region of Ets-1 folds back, forming interactions with the N-terminal region of HI-2 (Fig. 2B). It is interesting to note that the negatively charged Glu-343 is not conserved in Ets-1 (the equivalent residue being Asn-315) and, instead of forming a salt bridge, forms a hydrogen bond with a main chain residue within HI-2 (Ala-324). Other smaller differences can be observed in the relative positioning of HI-1, which is shifted by ∼20°, and the conformation of the β3-β4 loop, although the latter makes a number of contacts to the phosphodiester backbone of the DNA in the Ets-2 structure.</p><!><p>Structure of the autoinhibited form of Ets-2. A, schematic model of the interface between the core Ets domain (cyan) and the autoinhibitory module (gray). Key hydrophobic interacting residues are labeled and shown in the stick format. B, comparison of Ets-2 (Chain A, cyan) with the autoinhibited form of Ets-1 (shown in gray), with residues possibly contributing to the conformational difference of the HI-1-HI-2 loop shown in the stick format. C, 2Fo − 1Fc (shown in blue) and Fo − Fc (shown in green) electron density maps contoured at 1.0 and 2.5 σ, respectively, showing the partial electron density for the autoinhibitory module in the vicinity of chain D (shown in red). A copy of chain A is shown superposed for reference. D, stereo view of a comparison of the three Ets-2 chains in the asymmetric unit shown in the schematic representation with key residues forming intramolecular salt bridges in chain A but not in the other two chains highlighted in the stick format.</p><!><p>For the remaining chains in the asymmetric unit (chain D and G) the entire autoinhibitory module was either completely or partially disordered and was not modeled in final structure. Residual electron density, of insufficient quality to build into, can be observed in regions that would correspond to the expected positions of H1–2, H4, and H5 in chain D (Fig. 2C), suggesting that the disorder in this chain is less extensive. The remainder of the protein corresponding to the Ets core (residues 361–444) is overall highly similar to that of the autoinhibited form (∼0.6 Å root mean square deviation >85 aligned Cα residues), although there are some regions of significant difference between the two forms. Most notably the H1-β1 and the β1-β2 loops, which in the autoinhibited form contact the HI-1 and HI-2 loop and H5, respectively, adopt different main chain conformations. There are also a large number of side-chain residues that transition from being ordered in the autoinhibited form to being disordered in the other two chains; a significant proportion of these (Asp-387, Arg-401, Arg-406, Lys-436, and Arg-441) participate in intramolecular salt bridges in the autoinhibited form (Fig. 2D). Although distant from the recognition helix (H3), the even distribution of these residues around the molecule together with the fact that they generally link distant elements of secondary structure suggest that the formation of these interactions may be a way in which the presence/absence of the autoinhibitory module is transmitted allosterically. Comparing the crystallographic B factor values for the conserved regions of chains A, D, and G reveal a general pattern of significantly higher B values for regions contacting the autoinhibitory module in chains D and G, whereas the recognition helix is relatively unchanged (Fig. 3A).</p><!><p>A, stereo view of the Ets-2 Ets domain in complex with DNA, showing the differences in B-factor between chain A and chains D and G (average of the two values used for calculation) plotted on a per-residue basis onto the structure. B, comparison of the geometry of the pseudo-continuous DNA strand, formed by the interface of the blunt ends of DNA molecules bound to chains D and G in the Ets-2 structure, with a similar length of ideal B-form DNA.</p><!><p>An examination of the environment of the various chains in the asymmetric unit provides an explanation as to why two different forms are present. Although chain A does not form any significant interfaces with other Ets-2 molecules, chains D and G are closely associated, with each other being located on adjacent major grooves on the same face of the DNA in a manner highly reminiscent of other dimeric helix turn helix transcription factors. The two DNA molecules that form part of this interface stack their blunt ends together with almost exactly the same geometry as a continuous DNA double helix (Fig. 3B). Thus the arrangement of chains D and G in the crystal is very close to what would be expected from a palindromic DNA sequence in which the Ets binding sites are separated by six bases.</p><!><p>Given the fact that we observe the autoinhibited form of Ets-2 bound to the specific recognition sequence in the Ets-2 structure, we decided to investigate the autoinhibitory properties of Ets-2 and compare this activity with Ets-1. We have measured the binding affinity of three different length Ets-2 constructs on a single site Ets-2 consensus DNA binding sequence directly alongside the equivalent constructs of Ets-1 (for technical reasons we have used the murine Ets-1 gene, which differs from the human gene in only one position, S288Y, over the length of the constructs used). These constructs correspond to the equivalent of viral Ets-1 (lacking the entire N-terminal autoinhibitory module), the construct used for Ets-2 crystallization, and a longer construct containing an additional 20 residues N-terminal to HI-1 that are believed to be disordered and contain a serine-rich sequence. First, the overall binding affinity of the shortest constructs, which should not be subject to any kind of autoinhibition, is significantly higher for Ets-1 than Ets-2, with an ∼4-fold difference in affinity when tested on the same substrate (0.1 ± 0.06 nm versus 0.4 ± 0.04 nm). Given the two proteins share ∼90% sequence identity over this region and none of the substitutions could be expected to have any direct effect on DNA binding from the crystal structure, these differences indicate that some subtle aspects of the energetics of the interaction of Ets-1 and Ets-2 with DNA are not understood.</p><p>We have also found significant differences between Ets-1 and Ets-2 in the nature of their autoinhibition. In Ets-1 two mechanisms of autoinhibition have been found to be present, a modest effect caused by the association of the autoinhibitory module (2–3-fold inhibition) (4–6, 8, 9) and a more marked inhibition provided by an intrinsically disordered serine-rich sequence N-terminal to this (13–15). Consistent with previous studies, we were able to demonstrate both types of inhibition on Ets-1 (∼5- and 2.6-fold, respectively), but only the latter mechanism of inhibition seems to apply to Ets-2 (∼8-fold inhibition; Fig. 4).</p><!><p>Analysis of the autoinhibition of Ets-2. A, representative electrophoretic mobility shift assays of three Ets-2 constructs binding to DNA substrates with a single copy of the consensus Ets-2 binding sequence. The constructs from left to right correspond to Ets-2360–464 (core Ets domain only), Ets-2325–464 (core Ets and autoinhibition domains), and Ets-2308–469 (core, autoinhibition and N-terminal serine-rich sequence). B, representative electrophoretic mobility shift assays of the equivalent domains of Ets-1 with the same DNA sequence. C, quantification of the data presented in panel A; error bars are plotted as ±S.E. from at least three independent experiments, and the data are plotted for Kdapp. D, quantification of the Ets-1 binding data presented in panel B.</p><!><p>Cooperative binding to palindromic EBS repeats has been the focus of numerous studies using both Ets-2 and Ets-1 (9, 16, 20, 21), with the majority of studies focusing on palindromic arrangements such as that in the stromelysin promoter in which the core GGA(A) motif is separated by four base pairs. Given the differences observed in the autoinhibition between Ets-1 and Ets-2 and the finding of an arrangement in the crystal structure that mimics a palindromic arrangement separated by 6 bp, we have decided to perform electrophoretic mobility shift assays using the short and medium length Ets-2 constructs. These were incubated with a variety of DNA substrates containing inverted repeats of the consensus Ets binding motif separated by a variable 4–8-bp spacer.</p><p>The binding of Ets-2 to substrates with a palindromic arrangement was different between the two constructs, with the Ets-2 325–469 construct binding exclusively in a cooperative manner (forming a super-shifted band on the gel, which we presume represents an Ets-2 DNA complex with 2:1 stoichiometry) to substrates with a 4-bp spacer, and exclusively in a non-cooperative manner to substrates with a 5-bp spacer (Fig. 5A). Substrates containing 6- or 7-bp spacers appeared to be able to form both single and super-shifted species with both constructs (Fig. 5A). We performed a crude quantification of these data, taking the disappearance of the free DNA as a proxy for the total binding, and the appearance of single- and double-shifted bands to represent 1:1 and 2:1 interaction stoichiometries (Fig. 5, A and B). Although the data do not reach saturation it is clear that some degree of cooperativity is in effect in the binding of Ets-2 to the 6-bp substrate, which clearly shows significant amounts of the double-shifted band at lower protein concentrations than the 7-bp substrate (which we infer to be independent binding events). Data for substrates with 8-bp spacer sequences were found to be almost identical to the 7-bp data and for the sake of brevity are not shown. The extent of the cooperativity on the 6-bp substrate is significantly less marked than observed with the 4-bp substrate, and the fact that it is observed (although possibly to different extents) in both constructs suggests it is not dependent on residues in the autoinhibitory region. It is important to note, however, that in all cases the apparent affinity for the palindromic sequences is significantly lower than for the single-site substrate, perhaps indicating a sequence dependence on DNA binding.</p><!><p>Analysis of cooperative DNA binding by Ets-2. A, cooperative DNA binding properties of Ets-2325–464 (core Ets and autoinhibition domains) binding to a variety of DNA substrates with palindromic repeats, with a variable length spacer between the GGAA core. DNA sequences used are shown on the top, representative electrophoretic mobility shift assays are shown in the center, and a quantification plot, which expresses the amounts of free DNA (red triangles), single-shifted species (black circles), and double-shifted species (black squares) as a percentage of the total material in each lane, is shown on the bottom. Error bars are plotted as ±S.E. from two independent experiments, although additional biological replicates were performed with the same results. B, cooperative DNA binding properties of Ets-2360–464 (core Ets domain only). The data are presented as for A with the DNA substrate used being the same as that used above.</p><!><p>Using the DNA-bound structures of Ets-2 as a guide it is possible to model the potential intermolecular interactions formed on these substrates. In agreement with observations from crystal structures of Ets-1 in complex with the stromelysin promoter DNA (19, 20), positioning two copies of Ets-2 on substrates with a 4-bp spacer reveals a small but significant protein interface area (∼370 Å2) created by the HI-2-H1 loop contacting the H2-H3 loop in the adjacent chain, with the potential to form the same hydrogen bond between Asn-408 and Gly-361 as found in the structure of Ets-1 bound to the stromelysin promoter (19, 20) (Fig. 6A). Crucially this interface is unlikely to be formed in the shorter construct which lacks HI-1 and HI-2. Positioning Ets-2 on substrates with a 5-bp separation reveals significant steric clashes that occur between the two molecules involving primarily HI-1 and HI-2 but also regions of the core Ets domain (Fig. 6B), explaining the complete lack of formation of the super-shifted band in either construct. The likely arrangement of Ets-2 on a substrate with binding sites separated by 6 bp is revealed by chains D and G in the Ets-2 structure, and positioning two copies of the autoinhibited form onto these chains reveals a steric clash occurs between the C-terminal ends of HI-1 and HI-2 and their equivalents on the neighboring subunit (Fig. 6C). Nevertheless the fact that this arrangement is present in the Ets-2 crystals (which were obtained using the longer Ets-2 325–469 construct) and appears, from the EMSA analysis, to form preferentially in the longer construct but not the short, suggests that this clash can be accommodated presumably by the introduction of disorder in the N-terminal inhibitory module. Thus the structure suggests that there is a direct steric requirement for at least one of the autoinhibitory modules to be disordered when binding to this type of substrate.</p><!><p>Modeling of the Ets-2 structures on to three DNA substrates in which the core GGA(A/T) motif is separated by 4 (A), 5 (B), and 6 base pairs (C) viewed with the 2-fold symmetry axis vertical (upper row) and in the plane on the page (lower row).</p><!><p>We have also noticed through this superposition that, due to the fact that equivalent regions of the HI-2-H1 loop overlap close to residues Gly-361 and Pro-362, it is possible that the N-terminal autoinhibitory modules may adopt a domain-swapped conformation (Fig. 7A). Although the electron density is too poor to determine whether this is happening in the crystal (Fig. 7B), the fact that numerous examples of domain swapping have also occurred in various Ets-1 structures suggests that this phenomenon may be worthy of a more detailed investigation. Modeling of Ets-2 structures bound to substrates with Ets binding sites separated by seven or more base pairs reveals that there is no possibility of forming a significant protein-protein interface, and thus the binding at the two sites would be expected to be largely independent.</p><!><p>Possible domain swapping in Ets-2. A, superposition of Ets-2 Ets domain chain A onto chains D and G reveals the potential for domain swapping of the entire N-terminal autoinhibitory module to occur. B, view of the 2Fo − 1Fc electron density map in this region contoured at 1.0 σ. The black ribbon diagram represents the path of the molecule for the non-domain-swapped species formed by superposition of chain A onto chain D.</p><!><p>Previous structural studies of the closely related Ets-1 established a model for autoinhibition in which the unfolding of HI-1 upon binding DNA (a process that presumably has a positive ΔG) reduces the affinity of the autoinhibited form. It had been presumed that this mechanism may also apply to Ets-2; however, we failed to demonstrate any noticeable difference in DNA binding affinity on single Ets binding sites between Ets domain constructs with and without the N-terminal autoinhibitory regions. In contrast we have shown an ∼5-fold reduction in affinity when comparing the equivalent constructs of Ets-1, which is in agreement with previous studies (4–6, 8, 9, 30). Comparison of the two DNA complex structures reveals an almost identical protein DNA interface, with the sequence of the recognition helix and all DNA contacting residues being completely conserved between the two proteins, indicating that the differences in affinity may come from the dynamics of the association of the autoinhibitory module. Instead the serine-rich region upstream of the N-terminal autoinhibitory region, which has been shown to play a role in Ets-1 inhibition (10, 13, 15), appears to be the sole region responsible for the autoinhibition of Ets-2, causing an ∼10-fold inhibition. It is possible that this inhibition may be further enhanced by phosphorylation as is the case for Ets-1 (15).</p><p>We were also surprised to find two distinct forms for the Ets domain of Ets-2 in our crystals, one in which the N-terminal autoinhibitory regions are largely disordered and another with the entire autoinhibitory region ordered. The fact that both of these are bound to DNA and appear to make an identical protein DNA interface again suggests that the established model of Ets-1 autoinhibition is not directly applicable to Ets-2. Nevertheless a comparison of the two forms of Ets-2 gives insights into how the presence or absence of the autoinhibitory module may be transmitted to the rest of the Ets domain, with a number of intramolecular salt bridges formed in the autoinhibited form (presumably stabilized by the presence of the autoinhibitory module) which are evenly distributed throughout the molecule and may serve to subtly alter the conformation or dynamics of the residues forming the DNA interface.</p><p>We have also investigated the cooperativity of DNA binding by Ets-2 and in this respect find the activity of Ets-2 to be largely similar to Ets-1. A significant cooperative DNA binding effect was observed on DNA substrates where the Ets binding sites were separated by 4 bp, which was dependent on the presence of the N-terminal autoinhibitory helices. The length dependence of the DNA spacer was also investigated, and it was found that Ets-2 was unable to bind cooperatively to sites separated by 5 bp. Some degree of cooperativity in binding was observed for sites separated by 6 bp, which in contrast to the situation in the 4-bp-spaced sequences was effected by, but not dependent on the presence of the autoinhibitory region. Modeling of Ets-2 onto these various substrates revealed the possible mechanism by which cooperativity is conferred due to the potential to form favorable/unfavorable interactions and order-disorder transitions of the N-terminal autoinhibitory helices.</p><!><p>Atomic coordinates and structure factors were deposited in the Protein Data Bank with the accession number PDB 4BQA (Ets-2-DNA).</p><!><p>This work was supported by the Structural Genomics Consortium, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust (092809/Z/10/Z).</p><p>The atomic coordinates and structure factors (code 4BQA) have been deposited in the Protein Data Bank (http://wwpdb.org/).</p><p>E26 transformation-specific</p><p>Ets binding site</p><p>2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.</p>

PubMed Open Access

A FRET-Based Near-Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria

We report a near-infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near-infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18-fold and the fluorescence of the rhodamine acceptor decreased moderately by 45% under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.

a_fret-based_near-infrared_fluorescent_probe_for_ratiometric_detection_of_cysteine_in_mitochondria

Introduction<!>Synthesis<!>Optical responses of probe A to cysteine<!>Theoretical calculations<!>Kinetic and thermodynamic study<!>Selectivity, photostability, and pH effects<!>Cell viability and confocal fluorescence imaging of HeLa cells<!>In vivo experiments with D. melanogaster first-instar larvae<!>Conclusion<!>Instruments and chemicals:<!>Optical measurement method:<!>Cell culture and MTT cytotoxicity assay:<!>Cell confocal fluorescence microscopy imaging:<!>In vivo experiments with D. melanogaster first-instar larvae:<!>Synthesis of 3-(4-(4-acetylphenyl)piperazine-1-carbonyl)-7-(diethylamino)-2H-chromen-2-one (3):<!>Synthesis of N-(4-(2-carboxyphenyl)-2-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)piperazin-1-yl)phenyl)-7H-chromen-7-ylidene)-N-ethylethanaminium (5):<!>Synthesis of fluorescent probe A:

<p>Biothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) exert important roles in redox homeostasis, metabolism, protein synthesis, signal transduction, post-translational modification, and metabolism.[1] Biothiol deficiency is associated with different disorders such as liver damage, lethargy, neurotoxicity, slow growth, hematopoiesis, skin lesions, as well as neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.[1d,e] Many fluorescent probes have been developed to detect biothiols.[1b,2] Most of them are intensity-based probes and suffer from systematic errors caused by variations in the probe concentration, fluctuations of the radiation light, as well as different functioning locations and environments within cells.[2a–d] Ratiometric fluorescent probes have been developed to overcome systematic errors by introducing built-in internal fluorophores and using FRET or through-bond energy-transfer (TBET) strategies.[3] The near-infrared fluorophores have the advantage of unique ratiometric and near-infrared imaging features such as deep-tissue penetration, low cellular and tissue fluorescence background, and reduction of probe photobleaching.[3a,4] However, ratiometric fluorescent probes for cysteine detection with near-infrared emissions are currently limited to a few examples.[5] Herein, we detail a near-infrared fluorescent probe based on the FRET strategy for the ratiometric selective detection of Cys and Hcy over GSH in live cells by employing coumarin as a donor and near-infrared rhodamine as an acceptor with a piperazine-tethered spacer. We chose coumarin and the near-infrared rhodamine derivative because of their excellent optical properties, including outstanding photostability, high absorption coefficients, and high fluorescence quantum yields.[6] Ratiometric fluorescence sensing of Cys and Hcy is achieved by manipulating changes in the π-conjugation of the rhodamine acceptor with a phenyl thioester molecular switch in response to biothiols, since the phenyl thioester rapidly binds to Cys and Hcy through a substitution reaction with the phenyl thioester to form spirolactam ring configurations (Scheme 1). In the absence of Cys or Hcy and when excited at 440 nm, probe A shows two well-defined fluorescence peaks corresponding to the weak fluorescence from the coumarin donor and a strong fluorescence from the rhodamine acceptor, presumably because of highly efficient energy transfer from the coumarin donor to the rhodamine acceptor. The gradual addition of Cys results in a significant decrease in the fluorescence from the rhodamine acceptor and a corresponding increase in the fluorescence from the coumarin donor because the nonfluorescent closed-ring spirolactam structure is formed on the rhodamine acceptor and the reaction with Cys results in a fluorescence quenching of the rhodamine acceptor. This also effectively prevents energy transfer from the coumarin donor to the rhodamine acceptor. The probe displays less-sensitive ratiometric fluorescence responses to Hcy and insignificant responses to glutathione (Scheme 1). The probe has been applied to determine changes in the cysteine concentration in HeLa cells and Drosophila melanogaster larvae.</p><!><p>The synthetic route to probe A is shown in Scheme 2. 7-(Diethylamino)coumarin-3-carboxylic acid (2) was coordinated to 4'-piperazinoacetophenone (1) through an amide bond, thereby affording compound 3. A near-infrared rhodamine derivative bearing a coumarin donor (5) was prepared by condensation of 3 with 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid (4) in sulfuric acid at high temperature.[7] Our approach effectively overcomes a challenging separation issue by preparing a highly polar near-infrared rhodamine derivative bearing a piperazine residue.[6] Probe A was prepared by introducing the biothiol-sensing switch of a phenyl thioester into the rhodamine acceptor by coupling 3,5-bis(trifluoromethyl)benzenethiol (6) with dye intermediate 5. We chose dye 5 to develop the ratiometric fluorescent probe A for cysteine because it has a much greater fluorescence quantum yield than other near-infrared coumarin hybrid dyes.[8] Probe A was characterized by NMR spectroscopy and high-resolution mass spectrometry (Figures S1–S5 in the Supporting Information).</p><!><p>Probe A shows two strong well-defined absorption peaks at 420.7 and 606.9 nm, which correspond to the absorption wavelengths of the coumarin donor and the rhodamine acceptor, respectively (Figure 1 A). The gradual addition of Cys leads to a gradual decrease in the absorbance of the rhodamine acceptor and almost no change in the absorbance of the coumarin donor because of the formation of a closed spirolactam ring on the rhodamine acceptor, which significantly reduces the π-conjugation in the rhodamine moiety. The probe displays two well-defined fluorescence peaks, with a strong fluorescence for the rhodamine acceptor at 645 nm and a weak fluorescence peak for the coumarin donor at 467 nm in the absence of Cys (Figure 1 B) because of the highly efficient energy transfer from the coumarin donor to the rhodamine acceptor. The efficiency of this energy transfer was calculated to be 95.1 %. The probe shows a large pseudo-Stokes shift of 224 nm, that is, the difference between the coumarin absorption and the rhodamine fluorescence peaks. Upon gradual addition of Cys, the fluorescence of the rhodamine acceptor gradually decreases while there is a concomitant gradual increase in the fluorescence of the coumarin donor. This sensitive ratiometric change in the fluorescence response of the probe to Cys arises from a suppressed energy transfer from the coumarin donor to the rhodamine acceptor as a result of the formation of a closed spirolactam ring on the rhodamine acceptor after it reacts with Cys (Scheme 1). The fluorescence intensity ratios of rhodamine to coumarin increase from 0.235 to 4.237, with a final enhancement factor of 18-fold upon addition of Cys. The probe shows similar ratiometric responses to Hcy, with much smaller changes in the absorbance and a decrease in the fluorescence of the rhodamine acceptor, as well as an increase in the fluorescence of the coumarin donor (Figure S6). This observation was further confirmed by the fluorescence spectra of the probe in the absence and presence of Cys and Hcy under excitation of the rhodamine acceptor at 560 nm (Figures S7 and S8). Probe A also exhibits insignificant responses to GSH, since the reaction of the probe with GSH retains the same thioester bond without changing the π-conjugation of the rhodamine acceptor and affecting the efficiency of the energy transfer from the coumarin donor to the rhodamine acceptor. The reaction products of the probe with Cys, Hcy, and GSH were confirmed by mass spectrometry (Figures S9–S11).</p><!><p>We also examined the electronic properties of probe A and its reaction products with Cys and GSH through theoretical calculations, using an exchange correlation (xc) functional DFT/ TPSSH[9] and with atoms defined at the split-valence triple-ζ plus polarization function (TZVP[10]) implemented, using the Gaussian 16[11] suite of programs. Interestingly, reasonable agreement between theoretical and experimental data were obtained; for probe A, calculated (expt.) at 552 (607), 393 (421); A-1 at 393 (421); A-3 at 563, 393 nm. These values are within the expected error range of 0.20–0.25 eV.[12] Full details are available in the Supporting Information (Figures S12–S23). The isolated nature of the transitions can be observed in the difference density illustrations shown in Figure 2. In probe A, the ES2 transition at 552 nm has a 95.9% contribution from the rhodamine moiety, and at 393 nm, that is, ES8, 88.4% is contributed from a transition localized on the coumarin sector. A-1 has only one transition with a significant oscillator strength, and this was localized on the coumarin moiety with a 96.4% contribution. For A-3, ES2 (Figure 2) is localized on the rhodamine moiety at 95.2%, but for ES10, 17.3% is due to a transition from the coumarin to the rhodamine moiety, while 5.2% is from an LCAO on rhodamine to coumarin, and 74.4% is localized solely on the coumarin. The results of the calculations confirm that for probe A, excitation at 440 nm (Figure 1), which leads to fluorescence from the rhodamine moiety, does not occur as a consequence of orbital transmission through bonds.</p><!><p>Probe A responds quickly to Cys and Hcy; the fluorescence ratio values of the rhodamine acceptor to the coumarin donor plateaus within 20 minutes in the presence of 30 equivalents of Cys or Hcy (Figure 3 A). The pseudo-first-order rate constants (k) for Cys and Hcy were determined as 0.08572±0.00622 min−1 and 0.07364±0.00630 min−1, respectively (Figure S24). The reaction rate constant for Hcy is lower than that of Cys due to the facile formation of a five-membered ring intermediate by Cys attack being more favored than the intra-molecular attack by an amino group and formation of a six-membered ring compound with Hcy. The fluorescence ratio values of the rhodamine acceptor to the coumarin donor show a linear relationship with Cys concentrations from 25 to 150 μm at 20 min, with a detection limit of 1.5×10−6m (Figure 3B, Figure S25B). Furthermore, the detection limit of Hcy is calculated to be 1.3×10−5 m (Figure S25 A), which is much higher than that of Cys, thus indicating that probe A could serve as a ratiometric fluorescent tool to selectively detect Cys over other biothiols.</p><!><p>We also investigated the selectivity of the probe to Cys or Hcy in the presence of different biological species, such as amino acids (Figure S26), anions (Figure S27), and cations (Figure S28). The results suggest that the probe displays high selectivity to Cys and Hcy over other species. We further evaluated the photostability of fluorescent probe A in a time-dependent fluorescence experiment. The fluorescence ratio between the coumarin donor and rhodamine acceptor exhibited only a very small change after illumination at 400 nm for 2 h, with a decrease of less than 8% compared to the initial intensity (Figure S29). The fluorescence intensity of the rhodamine acceptor showed a less than 5% decrease after 2h excitation at 560 nm (Figure S30). Thus, probe A is highly resistant to photo-bleaching and has good photostability. We also studied the effect of the pH value on cysteine sensing in physiological ranges. The results presented in Figures S31 and S32 indicate that there are small and insignificant changes in the probe fluorescence and, more importantly, the fluorescence responses of the probe to cysteine are not significantly affected by the pH value between 5.0 and 8.5. Therefore, probe A can accurately sense cysteine within this pH range from 5.0 to 8.5.</p><!><p>We further investigated the cytotoxicity of the probe to HeLa cells by standard MTT assays. The cell viability is more than 90% on treatment with 50 μm probe A (Figure S33), thus affirming that probe A displays low cytotoxicity toward HeLa cells.</p><p>As probe A is positively charged, we suspected that it may target mitochondria through electrostatic interactions with negative electric potentials across the inner mitochondrial membranes. To test this hypothesis, we carried out intracellular colocalization experiments by co-staining HeLa cells with the probe and a mitochondria-targeting cyanine dye (IR-780). Strong cellular fluorescence of the probe's rhodamine acceptor was observed under excitation at 405 nm and 559 nm, and weak fluorescence of the probe's coumarin donor was observed under excitation at 405 nm (Figure 4). The weak fluorescence of the donor is due to the low intracellular Cys concentration in live cells. The Pearson colocalization coefficient of the probe with a cyanine dye (IR-780) is high at 0.94, which indicates that probe A stays in the mitochondria with the cyanine dye IR-780.</p><p>We investigated whether the probe can detect Cys in live cells by incubating HeLa cells with 10 μm of the probe at 37°C for 30 min. Both the blue fluorescence from the coumarin donor and the near-infrared fluorescence (red and yellow channels) were observed (Figure 5). However, when HeLa cells were incubated with different concentrations of Cys from 100 to 500 μm under the coexistence of endogenous Cys, followed by further incubation of the cells with probe A, the cellular blue fluorescence of the coumarin donor significantly increased proportionally with the increased Cys concentration, while the near-infrared fluorescence from the rhodamine acceptor decreased significantly. In addition, overlapped images of the fluorescence in the blue and red channels show significant color changes from pink to blue when the Cys concentration is increased from 100 μm to 500 μm. Ratiometric images of the probe (where coumarin fluorescence is divided by the rhodamine fluorescence) show considerable color changes from bluish red to white before and after cysteine treatment, respectively (Figure S34). These findings suggest that exogenous cysteine transported to mitochondria further reacts with the probe in mitochondria, thereby resulting in a closed spirolactam ring structure in the rhodamine acceptor and leading to an increase in the coumarin fluorescence and decrease in the rhodamine fluorescence under cysteine treatment. The cellular fluorescence responses of the probe to Cys are similar to the fluorescence responses in aqueous solution (Figure 1). We also conducted a control experiment by pre-treating HeLa cells with 1.0 mm NEM (N-ethylmaleimide) to remove intracellular endogenous biothiols, before the cells were incubated with the probe. A slight decrease in the blue fluorescence of the probe's coumarin donor was observed with a significant increase in the near-infrared fluorescence of the probe's rhodamine acceptor in the red and yellow channels. This indicates that NEM treatment causes a decrease in the biothiol concentration through an addition reaction of NEM with a mercapto group on the endogenous biothiols (Figure 5). We also investigated whether probe A could detect Hcy in HeLa cells. Similar results to sensing cysteine in HeLa cells were obtained (Figure S34), which is in agreement with the fluorescence responses of the probe to Hcy in buffer solutions (Figure 3).</p><p>We further studied concentration changes of Cys on HeLa cells that were oxidatively stress-induced by applying different concentrations of hydrogen peroxide (Figure 6). Gradual increases in the concentration of hydrogen peroxide from 20 to 100 μm led to a significant decrease in the cellular blue fluorescence from the coumarin donor, and a considerable increase in the cellular near-infrared fluorescence of the rhodamine acceptor in the red and yellow channels under excitation at 405 and 559 nm. Additionally, ratiometric images of the blue channel over the red channel also show significant color changes from bluish pink to an extremely weak blue. This is due to decreases in the cysteine concentration under oxidative stress when the hydrogen peroxide concentration is increased from 20 to 100 μm. Hydrogen peroxide converts the mercapto group on cysteine into a disulfide group through oxidation, resulting in a decrease in the concentration of endogenous cysteine in live cells. As a result, the coumarin fluorescence decreases in the blue channel while the near-infrared rhodamine fluorescence in the red channel increases under oxidative stress on treatment with hydrogen peroxide (Figure 6).</p><p>The decreases in the endogenous Cys concentration under oxidative stress in the presence of nitric oxide can lead to the effective oxidization of biothiols. Moreover, we studied changes in the intracellular Cys concentration under the stimulus of lipopolysaccharide (LPS), as it was reported that lipopolysaccharide treatment can generate nitric oxide and create oxidative stress in live cells to oxidize biothiols.[13] LPS treatment of HeLa cells results in a decrease in the coumarin fluorescence and an increase in the rhodamine fluorescence as a result of a decrease in the endogenous cysteine concentration due to the oxidation of cysteine by nitric oxide. Ratiometric images of the blue channel over the red channel show dramatic color changes from reddish blue to weak blue before and after LPS treatment (Figure 7). These results convincingly demonstrate that the probe possesses high cell permeability and is capable of detecting intracellular Cys changes ratiometrically with visible and near-infrared channels (Figures 5–7).</p><!><p>Finally, we conducted fluorescence imaging of live Drosophila melanogaster first-instar larvae in the absence and presence of different concentrations of cysteine (Figure 8). D. melanogaster does not have a fluorescence background and it possesses a very low cysteine concentration: probe A reveals very weak fluorescence from the coumarin donor and highly intense fluorescence from the rhodamine acceptor. The probe locates in the epidermis and tracheae (Figure 8). However, gradual increases in the incubation concentrations of Cys with the larvae result in increases in the coumarin fluorescence and decreases in the fluorescence from the rhodamine acceptor because intake cysteine into the larvae further reacted with the probe, thereby resulting in a closed nonfluorescent spirolactam ring of the rhodamine acceptor and preventing effective FRET from the coumarin donor to the rhodamine acceptor (Figure 8). Ratiometric images (ratio of blue channel to red channel) of the larvae incubated with probe A and increasing cysteine concentrations show dramatic pseudocolor changes from reddish blue to reddish white (Figure S41), thus indicating that increases in the cysteine concentrations lead to the increases in the coumarin fluorescence and decreases in the rhodamine fluorescence. The coumarin fluorescence of the probe in the blue channel decreases and the rhodamine near-infrared fluorescence in the red and yellow channel increases after the larvae were first incubated with cysteine and the probe, and then further treated with hydrogen peroxide (Figure S42). This suggests that treatment with hydrogen peroxide also reduces the cysteine concentration in the larvae. These results indicate that probe A can be successfully applied to live tissues to detect Cys concentrations.</p><!><p>We have developed a fluorescent probe A based on coumarin as a donor and a near-infrared rhodamine acceptor for the sensitive ratiometric detection of Cys. The probe offers ratiometric detection of the Cys concentration and fluctuation in live cells with a self-calibration capability under oxidative stress through treatment with hydrogen peroxide and LPS, and can be utilized for the visualization of cysteine changes in D. melanogaster larvae in the visible and near-infrared channels.</p><!><p>A 400 MHz Inova NMR spectrometer was employed to record 1H NMR spectra at 400 MHz and 13C NMR spectra at 100 MHz. Chemical shifts of intermediates and probes were determined by using solvent residual peaks as internal standards (1H: δ = 7.26 for CDCl3, δ = 2.50 for [D6]DMSO; 13C: δ = 77.3 for CDCl3). High-resolution mass spectra were recorded on an electrospray ionization mass spectrometer. Absorption spectra were obtained using a PerkinElmer Lambda 35 UV/Vis spectrometer, and conventional fluorescence spectra were obtained using a Jobin Yvon Fluoromax-4 spectrofluorometer. IR spectra were obtained using a PerkinElmer FT-IR Spectrometer. The MTT assay was performed on a BioTek ELx800 absorbance microplate reader. An Olympus IX81 inverted microscope was used for cellular imaging. All reagents and solvents were purchased from commercial sources and used without further purification.</p><!><p>The UV/Vis absorption spectra of probe A in the presence of thiols, for the first-order kinetic plot, and to establish selectivity, photostability, and linear ratio relationship measurements were obtained in the range 300 to 800 nm with increments of 1 nm. The corresponding fluorescence spectra were collected at an excitation wavelength of either 400 nm for donor or 560 nm for acceptor excitation. The concentration of the probe in each sample was 5 μm. Cresyl violet (Φf=0.56 in EtOH) was used as the reference standard to determine the fluorescence quantum yields of probe A in ethanol and buffer solutions. Quinine sulfate (Φf=0.546 in 1 n H2SO4) was used as the reference standard to determine the fluorescence quantum yield of probe A after reaction with thiols. All samples and references were freshly prepared under similar conditions. The fluorescence quantum yields were calculated using Equation (1): (1)ΦX=ΦstGradXGradstηX2ηst2</p><p>The subscripts "st" and "X" stand for standard and test, respectively, Φ is the fluorescence quantum yield, "Grad" means the gradient from the plot of integrated fluorescence intensity versus absorbance, and η is the refractive index of the solvent.</p><!><p>HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Fisher Scientific) at 37°C in a humid atmosphere containing 5% CO2. HeLa cells were subcultured at 80% confluence using 0.25% trypsin (w/v) (Fisher Scientific) every other day. A standard MTT assay was applied to determine the cytotoxicity of probe A. In detail, the cells were seeded in 96-well plates at an initial density of 4000 cells per well, with 100 μL DMEM medium per well. After seeding for 24 h in the 96-well plate, the medium was replaced by different concentrations of probe A (0, 5, 10, 15, 25, 50 μm solutions in fresh culture medium, 100 μL/well) for 48 h. After that, the cells were incubated for 4 h with the tetrazolium salt dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at a final concentration of 500 μg mL−1, whereupon metabolically active cells reduced the dye to the water-insoluble purple formazan dye. The dark purple crystals were dissolved with DMSO and the cell viability rate determined by measuring the absorbance at 490 nm (BioTek ELx800). The cell viability rate was calculated by Vrate = (A–AB)/(AC–AB) × 100%, where A is the absorbance of the experimental group, AC is the absorbance of the control group (cell medium used as control), and AB is the absorbance of the blank group (no cells). Data were illustrated graphically, with each data point calculated from an average of three wells.</p><!><p>Cells were seeded in confocal glass-bottom dishes with 105 cells per dish and cultured for 24 h. Probe A was added to each dish and cultured for 30 min. Cells were then washed with PBS (pH 7.4) twice, and 1 mL of PBS was then added before imaging. The fluorescence of probe A was determined based on excitation with a 405 nm or 559 nm laser, with emission spectra recorded at 450–500 or 630–680 nm, respectively. The fluorescence of the cyanine dye IR-780 channel was determined under excitation at 635 nm and its emission collected between 750 and 800 nm. A confocal fluorescence microscope (Olympus IX 81) was used to take images of the HeLa cell and an Olympus FV10-ASW 3.1 viewer, ImageJ, and Image Pro 6 were used to process the image data.</p><!><p>In order to test the probe in D. melanogaster, a nine-well glass viewing dish was used. The larvae were treated in four different ways: 1) Larvae were submerged in 500 μL distilled water for 4h and washed three times in 500 μL distilled water for the negative control. 2) Larvae were incubated with 20 μm probe for 2 h and washed three times with 500 μL distilled water. 3) larvae were submerged in 500 μL 1 mm cysteine for 2 h, washed three times with 500 μL distilled water, and incubated for an additional 2 h in distilled water. 4) Larvae were submerged in 500 μL of 50 pM, 100 pM, 200 μm, or 1 mM cysteine for 2 h, washed three times in 500 μL distilled water, incubated for an additional 2 h with 20 μm probe, and then washed three times in 500 μL distilled water. For each sample, ten freshly hatched first-instar larvae were used. After the incubation, the larvae were transferred with water onto a microscope slide and then covered with a cover slip. The larvae were then immediately analyzed by confocal fluorescence microscopy (Olympus IX 81). The confocal microscopy conditions of the channels were identical to those utilized for the HeLa cells images.</p><!><p>A mixture of 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (312 mg, 3 mmol), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (861 mg, 3.3 mmol), BOP reagent (1.46 g, 3.3 mmol), and trimethylamine (1 mL) in 20 mL anhydrous CH2Cl2 was stirred for 8 h at room temperature (Scheme 2). The mixture was then washed with water and then brine, dried with anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography under gradient elution with hexanes/ethyl acetate (1:1) to yield compound 3 as a yellow solid. 1H NMR (300 MHz, CDCl3): δ = 7.89-7.78 (m, 3H), 7.27 (d, J = 6.7 Hz, 1 H), 6.83 (d, J = 6.8 Hz, 2 H), 6.56 (dd, J = 6.6, 1.8 Hz, 1 H), 6.46-6.40 (m, 1 H), 3.85 (s, 2H), 3.52 (d, J = 12.2 Hz, 2H), 3.39 (q, J = 5.2 Hz, 8H), 2.48 (s, 3H), 1.21-1.13 ppm (m, 6H); 13C NMR (75 MHz, CDCl3): δ = 196.63, 165.27, 159.32, 157.46, 153.89, 151.93, 145.81, 130.55, 128.19, 115.79, 113.99, 109.63, 107.89, 97.04, 47.23, 45.26, 42.25, 26.52, 12.75 ppm. LCMS(ESI): calcd for C26H29N3O4: 447.2 [M]+; found: 448.1 [M+H]+.</p><!><p>After compounds 3 (447 mg, 1 mmol) and 4 (313 mg, 1 mmol) had been added to methanesulfonic acid (6 mL), the reaction mixture was stirred at 100 °C for 6h under argon (Scheme 2). The mixture was then added to 20 mL water and extracted with CH2Cl2 (3×100 mL). The organic layers were collected, dried over Na2SO4, and evaporated under reduced pressure. The mixture was purified by flash column chromatography using CH2Cl2/menthol (40:1) to yield compound 5[6] as a blue solid.</p><!><p>Compound 5 (75.3 mg, 0.1 mmol), compound 6 (26.9 mg, 0.11 mmol), EDC (21 mg, 0.11 mmol), and DMAP (13.4 mg, 0.11 mmol) were added to anhydrous CH2Cl2 (20 mL) and stirred for 8 h at room temperature. The mixture was then washed with water, then brine, dried with anhydrous Na2SO4, filtered, and then evaporated to dryness under reduced pressure. The resulting residue was purified by flash column chromatography using a mixture of CH2Cl2/methanol (30:1) as eluent to give probe A as a blue solid (Scheme 2). 1H NMR (300 MHz, CDCl3): δ = 8.24–8.09 (m, 3H), 7.93–7.84 (m, 2H), 7.86–7.73 (m, 2 H), 7.71–7.63 (m, 3H), 7.46 (s, 1 H), 7.33–7.25 (m, 2H), 7.13–6.94 (m, 4H), 6.59 (dd, J = 6.7, 1.8 Hz, 1 H), 6.46 (d, J = 2.4 Hz, 1 H), 3.87 (s, 2 H), 3.77–3.51 (m, 9H), 3.42 (q, J = 7.1 Hz, 4H), 1.31 (t, J = 5.0 Hz, 6H), 1.21 ppm (t, J = 5.0 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ = 188.70, 166.97, 165.53, 159.50, 159.38, 158.22, 157.54, 155.05, 154.92, 152.07, 145.99, 135.71, 134.88, 133.74, 132.87, 132.53, 131.61, 131.20, 130.25, 129.25, 127.62, 117.32, 115.51, 114.87, 114.39, 109.73, 109.49, 107.98, 97.14, 46.27, 45.28, 42.20, 12.82, 12.76 ppm. LCMS(ESI): calcd for C52H47F6N4O5S: 953.31659 [M]+; found: 953.31607.</p>

PubMed Author Manuscript

Spatial and temporal distributions of polycyclic aromatic hydrocarbons in sediments from the Canadian Arctic Archipelago

Highlights• The polycyclic aromatic hydrocarbon (PAH) concentrations in Canadian Arctic sediments are low • The PAH input to sediments has remained constant throughout the last century • The PAHs in Canadian Arctic sediments mainly originate from natural sources Abstract 1. The concentrations of 23 polycyclic aromatic hydrocarbons (PAHs; 16 parent and 7 alkylated PAHs) in 113 surface marine sediment samples, 13 on-land sediment samples and 8 subsampled push cores retrieved from the Canadian Arctic Archipelago (CAA) were calculated. PAHs were extracted via accelerated solvent extraction (ASE) and quantified via gas chromatography-mass spectrometry (GC-MS). The sums of the concentrations 16 PAHs in the surface sediments ranged from 7.8 to 247.7 ng g -1 (dry weight [dw]) basis). The PAH inputs to the sediments have remained constant during the last century and agree with the results obtained for the surface sediments. Diagnostic ratios indicated that the PAHs in the CAA mainly originate from natural petrogenic sources, with some pyrogenic sources. Temporal trends did not indicate major source shifts and largely indicated petrogenic inputs.Overall, the sediments retrieved from the CAA have low PAH concentrations that are mainly natural.

spatial_and_temporal_distributions_of_polycyclic_aromatic_hydrocarbons_in_sediments_from_the_canadia

Introduction<!>PAH sources<!>Environmental fate<!>Study site<!>Materials and reagents<!>Sediment samples and chronology<!>PAH extraction and analysis<!>Quality control and quality assurance (QC/QA)<!>Data processing<!>FCM clustering analysis<!>Distribution of the PAHs in recent sediments<!>Historical trends of the PAH inputs into the sediments<!>Principal component analysis (PCA)<!>Risk assessment of the PAHs 602<!>Conclusions

<p>Within the context of climate change, the Arctic is undergoing major perturbations, and many studies have focused on sea ice conditions and navigability projections in the Arctic Ocean (Lasserre et al., 2010;Askenov et al., 2017). Because the summer sea ice extent is rapidly decreasing, leading to a seasonally ice-free Arctic Ocean, it has been speculated that maritime traffic could increase within the Canadian Arctic Archipelago (CAA); for example, the northwest passage could open to cargo transportation for a longer time period each year by the middle of the century (Lasserre et al., 2010;Smith and Stephenson, 2013). This shipping route connecting Asia and Europe is shorter than the Suez Canal, the Panama Canal or the Cape of Good Hope (Lasserre et al., 2010;Askenov et al., 2017). Hence, maritime companies have shown interest in traveling through the Arctic since this would allow time and fuel savings and consequent cost reductions. However, maritime traffic and oil exploration within the Arctic could also increase the anthropogenic pressure and pollution load in Arctic ecosystems (Jörrundsdóttir, 2014).</p><p>Shipping-related fuel combustion and anthropogenic activities are local sources of polycyclic aromatic hydrocarbons (PAHs), which constitute a wide class of organic compounds consisting of more than one benzene ring (C6H6) fused in a variety of conformations (AMAP, 2017;Haritash and Kaushik, 2009). Hundreds of these compounds are found in the environment, but since the mid-70s, 16 PAHs have been listed as priority environmental pollutants by the Environmental Protection Agency of the United States (US EPA) and are therefore closely monitored (Keith et al., 2014;Pampanin and Sydnes, 2017).</p><p>Hence, PAHs are pollutants of great concern, especially since the emissions originating in developed countries have decreased while those originating in developing countries have increased (Zhang and Tao, 2009;Wang et al., 2010).</p><p>PAHs are introduced to the environment via natural or anthropogenic sources (Lima et al., 2005;Foster et al., 2015;Chen et al., 2018), and anthropogenic activities are major sources of the PAHs occurring in the biosphere (Yanik et al., 2003;Morillo et al., 2008).</p><p>Seven PAHs have been classified as probably carcinogenic for humans by the International Agency for Research on Cancer (IARC) of the World Health Organization because of the reactivity of their metabolites (IARC, 1987). Inuit communities within the Arctic might be exposed to PAHs via the consumption of traditional foods such as mollusks (Rapinski et al., 2018). However, the Arctic is a region where the seafloor composition is the least studied and understood. Indeed, the vast majority of the channels within the CAA and their adjoining continental shelves and slopes exhibit a substantial knowledge gap regarding sediment composition and associated contaminants (Stein, 2008). Moreover, studies have focused on specific areas of the Arctic (e.g., Beaufort Sea or Baffin Bay; Yunker et al., 1995Yunker et al., , 2002aYunker et al., , 2002b;;Foster et al., 2015) or sites near anthropogenic influences (Boitsov et al., 2009a,b;Zaborska et al., 2011). To our knowledge, no complete baseline information on the PAHs in recent sediments or historic tendencies of the PAH inputs to sediments are available within the CAA.</p><!><p>Pyrogenic PAHs are produced during the incomplete combustion of organic matter, which includes forest and bush fires and fossil fuel and coal combustion (Chen et al., 2018;Yu et al., 2019). These PAHs are mainly emitted into the atmosphere and could occur either in the gaseous phase or bonded to the particulate phase (i.e., mineral dust and salt) (Tobiszewski and Namieśnik, 2012). Owing to their low vapor pressures, the majority of these semivolatile compounds undergoes repeated cycles of volatilization-deposition, travel across long distances and eventually end up in waters, soils and sediments via deposition (AMAP, 2017;Chen et al., 2018;Balmer et al., 2019). The PAHs produced at mid-latitudes could thus reach the Arctic, as shown by modeling studies (Wang et al., 2010;Sofowote et al., 2011). Petrogenic PAHs are hydrocarbons stemming from losses or seepage of oil and petroleum deposits, crude oil spills or rock weathering and are therefore naturally present in sediments and water bodies, and these PAHs are not of great concern because of their very low concentration (Lima et al., 2005;Pampanin and Sydnes, 2017;Chen et al., 2018). They are readily dispersed via water runoff, and since petrogenic PAHs are not directly emitted into the atmosphere, they are slightly influenced by long-range atmospheric transport (Pampanin and Sydnes, 2017).</p><!><p>In the Arctic, PAHs stemming from distant sources may enter the marine environment via river discharge, but atmospheric long-range transport is believed to be a significant input process (Sofowote et al., 2011;Yu et al., 2019). Regarding the environmental fate of atmospheric PAHs, Lammel et al. (2009) showed via a modeling approach that between 0.5% and 12.8% of the total environmental burden of PAHs might be stored within Arctic ecosystems (i.e., air, soil, vegetation and ocean) depending on the chosen gas/particle partitioning scenario. In addition to the already existing natural PAHs in Arctic soils and sediments, atmospheric deposition of PAHs originating from remote sources, in addition to new local sources such as ship traffic and oil exploration/exploitation, are PAH sources in the CAA (Balmer et al., 2019). PAHs are not easily degraded under natural conditions and are therefore slightly persistent (Pelletier et al., 2008;Haritash and Kaushik, 2009).</p><p>Photooxidation of PAHs is a chemical pathway of degradation, but biological degradation by bacteria, fungi and algae is accepted as the main process (Roslund et al., 2018;Haritash and Kaushik, 2009;Balmer et al., 2019). Because most vertebrates (e.g., fishes, birds and mammals) readily metabolize PAHs, they do not tend to experience biomagnification through the food chain (Xue et Warshawsky, 2005;Haritash et Kaushik, 2009;AMAP, 2017). However, PAHs could accumulate in benthic species such as clams and mussels (Balmer et al. 2019), and these organisms are an important food source for northern communities (Jörrundsdóttir et al., 2014;Rapinski et al. 2018). If deposited on land, PAHs could leach through soils or could be transported via water runoff and eventually reach aquatic ecosystems (Wang et al., 2007;Klungsøyr et al., 2010). They are poorly soluble in water because of their hydrophobicity and lipophilicity (Chen et al., 2018;Zhao et al., 2016).</p><p>Consequently, PAHs exhibit a relatively high affinity for suspended and particulate matter and sediments, which is why the latter are considered the main sink of PAHs (Chen et al., 2018).</p><p>Considering that PAHs are pollutants of interest that could be released by an increase in anthropogenic activities in the Arctic and that could accumulate in sediments and considering that the Arctic sediment PAH composition is not completely known, it is essential to determine the actual baseline. The aim of this study is to (1) characterize the modern spatial distribution patterns of the PAHs within the CAA, (2) determine the temporal trends of the PAH concentration based on 210 Pb-dated box cores collected across the CAA, and (3) establish the origin of PAHs (i.e., petrogenic or pyrogenic) according to two diagnostic ratios, namely, fluoranthene over the sum of fluoranthene and pyrene (Fla/ [Fla+Pyr]) and benz(a)anthracene over the sum of benz(a)anthracene and chrysene (BaA/[BaA+Chr]).</p><!><p>Since pyrogenic PAHs traveling via long-range atmospheric transport are deposited in soil or water and petrogenic PAHs mainly originate from rock weathering and oil reserves, they both occur in sediments, i.e., sediments are a sink for organic pollutants such as PAHs, and their affinity for fine and organic-rich sediments is well documented (Chiou et al., 1998;Gschwend and Hites, 1981;Stark et al., 2003). Letaïef (2019) reported that the sediments within the CAA ranged from clay (2 µm) to fine silt (4 to 8 µm). The total organic carbon (TOC) content is lower than 2% in most surface sediments retrieved from the CAA (e.g., Letaïef, 2019). More specifically, the Mackenzie Shelf and Delta and the Beaufort Sea/Canada Basin exhibit TOC values ranging from 0.5% to 1.9% (Yunker et al., 2011;Letaïef, 2019). The Queen Maud Gulf and the M'Clintock Channel, central CAA, exhibit relatively low TOC contents, with values ranging from 0.2% to 0.5 % (Letaïef, 2019).</p><p>Finally, the TOC content in Baffin Bay sediments ranges from 0.2% to 1.5% (Stein, 1991;Madaj, 2016). The CAA counts approximately 36500 islands and numerous waterways, straits, channels and sills formed by glacial action under past climate conditions (Melling et al., 2002;Michel et al., 2006). The recent sedimentary dynamics within the CAA are controlled by the sediment supply stemming from river discharge in the west and central CAA, whereas the east CAA is more influenced by sea ice and coastal erosion (Letaïef, 2019). Indeed, the Mackenzie River alone annually discharges approximately 420 km 3 /yr of sediments onto the continental shelf of the Beaufort Sea and is therefore a major source of continental PAHs (Wagner et al., 2011). Other small rivers exert a cumulative significant impact on the sediment load, such as the Coppermine River, the Ellice and Back Rivers and the Cunningham River, with a total contribution of approximately 110 km 3 /yr (Alkire et al., 2017). Once in the marine ecosystem, sediments are entrained throughout the archipelago by sea ice via suspension freezing and ice anchoring (Reimnitz et al., 1993;Darby et al., 2003Darby et al., , 2011;;Stein, 2008). Coastal erosion by seasonal sea ice, glaciogenic debris flows, meltwater plumes, mass movements along submarine canyons and sea lifting are other dominant sedimentary processes in glacial environments contributing to the dispersal of sediments across great distances within the CAA (Hiscott et al., 1989;Ó Cofaigh et al., 2003;Harris, 2012;Dowdeswell et al., 2015;Lai et al., 2016). Processes involving sea ice are mostly active during the sea ice formation season, and sediments are discharged elsewhere during summer melting (Darby et al., 2011). All of these processes contribute to the dispersion of PAHs originating from distant sources within the CAA before they become trapped in marine sediments.</p><!><p>All reagents were of analytical or high-performance liquid chromatography (HPLC) grade. Hexanes and dichloromethane were obtained from Anachemia, methanol was acquired from Millipore and 2-propanol was obtained from Fisher Chemicals. Nitric acid (HNO3) and hydrochloric acid (HCl) were acquired from VWR Analytical. Silica gel (technical grade, 70-230 mesh) and copper powder (<425 µm) were obtained from Sigma-Aldrich.</p><p>Diatomaceous earth (celite 566) was acquired from UCT Enviro-Clean. Standard reference material NIST-1944 was purchased from the National Institute of Standards and Technology (NIST). PAH Mix manufactured by AccuStandard was adopted for the generation of calibration curves, combined with an alkylated PAH homemade mix (2,6dimethylnaphthalene and 9,10-dimethylanthracene were obtained from Sigma-Aldrich, while 2,3,5-trimethylnaphthalene, 1-methtylnaphthalene and 3,6-dimethylphenanthrene were acquired from Fisher Chemicals). Before analysis, every sample was spiked with deuterated 1-methylnaphthalene and benz(a)anthracene purchased from Sigma. A mixture of deuterated naphthalene (Sigma-Aldrich), anthracene (Cambridge Isotope Laboratories) and perylene (Sigma-Aldrich) was added as an internal standard for quantification purposes. The targeted compounds in this study included 16 parent PAHs and 7 alkylated PAHs: naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, 2,6-dimethylnaphthalene, acenaphthylene, acenaphthene, 2,3,5-trimethylnaphthalene, fluorene, phenanthrene, anthracene, 1-methylphenanthrene, 3,6-dimethylphenanthrene, fluoranthene, pyrene, 9,10dimethylanthracene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-c,d)pyrene, dibenz(a,h)anthracene and benzo(g,h,i)perylene.</p><p>Silica gel was activated at 450°C for 2 h and stored in a desiccator. Copper powder, for sulfur removal from sediments, was activated as follows: in a Teflon tube, copper powder was covered with hydrochloric acid (HCl) 6N and shaken for 3 min. It was then rinsed with distilled water until a neutral pH was attained. The copper powder was then washed 3 times with both methanol and dichloromethane and was finally stored in a Teflon tube covered with dichloromethane.</p><!><p>A total of 126 sediment samples was analyzed in this study: 113 marine surface sediment samples and 13 terrestrial sediment samples collected from glaciers and rivers (Fig. 1). Additionally, 8 push cores were subsampled to determine temporal trends (Fig. 1). All samples were retrieved from box cores collected between 2016 and 2019 across a large area covering the Canadian Beaufort Sea to Baffin Bay during the ArcticNet summer expeditions onboard the Canadian Coast Guard Ship (CCGS) icebreaker Amundsen. Samples were collected from glaciers and rivers using the ship helicopter as CCGS Amundsen traveled through the CAA. All marine coring sites were targeted using high-resolution seismic profiles, which indicated that Late Holocene sediment accumulation was not influenced by mass wasting events (Montero-Serrano et al., 2016, 2017, 2018, 2019). Surface sediments (uppermost 0.5 to 1 cm) were collected with a spoon and stored in plastic bags (WhirlPack) at 4°C until further analysis. Push cores were collected by pushing a plexiglass tube (10-cm diameter) into sediments. The cores were stored at 4°C until further subsampling. Sediment subsamples were retrieved from the push cores from 0 to 10 cm at 1cm intervals. Age-depth models of the box cores have been previously published in Letaïef (2019), constructed using 210 Pb measurements combined with the constant rate of supply (CRS) model (a constant rate of the 210 Pb supply; Appleby and Oldfield, 1983). The age is reported as common era (CE) and before common era (BCE) hereafter. The sedimentation rates and ages at ~10 cm depth are provided in Table 1 and Fig. S1. Note that no dates are available for core AMD1902-05BC recovered in the Robeson Channel (Fig. 1). However, based on the sedimentation rate (4 cm ka -1 ) determined for a well-dated neighboring core (HLY03-05GC; Jennings et al., 2011), we inferred that the age at a core depth of ~10 cm is approximately 2.5 cal ka BP (550 BCE). All samples were sieved through a 150-µm Nitex® mesh using distilled water. The <150 µm sediment fraction was then stored in a 50-mL Falcon® tube successively rinsed with tap water and soap, distilled water, nitric acid (HNO3) 5% (3 times), distilled water and 2-propanol (3 times). The sediment samples were then frozen at -80°C for at least 12 h and freeze dried. The samples were finally crushed using an agate mortar. Aliquots weighing 5 g of these homogenized sediment samples were used for PAH analysis.</p><!><p>PAH extraction was conducted via accelerated solvent extraction (ASE) following the method developed by Choi et al. (2014). Briefly, 22-mL stainless-steel extraction cells were loaded as follows, from bottom to top: cellulose filter, diatomaceous earth, activated silica gel (5 g), activated copper (5 g), freeze-dried sediment sample (5 g) and diatomaceous earth to the top (Fig. S2). A PAH spike (1-methylnaphthalene-d10 and benz(a)anthracene-d12) was directly added onto the sediment sample. The cells were maintained open and protected from dust contamination at room temperature for 2 h to acclimate. Blanks were prepared similar to the samples but with no sediments. To confirm the accuracy of the method, 0.5 g of standard reference material NIST-1944 was processed as a sample. One blank, one standard reference material sample and one duplicate were tested for every 12 samples.</p><p>The addition of activated silica gel and copper directly to the cell enabled one-step extraction and cleanup. The extraction was performed with a Dionex ASE 200 system (Thermo Co., Sunnyvale, CA, USA). The temperature and pressure were set to 100°C and 1700 psi, respectively. The flush volume and purge time were set to 60% and 100 s, respectively. The extraction was performed with a mixture of hexane and dichloromethane (at a ratio of 4:1 v/v) and two static cycles of 5 min. Extracts were collected in 60-mL clear collection vials (previously rinsed with tap water and soap, distilled water, hexane and dichloromethane (4:1 v/v) mixture and propanol). The extracts were then evaporated to approximately 5 mL with a rotating evaporator and then evaporated to exactly 0.5 mL with a nitrogen stream at room temperature. In regard to standard material NIST-1944, the extracts were evaporated to exactly 1.5 mL.</p><p>PAH analysis was performed via gas chromatography (GC, Agilent Technologies 6850 series II; Santa Clara, CA, USA) coupled with mass spectrometry (MS, Agilent Technologies 5975B VL MSD) using total ion count (TIC). The injection was performed with an Agilent Technologies 6850 series autosampler. The capillary column used was an Rxi®-5 ms (30 m x 0.25-mm inner diameter (ID) x 0.25 µm ft, 5% diphenyl and 95% polysiloxane from RESTEK). The oven temperature was set as follow: 50°C for 2 min, 15°C/min until 275°C, held for 2 min, 15°C/min until 325°C, held for 15 min, and a postrun of 2 min at 300°C. A sample volume of 1 µL was injected at a temperature of 250°C under splitless injection with helium as the carrier gas at a flow rate of 1 mL/min.</p><!><p>The procedural blanks did not reveal contamination. Hence, the results were not blank corrected. The spike recoveries were 73.7% ± 15.0% for 1-methynaphthalene-d10 and 83.5% ± 23.2% for benz(a)anthracene-d12. Some samples did not meet the generally accepted QC/QA recovery criteria of 70% to 103%. All samples were hence spike corrected. The efficiency of the method was confirmed with standard reference material NIST-1944. The mean recoveries obtained are compared in Table S2 to those obtained by Choi et al. (2014), who developed the ASE method applied in this study. The method detection limit (MDL) for each PAH was calculated as suggested by the US EPA (Oblinger Childress et al., 1999). Briefly, 7 replicates of a spiked solution at the second lowest calibration point were analyzed.</p><p>Hence, the MDL was determined as 3.143 times (Student's t value for 6 degrees of freedom and the 99% confidence level) the standard deviation of the measured concentration for each compound. The MDL ranged from 8.1 ng/g for benz(a)anthracene to 155.9 ng/g for acenaphthylene (Table S2).</p><!><p>Prior to all multivariate analyses, the values below the detection limit (VBDLs) were imputed via multiplicative lognormal replacement with R package zCompositions (Palarea-Albaladejo and Martin-Fernandez, 2015). This method preserves the geometry of the compositional data while accounting for corresponding detection limit thresholds. However, -Fernandez, 2015). Next, a log-centered (clr) transform was applied to the data (Aitchison, 1990). This operation removed the statistical constraints on the compositional variables, such as the constant-unit sum, and enabled the valid application of classical (Euclidean) statistical methods to the compositional data (Aitchison, 1986(Aitchison, , 1990;;Montero-Serrano et al., 2010). We applied fuzzy c-means (FCM) clustering analysis (Kaufman and Rousseeuw, 2009) to identify samples possessing similar PAH compositions within the CAA. We adopted the Aitchison distance as a measure of similarity between the samples and the Ward method (minimum-variance method) for agglomerative calculation purposes. The FCM algorithm requires in-advance specification of the overall number of clusters to be detected. R package NbClust (Charrad et al., 2014) was employed to apply 23 indices and to determine the optimum number of clusters. The FCM clustering results are visualized in silhouette and principal coordinate ordination plots (Kaufman and Rousseeuw, 2009). The silhouette plot allows visualization of the robustness of clusters, where negative values indicate an incorrect and/or questionable assignment (Borcard et al., 2011). Moreover, principal component analysis (PCA) was performed using the PAH data and FCM clustering results with the goal of determining PAH associations with similar relative variation patterns (von Eynatten et al., 2003;Montero-Serrano et al., 2010). FCM clustering analysis was conducted with R software (R Core Team, 2020) using the compositions (van den Boogaart and Tolosana-Delgado, 2008) and cluster packages (Maechler et al., 2019). PCA was conducted with Compositional Data Package (CODAPAK) software (Comas and Thió-Henestrosa, 2011). Finally, the FCM clustering results and PAH concentrations were analyzed to produce distribution maps using Ocean Data View software (Schlitzer, 2015).</p><p>These maps were generated using a weighted-average gridding algorithm with a quality limit of 1.5. Diagnostic ratios of fluoranthene over the sum of fluoranthene and pyrene (Fla/[Fla+Pyr]) and benz(a)anthracene over the sum of benz(a)anthracene and chrysene (BaA/[BaA+Chr]) were considered to draw boxplots and discriminate PAH sources (i.e., pyrogenic vs petrogenic).</p><!><p>The FCM clustering analysis results indicate that there are three regional PAH clusters within the CAA (Figs. 2A and S3). Cluster 1 (PAH C#1, red) is mostly representative of the western CAA. Yunker et al. (1996) showed that the Mackenzie River imposed a dominant influence on the sedimentary dynamics in this region, namely, all their samples collected from the Mackenzie River, the Mackenzie Shelf and the Beaufort Sea Shelf edge clustered together, agree with our results. This cluster also seems to be dominated more by both medium molecular weight PAHs (MMW = 4-5 rings) and light molecular weight PAHs (LMW = 2-3 rings). This cluster exhibits a higher influence of high molecular weight PAHs (HMW= 6 rings) than the other clusters; however, HMW PAHs are minor contributors to the clusters (Fig. 2B). Clusters 2 (PAH C#2, green) and 3 (PAH C#3, blue) are slightly less defined. However, cluster 2 tends to be more represented by LMW PAHs and samples from the eastern CAA, while cluster 3 is more represented by LMW to MMW PAHs (and samples retrieved from the central CAA, as shown in Fig. 2B). The ordination diagram (Fig. 3A) and silhouette plot (Fig. 3B) show the robustness of the above clusters, revealing that the samples can be divided into 3 clusters: (1) western CAA, (2) eastern CAA and (3) central CAA. The negative values in Fig. 3B indicate an incorrect and/or questionable assignment. The second group contains the most samples (n=6) that might belong to another group, whereas only 3 samples of the other groups might be incorrectly assigned. Indeed, those samples show a greater mix between the 3 clusters, likely due to their PAHs assemblages and sources (Fig. S4).</p><!><p>The sums of the concentrations of the 16 priority PAHs (Σ16PAHs, dry weight [dw]) designated by the US EPA in the surface sediments of the CAA ranged from 7.8 to 247.7 ng g −1 with a mean value of 56.8 ng g −1 (Fig. 4B; Table 2). The highest values of Σ16PAHs are found in the western CAA, with values ranging from 15.7 to 247.7 ng g −1 and a mean value of 107.9 ng g −1 (Fig. 4B). According to the values reported in the literature (Table 2), it seems that the seafloors near the Mackenzie River are naturally rich in PAHs, which has been previously explained by the river discharge of the Mackenzie River (Yunker et al., 2002a). Indeed, the river alone discharges an annual flux of 49 ± 8 tons of both particulate and dissolved PAHs onto the Mackenzie Shelf (Yunker et al., 1991). Additionally, the western CAA is well known for three areas of natural hydrocarbon seeps (oil and/or gas) along the Mackenzie River, Delta and Shelf (Thomas, 1979;Janicki, 2001;Yunker et al., 2002a). The Beaufort Shelf and Mackenzie Shelf are also known for their pockmarks and mud volcanoes releasing fluids and gas into the water column (Blasco et al., 2006;Walsh et al., 2006). Certain on-land seeps, such as the Smoking Hills (Cape Bathurst, Northwest Territories), also release smoke clouds and fumaroles containing PAHs that are then transported by wind (Klungsøyr et al., 2010).</p><p>The western CAA also has a past history of petroleum exploration. Indeed, from the 1960s to the 1990s, extensive drilling was performed in the Mackenzie/Beaufort Basin, and many sumps for drilling wastes were built, which have leaked since their abandonment. Hence, accidental oil spills have occurred, but the total inputs are much lower than those from other sources (Klungsøyr et al., 2010). This natural hydrocarbon-rich background and petroleum exploration/extraction activity could explain the relatively high concentrations observed in the Mackenzie River area. Overall, the surface sediment concentrations of Σ16PAHs reported in other studies for the Canada Basin (58.9 -75.9 ng g −1 ; Ma et al., 2017) and the Chukchi Sea/Canada Basin (102 ng g −1 ; Yunker et al., 2011 and 8.8 -78.3 ng g −1 ; Ma et al., 2017) are comparable to those reported here for the western CAA but lower than those reported for the Mackenzie Shelf (495 -755 ng g −1 ; Yunker and MacDonald, 1995). In the two other regions of the CAA, the value of Σ16PAHs (dw) remains low: 7.8 to 100.7 ng g −1 with a mean value of 40.8 ng g −1 for the eastern CAA and 9.3 to 79.2 ng g −1 with a mean value of 35.5 ng g −1 for the central CAA (Fig. 4B). These results are similar to other Arctic regions, such as the Kara Sea, the Barents Sea or the Svalbard coast, but higher than the reported values for the Makarov Basin or the Central Arctic Ocean (Table 2). Dong et al. (2015) pointed out a decreasing tendency of the PAH concentration with increasing latitude, which could explain why higher PAH concentrations occur in the CAA than those at other northerly sites (e.g., the Canada and Makarov Basins). Another known hydrocarbon seep is located in the eastern CAA along Baffin Island at Scott Inlet (Levy, 1978), but the sample collected near this area does not exhibit a higher concentration than those exhibited by the other samples collected from the eastern CAA. The values of Σ16PAHs (dw) for the terrestrial samples (i.e., glacier and river samples) are consistent with those for the marine sediments, with values ranging from 22.1 to 108.8 ng g −1 and a mean value of 71.1 ng g −1 (Fig. 4B), which are fairly low values. However, a major exception of 356.1 ng g −1 is found for the sample collected near the Sydkap Glacier, located approximately 60 km west of the Grise Fiord, the northernmost Inuit community in the CAA (Fig. 4A). It is the highest result among all the samples. No anthropogenic activities or historical accidental spills have been recorded in this area. However, Ellesmere Island is known for its numerous coal deposits (Ricketts and Embry, 1984;Kalkreuth, 2004;Harrison et al., 2011). Coal layers outcrop along the Stenkul Fiord, literally meaning the Coal Fiord, which is part of the Eureka Sound Group coals and occurs approximately 60 km north of the Grise Fiord (Kalkreuth et al., 1996). Thus, such outcropping along the watershed and river shores surrounding the Sydkap area could explain the high PAH concentrations observed in this area. In general, the inputs of PAHs during the last century seem to have remained relatively constant. The value of Σ16PAHs (dw) stays within the general concentrations observed in the surface sediments of the CAA, and none of them is higher than the maximum value of 246.6 ng g −1 encountered in the marine sediments in this study. In regard to the surface sediments, the highest sums are found in the western CAA, especially for AMD1603-408, with a mean value of 156.2 ng g −1 . Very low values are obtained for AMD1902-05 (a mean value of 20.0 ng g −1 ), a core collected near Alert (Nunavut, Canada) at the very extreme north of Ellesmere Island. This is consistent with the general trend of the decreasing PAH concentration in the sediments with increasing latitude, since remote locations are far from industrial activities, and the PAH inputs stemming from remote sources are only influenced by long-range atmospheric transport (Dong et al., 2015;Balmer et al., 2019). Additionally, it should be noted that in marine sediments, trends commonly tend to be less defined mainly because of ocean perturbations (e.g., currents or ship traffic; AMAP, 2017). Therefore, variations due to worldwide fluctuations might indicate a time shift, especially because processes involving ocean currents could last years, whereas atmospheric processes are more common on a daily scale (Klungsøyr et al., 2010). The PAH inputs occurring during the last century are therefore relatively stable. In sediments collected from the Barents Sea, Boitsov et al. (2009b) measured a 10-fold increase in the PAH concentration in marine sediments corresponding to the 1910-1940 period, while the inputs prior to 1850 remained constant.</p><!><p>After approximately 1980, the concentration slightly decreased. This general decreasing tendency has been associated with a reduction in the worldwide PAH emissions since 1995 (Shen et al., 2013). However, we do not observe this situation in our results. Additionally, Foster et al. (2015) studied pre-1900 and post-1900 sediments retrieved from the Baffin Bay area. The majority of their results is within a factor of 10 from those obtained for the post-1900 sediments, indicating a constant PAH concentration over time, which is consistent with our results. preindustrial sediments (e.g., Yunker et al., 2002b;Foster et al., 2015). Typically, Fla/(Fla+Pyr) ratios below 0.4 are representative of petrogenic PAHs, those between 0.4 and 0.5 are representative of fossil fuel combustion and those above 0.5 are representative of biomass combustion (Yunker et al., 2002b). In regard to the ratio benz(a)anthracene over the sum of benz(a)anthracene and chrysene (BaA/[BaA+Chr]), a ratio below 0.2 indicates a petrogenic source, a ratio ranging from 0.2 and 0.35 indicates a mixed source (i.e., either fossil fuel or biomass combustion) and a ratio above 0.35 indicates a pyrogenic source (Yunker et al., 2002b). In regard to the surface samples retrieved from the western CAA, the Fla/Fla+Pyr values ranged from 0.14 to 0.56, and the BaA/(BaA+Chr) values ranged from 0.13 to 0.33, indicating a mainly petrogenic origin (Fig. 6A-B) with a small influence of mixed combustion origins. This is in agreement with the results reported for the Mackenzie River basin (Yunker et al., 2002a(Yunker et al., , 2011)), suggesting that erosion of the organic-rich rocks of the Devonian Canol formation in the lower Mackenzie River valley contributes large amounts of petrogenic hydrocarbons to the shelf. Additionally, the hydrocarbon sources of the Mackenzie Shelf and Canada Basin sediments exhibit a strong signal originating from vascular plants and petrogenic input that is likely to overwhelm a possible combustion signal, leading to a petrogenic signal (Yunker et al., 2011). In the western Arctic Ocean, Ma et al.</p><p>(2017) also reported a mixed petrogenic and pyrogenic source for the PAHs in the surface sediments of the Chukchi Sea and Canada Basin, in line with our results. In the global Arctic</p><p>Ocean, it has been reported that the natural background signature of petrogenic PAHs seemed to dominate the signal in sediments (Yunker et al., 2011).</p><p>Similarly, the surface samples collected from the central CAA appeared to largely exhibit petrogenic signatures, as the Fla/(Fla+Pyr) values ranged from 0.13 to 0.50 (Fig. 6A).</p><p>However, the BaA/(BaA+Chr) values ranged from 0.20 to 0.6, suggesting a mixed source/pyrogenic source. Regarding the surface samples retrieved from the eastern CAA, the Fla/(Fla+Pyr) values ranged from 0.13 to 0.67, and the BaA/(BaA+Chr) values ranged from 0.20 to 0.50, indicating a well-mixed origin from both petrogenic and pyrogenic sources (Fig. 6A-B). Thus, the central and eastern CAA exhibit a greater pyrogenic influence than the western CAA. It was previously established that the diagnostic ratios for sediments from remote areas might reflect a more pyrogenic influence because the main PAH sources are atmospheric deposition followed by sedimentation (Tsapakis et al., 2003;Tobiszewski and Namieśnik, 2012), which could explain our results. Ma et al. (2017) and Zhao et al. (2016) also reported that the PAHs in samples retrieved from the Arctic Ocean and the Makarov Basin originated from a mixture of petroleum and biomass combustion. However, although diagnostic ratios are useful for discriminating the origins of PAHs, they should be interpreted with caution due to different environmental processing of the isomers during transport processes (e.g., Galarneau, 2008;Yunker et al., 2002aYunker et al., ,b, 2011)). For example, degradation and/or transformation occurring during atmospheric processes and transport through the water column of the less stable fluoranthene and benz(a)anthracene might contribute to bias in old sediments (Yunker et al., 2002a(Yunker et al., , 2002b(Yunker et al., , 2011)). Tobiszewski and Namieśnik (2012) also suggested that the ratio of Fla/(Fla+Pyr) was more conservative than other ratios (e.g.,</p><p>BaA/[BaA+Ch] and anthracene over the sum of anthracene and phenanthrene;</p><p>Ant/[Ant+Phe]) during atmospheric photoreactions.</p><p>Pyrogenic PAHs stemming from anthropogenic combustion have been widely</p><p>detected in atmospheric samples retrieved from remote areas in the Arctic, such as Alert (Yu et al., 2019). Modeling studies have shown that long-range atmospheric transport of PAHs from urban areas to remote Arctic regions occurs (Chen et al., 2018). More specifically, air mass trajectory modeling performed at Alert (Nunavut, Canada) has suggested that the atmospheric PAHs in this region mainly originate from Eurasia, North Europe and North America, while East China is a minor contributor (Wang et al., 2010). Hence, the main anthropogenic PAH source in the Canadian Arctic is the atmospheric deposition of PAHs stemming from worldwide hydrocarbon consumption (Klungsøyr et al., 2010;Yunker et al., 2011). It has also been proposed that local anthropogenic sources are actually negligible compared to deposition from remote sources (e.g., Rose et al., 2004;Wang et al., 2010).</p><p>In addition, forest fires are also a contributor to atmospheric pyrogenic PAHs, with an annual budget of approximately 9 tons in the Canadian Arctic, and pyrogenic PAHs have become more frequently detected in atmospheric samples since 2005 (Klungsøyr et al., 2010;Yu et al., 2019). Ma et al. (2017) found a pyrogenic influence in deep ocean sediments of the central Arctic Ocean stemming from forest fire events. These events could explain the pyrogenic signal observed in the central and eastern CAA, especially since forest fire events had increased in Canada. For example, the average annual burned area was approximately 1 million hectares in the early 1920s and reached 2 million hectares in the 2010s, with a maximum area of 2.75 million hectares in the 1990s (Wildland Fire Management Working Group, 2013). In Frobisher Bay, near Iqaluit, it appears that anthropogenic activities could locally contribute to pyrogenic PAHs in the bay (Fig. S6). The city, home to more than 7700 people, produces its electricity via imported diesel fuel (Government of Canada, 2017). In 2017, its main greenhouse gas emission sectors were the transportation, industry and electricity sectors (Government of Canada, 2017), all contributing to a pyrogenic signature.</p><p>Waste burning in Iqaluit is also a common practice (Giroux, 2014), and episodic landfill fire events might contribute to a local PAH input: in 2010, a landfill fire lasted 6 weeks (Harvey, 2018), while another major fire occurred between May and September in 2014 (Weichenthal et al., 2015). Finally, the petrogenic signature recorded near Sydkap Glacier confirms a coal origin (Fla/[Fla+Pyr] = 0.16) rather than an anthropogenic source, as previously reported (Fig. S6).</p><p>Regarding the core samples, they all seem to exhibit a mainly petrogenic signature combined with mixed sources: the Fla/(Fla+Pyr) values range from 0.13 to 0.88, and the BaA/(BaA+Chr) values range from 0.16 to 0.50 (Figs. 6A-B and S5). These values are consistent with the results obtained for the surface samples, except for the eastern CAA core samples, in which the pyrogenic side is more abundant. Overall, it seems that the PAH sources over time have remained relatively constant since the core sample results are consistent with the surface sample results but feature a greater pyrogenic influence. Finally, the terrestrial samples exhibit Fla/Fla+Pyr values ranging from 0.14 to 0.38, which indicate a petrogenic source, whereas the BaA/(BaA+Chr) values range from 0.12 to 0.50, indicating a relatively wide range of sources, from petrogenic to mixed/pyrogenic sources. Since only 30% (n=4) of the terrestrial samples attained a BaA/(BaA+Chr) value compared to 85% (n=11) of the samples in regard to Fla/(Fla+Pyr), this pyrogenic influence might not be representative of all the terrestrial samples. However, if it is representative of the 4 samples, it might indicate a more direct connection between atmospheric pyrogenic PAHs and soils since the sedimentary processes do not occur and that soils are mainly influenced by atmospheric deposition (Mostert et al., 2010).</p><p>Finally, the results given by the diagnostic ratios should be considered with care given the unknown effect of environmental processes occurring between the emission and deposition of the PAHs (Tobiszewski and Namieśnik, 2012). Katsoyiannis and Breivik (2014) illustrated that basic conditions, such as distance from the sources and ambient temperature, have a significant influence on the molecular ratios. Additionally, diagnostic ratios established for a certain type of sediment in urban area might not be directly applicable to remote sediments; indeed, old basin sediments in the Artic Ocean are depleted in reactive and LMW PAHs from combustion related sources, and only fluoranthene, pyrene and PAHS with molecular weights greater than 252 could provide usable source ratios (Yunker et al., 2011). Hence, the contradictory results obtained in this study do not mean that they are wrong: a combination of degradation during atmospheric processes and the remote locations might explain why the Fla/(Fla+Pyr) and BaA/(BaA+Chr) values are not exactly the same (Tobiszewski and Namieśnik, 2012).</p><!><p>PCA based on the Σ16PAH data for all samples (surface, core and terrestrial samples) revealed that PC-1 (24% of the total variance) was positively correlated with 9,10-dimethylanthracene, 3,6-dimethylphenanthrene and pyrene, whereas PC-2 (15% of the total variance) was positively correlated with chrysene, 1-methylphenanthrene and pyrene (Fig. 7A). Finally, PC-3 (14% of the total variance) was positively correlated with 1-methylnaphthalene, 2-methylnaphthalene and acenaphthene (Fig. 7B). Parent PAHs are typically more closely associated with combustion processes, while alkylated PAHs are generally derived from petrogenic PAHs (Yunker and Macdonald, 1995;Lima et al., 2005, Balmer et al., 2019). Hence, each score indicated a mostly petrogenic influence.</p><p>Additionally, the PC-1 and PC-3 scores were negatively correlated with at least one parent PAH composed of four rings (e.g., pyrene or chrysene). Unsubstituted PAHs containing four to six rings are mainly associated with combustion sources (Laflamme and Hites, 1978).</p><p>Hence, PCA confirms a mainly petrogenic influence with a small pyrogenic contribution to the PAHs occurring in the surface, core and terrestrial sediments within the CAA.</p><p>Overall, both diagnostic ratios support the predominant petrogenic nature of the PAHs in the surface sediments of the CAA (Thomas and MacDonald, 2005;Yunker et al., 2011;Foster et al., 2015;Yu et al., 2019). These petrogenic sources are presumably derived from hydrocarbon seeps, weathering of organic-rich rocks, and coastal terrestrially derived material. Strong pyrogenic influences are observed in the central and eastern CAA and are likely due to forest fire events plus long-range atmospheric transport and deposition of PAHs originating from distant sources. Other pyrogenic influence might occur because the remote locations are mainly influenced by deposition of atmospheric pyrogenic PAHs, as previously mentioned. Our results are comparable to those of previous studies pointing to petrogenic sources of the PAHs in the surface sediments of the Mackenzie River/Delta, Beaufort Sea, Nansen Basin, and North Baffin Bay (Yunker et al., 2011, Foster et al., 2015) and mixed sources in the surface sediments of North Baffin Bay, Greenland Sea and north Barents Sea (Yunker et al., 2011;Foster et al., 2015).</p><!><p>The potential ecological risk of the PAHs in sediments can be determined based on 603 guideline values, such as the effects range-low (ERL, the probability of adverse biological 604 effects is <10%) and effects range-median (ERM, the probability of adverse biological 605 effects is >50%) values, as proposed by Long et al. (1995). The PAH content ranges in the 606 CAA sediments are almost all below the ERL and ERM values (Table 3), indicating that the 607 measured PAHs pose a low ecological risk to the benthic organisms or other organisms living 608 near the sediments. Only fluorene might be an exception and would require greater attention. 609</p><p>The sample with a result of 23.7 ng g -1 is located in the Amundsen Gulf. This result excludes 610 the next highest value for fluorene, 15.9 ng g -1 , and all samples are therefore below the above 611 ERL and ERM values. 612 613 Long et al., 1995. 615 616</p><!><p>This study provides a robust baseline record of the PAHs in surface and core marine sediments and on-land sediments retrieved from the CAA. The results of this research yield the following generalizations and conclusions: 2. The sums of the concentrations of the 16 priority PAHs designated by the US EPA in both the marine and terrestrial sediments of the CAA are fairly low and comparable to other sediment levels reported for Arctic remote regions. Indeed, our results reveal Σ16PAH values ranging from 7.8 to 247.7 ng g −1 for the total CAA, while the terrestrial sediments exhibit values ranging from 23.1 to 108.8 ng g −1 . Overall, regarding Σ16PAHs, the different regions are classified as follows: central CAA < eastern CAA < terrestrial sediments < western CAA.</p><p>Additionally, the values of Σ16PAHs in the sediments are all below the ERL and ERM guidelines, except for a single sample retrieved from the Amundsen Gulf, which exhibits a fluorene content above the ERL value.</p><p>3. The inputs throughout the last century have remained relatively stable and below the maximum sum obtained for the surface sediment samples, with the Σ16PAH values in the core samples ranging from 8.1 to 191.1 ng g −1 with a very low Σ16PAH value in the northernmost core located in the Robertson Channel.</p><p>4. The diagnostic ratios of Fla/(Fla+Pyr) and BaA/(BaA+Chr), in addition to the PCA results of the PAH data, suggest that the PAHs mainly have a natural petrogenic origin, but the central and eastern CAA areas also contain PAHs originating from both fossil fuel and biomass burning, likely because of the increase in forest fire events in northern Canada in recent decades and long-range atmospheric deposition of PAHs stemming from urban areas located further south. However, diagnostic ratios should be used with care when applied to sediments from remote locations: PAHs might undergo major environmental processes before their deposition, which could lead to bias and complications in interpreting diagnostic ratio values.</p>

ChemRxiv

Design and synthesis of selective, small molecule inhibitors of coactivator-associated arginine methyltransferase 1 (CARM1)\xe2\x80\xa0\xe2\x80\xa1

Coactivator-associated arginine methyltransferase 1 (CARM1) is a type I protein arginine methyltransferase (PRMT) that catalyzes the conversion of arginine into monomethylarginine (MMA) and further into asymmetric dimethylarginine (ADMA). CARM1 methylates histone 3 arginines 17 and 26, as well as numerous non-histone proteins including CBP/p300, SRC-3, NCOA2, PABP1, and SAP49, while also functioning as a coactivator for various proteins that have been linked to cancer such as p53, NF-\xce\xba\xce\xb2, \xce\xb2-catenin, E2F1 and steroid hormone receptor ER\xce\xb1. As a result, CARM1 is involved in transcriptional activation, cellular differentiation, cell cycle progression, RNA splicing and DNA damage response. It has been associated with several human cancers including breast, colon, prostate and lung cancers and thus, is a potential oncological target. Herein, we present the design and synthesis of a series of CARM1 inhibitors. Based on a fragment hit, we discovered compound 9 as a potent inhibitor that displayed selectivity for CARM1 over other PRMTs.

design_and_synthesis_of_selective,_small_molecule_inhibitors_of_coactivator-associated_arginine_meth

Introduction<!>Results and Discussion<!>Conclusions

<p>Coactivator-associated arginine methyltransferase 1 (CARM1, also known as PRMT4) is a type I protein arginine methyltransferase (PRMT) along with PRMT1, PRMT3, PRMT6 and PRMT8, which are responsible for converting arginine into monomethylarginine (MMA) and further into asymmetric dimethylarginine (ADMA).1 CARM1 methylates histone 3 arginines 17 and 26,2, 3 as well as numerous non-histone proteins including CBP/p300, SRC-3, NCOA2, PABP1, SmB, HuR, HuD, CA150, SAP49, and U1C.1 Therefore, it plays a role in cellular processes such as transcriptional activation,4 RNA splicing,4, 5 cellular differentiation,6 cell cycle progression7 and DNA repair.8 It has been shown that the enzymatic activity of CARM1 is essential for most of its in vivo functions.9 CARM1 functions as a coactivator for various proteins that have been linked to cancer including p53, NF-κβ, β-catenin, E2F1 and steroid hormone receptor ERα.1, 10–12 Overexpression of CARM1 in several human cancers including breast,7 colon,13 prostate14 and lung15 cancers has been reported. Thus, CARM1 is a potentially attractive therapeutical target for cancer and as a result there have been numerous studies directed toward the discovery of small molecule CARM1 inhibitors.16, 17</p><p>Several high throughput screening (HTS) campaigns resulted in the identification of pyrrazole-amide as well as benzo[d]imidazole CARM1 inhibitors.18, 19 These initial reports were followed by structure-activity relationship (SAR) studies, which led to the discovery of small molecule inhibitors 1 and 2 with IC50 values around 30 nM. These inhibitors displayed selectivity for CARM1 over PRMT1 and PRMT3 while the selectivity over other PRMTs was not reported (Figure 1).20–23 We have recently reported a type I PRMT chemical probe, MS023 (3), which is potent against all type I PRMTs, but inactive against type II and type III PRMTs as well as other methyltransferases.24 A series of adenosine-based CARM1 inhibitors with high potency and selectivity over PRMT1 and PRMT6 was also published.25 Herein we report the discovery of selective CARM1 inhibitors and describe their design, synthesis and biological evaluation.</p><!><p>The alkyl-diamino tail, shown to be bound in the substrate-binding site of CARM1,21 is the shared feature of inhibitors 1-3 (Figure 1) as well as of a recently published PRMT6 inhibitor.26 In a recent study, a commercially available, diverse fragment library of compounds mimicking this alkyl-diamino tail were tested against PRMT6 and compound 4 was reported as a PRMT6 inhibitor with IC50 of 300 ± 40 nM (Figure 2).27 Compound 4 was 3- and 7-fold less potent for CARM1 (IC50 = 1,000 ± 40 nM) and PRMT8 (IC50 = 2100 ± 200 nM), respectively. It was not very potent against PRMT1 and PRMT3 (>40-fold less potent) and did not inhibit other methyltransferases. We sought to discover potent CARM1 selective inhibitors by using the fragment hit 4 as a starting point. Our initial SAR studies resulted in compounds 5 and 6 as promising CARM1 inhibitors (Figure 2). Compound 5 shares the (piperidinyl)ethan-1-amine core of 4, but it is connected to the phenyl group via a methylene-amine (-CH2NH-) linker instead of methylene (-CH2-) linker. It was found to be 10-fold more potent for CARM1 (IC50 = 471 ± 36 nM) over PRMT6 (IC50 = 4,564 ± 1210 nM), exhibiting some selectivity (Figure 2). In addition, it had no appreciable inhibitory activity against PRMT1, PRMT3 and PRMT8 as well as PRMT5 and PRMT7 (IC50 > 50,000 nM for all). On the other hand, compound 6 contains an (azetidinyl)ethan-1-amine core and –CH2O- connection to the aryl ring (Figure 2). This inhibitor displayed good potency for CARM1 (IC50 = 144 ± 37 nM) and was less than 10-fold selective over PRMT6 and PRMT8 (IC50 = 1,079 ± 75 and 1,200 ± 70 nM, respectively) while it was inactive against other PRMTs.</p><p>Since inserting a nitrogen between the piperidine and benzyl groups of compound 4 (compound 5, Figure 2) resulted in some selectivity for CARM1 over PRMT6, we further explored this scaffold and synthesized derivatives of 5 by adding substituents on the phenyl ring (Table 1). We mainly investigated meta-substitution based on the encouraging potency of compound 6 for CARM1. Among the meta-fluoro (7), meta-chloro (8) and meta-bromo (9) derivatives, 9 displayed the best potency for CARM1 with IC50 of 94 ± 23 nM as well as selectivity over PRMT6 (around 20–fold, IC50 = 2,160 ± 68 nM). The electron-withdrawing trifluoromethyl (10), or electron-donating triflourometoxy (11) substitution did not result in a more potent or selective inhibitor than 9. The phenyl-substituted derivative (12) reversed the selectivity in favour of PRMT6 (IC50 = 120 ± 13 nM) over CARM1 (IC50 = 330 ± 43 nM).</p><p>We then focused our attention on the (azetidinyl)ethan-1-amine sca1fold represented by compound 6 (Figure 2). Since this scaffold with an oxygen linker did not show good selectivity over either PRMT6 or PRMT8, we utilized the methylene-amine (-CH2NH-) linker as in the piperidinyl series and synthesized substituted phenyl derivatives 13-18 (Table 2). While there was an overall increase in potency of these inhibitors as compared to compounds 7-12 for CARM1, the potency for PRMT6 was even greater in comparison and thus, the selectivity over PRMT6 suffered significantly. For example the meta-chloro (14) and meta-bromo (15) derivatives were potent for CARM1 with IC50 of 50 ± 14 and 67 ± 9 nM, respectively. However, 14 and 15 displayed high potency for PRMT6 with IC50 of 133 ± 8 and 249 ± 22 nM respectively, resulting in only 3 to 4-fold selectivity for CARM1 over PRMT6.</p><p>The synthetic route for the preparation of compounds 7-12 is shown in Scheme 1. The synthesis started with a nucleophilic displacement reaction between 4-amino-1-Boc-piperidine (19) and meta-substituted benzyl bromides (20). The resulting diamines were then treated with methanolic HCl to remove the tert-butoxy carbamate group. The reductive amination of amines 21 with N-Boc-(methylamino)acetaldehyde (22), followed by a deprotection reaction, afforded the desired compounds (5 and 7-12). A similar synthetic route was employed for the synthesis of compounds 13-18 starting with 3-amino-1-Boc-azetidine (see supporting information for detailed procedures).</p><p>Among all the derivatives synthesized, compound 9 emerged as the best inhibitor with good potency and around 20-fold selectivity toward CARM1 over PRMT6. In addition, we have tested Inhibitor 9 against other PRMTs in biochemical assays and found that it showed excellent selectivity for CARM1 over other type I PRMTs, PRMT1, PRMT3 (IC50 > 50,000 nM) and PRMT8 (IC50 = 9,200 ± 500 nM) as well as the type II and type III PRMTs, PRMT 5 and 7 (IC50 > 50,000 nM).</p><p>To assess the mechanism of action (MOA) of inhibitor 9, we evaluated the effect of the peptide substrate and cofactor S-adenosyl-L-methionine (SAM) concentrations on IC50 values of 9 against CARM1. As shown in Figure 3, increasing the peptide substrate or SAM concentrations had no effect on the IC50 values of 9 against CARM1, suggesting that this inhibitor is noncompetitive with both the cofactor SAM and peptide substrate. It has been previously reported that active site binding inhibitors can display noncompetitive behavior in MOA studies.28, 29 We and others have observed this phenomenon with PRMT inhibitors.24, 26 For example, MOA studies of MS023 (3) suggested that the inhibitor was noncompetitive with both SAM and peptide substrate while the X-ray crystal structure of MS023 in complex with PRMT6 clearly showed that it occupies the substrate binding pocket. Therefore, based on the literature precedents, it is very likely that inhibitor 9 interacts with the substrate binding site of CARM1 to assert its inhibitory effect.</p><!><p>Starting from a reported fragment hit (4), a PRMT6 inhibitor, we discovered compound 9, a potent and selective inhibitor of CARM1, through a SAR study. In biochemical assays, 9 displayed high potency (IC50 = 94 ± 23 nM) for CARM1 and was around 20-fold selective for CARM1 over PRMT6 and highly selective over PRMT1, PRMT3, PRMT8 as well as PRMT5 and PRMT7. We believe that inhibitor 9 could be a useful tool for studying the role of CARM1 in health and disease. We anticipate that the work described here will facilitate further development of CARM1 selective inhibitors and chemical tools.</p>

PubMed Author Manuscript

Contact Line Pinning Is Not Required for Nanobubble Stability on Copolymer Brushes

Whereas nanobubble stability on solid surfaces is thought to be based on local surface structure, in this work, we show that nanobubble stability on polymer brushes does not appear to require contact-line pinning. Glass surfaces were functionalized with copolymer brushes containing mixtures of hydrophobic and hydrophilic segments, exhibiting water contact angles ranging from 10 to 75\xc2\xb0. On unmodified glass, dissolution and redeposition of nanobubbles resulted in reformation in mostly the same locations, consistent with the contact line pinning hypothesis. However, on polymer brushes, the nucleation sites were random, and nanobubbles formed in new locations upon redeposition. Moreover, the presence of stable nanobubbles was correlated with global surface wettability, as opposed to local structure, when the surface exceeded a critical water contact angle of 50 or 60\xc2\xb0 for polymers containing carboxyl or sulfobetaine groups, respectively, as hydrophilic side chains. The critical contact angles were insensitive to the identity of the hydrophobic segments.

contact_line_pinning_is_not_required_for_nanobubble_stability_on_copolymer_brushes

<p>Surface nanobubbles are small, stable gas pockets that may be present at a solid–liquid interface, with dimensions on the order of tens of nanometers in height and hundreds of nanometers in diameter.2,3 The presence of gas has been confirmed by a variety of characterization methods, including atomic force microscopy,1,4 attenuated total reflectance infrared spectroscopy,5 total internal reflection microscopy (TIRF),6 and others.7–12 Nucleation of surface nanobubbles generally occurs through supersaturation of gas at a liquid–solid interface, through either direct immersion of a surface in water4 or solvent exchange from a solvent with higher gas solubility.13 This process is followed by growth from dissolved gas controlled by either gas saturation or temperature.14–16 For a small radius of curvature, the high Laplace pressure would predict that very high supersaturation ratios would be required for stability, yet despite Laplace pressures estimated at tens of megapascals, surface nanobubbles have been recorded to be stable for days at a time,2 thus appearing to defy conventional interfacial thermodynamics.3,17,18</p><p>One of the key hypotheses for nanobubbles' exceptional stability is pinning of nanobubble contact line due to surface topography or the presence of contaminants.18 Pinning, or fixation of the contact line by a topological defect, is a commonly found when liquid is added to an air–solid interface through either liquid transfer or nucleation.19–21 Under supersaturated conditions,22 one might expect a coarsening process, as observed for bubbles dispersed in a bulk liquid, where large (less-curved) bubbles grow at the expense of small (more-curved) bubbles and there is no stable bubble size. However, if a bubble's contact line is pinned, then bubble growth increases curvature. Experimentally, it has been rigorously shown that nanobubbles can be stable under these conditions.22,23</p><p>On solid surfaces, there is considerable experimental evidence to support the pinning hypothesis.16,24 For example, whereas surface-bound nanobubbles would be expected to be spherical caps, irregular shapes have also been imaged that could be explained by topography in the underlying surface structure.1 Pinning would also help to explain apparent discrepancies in observations of nanobubble stability on various solid surfaces. In general, nanobubbles have been found to be stable on hydrophobic and unstable on hydrophilic surfaces;10 however, various groups have reported the presence of stable nanobubbles on gold surfaces with receding contact angles ranging from 107 (hydrophobic) to 15° (hydrophilic).25,26 Many studies have shown that whereas alkylated silica serves as a convenient surface for nanobubble study, nanobubbles have also been detected on bare glass.6 The effect of surface roughness is also debatable: Although defect sites appear to promote stability, nanobubbles have also been found on hydrophilic, flat mica.27 In addition, nanobubbles have been observed on highly ordered pyrolytic graphite (HOPG) but not amorphous glassy carbon.28 Interestingly, McKinley and coworkers showed that nanobubbles could be confined to hydrophobic domains on a photopatterned polymer surface,29 but to our knowledge no systematic study has been performed describing the effect of copolymer structure on nanobubble stability.</p><p>In this work, we present evidence that nanobubbles can indeed be stabilized on homogeneous dynamic copolymer brush surfaces, even in the absence of strong pinning sites. Because macromolecules can continuously reconfigure their conformations to balance osmotic pressure and chain entropy, structural defects necessary for pinning are transient, if present at all. Thus, whereas nanobubbles repeatedly form in the same locations on silane-functionalized glass (presumably at defect sites that promote strong pinning), here we show that on polymer surfaces they form in random locations with no correlation to previous nucleation events. The hydrophobicity of the brushes was systematically adjusted by synthesizing copolymers with a controlled ratio of hydrophilic to hydrophobic side chains. Interestingly, we found that a critical hydrophobicity (as determined by water contact angle) was required for nanobubble stability on these polymer surfaces, the value of which depended on the underlying polymer structure. Above this contact angle, hundreds of nanobubbles were imaged over a 1000 μm2 area, but below this angle no nanobubbles were found. Even partially wetting surfaces, in some cases with receding contact angles <50°, were found to stabilize nanobubbles. Thus, in contrast with typical solid surfaces that possess defect sites that are unpredictable and difficult to control, the conditions under which nanobubbles are stable on copolymer brushes are repeatable and tunable. The ability to understand and control surface nanobubble stability will have an impact on the design of materials for a wide range of important applications, including froth flotation,30–33 surface cleaning,34,35 hydrodynamic boundary slip,36 photoacoustic imaging,37 enhanced adhesion, and nonfouling surfaces.38,39</p><p>To obtain fine control and consistency over surface properties, polymers were grown directly from surfaces using atom transfer radical polymerization (ATRP). ATRP utilizes a copper-bromide equilibrium to eliminate termination steps, thereby assuring a copolymer brush with consistent composition. Polymers grown on a surface by ATRP have been shown to be consistent in composition and molecular weight dispersity with those found in the same polymers synthesized in bulk, so accurate predictions can be made about the resultant surface properties.40 To allow polymerization from the surface, cleaned glass slides were functionalized with 3-aminopropyltriethoxysilane (APTES) and α-bromoisobutyryl bromide (BIBB) in successive steps, resulting in the formation of surfaces with θ ≈ 60 and 75°, respectively. Successful conjugation was also confirmed by ellipsometry measurements of analogous reactions on a silicon wafer, which showed successive increases in surface thickness of 2.0 to 3.5 and 1.3 nm for APTES and BIBB, respectively. Each advancing contact-angle measurement was run in at least three locations on each surface, where error was ~5° for most surfaces.</p><p>Once the surfaces were prepared, nanobubbles were formed by a solvent-exchange method on functionalized glass surfaces and imaged in situ by TIRF microscopy, which allowed imaging directly in the flow chamber during solvent exchange (Figure S1). To confirm the presence of nanobubbles, glass coverslips (40 × 22 × 0.1 mm) were first methylated with hexamethyldisilazane (HMDS) to create a hydrophobic surface that has been reported to support nanobubble stability.10,41 The coverslip was then placed in a flow chamber and mounted on the TIRF microscope stage (Figure S1). An air-saturated ethanolic solution of AlexaFluor 488 (AF488) was flowed onto a glass surface, followed by exchange with air-saturated water. The rapid decrease in air solubility from ethanol to water caused nucleation of gas pockets on the surface. These gas pockets prevented AF488 intrusion, which created voids in the TIRF microscope image (Figure 1 and Figure S2). AF488 was an ideal choice because of its net negative charge, low surface activity, and stability against photobleaching. Several previous reports utilized Rhodamine 6G as a fluorescent contrast marker;6,42–44 however, in our experiments, the positively charged Rhodamine 6G appeared to adhere to the negatively charged glass surfaces, and it was difficult to determine whether the nanobubbles were forming on the functionalized glass or on adsorbed rhodamine islands (Figure S3). Images were analyzed using an edge detection MATLAB code. Because of concerns about achieving accurate nanobubble size measurements near the diffraction limit, nanobubble count per area was used as a metric for evaluating nanobubble stabilization rather than area fraction or nanobubble size.</p><p>The differences between nanobubbles formed on organosilane-functionalized glass as compared with polymer brushes were elucidated by a series of repeated nanobubble deposition and dissolution experiments. After nanobubbles were deposited by solvent exchange from air-saturated ethanol to water, the process was reversed by exchanging with ethanol again, followed by an exchange with degassed water, which was expected to result in bubble dissolution. No nanobubbles were found after surface immersion in degassed water, which was consistent with previous reports.10 This observation confirms that the features observed in microscope images were indeed composed of gas rather than a contaminant or silicone oil from the syringe and tubing assembly.10,45 When the methylated glass surfaces were subsequently exposed to air-saturated ethanol and air-saturated water, the nanobubbles reformed in the same or similar locations as prior to degassing, as shown in representative images (Figure S4). Using an algorithm to colocalize the two images, the Pearson correlation coefficient (PCC) for the nanobubbles reforming on a methylated glass surface was found to be 0.69 (Figure 2), indicating a strong inclination to form bubbles in the same locations. This observation was consistent with studies by Lohse and coworkers, who showed that nanobubbles appeared in the same locations on methylated glass surface even after cavitation with high-intensity focused ultrasound.46 Importantly, the same solvent-exchange process performed for surfaces with methacrylic acid-co-methyl methacrylate (MAA-co-MMA) polymer brushes led to the formation of nanobubbles in apparently random locations (Figure 2, PCC = 0.16). We hypothesize that defect sites on silane-modified silica surfaces were present that were conducive to nanobubble stabilization, and these isolated sites consistently stabilized bubbles in independent depositions. In contrast, polymer brushes fully covered the surface, as supported by film thicknesses from 7 to 80 nm (Table S1), and provided a much more homogeneous substrate, consistent with the molecular-level dynamic nature of polymer chains; any defects that might have stabilized nanobubbles were expected to be transient and to heal over time.</p><p>To determine the relationship between nanobubble stability and surface hydrophobicity, we synthesized brushes composed of random copolymers of methacrylic acid (MAA) and methyl methacrylate, adjusting the feed ratios from 0 to 100 to achieve a range of receding water contact angles (θ) from 30 to 70°, respectively (Figure S5). Dynamic contact-angle measurements showed a difference of about 25–35° between advancing and receding contact angles, which is consistent with literature reports for measurements on copolymer surfaces (Figure S6).47,48 When surfaces with MAA-co-MMA were subjected to solvent exchange, nanobubble stability was only supported once a critical surface hydrophobicity was reached (Figure 3). For ten MAA-co-MMA surfaces with θ < 44°, none had more than one nanobubble. In contrast, for the 13 surfaces analyzed for θ > 50°, all but one had at least 450 nanobubbles per 1000 μm2. The presence of the AF488 did not affect the measured contact angle (Figure S7). The four surfaces measured between 44 and 50° showed various nanobubble counts over a 1000 μm2 area, including (44°, 10), (47°, 249), (50°, 0), and (51°, 177); from these results, we assign a critical contact angle of 48 ± 5°. Data obtained using different synthesis batches and on different days still exhibited the same critical surface hydrophobicity, and nanobubbles were detected at the same locations 3 h postdeposition (Figure S8).</p><p>To ascertain the effects of polymer chemistry on nanobubble stability, polymers with zwitterionic side chains were employed in place of negatively charged MAA monomers; sulfobetaine methacrylate (SBMA) was copolymerized with MMA to form SBMA-co-MMA copolymers in a range of contact angles from 5 to 90° (Figure S5). When nanobubbles were formed on these surfaces, a similar trend appeared as for the MAA-co-MMA surfaces. In particular, for θ < 55°, nine surfaces showed zero or one nanobubble, and one surface had 30 per 1000 μm2 (Figure 3). When θ > 60°, six of seven surfaces showed >270 nanobubbles per 1000 μm2, whereas the other had only 36 nanobubbles per 1000 μm2. As for the previous copolymer study, this result was obtained over different synthesis batches and performed on different days, showing the reproducibility and self-consistency of this system. Taking contact-angle measurement error into account, these surfaces appear to show a critical contact angle of ~62 ± 5°, which was 10–15° higher than for the MAA-co-MMA.</p><p>To test the potential effects of surface charge, we synthesized copolymers containing positively charged 2-(dimethylamino)-ethyl methacrylate (DMAEMA) as a hydrophobic component. Although DMAEMA contains a basic amine group, which has been utilized by others for inclusion of positive charge at neutral pH,49 we found that DMAEMA copolymerization with either MAA or SBMA increased surface hydrophobicity, likely due to the increased alkyl fraction of DMAEMA relative to the hydrophilic monomers. The results with DMAEMA matched closely with those using other copolymers. SBMA-co-DMAEMA exhibited a critical contact angle of ~62 ± 5°, consistent with SBMA-co-MMA. For comparison, surfaces functionalized with MAA-co-DMAEMA showed a critical contact angle between 40 and 55°. Unfortunately, this value could not be narrowed further due to contact-angle measurement uncertainty near this value, but this contact angle was generally consistent with the results found on MAA-co-MMA brushes. The self-consistency between experiments suggests that the hydrophilic component in the copolymer played a significant role in determining nanobubble stability on copolymer surfaces. As to the effect of hydrophilic side chain structure, Zhang and coworkers have suggested that water adsorbs strongly to SBMA zwitterions; perhaps this adhesion may induce an additional enthalpic penalty to water removal during nanobubble formation, thus requiring additional surface hydrophobicity to stabilize nanobubbles.50 Whereas we do not have additional evidence to support this hypothesis, this theory is consistent with the observation that SBMA surfaces (θ < 10°) are more hydrophilic than MAA surfaces (θ ≈ 30°) (Figure S5).</p><p>Finally, the effect of surface charge was investigated as a potential factor in nanobubble stabilization, which is a plausible hypothesis given that bare bubbles in solution possess a negative zeta potential due to water ordering at the surface.51–55 Because we prepared brushes with a wide range of cationic, neutral, and anionic side changes, we were able to empirically differentiate the potential effects of hydrophobicity and charge. The polymer brush surfaces were synthesized on standard glass microscope slides to allow measurement of surface zeta potential. By varying monomer composition, we obtained a large range of zeta potentials, from −40 to +25 mV (Figure S9). Interestingly, there did not appear to be a common critical zeta potential, suggesting that the charge was not as important a factor as surface hydrophobicity (Figure 4).</p><p>In this work, copolymer brushes were synthesized on surfaces to show how changes in surface hydrophobicity and surface charge affect nanobubble stability. Copolymers consisting of hydrophobic and hydrophilic monomers were grown from surfaces using ATRP. Nanobubbles were formed on these surfaces using a solvent-exchange method. After removing nanobubbles by water degassing, the new nanobubbles were found to form in different spots than those found originally, indicating that nanobubble stability was not dependent on underlying surface structure. By tuning the ratio of hydrophobic to hydrophilic monomers in the copolymers, it was found that nanobubbles required a critical hydrophobicity, as determined by contact angle, to maintain stability, and that the critical value of contact angle depended primarily on the detailed chemistry of the hydrophilic side chain. Surface charge was not correlated to nanobubble stability. This study suggests that nanobubbles on dynamic polymer surfaces do not require pinning for stability, which indicates a mechanism separate from nanobubbles on solid surfaces. Currently, we hypothesize that the dynamic polymer chains are able to distort their equilibrium packing to allow the nanobubble to adopt very low curvatures while preserving an equilibrium contact angle; however, testing this hypothesis will require further study. Taken together, these results have important implications for surface adhesion, membrane fouling, and other applications.</p>

PubMed Author Manuscript

Method development and survey of Sudan I\xe2\x80\x93IV in palm oil and chilli spices in the Washington, DC, area

Sudan I, II, III and IV dyes are banned for use as food colorants in the United States and European Union because they are toxic and carcinogenic. These dyes have been illegally used as food additives in products such as chilli spices and palm oil to enhance their red colour. From 2003 to 2005, the European Union made a series of decisions requiring chilli spices and palm oil imported to the European Union to contain analytical reports declaring them free of Sudan I\xe2\x80\x93IV. In order for the USFDA to investigate the adulteration of palm oil and chilli spices with unapproved colour additives in the United States, a method was developed for the extraction and analysis of Sudan dyes in palm oil, and previous methods were validated for Sudan dyes in chilli spices. Both LC-DAD and LC-MS/MS methods were examined for their limitations and effectiveness in identifying adulterated samples. Method validation was performed for both chilli spices and palm oil by spiking samples known to be free of Sudan dyes at concentrations close to the limit of detection. Reproducibility, matrix effects, and selectivity of the method were also investigated. Additionally, for the first time a survey of palm oil and chilli spices was performed in the United States, specifically in the Washington, DC, area. Illegal dyes, primarily Sudan IV, were detected in palm oil at concentrations from 150 to 24 000 ng ml\xe2\x88\x921. Low concentrations (< 21 \xce\xbcg kg\xe2\x88\x921) of Sudan dyes were found in 11 out of 57 spices and are most likely a result of cross-contamination during preparation and storage and not intentional adulteration.

method_development_and_survey_of_sudan_i\xe2\x80\x93iv_in_palm_oil_and_chilli_spices_in_the_washingt

Introduction<!>Standards<!>Samples<!>Palm oil<!>Chilli spices<!>Solid-phase extraction (SPE) cleanup and preparation of extracts<!>Liquid chromatography (LC)<!>MS/MS<!>Diode array detector (DAD)<!>Palm oil<!>Chilli spices<!>Palm oil<!>Spices<!>Discussion

<p>Sudans I–IV are oil-soluble azo dyes used for colouring textiles, plastics, wax, floor polish, and as a biological stain for lipids, triglycerides and lipoproteins (Hunger et al. 2000). They are labelled category 3 carcinogens by the IARC (IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans 1987) and are not permitted to be used in food in most countries including the United States and the European Union (Title 21 –Food and Drugs 2014; European Commission 1994, 2005). Because these dyes are bright in colour and low in cost, they have been used intentionally to adulterate palm oil and spices to enhance the perceived quality of the product (Arora & Bharti 2005). High-quality chillies naturally have a bright red colour and receive higher prices at market than those that are dull or orange and yellow (Arora & Bharti 2005). Adulteration with dyes has been used to cover up products in which the natural colour has been lost due to improper drying, storage or fungal infestations where pigment is degraded (Arora & Bharti 2005; Mishra et al. 2007). In 2003 the first detection of Sudan I in chilli powder in the European Union was reported, and the source of contamination was traced back to an adulterated chilli powder imported from India with a concentration of 4000 mg kg−1 (RASFF 2003; ASTA 2005). At this time, a decision (2003/460/EC) was made that required all imports from fruit produced from plants under the genus name Capsicum be accompanied by an analytical report indicating the sample was free of Sudan I (European Commission 2003). In 2004, this requirement was expanded to Sudans II–IV, and in 2005, it was expanded to include palm oil and the genus Curcuma (turmeric) (European Commission 2005). In the UK in 2005, Sudan I was detected in a Worcester sauce (3 μg ml−1) that was found to contain adulterated chilli powder (80 mg kg−1) (RASFF 2005). This sauce had been used to make a wide variety of foods including soups, mince and sausage ready-meals, seafood sauces, pate, salad dressings and sauces (Sudan I Consolidated Product List from February 2005). As a result, close to 500 food products were recalled, making it the largest recall in the UK history to date. Unauthorised colours continue to be reported in the RASFF portal, with a total of 16 notifications in 2014 and 2015 (RASFF 2015).</p><p>Once the European Union began to require analytical reports with imported products, there came a need for analytical methods for quantifying Sudan dyes in a wide variety of foods. Many methods were developed using LC combined with UV-vis absorbance, photodiode array (PDA) detectors, or mass spectrometric detectors (MSD) (Rebane et al. 2010). For MS both electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) were used along with several mass analysers including Q-TOF, TOF, ion trap, single quadrupole and triple quadrupole (Rebane et al. 2010). For chilli powders, multiple methods were developed (Rebane et al. 2010) including a method validated for the Belgium monitoring programme that involved extraction of the spices into acetonitrile followed by LC-diode array detection (DAD) analysis (Cornet et al. 2006). This method used matrix-matched standards for calibration and the LOQs for the Sudan dyes ranged from 1.5 to 2 mg kg−1 (Cornet et al. 2006). Without matrix-matched standards, the lowest LODs for spices were reported at 0.5–10 μg kg−1 for Sudans I and III, and 5–100 μg kg−1 for Sudans II–IV using LC-MS/MS (Schummer et al. 2013). Both methods involved a simple extraction with acetonitrile followed by filtration and dilution prior to analysis. For palm oil, only a few methods are published (Guffogg et al. 2004; Uematsu et al. 2007) including a qualitative TLC method used in monitoring by the Food Standards Agency (FSA) (Guffogg et al. 2004) and a method using DAD and clean-up by conversion of the oil to fatty acid methyl esters (FAMEs) and further clean-up by silica gel chromatography (Uematsu et al. 2007) In order for the USFDA to monitor and assess potentially contaminated palm oil samples, there was a need to develop a simple quantitative method using LC-MS/MS that could provide structural confirmation of Sudan dyes in palm oil.</p><p>Since many methods have been published on the analysis of Sudan dyes in spices using LC-MS/MS, there was no need to develop a new method and a previously developed method was validated (Tran et al. 2005). Additionally, the detection of Sudan dyes in both chilli powders and palm oil using LC-DAD was examined for strengths and weaknesses in identifying adulterated samples, since in a regulatory laboratory setting LC-DAD could be a useful screening tool for large numbers of samples. Using these methods, a survey of Sudans I–IV in palm oil and chilli spices sold in the Washington, DC, area and from online retailers was performed. The objectives of this research were: to develop a simple extraction method for Sudan dyes in palm oil; to validate an LC-MS/MS method and an LC-DAD method for palm oil and chilli spices; to investigate the suitability of LC-DAD and LC-MS/MS for Sudan dye analysis; and, for the first time, to investigate the concentrations of Sudan dyes present in these foods in the United States.</p><!><p>Sudan I–IV analytical standards with purity > 96% were purchased from Sigma Aldrich (St. Louis, MO, USA) and deuterium-labelled d6-Sudan III for use as an internal standard was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Stock solutions were prepared in methanol with 10% tetrahydrofuran (THF) to ensure dissolution of the Sudan dyes. It is known that E-Z photo-isomerisation can occur when Sudans III and IV are exposed to light. The isomers formed produce additional 'fast peaks' in the chromatogram that elute much earlier than Sudans III and IV (Mölder et al. 2007). Unless the entire analytical process is performed in the dark, it is difficult to avoid the formation of these isomers. Mölder et al. (2007) found that once the vials were wrapped in aluminium foil and placed in the dark for up to 4.5 h, the isomers of the 'fast peaks' reverted back to their more stable form and were no longer present in the chromatogram. Therefore, in this study, all calibration standards and samples were stored in amber vials and kept in the autosampler tray in complete darkness for at least 1 h prior to analysis. This prevented the formation of 'fast peaks' that could potentially underestimate the concentrations of Sudans III and IV in samples.</p><!><p>Red palm oil samples were collected at supermarkets in the Washington, DC, area with a focus on those specialising in African foods. It was assumed that these oils were unrefined and the red colour was due to naturally occurring carotenes. In this case, adulteration with Sudan dyes would enhance the red colour to make the palm oil appear nutritionally superior. Based on the smell and texture of these oils, they appeared to be palm oil and not a cheaper oil substitute, but this was not investigated in this study. Additional samples were obtained through online retailers with a focus on red palm oil samples used as dietary supplements. In total 30 samples were collected including 23 red palm oils and seven red palm oil dietary supplements sold as capsules. A majority of these samples were imported from West Africa (17), with a few samples coming from Malaysia (2) and South America (2). The country of origin of the remaining nine samples could not be identified from the labels.</p><p>Spices were collected at local and international markets in the Washington, DC, area that included crushed or minced chilli, ground chilli powder, ground cayenne pepper, paprika powder, and turmeric or other chilli products that are from the genera Capsicum and Curcuma. In total, 59 spices were collected and represent imports from Africa, Korea, China and India.</p><!><p>Palm oil bottles were heated in a 66°C oven for approximately 5–10 min until the oil liquefied. Then, 100 μl of the palm oil were added to 900 μl of hexane to make a 1 ml solution.</p><!><p>For the analysis of spices, a previously developed extraction method was used (Tran et al. 2005). Briefly, 1 g of the spice and 10 ml of acetone were added to a 50 ml polypropylene Falcon tube. The vial was shaken using a Glas-Col Digital Pulse Mixer (Terra Haute, IN, USA) for 5 min at a speed of 1200 rpm. The vial was then centrifuged for 5 min at 1800 rcf. An aliquot of the supernatant (1 ml) was removed and reduced to dryness using nitrogen. The sample was then brought up to 1 ml in hexane in preparation for SPE. For turmeric, low recoveries (approximately 5%) were found when the sample was brought up in hexane, likely due to the low solubility of the turmeric residue in hexane. Therefore, the sample was brought up in 1 ml of ethyl ether instead followed by a modified SPE clean-up. This improved the average recoveries to an acceptable range of 61–119%.</p><!><p>SPE was performed using LC-Alumina-B SPE tubes (1 g/3 ml) from Supelco (St. Louis, MO, USA) and the conditioning and elution steps were modified from a method for spices and chilli oil (Tran et al. 2005). The cartridge was conditioned with 6 ml of methanol, 6 ml of ethyl acetate and 6 ml of hexane. Then, 1 ml of the sample solution was added to the SPE column. The sample was washed with 6 ml of hexane and 6 ml wash of ethyl ether (palm oil only), followed by 2 ml of ethyl acetate The Sudan dyes were then eluted from the column using 8 ml of 90:10 ethyl acetate:methanol solution. For turmeric, the conditioning steps were the same and the wash step consisted of 2 ml of ethyl acetate followed by elution with 90:10 ethyl acetate:methanol. The elution solvent was blown to dryness using nitrogen and brought up to 1 ml in methanol. The extract was filtered through a 0.2 μm PTFE filter and internal standard was added (10 μl of 1 ng/μl d6-Sudan III solution) prior to LC-MS/MS analysis. The addition of internal standard was necessary to correct for matrix effects based on the slopes of the calibration curves with and without internal standard correction. Both d5-Sudan I and d6-Sudan III were investigated as internal standards and it was determined that d6-Sudan III was just as effective as d5-Sudan I for correcting for matrix effects for Sudans I and II. Therefore, just d6-Sudan III was used as an internal standard. For LC-DAD analysis, no internal standard was added since in DAD the internal standard and native peaks are not separated. However, the addition of a small amount of internal standard (10 ng ml−1) to the final extract will have little effect on the measured concentration of Sudan III using LC-DAD since this concentration is well below the lower limit of quantification (LLOQ) for LC-DAD analysis (25 ng ml−1) and since adulterated samples have significantly higher concentrations (ppm range).</p><!><p>LC was performed using a Prominence UFLC XR (Shimadzu, Kyoto, Japan). Isocratic elution was used to separate the Sudan dyes (I–IV) with an Agilent Eclipse 5 μm XDB-C18 (4.6 mm × 150 mm) column at 40°C and a flow rate of 0.8 ml min−1 for 30 min. For LC separation with DAD detection, a 150 mm column along with a flow rate of at least 0.5 ml min−1 is needed for good separation between the natural colours and Sudan dyes. For analysis using MS/MS without the DAD detector in series, a shorter column with a smaller particle size and lower flow rate would reduce solvent and the run time and maximise sample throughput. The mobile phase was 95/5 v/v methanol: water buffered with 5 mM ammonium formate and 0.1% formic acid with an injection volume of 5 μl. A 5-mincolumn wash was added at the end of each run using 98/2 v/v isopropanol:water solution buffered with 2 mM ammonium formate and 0.03% formic acid followed by a 5-minequilibration with mobile phase solvents. Additionally, during DAD analysis, a wash of 100% isopropanol was added at the end of each sample set to remove the buffered mobile phase from the DAD detector. The LC conditions (flow rate, column selection) were optimised for use with a DAD and MS/MS detector in series on the same instrument in order to develop a method that could be easily transferred among instruments with different detectors for testing adulterated samples.</p><!><p>An AB Sciex 4500 QTRAP hybrid triple-quadrupole/linear ion-trap MS with an electrospray ESI ion source (AB Sciex, Toronto, ON, Canada) was used for MS/MS detection for the palm oil samples. Initially, the chilli powder samples were analysed using the AB Sciex 4500, but the concentrations in the samples were quite low, so the analysis was moved to the AB Sciex 5500 QTRAP hybrid triple-quadrupole/linear ion-trap MS. Individual standards for Sudans I–IV were infused into the MS to optimise MS parameters (entrance potential, CE and CXP) for individual compounds. The two most abundant MRM transitions were monitored for each analyte, except in the case of Sudans I and II where two transitions were not chosen (249 ← 93 and 277 ← 121) due to interferences. The monitored transitions were: 249 ← 156, 249 ← 128 for Sudan I, 277 ← 156, 277 ← 128 for Sudan II, 353 ← 197, 353 ← 120 for Sudan III, 381 ← 225, 381 ← 106 for Sudan IV, and 359 ← 197, 359 ← 162 for d6-Sudan III. The MS source/gas parameters were: curtain gas 20 arbitrary units (au), collision gas medium, ion spray voltage 4000 V, source temperature 400, ion source gas 160 au, ion source gas 270 au, and declustering potential 75 V. A seven-point linear calibration curve (r2 > 0.999) for MS/MS analysis using the AB Sciex 4500 ranged from 0.5 to 100 ng ml−1 on column. The seven-point calibration curve for MS/MS analysis on the AB Sciex 5500 ranged from 0.1 to 10 ng ml−1 on column. Since the samples were diluted 10 times prior to analysis, the concentration range measured in the palm oil samples was 5–1000 ng ml−1 and in chilli spices was 1–100 μg kg−1. The range for the calibration curve for chilli spices was adjusted to measure accurately the low concentrations in the survey samples.</p><!><p>A UV/vis photodiode array detector (DAD) Prominence by Shimadzu (SPD-M20A) was used for the detection of the Sudan dyes. The acquisition parameters included using both a D2 and W lamp wavelength scan from 400 to 600 nm, a wave step of 8, slit width of 8, cell temperature of 40°C, and a period of 2 s (frequency of 4.167 Hz). The λmax (nm) was 480 nm for Sudan I, 496 nm for Sudan II, 504 nm for Sudan III, and 520 nm for Sudan IV. For the DAD analysis, a six-point linear calibration curve (r2 > 0.99) ranged from 25 to 1000 ng ml−1 on column. Because of the 10:1 sample dilution, this corresponded to a concentration range of 250–10 000 ng ml−1 in palm oil and 250–10 000 μg kg−1 in chilli spices.</p><!><p>The analytical method was validated in palm oil by performing spike and recovery experiments in three different brands of palm oil at three spikes close to the LLOQ. For the MS/MS method, spikes were performed at 10, 20 and 30 ng ml−1 in palm oil and for the DAD analysis the spikes were performed at 350, 450 and 550 ng ml−1 in palm oil. The chromatograms palm oil spiked at 1 ng ml−1 can be seen in Figure 1 for LC-MS/MS analysis and in Figure 2 for LC-DAD analysis. Results of the method recoveries can be seen in Figure 3. The average recoveries among all three matrices ranged: 77–84% for Sudan I, 57–62% for Sudan II, 73–92% for Sudan III, and 64–76% for Sudan IV for the MS/MS analysis; and: 66–83% for Sudan I, 60–74% for Sudan II, 67–81% for Sudan III, and 65–68% for Sudan IV for the DAD analysis. Since these recoveries were determined close to the LOD, they fall within the acceptable recovery range of 50–120% (Codex Alimentarius Commission 1993). Method recovery spikes at the concentration of suspected adulteration (approximately 1 μg ml−1) were higher and averaged 71–99% for Sudan I–IV in palm oil. The method detection limits (MDLs) were calculated by performing seven low-level spikes at 10 ng ml−1 in oil (1 ng ml−1 on column, S:N = 3:1–8:1) for LC-MS/MS analysis and 350 ng ml−1 in oil (35 ng ml−1 on column, S:N = 3:1–8:1) for LC-DAD analysis and multiplying the standard deviation of the replicates by 3.14 (t-value for seven replicates where 1 − α = 0.99) (40 CFR Appendix B part 136) (Definition and Procedure for the Determination of the Method Detection Limit –Revision 1.11). The MDLs in palm oil were 2.4 ng ml−1 for Sudan I, 4.4 ng ml−1 for Sudan II, 2.0 ng ml−1 for Sudan III, and 3.7 ng ml−1 for Sudan IV with LC-MS/MS analysis. The MDLs for LC-DAD analysis were 184 ng ml−1 for Sudan I, 232 ng ml−1 for Sudan II, 193 ng ml−1 for Sudan III, and 237 ng ml−1 for Sudan IV in palm oil.</p><!><p>Method validation was performed for the Sudan dyes in spice samples by performing spike and recovery experiments in three different spices at concentrations of 1, 2 and 3 μg kg−1 in the spices and for the diode array analysis at 8, 9 and 10 mg kg−1 in the spices. The percentage recoveries for the LC-MS/MS method can be seen in Figure 3, and ranged: 88–100% for Sudan I, 89–104% for Sudan II, 89–93% for Sudan III, and 66–79% for Sudan IV. The recoveries for the diode array analysis are also shown in Figure 3, and ranged: 76–83% for Sudan I, 76–81% for Sudan II, 80–86% for Sudan III, and 70–76% for Sudan IV. The MDLs for Sudan dyes in chilli spices was calculated by performing seven low-level spikes at 1 μg kg−1 in chilli spices (0.1 ng ml−1 on column) and multiplying the standard deviation of the replicates by 3.14 (t-value for seven replicates where 1 − α = 0.99) (40 CFR Appendix B part 136) (Definition and Procedure for the Determination of the Method Detection Limit – Revision 1.11). The MDLs in chilli spices were 0.7 μg kg−1 for Sudan I, 0.5 μg kg−1 for Sudan II, 0.7 μg kg−1 for Sudan III, and 1.0 μg kg−1 for Sudan IV for LC-MS/MS analysis. The analysis of spices by LC-DAD is more challenging than in palm oil due to the large amounts of natural colours present with similar polarities as the Sudan dyes. For example, in Figure 2, the DAD chromatogram for palm oil extract spiked at 1 mg kg−1 is much cleaner than the chilli powder spiked at 1, 5 and 10 mg kg−1. A spiked concentration of at least 5 mg kg−1 is needed in order to quantify the peaks based on retention time and spectra quality. Also, spices vary in the amounts and types of natural colours present, as seen in Figure 4. The DAD chromatograms of the chilli powder and crushed red pepper spiked at 10 mg kg−1 is much cleaner than the paprika chromatogram, which has a colour eluting just under 6 min that results in a maximum absorbance around 250 mAU compared with 40 mAU. Previous research has identified higher LODs (1.5–2 mg kg−1) of Sudan dyes in spices using LC-DAD with matrix-matched calibration curves (Cornet et al. 2006) as well as interferences with paprika and turmeric (Tateo & Bononi 2004). Additionally, carotenoids present in Capsicums can absorb in the same range as Sudan dyes and give false-positives (Hoenicke 2006). Because of these natural colours, the method LODs were much higher for chilli spices than that of the palm oil and calculated to be 3.6 mg kg−1 for Sudan I, 4.1 mg kg−1 for Sudan II, 3.1 mg kg−1 for Sudan III, and 4.6 mg kg−1 for Sudan IV in chilli spices based on an 8 mg kg−1 spike. This amount was chosen for MDL calculations based on the spice (paprika) with the DAD chromatogram with the most interferences (Figure 4). Studies have shown that at least 120 mg kg−1 of Sudan I would be needed to increase the extractable colour one ASTA colour unit using the ASTA method 20.1 (ASTA 2005), and researchers in Europe have found spices identified as adulterated ranging in concentration from 25 to 3500 mg kg−1 (Hoenicke 2006). Therefore, this method with MDLs from 3.1 to 4.6 mg kg−1 would be an appropriate screening method for spice samples that are potentially contaminated. Suspected contaminated samples should be verified using LC-MS/MS to confirm there are no false-positives.</p><!><p>Twenty-two red palm oil samples and eight red palm oil dietary supplements were analysed for Sudans I–IV. Since one sample was highly contaminated (Ivory Coast 1), two additional bottles from different retailers were also purchased to determine if the concentrations were consistent (Ivory Coast (2) and (3)). In total, 20 different brands of palm oil were tested and, of those, four different brands had detections of Sudan IV. The concentrations ranged from 150 to 24 000 ng ml−1 by LC-MS/MS detection and from 240 to 25 000 ng ml−1 by LC-DAD detection (Table 1). Based on the high concentrations measured in contaminated samples, both methods are suitable for analysis. In four out of five of the samples, low level detections of Sudans I and III were found (0.5–30 ng ml−1), which are likely attributed to impurities in the Sudan IV dye. Although the first three samples were purchased from different retailers, they were the same brand name and all imported from Ivory Coast. It is unclear from their labels whether or not they come from the same lots. The sample from Ivory Coast (1) had the highest contamination of Sudan IV at 24 000 ng ml−1, while the other two samples had lower concentrations at 5000–7000 ng ml−1. The other samples that were contaminated were two samples from Ghana with concentrations of 6400 and 1500 ng ml−1 and in another sample from West Africa at 150 ng ml−1 of Sudan IV, but the exact country of origin is unknown. Of these four contaminated samples, three were purchased from retail stores in the Washington, DC, area that contain grocery items primarily imported from Africa, and one of the contaminated samples was purchased from an online retailer. Although the presence of any colour additive not approved for use in food (European Commission 1994) is considered adulteration, it was proposed in 2006 at a European Union meeting of the Standard Committee on the Food Chain and Animal Health that an action limit of 500 ppb be adopted. This was due to very low concentrations of Sudan dyes found in products that were thought to be cross-contamination and not intentional adulteration. In this case, three out of these four samples would exceed this limit.</p><!><p>Spices that fell into the categories that would require an analytical report to accompany import into the European Union were collected from local grocery stores including those specialising in international foods. Out of 59 samples, 11 were contaminated with at least one illegal dye with concentrations less than 21 μg kg−1 (Table 2). These concentrations were determined using LC-MS/MS with an AB Sciex 5500 QTRAP hybrid triple-quadrupole/linear ion-trap MS, since on the AB Sciex 4500 only one out of 11 of the results was above the LOQ. Additionally, the LC-DAD could not be used for this analysis since the MDL in spices is close to 1 mg kg−1. In total, four spices were contaminated with Sudan I, none with Sudan II, two with Sudan III, and eight with Sudan IV. Since these concentrations are so low, it is unlikely that any of these spices were intentionally adulterated. The concentrations of illegal dyes in adulterated samples detected in the European Union ranged from 25 to 3500 mg kg−1 (Hoenicke 2006), and in order to enhance visually the colour of the spice, 100–1000 mg kg−1 of dye needs to be added (ASTA 2005). An analytical laboratory in Germany also detected very low levels of Sudan dyes in spices. In order to investigate the cause of the concentrations, they performed two case studies. One identified μg ml−1 concentrations of Sudan I in a red lubricant used for greasing extraction plants as the source of Sudan I measured in spices ranging from 10 to 120 μg kg−1 (Hoenicke 2006). The second case study found mg kg−1 amounts of Sudan IV in red mesh bags used for storing and transporting chilli pods. In the corresponding samples, the concentration of Sudan IV was 10–20 μg kg−1 (Hoenicke 2006). As a result, it appears that the most likely source of the low concentrations measured in the spice samples obtained in the Washington, DC, region is related to cross-contamination during sample processing and storage.</p><!><p>A method for the extraction and analysis of Sudans I–IV in palm oil using LC-MS/MS was developed and validated. The benefits of this palm oil method compared with previous methods (Guffogg et al. 2004; Uematsu et al. 2007) are the simple preparation and extraction steps, low levels of detection (2.0–4.4 ng ml−1), and structural confirmation by MS/MS. The use of LC-MS/MS is preferred for confirmation, but the LC-DAD results are comparable (Table 1) and had clean chromatograms (Figure 2) with the disadvantage of higher LODs (184–237 ng ml−1). For the spices, the LC-MS/MS method is clearly advantageous due to the many natural colours present that result in a LC-DAD chromatogram with a poor background, potential interferences and false-positives. But considering the high concentrations typically found in adulterated spices (> 25 mg kg−1), the LC-DAD method performs well at these concentrations and would provide a suitable screening method for routine analysis. Previous methods that involve the extraction of spices with acetonitrile followed by filtration and dilution are excellent for use with LC-MS/MS (Schummer et al. 2013). For DAD analysis, it was determined that SPE clean-up was necessary to reduce the interferences and improve LODs. The validation of these methods for the analysis of illegal dyes in palm oil and chilli spices will allow the regulatory analysis of samples imported into the United States using either LC-MS/MS or LC-DAD detection. Based on the survey results in the Washington, DC, area, adulterated palm oil is present in the marketplace at concentrations up to 24 000 ng ml−1. Red palm oil is popular in the diets of people from West Africa, the Caribbean and South America, and can be consumed in large amounts (several cups) daily in the form of soup or stew. As a result, these populations are at a higher risk of being exposed to unapproved and, therefore, illegal dyes in their diets. Additionally, red palm oil for use as a dietary supplement has the potential to be adulterated. In this limited survey, Sudan dyes were not detected above the MDL in any of the dietary supplements. The amount of illegal dyes found in the spices purchased in the Washington, DC, area was less than 21 μg kg−1, which is well below the 500 μg kg−1 proposed action limit in the European Union for Sudans I–IV in spices and palm oil. Considering these samples represent a regional subset of palm oil and chilli products available in the US market, the validation of the LC-DAD and LC-MS/MS methods presented ensures that future adulterated products can be tested and regulated.</p>

PubMed Author Manuscript

Synthesis of medicinally relevant terpenes: reducing the cost and time of drug discovery

Terpenoids constitute a significant fraction of molecules produced by living organisms that have found use in medicine and other industries. Problems associated with their procurement and adaptation for human use can be solved using chemical synthesis, which is an increasingly economical option in the modern era of chemistry. This article documents, by way of individual case studies, strategies for reducing the time and cost of terpene synthesis for drug discovery. A major trend evident in recent syntheses is that complex terpenes are increasingly realistic starting points for both medicinal chemistry campaigns and large-scale syntheses, at least in the context of the academic laboratory, and this trend will likely penetrate the commercial sector in the near future.

synthesis_of_medicinally_relevant_terpenes:_reducing_the_cost_and_time_of_drug_discovery

<!>Identifying the problem<!>Stereochemistry-based strategies: the power of substrate relay<!>Englerin A<!>Artemisinin<!>Structure-goal strategies: the power of \xe2\x80\xb2semi-synthesis\xe2\x80\xb2<!>Cortistatin A<!>Ouabagenin<!>Cyclopamine<!>Transform-based strategies: the power of the \xe2\x80\x98key step\xe2\x80\xb2<!>Neothiobinupharidine<!>Hyperforin<!>Ingenol<!>FG-based strategy: the power of new methods<!>Amphilectene & the isocyanoterpenes<!>Pumilaside aglycon<!>Peyssonol A<!>Frondosin B<!>Conclusion<!>Future perspective

<p>Terpenes (or terpenoids or isoprenoids) constitute a major class of organic molecules produced by diverse organisms to perform an assortment of biological functions in varying ecological contexts. Although all terpenes originate from the same five-carbon building blocks (dimethylallyl pyrophosphate and isopentenyl pyrophosphate), the structures and functions of terpenes vary widely, and are highly tailored to the requirements of the source organism's environmental pressures and resources. As a consequence of the biological functions of terpenes (in humans and other organisms), these molecules have led to six major drug classes over the last century, namely steroids, tocopherols, taxanes, artemisinins, ingenanes and cannabinoids [1]. However, optimization of terpene structures for human use requires economical access to not only the carbon scaffold of the terpene class, but also embedded functional groups that allow specific modification of the molecule through a rational medicinal chemistry campaign. Whether a campaign starts from a complex isolate with an essentially intact scaffold (sometimes called semi-synthesis) or several smaller fragments (sometimes called total synthesis, vide infra), this exploration can be costly and lengthy. In this article, we document several strategies to reduce the cost and time of developing terpene-based therapeutics.</p><!><p>The biological role and medical use of terpenes must inform how the problem of chemical synthesis is addressed. Bioactive terpenes are isolated frequently; medically relevant terpenes less so, and distinguishing the latter is not always straightforward prior to clinical validation. One benefit of the isolation literature is the frequent use of phenotypic assays, rather than target-based assays, a preference that is receiving increased interest in the pharmaceuticals industry [2] as it provides important information about potency and selectivity in a cell, rather than on a single macromolecule. The obvious downside is that observation of phenotypic response leaves open the question of the biomolecular basis for that response. Thus, use of a phenotypic versus target-based assay neatly divides medicinally relevant terpenes into two subclasses depending on whether the mechanism of action is known or unknown.</p><p>Approximately 35,000 terpenes have been identified and the majority of possible functions of these molecules are unknown [1]. Consequently, synthesis efforts in this 'naive' phase of terpene research focus on procuring relatively small amounts of material or affinity-labeled material for target identification. That being said, a disproportionate number of terpenes receive attention in the synthesis literature because they possess known functions of medical importance, which leads to a focus on either scalability of synthesis, or exploration of analogous structures. A chemical synthesis should serve to address real problems associated with the target structure, which are largely dependent on knowledge of the molecule's mechanism.</p><p>So what constitutes the best approach to small molecule synthesis [3–5]? The answer is simple: it's complicated. For example, a coarse articulation of the ideal synthesis is one that creates homogeneous material in large scale at low cost, which includes low cost of labor, reactants and reagents, solvents, purification and waste disposal. However, even this rudimentary definition does not always hold: what if you do not know the small molecule structure in advance? If not, then these metrics of synthesis disintegrate. In medicinal chemistry, a synthetic route must be diversifiable and modular, and scalability is not a major issue or even a goal. Nor is yield or purification, which can often be performed rapidly on small scale by HPLC. Similarly, metrics that articulate the need for chemical diversity measure diversity only, even though medicinal chemistry is not a random walk into unknown regions of space, but rather a logical exploration of the relationship between structure and function (SAR) [6]. All this is to say that for a given molecule, there can be various goals of synthesis – access to bulk material (process), specific modification of structure (optimization of properties, e.g., medicinal chemistry) or diversification of structure (e.g., combinatorial chemistry) – all of which possess competing agendas rather than a grand, unified vision. This means that a synthesis must be tailored to the problem it is trying to solve, and therefore identification of the actual problem is crucial.</p><p>The one unifying feature between the three synthesis goals above (process, optimization and diversification; Figure 1) is the primacy of step count economy [5], which has the potential to minimize costs at multiple levels. Sometimes other features such as modularity requirements or purification problems override the benefits of a short step-count. However, in general, a shorter synthesis is a better synthesis, and therefore in this article we will highlight academic syntheses from the last 5 years where step counts are minimized relative to either prior syntheses or syntheses of similarly complex molecules. Nevertheless, to truly evaluate the quality of a synthesis, it is essential to identify its purpose, (i.e., the problem it is attempting to solve).</p><p>We should briefly note that a decreasing number of syntheses are undertaken just to show how a biologically produced molecule (natural product) might be synthesized in the laboratory by chemical means. This is primarily due to funding pressures, despite the inherent value of this pure science endeavor. The justifications for these syntheses are not necessarily biological/medicinal in nature, but are structural/chemical, and often result in innovations in reaction invention or strategies to access a general structure. This is certainly not to say that all complex molecule syntheses are justified or contribute to progress in chemistry. But rather, the decline of exercises in the pure science of synthesis as ends in themselves will likely parallel a decline in organic chemistry as a whole, and negatively impact the development of new drugs.</p><p>Below, we analyze several recent examples of terpene syntheses from the academic literature, illustrate how they solve problems associated with the target molecule, and in some cases discuss what problems remain to be solved. The syntheses of these terpenes are grouped according to the primary 'higher-level strategy' employed, although in most cases the syntheses utilize multiple strategies. Along the way, we discuss changes in focus that have accrued over time within the synthesis field, and attempt to project these trends into the future. One major trend evident in these recent efforts is that complex terpenes are increasingly realistic starting points for both medicinal chemistry campaigns and large-scale syntheses, at least in the context of the academic laboratory, and this trend will likely penetrate the commercial sector in the near future. Imitation of these syntheses' strengths and repudiation of their weaknesses will, in a general way, reduce the cost of developing terpene-based therapeutics.</p><!><p>One defining feature of terpenoid metabolites is their stereochemical complexity. In order to arrive at an efficient synthesis, the number of stereogenic steps must be minimized, and the level of stereocontrol at each of these steps must be maximized. A logical way to achieve these goals is to prioritize retrosynthetic transforms that efficiently set multiple stereocenters at once (see also 'Pumilaside aglycon' section). Furthermore, stereochemical relay from the substrate can be an effective tool to rapidly increase complexity, as long as the appropriate choreography of bond disconnections can be determined. Below, we document several recent examples of terpenoid synthesis and we focus on the stereochemical decisions made during retrosynthetic analysis. In many cases, the actual stereocontrol achieved is not perfect, even though the logic is good and reduces the length of the route. The lessons learned from these syntheses should inform future work in the same or related molecules.</p><!><p>It is estimated that roughly 65,000 people were affected by renal cancer in 2013 and a further 13,500 would die because of it [7]. Englerin A (1, Figure 2), a guaiane sesquiterpene, was isolated in 2008 from the extract of Phyllanthusengleri as a result of a positive hit in an National Cancer Institute 60-cell screen of natural product extracts that exhibit preferential selectivity for renal tumor cells [8]. Indeed, against several renal cancer cells lines (1) possesses nanomolar potency and in many cases, it was more potent than paclitaxel. Recent work has shown that englerin A activates PKCθ in renal cancer cells, which appears to have a two-pronged effect [9]. First, activation of PKCθ causes phosphorylation of IRS-1 and decreases insulin receptor-mediated glucose uptake. Second, PKCθ activation causes production of HSF1, which enhances the glucose dependence of the cells. Since englerin A decreases cellular glucose uptake while increasing cellular glucose dependence, the cancer cell is starved of energy. With a possible mechanism of action in hand, the goal for chemists should be to increase access to (1) within the cancer community, synthesize derivatives and develop SARs, all of which can be facilitated by an efficient synthesis.</p><p>As of this publication, there have been 13 syntheses of (−)-englerin A or its derivatives. Christmann and co-workers recorded the first synthesis and confirmed the absolute configuration of ent-(+)-englerin A in 2009. These authors published a follow-up synthesis of the natural enantiomer along with several analogs in 2011 [10,11]. Bicycle 2 is synthesized in 11 steps from (−)-nepetalactone and can be engaged in a transannular epoxide opening to generate the bridging ether with high regioselectivity. Christmann and co-workers prepared over 7 g of intermediate 3 and were able to make dozens of analogs, several of which had greater potency than englerin A itself.</p><p>In 2006, Echavarren and co-workers developed a novel gold (I)-catalyzed Prins cyclization cascade to synthesize (+)-orientalol F and other molecules that possess essentially the same tricyclic core as englerin A [12]. In 2010, both Echavarren and Ma recognized this relationship, targeted a similar core substructure, and independently published syntheses using similar synthetic routes (see 4→5) [13,14]. From the cascade cyclization product 5, several functional group interconversions are necessary to then elaborate this core substructure to englerin A (1).</p><p>But perhaps the most elegant and efficient synthesis to date was accomplished by Chain and co-workers in 2011 [15]. Using simple carbonyl chemistry, these authors synthesize englerin A in only eight linear steps. Realization that the esters of the cyclic core are nascent ketones in a 1,4 and 1,5 relationship prompts Chain to disconnect the tricyclic core by a Michael addition and an umpolung carbonyl-alkene radical cyclization. These disconnections are highly simplifying because they divide the molecule into two simple cyclopentenes, and the two targeted bonds lie between vicinal stereocenters, thus removing four chiral centers in just two steps. The logic behind this strategy is both powerful and general.</p><p>Chain executes his strategy by first reacting the enolate of 6 with citronellal-derived enal 7. A modest 2:1 ratio of 8 to all other isomers is obtained, but this mixture can be subjected to samarium (II) iodide in hexamethylphosphoramide to effect carbonyl-alkene cyclization in 43% yield (maximum 66% yield based on 2:1 diastereomeric mixture of starting material). Keto-alcohol 9 is then elaborated to englerin A in four steps, completing a synthesis of only eight steps from commercial materials in a remarkable 20% overall yield.</p><!><p>One of the more well-known and clinically validated terpenes for the treatment of an infectious disease is artemisinin (10, Figure 3), a plant-derived antimalarial that exhibits rapid clearance rates of Plasmodium parasites from infected patients [16]. Since malaria affects 300–500 million people per year, primarily in low-income countries, there is an ongoing search for means to provide bulk material at very low cost. Extraction of 10 from Artemesia annua is a time-tested successful strategy for procuring bulk material, but the low yield of this process and the vagaries of crop production have not allowed extraction to keep pace with demand [17]. Efforts in selective breeding of higher yielding Artemesia plants have not proven successful, and conversion of the more abundant metabolite artemisinic acid (11, see Figure 4) to 10 generally suffers from poor yields [18]. The combined approach of engineering the plant biosynthetic pathway of 11 into Saccharomyces cerevisiae followed by semi-synthetic conversion to 10 has been pioneered by the Keasling group, and may serve to stabilize the market price of artemisinin [19]. However, the ultimate cost of production using this route is still an open question, although early estimates are promising with Sanofi (Paris, France) reporting artemisinin production in 370 kg batches (60 tons estimated for 2014) [20].</p><p>Chemical synthesis may prove an effective strategy to provide 10 in large quantity and at low cost. A general 'blueprint' of a potential route to produce 10 from inexpensive building blocks was recently developed by the Cook laboratory at Indiana University (IN, USA) [21]. Based on knowledge gleaned from the semi-synthetic conversion of 11 to 10, the Cook group realized that three stereocenters (in green) of the stereochemically complex artemisinin core could be immediately cleared to achiral or racemic precursor carbons (Figure 3). Therefore, the only stereocenter necessary to control in 10 is the tert-alkyl endoperoxide (in blue), which would be favored based on the concavity of octalin 12. This concavity imparted by the ring-fused carbon of 12 (in blue) could be generated by a stereocontrolled annulation of cyclohexanone 13, which in turn might be formed by vicinal difunctionalization of the chemical feedstock, 2-cyclohexenone (14). Thus, seven stereocenters of artemisinin 10 could be relayed from a single stereocenter derived from asymmetric conjugate addition into 14.</p><p>Utilization of the Cook group's previous investigations into asymmetric vicinal difunctionalization reactions enables the synthesis of 13 in 61% yield and in very good diastereo- and enantio-selectivity (Figure 5) [22,23]. Ketone 13 is then elaborated in three steps to enal 15, which is engaged in a [4+2] ring annulation with a silyl ketene acetal to set an additional nonepimerizable stereocenter. After experimentation with several Lewis acids, dialkylaluminum chlorides were found to promote formation of adduct 12 in 95% yield and in preference to other side reactions (aldol, Michael addition, [2+2]). It is noteworthy that this reaction can be scaled to produce large quantities (up to 50 g) of 12, albeit as a 10:4:1:1 mixture of diastereomers. The authors suggest that the targeted diastereomer 12 is the major component of this mixture, but a rigorous assignment of stereoisomers has not been published. A subsequent Wacker oxidation of 12 with catalytic palladium chloride and aqueous hydrogen peroxide affords ketone 16 as a mixture of diastereomers in 61% yield. To convert 16 to artemisinin, various oxidations were attempted but proved fruitless. Eventually, it was found that generation of singlet oxygen with a molybdate catalyst and hydrogen peroxide allows for the selective oxidation of the enol ether olefin. Previous studies on the conversion of dihydroartemisinic acid to artemisinin have found that the efficiency of peroxidation is dependent on the ability to populate similar enol tautomers, so intermediate 12 may be useful for follow-up study [24]. Treatment of the mixture of oxidized products (17) with mild acid provides artemisinin (up to 1.26 g) in 29–42% yield after crystallization.</p><p>This proof-of-principle synthesis of artemisinin from chemical feedstocks in an academic laboratory highlights the increasing potential of chemical synthesis to provide meaning quantities of highly complex molecules. Key to the success of this approach is the judicious use of a stereochemical relay in the initial retrosynthetic analysis. Further reports on the stereochemical outcome of the key annulation reaction are crucial to evaluate the long-term and large-scale prospects of this approach.</p><p>It should be noted that chemical synthesis has the potential to contribute in a more deep-seated way to the artemisinin story. Unlike most small molecule therapeutics, artemisinin does not bind to a macromolecule target, but instead reacts with heme, a byproduct of hemoglobin metabolism in blood-stage malaria. Simple 1,2,4-trioxanes can be used to mimic this biological reactivity of artemisinin and can be procured by synthesis quite easily on large scale [25–27]. It will take time for these unnatural analogs to progress through the clinic, but they appear to be viable replacements for artemisinin therapy in the future. In the meantime, the lives saved every day by the current application of artemisinin against malaria still justify the continued investigation of its economical production.</p><!><p>Historically, there have been three major terms to characterize the chemical synthesis of a natural product: total synthesis, semi-synthesis and formal synthesis [28]. The term total synthesis arose from early justifications for undertaking the synthesis of a natural product: rigorous structural assignment, proof-of-concept for constructing 'biological' molecules by chemical means, or understanding basic reactivity of organic compounds, all of which can be thought of as the chemical analog of a mathematical proof. The term 'total' can also be taken to mean chemical synthesis starting from molecules so simple that they can be easily derived from the pure elements (C, H2, O2, N2 and so on), and therefore the possibility of synthesizing the target in a general way is proven. Currently, such rationale for synthesis is infrequently invoked: there is seldom any justification for synthesizing a complex molecule from elemental carbon. Instead, a natural product synthesis is now usually undertaken because of the relevance of the target molecule to biology, medicine and the drug discovery process. Given the increased ability of organic chemistry to procure these molecules on large scale for drug discovery or production, this justification is projected to only increase in the future.</p><p>The term 'total synthesis' is now most frequently used to connote a synthesis that begins from commercially available materials that do not contain the full, partial or rearranged skeleton of the target molecule (as opposed to 'semi-synthesis'; see below) [29]. However, commercially available materials vary widely in their cost and complexity, and often constitute an 'outsourcing' of the starting materials rather than an economical basis of the synthesis. To achieve the total synthesis of a molecule is not necessarily to produce it on a large scale or economically. If access to bulk material is a major problem, total synthesis is only one strategy among many.</p><p>The alternative approach to procuring a molecule chemically is semi-synthesis, where most of the molecular skeleton of the target is already intact. This approach can occasionally limit drug development since it inherently limits the number and diversity of analogs that can be easily accessed. However, semi-synthesis has a proven track record within the steroid class of terpenes, which outnumber all other terpenes among validated therapeutics. As a testament to its power, the semi-synthesis of progesterone by Russell Marker was responsible for reshaping all of society: it led to the development of the birth control pill, which partly fueled the sexual revolution of the 1960s [30]. Clearly, semi-synthesis has been the most useful means to access diverse analogs of some natural product classes for use in drug development.</p><p>It may be prescient to consider phasing-out the terms 'total' and 'semi-synthesis' in the current and future eras of chemistry, where advances in the field increasingly deal with practicality, economy and interface with the therapeutic sciences. These authors favor 'chemical synthesis' as a catch-all term to describe the construction of a molecule using largely nonenzymatic methods. The benefit of such a revision of common usage is that it hopefully encourages practitioners of chemical synthesis to focus on solving the specific problems possessed by a molecule, rather than becoming fixated on achieving feats of technical success. 'Total' synthesis does not totally solve a problem any more than 'semi-' synthesis partially solves it. Rather, the unique structural, biological, physical, ecological and economical problems associated with a given molecule dictate the tools that may best be applied, and chemists should think carefully about what approach should be taken.</p><!><p>The synthesis of cortistatin A by Baran and co-workers (Figure 6) is a recent example of the power of the 'semi-synthesis' of steroids in a modern context [31,32]. Cortistatin A belongs to a small family of marine-derived terpenoids that exhibit potent anti-angiogenic activity and therefore may be useful for the treatment of cancer, macular degeneration and other diseases [33]. The retrosynthesis was analyzed primarily from a structure-goal perspective, with an intact steroid skeleton as the terminal goal. Secondarily, the retrosynthesis was refined by exploring the oxidation states of carbons within the cortistatins, and these carbons were mapped onto several commercially available steroids. Of the inexpensive steroids, prednisone (1.2 US$/g) exhibited the closest similarity in oxidation state to the cortistatins; the oxidation state changes separating them are highlighted in Figure 6.</p><p>The A-ring of prednisone requires the most adjustment, so Baran and co-workers first modify the dienone into the fully stereo-defined cortistatin A-ring in five steps (two steps are also used to degrade the D-ring pregnane side chain). A novel geminaldi bromination procedure is utilized to access the correct oxidation state of the C19 methyl; the adjacent alcohol is protected in situ at low temperature as the trimethylsilyl ether to prevent backside attack of the now electrophilic carbon. Alkylation of the proximal ketone then affords as a single diastereomer the unusual bromocyclopropane 21, which is engaged in a ring expansion cascade using samarium (II) iodide and 2,4,4,6-tetrabromo-2,5-cyclohexadienone. The resultant α-bromoketone is then eliminated using lithium bromide and lithium carbonate to form dienone 22. Alane reduction followed by addition of potassium carbonate provides the amino tetraol. In the initial publication, a three-step sequence was required to install the tetrahydrofuran ring, but eventually the group found that treatment of the tetraol with bismuth trichloride could install this moiety directly. Deketalization is also accomplished in this step to give what is termed 'cortistatinone' 23, an intermediate from which arylated analogs can be derived. Installation of the naturally occurring isoquinoline was performed in three steps to give cortistatin A (24) in 16 steps overall. At the time of the publication of the second-generation synthesis, there were two contract companies preparing cortistatin A using the original synthetic route.</p><!><p>A similar semi-synthetic strategy was also pursued by Baran and co-workers in their synthesis of ouabagenin (30, Figure 7), whose glycoside, ouabain, is a cardiotonic steroid with a narrow therapeutic window that has resisted expansion despite some effort in medicinal chemistry [34–36]. Therefore, it was envisaged that semi-synthetic conversion of an inexpensive steroid might increase the opportunities for derivatization of the ouabain core and potentially lead to better tolerated treatments of, for instance, congestive heart failure.</p><p>Similar to the strategy pursued in cortistatin A, a simple mapping of oxidation states between ouabagenin and commercially available steroids identified adrenosterone 25 as a viable precursor. This molecule contains the appropriate functionality for elaboration to ouabagenin – as long as the appropriate transformations can be found to efficiently modify the oxidation state of several key carbons, including the isolated C19 methyl (compare with cortistatin strategy, above).</p><p>Like cortistatin, the synthesis of oubagenin focuses first on addressing the oxidation state changes necessary to convert the A-ring of 25 and its angular methyl (C19) to the polyhydroxylated ouabagenin core. Functionalization of C19 is accomplished using a solid-state irradiation technique for a Norrish type II reaction and allows 26 to be synthesized in two steps from adrenosterone. These atypical conditions (suspension in water and irradiation in a standard photoreactor) prevent Norrish type I fragmentation of the carbon skeleton. Several more oxidation events are used to convert 26 to 27 in six steps. Formation of an acetonide followed by reduction with lithium triethylborohydride affords the stable ethyl boronic ester 28, which is useful as a protecting group and is carried through subsequent steps. Installation of the butenolide and another formal C-H oxidation event (accomplished by ketone unsaturation, isomerization and Mukaiyama hydration) occurs over seven steps. Reduction of the final double bond and placement of the final stereochemistry of the butenolide proved to be a difficult task. Most reducing conditions delivered hydrogen from the convex face and resulted in incorrect stereochemistry for the natural product. Eventually, it was found that in situ-generated cobalt boride reduces the extended conjugated system to give the non-conjugated tetrasubstituted olefin. Treatment with Barton's base reforms the butenolide and provides a 3:1 mixture favoring the correct diastereomer. Final deprotection with acid affords ouabagenin in 20 overall steps, 0.56% yield and six net redox manipulations. To date, over 100 mg of ouabagenin has been synthesized using this route and the synthesis of analogs is now being investigated. Whether this route is able to address the narrow therapeutic index of ouabain remains to be seen, but certainly a foundation has been laid for more extensive work to address this worthy problem.</p><!><p>Another recent example of steroid semi-synthesis applied to a molecule of significant medical importance appeared when Giannis published his approach to the plant alkaloid cyclopamine (36, Figure 8) [37]. This remarkable compound antagonizes a G-protein-coupled receptor called Smoothened, an integral component of the Hedgehog (Hh) signaling pathway, which is overactive in basal cell carcinoma and other cancers [38– 40]. Numerous small molecule therapeutics are currently in development to target this pathway, including derivatives of cyclopamine itself, which was the first identified Hh signaling inhibitor. Unfortunately, (36) is metabolically unstable, as a consequence of the acid-labile allylic tert-alkyl ether functionality, which is readily cleaved via tetrahydrofuran ring-opening to yield a mixture of inactive compounds. Giannis anticipated that chemical synthesis of cyclopamine might allow identification of potent but metabolically stable derivatives.</p><p>Since 36 is a rearranged steroidal alkaloid, an inexpensive steroid skeleton was retrosynthetically targeted as a viable starting point, in the anticipation that a method could be developed to allow migration of the C-D ring-fusion bond to provide C-nor-D-homo skeleton of cyclopamine.</p><p>Starting from a commercially available steroid, dehydro-epi-androsterone (31), Giannis and co-workers access the 2-picolylimine derivative 32 in two steps. This directing group, in concert with tetrakis(acetonitrilo)copper (I) hexafluorophosphate and molecular oxygen, effects a C-H oxidation with excellent regio- and diastereostereo-selectivity. Installation of this oxidation state at C12 is critical to enable skeletal rearrangement of the C-D bond later in the sequence. The spirolactone of cyclopamine is installed over nine steps to give 34. The alcohol of 34 is then triflated, and under these conditions a Wagner–Meerwein shift occurs to give the rearranged product, 35, in high yield and with reasonable selectivity for the exo-alkene. Although the endo-alkene is the desired isomer in the final product, it was determined that the exo-alkene could be isomerized later on in the sequence with little difficulty. Giannis and co-workers were able to elaborate 36 to cyclopamine in eleven steps (20 steps overall, 1% overall yield).</p><p>Although this synthesis was an excellent proof of concept for chemical access to cyclopamine, the real fruit of this project was reported 2 years later. By conversion of the major exo-alkene isomer 35 to exo-cyclopamine (37, see Figure 9), Giannis was able to compare the activity of this close analog with cyclopamine itself, and found that the potency of 37 against Hh-signaling is tenfold greater than that of 36 [41,42]. Even more remarkably, 37 is stable over 24 h at pH 1.5, whereas cyclopamine degrades rapidly under the same conditions. The acid stability of 37 versus 36 is easy to rationalize on the basis of either decreased kinetic basicity of 37 (assuming protonation is the rate-determining step), or the greater stability of the carbenium ion derived from ionization of the C-O bond in cyclopamine 36 (assuming a late transition state and that ionization is the rate-determining step). Most importantly, 37 represents an important lead in developing systemically viable and potent cyclopamine-derived Hh-signaling inhibitors.</p><!><p>Analysis of a target structure occasionally identifies a key retrosynthetic transform (Tf. – the exact reverse of a synthetic reaction) that has the potential to dramatically simplify the synthesis of a complex scaffold. Determination of the best point within the retrosynthesis to apply this Tf. then allows a bidirectional search to connect the intermediates with the final target and with possible starting materials. In this section, we will discuss recent syntheses of bioactive terpenoids in which a key reaction enables a rapid build-up in structural complexity and leads to a highly efficient chemical synthesis.</p><!><p>The Nuphar alkaloids are fresh water plant metabolites that exhibit promising antimetastatic activity both in vitro and in mouse studies [43,44]. Although recent biological work has shown promise for these compounds as therapeutic agents, these studies relied on impure plant extracts due to the limited availability of the pure alkaloids [45]. Current biological work has focused on determination of the discrete mechanism of action associated with the Nuphar dimers, which may involve inhibition of NFκB signaling. An insightful proposal by LaLonde suggested that these dimers might arise in nature by a sulfurizing dimerization of quinolizidine monomers [46]. Realization of such a dimerization in the laboratory would vastly simplify the synthesis of these compounds. However, while the biosynthetic proposal was a useful guide, it is important to note that this dimerization pathway was not expected to be selective for the formation of a naturally occurring stereoisomer; in fact, conformational analysis predicts the formation of the only stereoisomer never isolated. Nevertheless, the level of simplification provided by the dimerization Tf. prompted the authors of this review to explore the corresponding transformation in the laboratory [47].</p><p>Chirality can be introduced in the synthesis (Figure 10), with high enantioselectivity using Buchwald's conditions for asymmetric enone reduction of 38, followed by palladium-catalyzed allylation [48]. It should be noted that this in-house procedure for reductive allylation of cyclopentenones is the only reaction that cleanly provides 39 in reasonable yield. Lactam 40 is synthesized in three more steps, and is primed for ring-closing metathesis followed by in situ protodesilylation in the manner of Vanderwal to give the bicyclic lactam 41 [49]. Installation of the furan using a modification of Fowler's procedure and N-oxidation provides amine-oxide 42 [50]. Treatment of 42 with trifluoroacetic anhydride effects a highly regioselective Polonovski elimination at only one of three possible positions, and subsequent addition of sodium tetrasulfide in DMSO yields dimeric intermediate 43. This intermediate proved difficult to isolate and purify, but in situ reduction with sodium borohydride provided neothiobinupharidine 44 in good yield and high stereoselectivity in preference to three other possible stereoisomers. The solvent for dimerization proved vital to good diastereo selection, which could be roughly correlated to the solvent's dispersion force component of its Hansen parameters [51]. Subsequently, 43 has been isolated in pure form, which sets the stage for exploration of its biological mechanism of action.</p><!><p>Hyperforin (52, Figure 11) is a terpenoid plant metabolite believed to be responsible for the antidepressant activity ascribed to St John's Wort (Hypericumperforatum L.) [52] Unfortunately, its off-target effects and inherent instability limit its immediate application as a small molecule therapeutic. Therefore, Shair and co-workers targeted a modular synthetic sequence with the aim of improving the biological properties of 52 [53]. If a single, key insight can be attributed to both the success and efficiency of the synthesis, it is probably the realization of latent symmetry within phloroglucinol core, which might be accessed by relaying the stereochemistry of a chiral sidechain into a symmetrical cyclohexane (red box, Figure 11). The implementation of this approach is shown below.</p><p>Starting from the inexpensive polyprenolgeraniol, Shair and co-workers are able to synthesize epoxide 45 in five steps. The anion derived from prenylcyclohexadiene 46 displaces the alkyl bromide of 45 and sets the stage for the key desymmetrization reaction. Exposure of 47 to trimethylsilyltriflate and 2,6-lutidine cleanly inverts the epoxide stereochemistry (as observed in related terpenoid systems [54]) and yields 48 as the only isolable product. It is noteworthy that two chiral all-carbon quaternary stereocenters are formed in this step, and the scalability of this sequence is demonstrated by the preparation of 60 g of 48. Thionocarbonate 49 is then prepared in four steps, and allows for installation of the allyl group using Keck allylation followed by olefin cross-metathesis with isobutene [55,56]. Shair and co-workers then install another two prenyl units over the next five steps to afford hyperforin in a longest linear sequence of 18 steps. Over 40 mg of hyperforin had been prepared at the time of their publication.</p><!><p>The mebutate ester of ingenol was recently approved by the US FDA for topical treatment of actinic keratosis, and clinical trials are in progress for treatment of basal cell carcinoma [57]. Ingenol itself (59, Figure 12) is a diterpenoid that was first isolated from Euphorbia ingens in 1968 [58]. The limited quantities of 59 available from plant extraction (390 mg, 59, from 2 kg dried seeds) [59] and the diverse phenotypic responses of cells to ingenol analogs indicate that the development a practical, diversifiable synthetic route to this metabolite might enable significant contributions to medicine and biology [59]. Three previous total syntheses of ingenol have been published, all requiring between 37 and 45 steps [60–63]. Although these exercises have made important contributions to understanding the reactivity of ingenanes, they are impractical for analog exploration or large-scale production. Part of the difficulty of synthesizing ingenol is developing a strategy to create its unique 'in, out' [4.4.1] bicycloundecane architecture. This unusual scaffold arises in nature via a 1,2-pinacol-type shift from a related (tigliane) [5.4.0] bicycloundecane skeleton [57], which was demonstrated by Cha to be easily reproduced by chemical methods [64]. Embedding this transform into a retrosynthetic analysis allows a bidirectional search for routes to synthesize the tigliane skeleton, and strategies to convert the pinacol product to ingenol. This strategy formed the basis of a remarkably concise synthesis of ingenol from the Baran laboratory.</p><p>Starting from the inexpensive monoterpene(+)-carene (53), Baran and co-workers install the requisite functionality for a Pauson–Khand cyclization, and use Brummond's conditions to effect the intramolecular variant of this reaction, producing dienone 56 [65]. Functional group interconversions over the next three steps provide 57, which is ready for skeletal rearrangement to install the 'in, out' [4.4.1] bicycloundecane framework. Initial attempts with various Lewis acid catalysts failed, but rigorous control of temperature (−78 to −40°C) during treatment with boron trifluoride etherate allows the desired vinylogous pinacol rearrangement to occur to provide ingenane 58. Over the next four steps, 58 is converted to ingenol 59, which ultimately arrives in only 14 steps from 53 and 1.4% overall yield.</p><!><p>Retrosynthetic analysis of a complex structure will identify multiple reasonable pathways that may lead to a viable laboratory synthesis. Choice of the 'best' pathway can be assisted by identifying the problem to be addressed (scalability, modularity and predictability; see above) and eliminating the routes that would not fulfill the correct criteria. Among the remaining routes, some stand out as more concise and elegant than others, usually as a result of judicious application of Tf.s) that efficiently remove complexity from the target molecule [66]. Occasionally, these Tf.s are purely theoretical, and correspond to chemical reactions that, while probable, have not been previously reported. In this section, we will highlight syntheses where new reactions are invented to 'jump the gaps' in the retrosynthetic logic. We will also cover syntheses that take advantage of newly invented methods that dramatically simplify the construction of complex terpenes.</p><!><p>Resistance to the front-line medication artemisinin (see above) has recently been reported in Southeast Asia, and therefore the search for new antimalarials is of utmost importance [67]. Over the last 40 years, a large family of marine terpenes has accrued in the literature, and has received attention for its unusual biosynthetic incorporation of nitrogen atoms derived from cyanide [68]. These secondary metabolites have also shown potent and selective activity against Plasmodium parasites (the causative agents of malaria) and due to the presence of one or more isonitrile functions in the molecules, it has been theorized that these compounds may disrupt heme detoxification in blood stage parasites (see also 'Artemisinin' section above) [69]. Unfortunately, the mechanism of action of these compounds is unknown, and in vitro experiments to support heme binding do not account for the efficacy of related terpenes that contain formamide instead of isonitrile groups.</p><p>Our group undertook the synthesis of the most potent member of the amphilectene isocyano terpenes (64, Figure 13) in order to access both bulk material and eventually photoaffinity-labeled analogs to discover the antimalarial mechanism of action of this class [70,71]. There were no efficient methods reported to install the key C-N bond, which clued us in to the potential for the development of a new method. Additionally, there were no efficient approaches to the carbon skeleton of the amphilectenes and related terpenes. To solve these problems, we invented a new type of linear polyene, which we termed a 'Danishef-skydendralene,' that is capable of undergoing iterative cycloadditions with high levels of regio- and stereo-selectivity [72]. To address the C-N bond, we recognized stereoinversion of a tertiary alcohol to be the shortest route to install the isonitrile pharmacophore, but no method existed for such a reaction. Therefore, we designed a Lewis-acid catalyzed solvolysis reaction to invert the stereochemistry of a chiral alcohol and simultaneously install the tert-alkyl isonitrile. The combined results of these methodological advances are illustrated in the synthesis of amphilectene 64 in Figure 13.</p><p>In the first two steps of the synthesis, our designed polyene, the parent [3]-Danishefsky-dendralene 60 reacts with double-dienophile 61 to provide the full amphilectene core 62 as the major stereoisomer. Four steps are required to elaborate this tricycle to the penultimate intermediate 63, which when subjected to our stereoinversion reaction provides the targeted molecule 64 with high diastereoselectivity. This final step likely proceeds via contact ion pair inversion (see 66), and can be used to install multiple nitrogenous functional groups since the isonitrile is easily degraded to the parent amine (see 67→68) [73]. This reaction can be extended to more challenging substrates that illustrate its potential to convert abundant terrestrial terpenes (bisabolol, 69) to scarce marine terpenes (70), or to selectively modify tertiary alcohols in the presence of secondary and primary alcohols – a selectivity which is orthogonal to standard SN2 reactions. It is anticipated that these conditions for Lewis-acid catalyzed solvolysis may lead to a small arsenal of stereoinversion reactions for tertiary alcohols.</p><!><p>Oxygenated terpenoids are usually biosynthesized by two main reaction pathways: capture of water to terminate cationic polycyclizations, and oxidation of terpenecarbocycles by cytochrome P450 enzymes [1] In the laboratory, chemical mimicry of either of these enzyme-mediated steps is possible, but far from general. Instead, use of the innate reactivity and/or 'charge affinity pattern' of an oxygenated carbocycle [74] can be used to easily dissect the molecule using the chemistry of carbonyls: aldol, Michael and Mannich transforms, among others. If the functional groups do not signal these facile maneuvers (the groups are 'dissonant,' to use Evans' terminology [74]), then dissection of the scaffold becomes more challenging and less uniform, which usually corresponds to more functional group interconversions and a longer step count.</p><p>With these challenges in mind, the Morken group sought to explore the application of their recently developed asymmetric diborylation technology (74→75, Figure 14) to the synthesis of complex oxygenated metabolites [75–77]. Remarkably, addition of a 1,4-dicarbonyl to the reaction mixture containing a chiral diboryl alkene results in tandem diallylation of the dicarbonyl to produce cyclic structures (76) containing four new stereocenters with high diastereo-and enantio-selectivity. These structures map well onto several known terpenoids, including pumilaside aglycon (80) [78]. The Morken group synthesizes this complex tricycle in only six steps first by diborylation of neryl-derived triene 77 followed by addition of 4-oxopentanal to provide cyclohexanediol 79 as the major stereoisomer. Elaboration of 79 to the target molecule requires only five steps of simple functional group interconversions, and more importantly, this method provides access to highly complex building blocks for terpene synthesis.</p><!><p>In contrast to terrestrial terpenes, marine terpenes are frequently subject to oxidative modification by halogenating enzymes that add bromine and chlorine atoms to their substrates. Given the abundance of halide anions in seawater, these halonium biosynthetic pathways are perhaps not surprising [79,80]. However, regio- and stereo-selective incorporation of halogens into terpenes scaffolds using chemical methods is by no means straightforward, and is the subject of considerable current research.</p><p>Peyssonol A (83, Figure 15) is a secondary metabolite isolated from Red Sea alga (Peyssonnelia sp.) and is likely biosynthesized via halonium-mediated polycyclization of a farnesyl chain [81]. This metabolite possesses allosteric inhibitory activity for HIV reverse transcriptase, paralleling the activity of related sesquiterpenes and avaroland avarone, which have been shown to inhibit HIV-1 transcriptase at concentrations as low as 300 nM [82]. Nevertheless, many compounds from this family suffer from high cytotoxicity. While the pharmaceutical relevance of this family of compounds remains uncertain, generation of analogs is crucial for understanding SAR and reducing the cytotoxicity of these molecules.</p><p>When replicating halonium cyclizations in the laboratory, a confounding feature is the ability of the counter anion to pre-emptively terminate the cyclization process [83]. Nevertheless, these polycyclization cascades can be vastly simplifying for the synthesis of polycyclic terpenoids because of the commercial availability of linear precursors derived from geraniol, nerol and farnesol. Snyder and co-workers developed bromo diethyl sulfonium bromo pentachloro antimonate (V) (BDSB) as a novel reagent for halonium ion promoted cyclizations [84,85]. This reagent benefits from its non-nucleophilic antimonate counter anion that does not interrupt the cation cyclization. Reactions are typically completed in less than 10 min and provide acceptable yields when using farnesyl-based substrates.</p><p>For the synthesis of peyssonol A, Snyder and co-workers subjected 2Z,6E-farnesyl carbonate 81 to BDSB, which causes efficient halonium-induced cyclization to provide tricycle 82. This intermediate is carried forward six steps to 83, which matches the spectroscopic data from the isolation literature and revises the original assignment of a cis-decalin topology of the natural product. Snyder and co-workers disclosed in follow-up work that they were able to use this technology to generate several simple analogs possessing antiviral activity against HIV in the low micromolar to high nanomolar range while having good therapeutic indices (>20), an excellent proof of principle for translating methodological innovation into synthetic application and on to medicinal chemistry exploration [86].</p><!><p>Bioassay-guided fractionation of the marine sponge Dysidea frondosa identified frondosin B (91, Figure 16) and several of its family members as potential anti-inflamatory compounds [87]. Initial work showed that frondosin B had micromolar inhibitory activity against IL-8Rα, IL-8Rβ and PKC-α. The authors suggested that the inhibitory activity of the frondosin family members might be a result of general reactivity of the compounds rather than true agonist/antagonist activity. A second report also suggested that other members of the frondosin family might have moderate, but variable, anti-HIV activity [88]. During the isolation process it was noted that several of the compounds have opposite optical rotation values and consequently they are thought to occur in nature as scalemic mixtures.</p><p>Frondosin B is characterized by a benzofuran ring system attached to a norsesquiterpenoid framework that contains a single stereogenic methyl group. Despite the seemingly simple framework, efforts to install the lone benzylic stereocenter have proved difficult, as evidenced by lengthy asymmetric syntheses. The first asymmetric synthesis of natural (+)-frondosin B was published by Danishefsky in 2001 and suffered from high step count and a low overall yield of 0.7% [89]. In 2002, Trauner published the synthesis ent-(-)-frondosin B and called into question the absolute configuration of the natural product. Trauner's synthesis also suffered from high step count, but a more reasonable overall yield of 7.3% was obtained [90,91]. More recently, Ovaska published an eight-step racemic synthesis of frondosin B in 2007 and followed up with a ten-step asymmetric synthesis of (-)-frondosin B in 2009 [92,93] .</p><p>MacMilllan and co-workers published a synthesis of (+)-frondosin B in 2010 [94], which relies on the laboratory's previous work in asymmetric catalysis [95]. The MacMillan laboratory previously reported the conjugate addition of aryltrifluoroborate potassium salts to chiral iminium ions generated by the condensation of aldehydes with amino acid-derived catalysts. These reactions provide chiral β-disubstituted aldehydes in good yield and excellent enantioselectivity. Application to the synthesis of 91 entailed reaction of commercially available boronic acid 87 with crotonaldehyde in the presence of catalyst 84 and hydrofluoric acid in ethyl acetate, to provide 88 in high yield and enantioselectivity. When 88 is treated with the alkenyl lithium reagent derived from the Shapiro reaction of 89 and t-BuLi, 90 is produced in good yield as an inconsequential mixture of diastereomers. A two-step process involving a molybdenum catalyst and BBr3 was initially used to synthesize (+)-frondosin B, but upon further experimentation it was discovered that treatment of 90 with BBr3 causes formation of the final seven-membered, demethylationthe phenoxy ether, and isomerization of the olefin to the desired endocyclic isomer, supplying (+)-frondosin B in only three steps (from 87) and an outstanding 50% overall yield.</p><!><p>Terpenoids provide an excellent foundation for the development of new small molecule therapeutics. The increasing focus in academia on generating concise and scalable routes to these molecules should allow a much broader range of terpene scaffolds to be considered viable foundations for medicinal chemistry campaigns. Similarly, the economical production of complex terpenes on multikilogram scale through chemical synthesis is increasingly possible as a less-expensive alternative to isolation from living organisms (this has been true of the monoterpene menthol for several years). Key to the development of a chemical solution to a given terpenoid structure is identification of the most important problem to be addressed by synthesis – diverse analog production, large-scale production, increased chemical/metabolic stability and generation of mechanistic probes, among others. In support of any of these goals, the development of a concise synthetic route that minimizes the number of steps is an additional, crucial agenda. The strategies listed above to minimize synthetic operations should provide a general approach to reduce the costs of terpene synthesis at multiple levels. It is anticipated that the brevity, practicality and favorable economies of modern chemical synthesis will reinvigorate the use of terpenes in the drug discovery process. Investment in the right scaffolds, synthesis strategies and methodological advances are likely to yield a high return for any drug discovery team.</p><!><p>Chemical synthesis – a central, foundational industry in society – is not anticipated to recede in the next decade. Instead, molecules of increasing complexity, including terpenes, will be increasingly accessible through synthesis and ever more applied to fields that interface with chemistry, especially medicine. A focus on simplicity of synthesis through the invention of operationally straightforward, predictable and air/water/cell-tolerant reactions will predominate. Most importantly, application of both new reactions and new molecules to modulate biological function will increase in power, breadth and predictability. The artificial, conceptual barrier separating 'natural product-like' from 'drug-like' molecules is likely to fall, as drugs become more complex, and intersperse more stereocenters, rotatable bonds and common metabolite motifs such as polyketide chains. Similarly, research in chemical synthesis will become equally liberated to address problems at the interface of other diverse fields, rather than remaining insular and self-serving. The prospects for discovery in organic chemistry are therefore both exciting and limitless.</p>

PubMed Author Manuscript

Transferase Activity of Lactobacillal and Bifidobacterial \xce\xb2-Galactosidases with Various Sugars as Galactosyl Acceptors

The \xce\xb2-galactosidases from Lactobacillus reuteri L103 (Lreu\xce\xb2gal), Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (Lbul\xce\xb2gal), and Bifidobacterium breve DSM 20281 (Bbre\xce\xb2gal-I and Bbre\xce\xb2gal-II) were investigated in detail with respect to their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products d-glucose and d-galactose, and certain sugar acceptors such as N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-galactosamine (GalNAc), and l-fucose (Fuc) under defined, initial velocity conditions. The rate constants or partitioning ratios (kNu/kwater) determined for these different acceptors (termed nucleophiles, Nu) were used as a measure for the ability of a certain substance to act as a galactosyl acceptor of these \xce\xb2-galactosidases. When using Lbul\xce\xb2gal or Bbre\xce\xb2gal-II, the galactosyl transfer to GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and GlcNAc used in equimolar substrate concentrations, Lbul\xce\xb2gal and Bbre\xce\xb2gal-II catalyzed the formation of N-acetyl-allolactosamine with the highest yields of 41 and 24%, respectively, as calculated from the initial GlcNAc concentration.

transferase_activity_of_lactobacillal_and_bifidobacterial_\xce\xb2-galactosidases_with_various_sugar

INTRODUCTION<!>Chemicals<!>Enzyme Preparation<!>\xce\xb2-Galactosidase Assays<!>GOS Synthesis<!>Intermolecular Galactosyl Transfer under Defined Initial Velocity Conditions<!>N-Acetyl Oligosaccharide Formation<!>Purification of N-Acetyl-oligosaccharides<!>NMR Measurements<!>RESULTS AND DISCUSSION<!>Hydrolysis versus Transgalactosylation Using Lactose as Substrate<!>Partitioning Analysis<!>Formation of GalNAc-Containing Transgalactosylation Products<!>Structural Characterization of GlcNAc Transgalactosylation Products

<p>β-Galactosidases (β-d-galactoside galactohydrolase, EC 3.2.1.23; βgal) have long been known to catalyze the hydrolysis of lactose into glucose and galactose, as well as the transfer of a galactosyl moiety to suitable acceptors. If lactose is present in excess, βgal will use lactose, or its hydrolysis products, glucose and galactose, as alternative galactosyl acceptors to form galacto-oligosaccharides (GOS) (Scheme 1). The source of βgal, lactose concentration, and working temperature influence the GOS type, GOS yield, and specific linkages formed, thus creating a wide array of GOS.1</p><p>β-Galactosidases have also been used to form hetero-oligosaccharides (HeOS) or other galactosylated compounds, with mannose, fructose, N-acetylneuraminic acid, glucuronic acid, and a number of aromatic compounds used as galactosyl acceptor.2–7 Using this approach, Sulfolobus solfataricus and Kluyveromyces lactis β-galactosidases were used to produce lactulose, a commercial prebiotic disaccharide, and galactosylated aromatic primary alcohols.5,8 Part of the human milk oligosaccharide (HMO) core structure or structurally related compounds can also be accessed, albeit not in pure form, through an approach based on β-galactosidase-catalyzed transglycosylation with lactose as donor (thus transferring galactose onto suitable acceptors) and N-acetylglucosamine (GlcNAc) as acceptor. Thereby, lacto-N-biose (LNB; Gal-β-1,3-GlcNAc) and N-acetyl-lactosamine (LacNAc; Gal-β-1,4-GlcNAc) together with their regioisomers can be obtained. β-Galactosidases from Bacillus circulans, K. lactis, and Lactobacillus bulgaricus were proved to be suitable biocatalysts for the formation of N-acetyl-oligosaccharides using lactose and GlcNAc as substrates.9–11 Although the feasibility of this transferase reaction has been shown, no study has dealt with a more detailed biochemical characterization of this reaction.</p><p>The intermolecular transfer of galactose to acceptors other than water typically presents the major pathway for the formation of GOS during lactose hydrolysis by retaining β-galactosidase (Scheme 1). As the sugar species in the mixture that can act as a nucleophile and hence as a galactosyl acceptor change constantly during the reaction, an exact prediction of product formation and degradation cannot be made. However, the partitioning of the galactosylated enzyme (which is formed as an intermediate in the enzymatic reaction; E-Gal in Scheme 1) between the reaction with water and, hence, hydrolysis and the reaction with a galactosyl acceptor can be studied under defined initial velocity conditions. When complete hydrolysis of the disaccharide lactose occurs, equimolar amounts of d-glucose and d-galactose are formed, and therefore the initial velocities at which the two sugars are released are identical and the ratio vGlu/vGal is 1.0. In the presence of an appropriate Nu (e.g., a sugar acceptor for galactose) vGlu/vGal will increase because some of the galactosyl moieties will not be released but transferred onto the acceptor. Richard et al.12 derived eq 1 from Scheme 1, where the rate constant ratio kNu/kwater is obtained as the slope from the linear correlation of vGlu/vGal with increasing concentrations of Nu: (1)vGlcvGal=1+kNukwater[Nu]</p><p>With regard to the process for the formation of HeOS that mimics HMO, the main candidates for galactosyl acceptors are N-acetylglucosamine, N-acetylgalactosamine, and l-fucose (which will be added to the reaction mixture), the substrate lactose, and its hydrolysis products d-glucose and d-galactose. The rate constant ratios determined for the different acceptors can therefore be used as a measure of the ability of a certain substance to act as a galactosyl acceptor (i.e., nucleophile), which in turn allows an estimation of the transgalactosylation products obtained for a known reaction mixture or the suitability of a certain enzyme for the efficient synthesis of HeOS.</p><p>In this paper, the propensity of the β-galactosidases from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (L. bulgaricus, Lbulβgal), L. reuteri L103 (Lreuβgal), and Bifidobacterium breve DSM 20281 βgal I (Bbreβgal-I) and βgal-II (Bbreβgal-II) to transfer galactosyl moiety to different acceptors such as lactose (Lac), glucose (d-Glc), galactose (d-Gal), l-fucose (Fuc), N-acetyl-d-glucosamine (GlcNAc), and N-acetyl-d-galactosamine (GalNAc) was determined to deepen the understanding of the relative extent of galactosyl transfer of a specific β-galactosidase to different sugar acceptors. The four β-galactosidases belong to glycoside hydrolase family 2 (GH 2) and are of the hetero-oligomeric LacLM type (Lreuβgal) or the homo-oligomeric LacZ type (Lbulβgal, Bbreβgal-I, Bbreβgal-II). The enzymatic synthesis of N-acetyl oligosaccharides using the two enzymes Lbulβgal and Bbreβgal-II will be also presented.</p><!><p>All chemicals and enzymes were purchased from Sigma (St. Louis, MO, USA), unless stated otherwise, and were of the highest quality available. The test kit for the determination of d-glucose was obtained from Megazyme (Wicklow, Ireland). Galacto-oligosaccharide standards of β-d-Galp-(1→3)-d-Glc, β-d-Galp-(1→6)-d-Glc, β-d-Galp-(1→3)-d-Gal, β-d-Galp-(1→4)-d-Gal, β-d-Galp-(1→6)-d-Gal, β-d-Galp-(1→3)-β-d-Galp-(1→4)-d-Glc, β-d-Galp-(1→4)-β-d-Galp-(1→4)-d-Glc, and β-d-Galp-(1→6)-β-d-Galp-(1→4)-d-Glc were purchased from Carbosynth (Berkshire, UK), whereas β-d-Galp-(1→3)-d-GlcNAc (lacto-N-biose I, LNB I) and β-d-Galp-(1→4)-d-GlcNAc (N-acetyl-d-lactosamine, LacNAc) were purchased from Dextra Laboratories (Reading, UK).</p><!><p>β-Galactosidase from L. reuteri L103 (Lreuβgal) was recombinantly produced in Escherichia coli and purified as reported previously,13 whereas the lacZ gene encoding βgal from L. bulgaricus DSM 20081 was expressed in L. plantarum WCFS1 and the corresponding protein was purified as described before.14 B. breve DSM 20231 βgal-I and βgal-II were prepared according to the method of Arreola et al.15</p><!><p>The measurement of β-galactosidase activity using o-nitrophenyl β-d-galactopyranoside (oNPG) or lactose as substrate was carried out as described previously.16 Briefly, these assays were performed in 50 mM sodium phosphate buffer of pH 6.5 at 30 °C, and the final substrate concentrations in the 10 min assay were 22 mM for oNPG and 600 mM for lactose. Protein concentrations were determined using the method of Bradford with bovine serum albumin (BSA) as a standard.</p><!><p>The ability of the four recombinant β-galactosidases to synthesize GOS was compared by carrying out discontinuous conversion reactions in a 2 mL scale. The activities (ULac/mL) of the recombinant βgals used were as follows: L. reuteri, 0.8; L. bulgaricus, 1.5; B. breve βgal-I 1.0; B. breve βgal-II, 2.5. Reaction conditions were 600 mM initial lactose concentration in sodium phosphate buffer (50 mM, pH 6.5) containing 1 mM Mg2+ with the incubation temperature set at 30 °C and continuous agitation at 300 rpm. At certain time intervals, samples were withdrawn and the reaction was stopped by heating at 95 °C for 5 min. The composition of the GOS mixture was analyzed by HPAEC-PAD following the method described previously.15 d-Glc, d-Gal, lactose, and GOS components were identified and quantified using the external standard technique.</p><!><p>Initial velocities of d-Glc or d-Gal release were determined using 50 mM sodium phosphate buffer (pH 6.5) at 30 °C using either 10 mM oNPG or 100 mM lactose as substrate. These substrate concentrations were a compromise between the practical requirement to measure the initial velocity of d-Gal (and/or d-Glc) formation and to maximize the transfer of d-Gal to the external added nucleophile but not to the substrate. The final enzyme concentration used was ≤1.0 U/mL. The relationship between [oNP] (or [d-Glc]) and [d-Gal] was found to be linear up to 30 min. Thus, the standard reaction time of 20 min was used. voNP, vGlc, and vGal were obtained from measuring the molar concentrations of oNP, d-Glc, and d-Gal, respectively. The ratios of voNP and vGal were measured in the absence and presence of d-glucose with its concentration varied between 2.5 and 20 mM.</p><p>The intermolecular transgalactosylation to lactose was performed with various initial lactose concentrations (9–600 mM), whereas galactosyl transfer to either GlcNAc, GalNAc, or l-fucose was determined using 100 mM lactose with acceptor concentrations varying from 12.5 to 200 mM. After incubation of the reaction mixture for 20 min at 30 °C, the reaction was stopped by heating for 5 min at 95 °C. The rate of formation of oNP (voNP) was measured using the standard β-galactosidase assay, whereas d-galactose (vGal) or d-glucose (vGlc) measurement was carried out by HPLC (Dionex, Chelmsford, MA, USA) using an Aminex HPX-87K column (300 × 7.8 mm; Bio-Rad, Hercules, CA, USA) equipped with a refractive index detector. Water was used as mobile phase at a flow rate of 0.80 mL min−1, and the column temperature was 80 °C.</p><!><p>N-Acetyl-oligosaccharide synthesis was carried out using lactose and GlcNAc (or GalNAc) as substrate with either Lbulβgal or Bbreβgal-II. The effects of temperature (30 and 50 °C), substrate concentration (0.6 and 1 M), molar ratio of donor to acceptor (1:2, 1:1 and 2:1), and enzyme concentration (2.5 and 5.0 U/mL) on the synthesis were also investigated. Substrates were dissolved in 50 mM sodium phosphate buffer (pH 6.5) containing 1 mM Mg2+. The enzyme was added, and the incubation was done at the required temperature at 300 rpm with a thermomixer (Eppendorf, Hamburg, Germany). Aliquots of samples were withdrawn at certain time intervals to determine the residual activities and carbohydrate content using either HPAEC-PAD as described by Splechtna et al.17 or an HPLC system consisting of a UV detector and the Hypercarb column (0.32 × 150 mm, inner diameter = 5 μm; Thermo Scientific). Ammonium formate buffer (0.3% formic acid, pH 9.0) was used as buffer A, and a gradient was performed from 0 to 35% acetonitrile within 35 min using a Dionex Ultimate 3000 pump (cap flow, 1 mL/min). The GlcNAc transgalactosylation yield was determined on the basis of the starting GlcNAc concentration and was calculated using eq 2.</p><!><p>For purification and identification of GlcNAc transfer products, a 10 mL discontinuous batch reaction using Bbreβgal-II (5 ULac/mL) was carried out at 30 °C using initial equimolar concentrations of lactose and GlcNAc (600 mM each) dissolved in 50 mM sodium phosphate buffer (pH 6.5) with 1 mM Mg2+. Agitation was at 300 rpm on a rotary shaker. After 4 h, the reaction was stopped by heating at 95 °C. GlcNAc-containing oligosaccharides were prepared also with Lbulβgal in a similar way, and here the reaction conditions were 2.5 ULac/mL with 1 M lactose and 1 M GlcNAc at 50 °C. Due to the complex course of transgalactosylation reactions, the reaction mixture was partially purified by gel permeation chromatography on Bio-Gel P2 (2.0 × 100 cm) equilibrated in water containing 5% (v/v) ethanol and 0.0015% (w/v) NaCl. The elution was followed by UV reading at 210 nm to detect the presence of GlcNAc and transgalactosylation products. The fractions containing the desired transgalactosylation products were pooled, freeze-dried, and redissolved in acetonitrile. The complete purification of the transgalactosylation products was obtained by using an HPLC system and the Hypercarb column as described above.</p><!><p>NMR spectra were recorded at 27 °C in 99.9% D2O on a Bruker Avance III 600 spectrometer (1H at 600.13 MHz and 13C at 150 MHz) equipped with a BBFO broad-band inverse probe head and z-gradients using standard Bruker NMR software. COSY experiments were recorded using the program cosygpqf with 2048 × 256 data points, respectively, per t1 increment. Multiplicity-edited HSQC spectra were recorded using hsqcedetgp with 1024 × 128 data points and 16 scans, respectively, per t1 increment. 1H NMR spectra were referenced to internal DSS (δ = 0); 13C NMR spectra were referenced to external 1,4-dioxane (δ = 67.4).</p><!><p>We studied in detail different β-galactosidases from probiotic strains of lactic acid bacteria (Lreuβgal and Lbulβgal) and bifidobacteria (Bbreβgal-I and Bbreβgal-II) with respect to their propensity to transfer galactosyl moieties onto certain sugar acceptors. Although the biochemical properties of these enzymes have been investigated in earlier works,14–17 detailed kinetic analyses of the formation of oligosaccharides based on the transfer constants (kNu/kwater) (Scheme 1) have not been reported.</p><!><p>The measurement of the d-Glc/d-Gal ratio as a function of the reaction time provides a good estimate as to what extent transgalactosylation (onto lactose, d-Glc, or d-Gal as acceptors) competes with hydrolysis during lactose conversion. This ratio, however, does not accurately reflect the true extent of lactose conversion because a transfer of the galactosyl moiety can occur via either intramolecular or intermolecular reaction.</p><p>The formation of d-Glc and d-Gal was monitored over the entire course of the conversion using an initial lactose concentration of 600 mM (Figure 1A). At all times, the ratio of d-Glc/d-Gal was higher for Lbulβgal when compared with that of Lreuβgal, Bbreβgal-I, and Bbreβgal-II, indicating that this enzyme shows a more pronounced transferase activity. The maximum value of the d-Glc/d-Gal ratio for Lbulβgal was 3.0 at 16% lactose conversion, where trisaccharides form predominantly. This had also been confirmed in our previous work showing that β-d-Galp-(1→6)-β-d-Galp-(1→4)-d-Glc and β-d-Galp-(1→3)-β-d-Galp-(1→4)-d-Glc are the main transgalactosylation products at the beginning of the reaction when using this enzyme. This ratio decreased to 2.71 at ~30% lactose conversion, then remained constant until lactose conversion was ~70%, and decreased dramatically with >90% lactose conversion. The same trend was observed for Bbreβgal-I, where the highest d-Glc/d-Gal ratio was observed at the initial stage of the reaction and further decreased as the reaction progressed. For Lreuβgal and Bbreβgal-II, maximum values of the d-Glc/d-Gal ratio were found over a rather broad range of lactose conversion (20–80%). At about 98–99% lactose conversion, the d-Glc/d-Gal ratio was close to 1.0 for Lreuβgal and Bbreβgal-I, suggesting that lactose and all GOS formed were extensively hydrolyzed. In contrast, the d-Glc/d-Gal ratio with Lbulβgal and Bbreβgal-II was still 1.76 and 1.42, respectively, at almost complete lactose conversion, implying that a significant amount of GOS resisted hydrolysis even when lactose conversion was almost 100%.</p><p>The ratio of d-Glc/d-Gal measured can be related directly to the level of GOS formation when these enzymes are used for the reaction with lactose. Lbulβgal, which exhibited the highest d-Glc/d-Gal ratio, showed the highest GOS yield at all lactose conversion levels compared to that of the other three βgals (Figure 1B). The yields as well as the type of GOS formed differ significantly among the βgals studied. Overall, Lreuβgal and Lbulβgal yielded the same mixture of GOS, which is different from those of Bbreβgal-I and Bbreβgal-II. Typical HPLC chromatograms of GOS formed by Lbulβgal and Bbreβgal-II are depicted in Figure S1.</p><!><p>The transfer constant kNu/kwater, which is obtained from the velocity ratios of vGlc/vGal or voNP/vGal, provides a useful tool to measure the ability of a certain substance to act as a galactosyl acceptor (i.e., a nucleophile, Nu), which in turn allows an estimation of the level of transgalactosylation products obtained from a known reaction mixture. Initial velocities were measured at 30 °C in 50 mM sodium phosphate buffer (pH 6.5). Plots of (voNP/vGal) or (vGlc/vGal) against [Nu] were linear for a specific range of the acceptor concentration. Deviation from linearity, which occurred mainly at low and high concentrations of the nucleophile, may be due to competition for binding to the nucleophile-binding site of the galactosyl-enzyme intermediate [E-Gal].18 The F test at 95% probability level confirmed the validity of the linear fit for the range of [Nu] as shown in Figure S2. Moreover, the fit of the lines as represented by r2 was usually >0.98. When kGlc/kwater was determined, Bbreβgal-II showed the lowest partitioning ratio (3.91 ± 0.44 M−1), whereas Lbulβgal gave the highest (9.36 ± 0.56 M−1) as shown in Table 1. Likewise, when lactose alone was used as the substrate, where the only possible galactosyl acceptors are lactose and its hydrolysis products, d-Gal and d-Glc, Bbreβgal-II showed the lowest kLac/kwater ratio (0.53 M−1), whereas that of Lbulβgal was the highest (2.79 M−1), again confirming the high transferase activity of this latter enzyme.</p><p>When kGlc/kLac was determined (obtained from the ratio of kGlc/kwater to kLac/kwater), Bbreβgal-II showed the highest ratio of 7.4 and Bbreβgal-I, a ratio of 4.2, whereas Lreuβgal and Lbulβgal showed lower ratios of 3.4 and 3.3, respectively. These values suggest that d-Glc is a far better galactosyl acceptor for the two bifidobacterial β-galactosidases than lactose when compared to the two lactobacillal β-galactosidases. Hence, disaccharides other than lactose will make up a large proportion of GOS mixtures formed with the two bifidobacterial enzymes. Figure 2A confirms that d-glucose is in fact a better galactosyl acceptor than d-lactose when looking at the ratio of total GalGlc disaccharides to Galβ-d-Galp-(1→4)-d-Glc trisaccharides formed. This was especially pronounced at the beginning of the reaction, where this ratio was ~5 for Bbreβgal-I or ~4 for Bbreβgal-II at 20% lactose conversion, whereas it was 0.44 for both Lbulβgal and Lreuβgal. We reported in our recent study that the predominant oligosaccharide product of both bifidobacterial enzymes was β-d-Galp-(1→6)-d-Glc (allolactose), accounting for approximately 45 and 50% of the GOS formed by transgalactosylation by Bbreβgal-I and Bbreβgal-II, respectively, at maximum total GOS yield,15 confirming the results predicted by the partionining coefficients.</p><p>Furthermore, the propensity of the four β-galactosidases to transfer the galactose moiety to either GlcNAc, GalNAc, or Fuc was determined using a fixed lactose concentration (100 mM) as galactosyl donor and the respective nucleophile in various concentrations as galactosyl acceptor. In the presence of 100 mM lactose and the absence of any external galactosyl acceptor, vGlc/vGal was found to be ~1.3 for Lbulβgal, suggesting GOS formation even at this low lactose concentration, whereas vGlc/vGal of the other three βgals was nearly 1.0, indicating that hydrolysis of lactose is the main reaction.</p><p>Both Lbulβgal and Bbreβgal-II effectively transferred the galactosyl moiety to GlcNAc rather than to water as indicated by the rate constant ratios kGlcNAc/kwater of 16.8 and 5.42 M−1 (Table 1), respectively. Bbreβgal-II and Lbulβgal also showed high preference to transfer galactosyl moiety to GlcNAc rather than to lactose, and GlcNAc is a ~10- and 6-fold better galactosyl acceptor than lactose for these two enzymes. The kGlcNAc/kGlc ratios of Lbulβgal and Bbreβgal-II (1.8 and 1.4, respectively) indicate furthermore that GlcNAc is also the preferred galactosyl acceptor over glucose. This altogether suggests that GlcNAc is an excellent acceptor for Lbulβgal and Bbreβgal-II and that the disaccharide Gal-GlcNAc (positional isomers thereof) will be the main products rather than tri-GOS or other GOS when using a mixture of GlcNAc and lactose as substrate.</p><p>To confirm this assumption, discontinuous conversion reactions using either Bbreβgal-II or Lbulβgal (2.5ULac/mL) were carried out with 600 mM lactose in the absence and presence of 600 mM GlcNAc. The formation of d-Glc, d-Gal, and oligosaccharides was determined at different time intervals. Figure 2B shows that when using lactose alone as the substrate, the maximum d-Glc/d-Gal ratios with Lbulβgal and Bbreβgal-II were 3.0 and 1.5, respectively, whereas the presence of GlcNAc with lactose as substrates resulted in significantly higher d-Glc/d-Gal ratios of 11.0 and 4.6, respectively. The 3-fold increase in the d-Glc/d-Gal ratio suggests that the transferase activity of the enzymes was significantly increased over hydrolysis and that the galactosyl moiety was transferred preferentially to GlcNAc rather than onto lactose or glucose. For example, the presence of GlcNAc resulted in a decrease of GalGal formation by Bbreβgal-II, particularly of 6′-galactobiose (β-d-Galp-(1→6)-d-Gal), as is shown in Figure 3A. Moreover, the synthesis of GOS trisaccharides by Bbreβgal-II decreased significantly from 24 to 13 g/L when GlcNAc was present together with lactose as substrates (Figure 3B). In addition, HPLC analysis showed the presence of a prominent, novel oligosaccharide peak when GlcNAc was added to the reaction mixture (see below). Overall, this indicates that GlcNAc can be an excellent galactosyl acceptor for certain β-galactosidases such as Lbulβgal and Bbreβgal-II.</p><p>N-Acetyl-d-galactosamine can also serve as galactosyl accept- or when using Lbulβgal as judged by the measured kGalNAc/kwater ratio (3.21 M−1). Bbreβgal-I, Bbreβgal-II, and Lreuβgal showed kGalNAc/kwater ≤ 1.0 M−1, suggesting that hydrolysis is the preferred reaction in the presence of GalNAc. l-Fucose, on the other hand, was shown to be a weak nucleophilic acceptor for all four enzymes based on the kFuc/kwater ratio (≤1.27 M−1).</p><p>The ratio of kGal/kwater would also be an essential kinetic parameter to measure the propensity to transfer the galactosyl moiety to another galactose unit. Unfortunately, kGal/kwater could not be determined because the amount of galactose released cannot be measured accurately in the presence of an excess of free galactose.</p><!><p>It was shown earlier that GalNAc can also serve as galactosyl acceptor when using Lbulβgal, based on the measured kGalNAc/kwater ratio (3.21 M−1). The formation of N-acetyl-oligosaccharides with lactose and N-acetyl-d-galactosamine (GalNAc) using Lbulβgal as biocatalyst was hence investigated. The maximum GalNAc transgalactosylation yield (29.2%) was obtained after 1 h with a donor/acceptor molar ratio of 2:1 and initial concentrations of 600 mM lactose and 300 mM GalNAc (Figure 4A). The HPLC profile showed that both di- and trisaccharides containing GalNAc were formed; however, the individual components were not structurally identified or quantified (Figure 4B).</p><!><p>The effects of enzyme concentrations and the molar ratios of the donor (lactose) to the acceptor (GlcNAc) were studied to obtain the maximum yield of GlcNAc-containing transgalactosylation products. The molar ratio of the donor (lactose) to the acceptor (GlcNAc) of 1:1 (0.6 M lactose and 0.6 M GlcNAc) was found to be optimal for both enzymes, βgal from L. bulgaricus and βgal-II from B. breve (data not shown). Hence, the optimal conditions for the transgalactosylation reactions for βgal from L. bulgaricus (2.5 ULac/mL with 1 M lactose and 1 M GlcNAc at 50 °C) and for βgal-II from B. breve (5.0 ULac/mL with 0.6 M lactose and 0.6 M GlcNAc at 30 °C) were employed for the formation of N-acetyl-oligosaccharides. The reaction at 50 °C was carried out only with Lbulβgal because Bbreβgal-II is not stable at higher temperature.</p><p>The products formed in these reactions were then separated using a Hypercarb column. This chromatographic column can also separate the anomeric forms of reducing sugars, and thus each oligosaccharide is represented by two peaks constituting the anomeric isomers.19 The chromatographic patterns and the compounds synthesized by Lbulβgal and Bbreβgal-II were found to be similar as judged by HPLC analysis (Figure S3).</p><p>The major product was purified as described under Materials and Methods. This major disaccharide product was found to be β-d-Galp-(1→6)-d-GlcNAc (N-acetyl-allolactosamine) as identified by the NMR data. Despite the presence of the anomeric forms of the reducing glucosamine unit (α/β ratio ~ 1.4:1), which led to two sets of spin-coupled systems, a full assignment could be achieved based on COSY and edited HSQC spectra (Figure S4; Table S1) and showed a low-field shift of carbon 6 of the reducing GlcNAc to 69.4 ppm. The data, when corrected for different referencing of chemical shifts, were in full agreement with published 13C NMR data of N-acetyl-allolactosamine.20–22 Peaks 2, 4, 6, and 7 (Figure S3) were not identified; however, on the basis of the elution rate, peaks 6 and 7 might be assigned to the trisaccharides GalGalGlcNAc, but the exact identities of these compounds have not been determined.</p><p>Under the optimal conditions, the maximum yields of N-acetyl-allolactosamine, calculated as the percentage of initial GlcNAc, were 41 and 24% with Lbulβgal and Bbreβgal-II, respectively (data not shown). The formation of disaccharides as a product of transgalactosylation of GlcNAc using β-galactosidases from different organisms has been reported, and the linkage preference of the transgalactosylation products varies for different enzymes. βgals from K. lactis, L. bulgaricus, and L. plantarum synthesized N-acetyl-allolactosamine as the major product and N-acetyl-lactosamine (LacNAc) as a minor product.9,11 βgals from Bifidobacterium bifidum and Bacillus circulans favored the formation of LacNAc over N-acetyl-allolactosamine,22–24 whereas N-acetyl-allolactosamine was exclusively synthesized with βgals from Penicillum multicolor, Aspergillus oryzae, and Bifidobacterium longum.22 The presence of higher DP N-acetyl-oligosaccharides in the reaction mixtures of transgalactosylation by using βgals from Sulfolobus solfataricus, A. oryzae, or E. coli was also observed, but they were not identified.25</p><p>In conclusion, kinetic analyses of β-galactosidases from L. reuteri, L. bulgaricus, B. breve (βgal-I and βgal-II) with various sugars as nucleophile provided an insight into the specificities of the given enzymes for transgalactosylation and formation of hetero-oligosaccharides. The transgalactosylation reaction with GlcNAc using Lbulβgal and Bbreβgal-II showed high yields of N-acetyl-oligosaccharides, of which N-acetyl-allolactosamine β-d-Galp-(1→6)-d-GlcNAc is dominant. Although the major product formed is not similar to the core structures of human milk oligosaccharides (HMO), which are lacto-N-biose (LNB, β-d-Galp-(1→3)-d-GlcNA, type I) or N-acetyl-lactosamine (LacNAc, β-d-Galp-(1→4)-d-GlcNAc, type II), this hetero-oligosaccharide might be of interest because of its potentially extended functionality in addition to galacto-oligosaccharides.</p>

PubMed Author Manuscript

Novel pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety: design, synthesis, molecular docking, and biological evaluation as potential multi-target cytotoxic agents

AbstractIn the present study, two new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moiety were designed, synthesised, and evaluated for their cytotoxic activity. The benzamide derivatives 16a–e showed higher cytotoxicity than their corresponding Schiff bases 15a–e. Compounds 16a,b,d also inhibited the growth of MCF-7/ADR cells with IC50 in the range of 0.52–6.26 μM. Interestingly, the new compounds were less cytotoxic against normal MRC-5 cells (IC50=0.155–17.08 μM). Mechanistic studies revealed the ability of compounds 16a,b,d to inhibit tubulin polymerisation and multiple oncogenic kinases. Moreover, compounds 16a,b,d induced preG1 and G2/M cell cycle arrest and early apoptosis in MCF-7 cells. The molecular docking analyses of compounds 16a,b,d into the active site in tubulin, CDK-2, and EGFR proteins revealed higher binding affinities compared to the co-crystallised ligands. These preliminary results suggested that compounds 16a,b,d could serve as promising lead compounds for the future development of new potent anticancer agents.HighlightsTwo new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moieties were synthesized.Compounds 16a,b,d displayed the highest cytotoxicity against the three cancer cell lines.Kinase profiling test revealed inhibition of multiple oncogenic kinases by compounds 16a,b,d.Compounds 16a,b,d exhibited weak to moderate inhibition of tubulin-polymerization.Compounds 16a,b,d induced preG1 and G2/M cell cycle arrest and early apoptosis in MCF-7 cells.Docking studies revealed high binding affinities for compounds 16a,b towards tubulin and CDK-2.

novel_pyrrolizines_bearing_3,4,5-trimethoxyphenyl_moiety:_design,_synthesis,_molecular_docking,_and_

<!>Introduction<!><!>Introduction<!><!>Introduction<!>Rational design<!><!>Rational design<!>Chemistry<!>General procedure (A) for the preparation of compound 15a–e<!><!>7-Cyano-N-phenyl-6-((3,4,5-trimethoxybenzylidene)amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (15a)<!>7-Cyano-N-(p-tolyl)-6-((3,4,5-trimethoxybenzylidene)amino) -2,3-dihydro-1H-pyrrolizine-5-carboxamide (15b)<!>7-Cyano-N-(4-methoxyphenyl)-6-((3,4,5-trimethoxybenzylidene)amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (15c)<!>N-(4-Chlorophenyl)-7-cyano-6-((3,4,5-trimethoxybenzylidene)amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (15d)<!>N-(4-Bromophenyl)-7-cyano-6-((3,4,5-trimethoxybenzylidene)amino)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (15e)<!>General procedure (B) for the preparation of compounds 16a–e<!>7-Cyano-N-phenyl-6-(3,4,5-trimethoxybenzamido)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (16a)<!>7-Cyano-N-(p-tolyl)-6-(3,4,5-trimethoxybenzamido)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (16b)<!>7-Cyano-N-(4-methoxyphenyl)-6-(3,4,5-trimethoxybenzamido)-2,3-dihydro-1H-pyrrolizine-5-carboxamide (16c)<!>N-(4-Chlorophenyl)-7-cyano-6-(3,4,5-trimethoxybenzamido) -2,3-dihydro-1H-pyrrolizine-5-carboxamide (16d)<!>N-(4-Bromophenyl)-7-cyano-6-(3,4,5-trimethoxybenzamido) -2,3-dihydro-1H-pyrrolizine-5-carboxamide (16e)<!>1-Cyano-N-phenyl-2-((3,4,5-trimethoxybenzylidene)amino)-5, 6,7,8-tetrahydroindolizine-3-carboxamide (20)<!><!>1-Cyano-N-phenyl-2-(3,4,5-trimethoxybenzamido)-5,6,7,8-tetrahydroindolizine-3-carboxamide (21)<!>Cell culture<!>MTT cell proliferation assay<!>Annexin V-FITC/PI apoptosis assay<!>Kinase profiling test<!>CDK-2 inhibitory assay<!>Cell cycle analysis<!>Tubulin polymerisation assay<!>Immunofluorescence staining<!>Molecular docking<!>Chemistry<!>Evaluation of cytotoxic activity against cancer cell lines<!><!>Evaluation of cytotoxic activity against cancer cell lines<!>Evaluation of cytotoxic activity against doxorubicin-resistant MCF-7/ADR cells<!><!>Evaluation of cytotoxic selectivity<!><!>Evaluation of cytotoxic selectivity<!>Structure–activity relationship<!><!>Structure–activity relationship<!>Annexin V-FITC/PI apoptosis assay<!><!>Annexin V-FITC/PI apoptosis assay<!>Kinase profiling test<!><!>Kinase profiling test<!><!>Kinase profiling test<!>CDK-2 inhibitory activity<!><!>CDK-2 inhibitory activity<!>Cell cycle analysis<!><!>Cell cycle analysis<!>Tubulin polymerisation assay (kinetic study)<!><!>Tubulin polymerisation assay (kinetic study)<!>Immunofluorescence staining<!><!>Molecular docking<!>Docking study into protein kinases<!><!>Docking study into protein kinases<!><!>Docking study into protein kinases<!><!>Docking study into protein kinases<!>Docking study into tubulin<!><!>Docking study into tubulin<!><!>Docking study into tubulin<!><!>Docking study into tubulin<!><!>Docking study into tubulin<!>Conclusions<!>

<p>Supplemental data for this article can be accessed here.</p><p>Two new series of pyrrolizines bearing 3,4,5-trimethoxyphenyl moieties were synthesized.</p><p>Compounds 16a,b,d displayed the highest cytotoxicity against the three cancer cell lines.</p><p>Kinase profiling test revealed inhibition of multiple oncogenic kinases by compounds 16a,b,d.</p><p>Compounds 16a,b,d exhibited weak to moderate inhibition of tubulin-polymerization.</p><p>Compounds 16a,b,d induced preG1 and G2/M cell cycle arrest and early apoptosis in MCF-7 cells.</p><p>Docking studies revealed high binding affinities for compounds 16a,b towards tubulin and CDK-2.</p><!><p>Despite the presence of clinically effective anticancer agents, cancer is still one of the most leading causes of death in the world1. In addition, the development of multidrug resistance to many of the currently used anticancer drugs represents another challenge in this field2. To overcome these problems, several approaches have been developed and proved better efficacy in cancer treatment. Of these approaches, combination therapy has gained momentum in the treatment of different types of cancers3. However, the high cost, toxicity, and high potential for drug–drug interactions have limited the widespread use of combination therapy in the treatment of cancers3,4.</p><p>Recently, multi-target anticancer agents also emerged as a new approach to cancer chemotherapy5–7. This approach attracted much attention as it could provide a better alternative for combination therapy with lower toxicity and fewer drug–drug interaction problems. Although most of the multi-target agents were discovered by serendipity, several rationally designed multi-target anticancer agents have also been reported7. The rational design of these agents was achieved by combining pharmacophoric groups of two or more different anticancer drugs in a single scaffold5–7. Accordingly, the resulting multi-pharmacophore scaffold could hit multiple targets, simultaneously.</p><p>Tubulin polymerisation is one of the promising targets in the development of new anticancer agents8–10. Combretastatin A-4 (CA-4) 1, and its analogues 2, and 3 (Figure 1) are examples of tubulin polymerisation inhibitors (TBIs) that exhibited potent anticancer activities. The mechanism of action of these compounds is mediated by their binding to the colchicine binding site in tubulin resulting in inhibition of the polymerisation8. In addition, the trimethoxybenzoyl derivatives 4–6 (Figure 1) were also reported as TBIs with potent anticancer activities8–10. The mechanism of action of compounds 4–6 depends also on the inhibition of tubulin assembly resulting in suppression of microtubule formation8. Considering the chemical structure of compounds 1–6, it was observed that they all have similar pharmacophoric features which include 3,4,5-trimethoxyphenyl (TMP) moiety attached through a linker of 1–3 atom length to another substituted phenyl ring. A 2D pharmacophore of these TPIs 1–6 is generated in Figure 1.</p><!><p>TMP bearing tubulin polymerisation inhibitors 1–6 and their pharmacophore features.</p><!><p>The TMP moiety is considered as a tubulin-binding moiety and plays an essential role in the antitubulin activity of compounds 1–6 [9]. In addition, the linker between the two aromatic rings could be an olefinic (ethenyl) group which restricts the free rotation and keep the two aromatic rings in cis-conformation. However, this type of restriction is absent in compounds 4–6, where the two phenyl rings can adopt different orientations relative to each other (Figure 1).</p><p>Incorporation of tubulin-binding moieties such as the TMP moiety into anticancer agents was succeeded as a new strategy to produce multi-target anticancer agents (Figure 2). Compound 7, a multi-target anticancer agent designed by combining the pharmacophoric groups which can target tubulin polymerisation and tyrosine kinase, simultaneously11. Biological evaluation of compound 7 revealed potent inhibitory activity against tubulin polymerisation, vascular endothelial growth factor receptor-2 (VEGFR-2), and platelet-derived growth factor receptor β (PDGFR-β). Compound 7 also displayed superior in vivo activity in reducing tumour size and vascularity compared to sunitinib, and docetaxel11.</p><!><p>Rationally designed multi-target anticancer agents incorporating tubulin binding moieties.</p><!><p>Compound 8, another multi-target anticancer agent designed by combining the pharmacophoric groups of the TBI (colchicine) with those needed to inhibit HDAC12. Compound 8 displayed potent cytotoxic activity (IC50=2–105 nM) against a panel of cancer cell lines mediated by moderate inhibition of HDAC and tubulin polymerisation and activity (Figure 2).</p><!><p>Recently, we reported compounds 9a,b (Figure 3) among a series of pyrrolizine derivatives with cytotoxic activity against MCF-7, A2780, and HT29 cell lines (IC50=0.10–4.16 μM)13. Mechanistic studies of compounds 9a,b revealed inhibitory activities against COX-2 (IC50=13.49 and 1.49 μM, respectively) and/or multiple oncogenic kinases. However, the results of the MTT assay revealed the ability of the two compounds to inhibit the growth of MCF-7 cells at a concentration lower than those required for COX-2 inhibition. In addition, compound 9b exhibited poor selectivity towards A2780 and HT29 cells (SI = 2.16 and 3.03, respectively). Accordingly, we perform the current study to optimise the cytotoxic potential of compounds 9b and investigate other potential targets which could contribute to their cytotoxic activities. However, to keep the ability of the new derivatives targeting oncogenic kinases, only small structural modifications in the scaffold of compound 9a were allowed.</p><!><p>Rational design and structural modifications of scaffold A.</p><!><p>To optimise the cytotoxic potential of the new compounds, scaffold A was designed by replacement of the 3-methylbutylidene/benzylidene moieties in compounds 9a,b by trimethoxy-benzylidene/benzoyl moieties (Figure 3). The new compounds designed bearing these pharmacophoric features will be evaluated for their ability to interfere with the activity of oncogenic kinases and tubulin polymerisation.</p><p>The study of structure–activity relationship (SAR) of scaffold A (Figure 3) was achieved through a series of structural modifications which include: (1) variation of the linker between the TMP moiety and pyrrolizine nucleus to restrict/allow the free rotation of the two moieties, (2) expansion of pyrrolidine ring to the six-membered piperidine ring, and (3) variation of the type of substituents (R) on the phenyl ring (A) to include electron-donating/electron-withdrawing groups. A set of 52 derivatives were designed based on these modifications (Supplementary data, Tables S1 and S2).</p><p>In addition, a preliminary docking study was performed to evaluate the binding affinities of the designed derivatives into the binding site of colchicine in tubulin protein. The derivatives which displayed high binding scores towards tubulin were also evaluated for their binding affinities towards two of the oncogenic kinases (CDK-2, and EGFR). The selection of these kinases was based on the results of the mechanistic study of compound 9a13. Moreover, the synthetic accessibility and drug-likeness scores of the designed analogues were also evaluated. The results of these studies are provided in Supplementary data (Tables S1 and S2). Based on these results, 10 of the designed derivatives which showed good scores were selected for the synthesis.</p><!><p>All the chemical reagents and solvents used were purchased from Sigma-Aldrich (St. Louis, MO). Solvents were dried according to the literature when necessary. The purity of the new compounds was checked with TLC using the benzene–ethanol mixture (9:1). Melting points (m.p.) are uncorrected and were determined by IA 9100MK-Digital melting point apparatus. BRUKER TENSOR 37 spectrophotometer (Billerica, MA) was used to perform the infra-red (IR) spectra of the new compounds, the spectra were recorded using KBr disc and were expressed in wavenumber (cm−1). The proton magnetic spectra were recorded on BRUKER AVANCE III at 500 MHz (Faculty of Pharmacy, Umm Al-Qura University, Mecca, Saudi Arabia) in CDCl3/DMSO-d6. The J constant is given in Hz. The 13C NMR spectra of the new compounds in CDCl3/DMSO-d6 were done at 125 MHz. Mass spectra were recorded on Shimadzu GCMS QP5050A spectrometer (Kyoto, Japan), at 70 eV (EI) at the regional centre for mycology and biotechnology, Al-Azhar University (Cairo, Egypt). Elemental analyses were done in the microanalytical centre, Cairo University (Giza, Egypt). Compounds 1114, 13a–e15,16, 14a–e17, 1815, and 1915 were prepared according to the previous report13. Copies of spectral data including IR, 1H NMR, 13C NMR, 13C NMR, DEPT C135, and mass spectra for each of the new compounds are provided in Supplementary (Figs. S1–S141).</p><p>For easy identification of different protons/carbons and their chemical shifts, the atoms were numbered as illustrated in scaffold A (Figure 3). The readers should also note that the indolizine nucleus is numbered in a reverse direction.</p><!><p>A mixture of pyrrolizine-5-carboxamides 14a–e (3 mmol) and 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol), 0.5 mL glacial acetic acid in absolute ethanol (30 mL) was stirred under reflux for 5 h (Scheme 1). The solvent was then evaporated under reduced pressure. The solid obtained was collected and recrystallised from acetone–chloroform (1:1).</p><!><p>Synthesis of compounds 15a-e and 16a-e.</p><!><p>The title compound was prepared from the reaction of compound 14a (0.8 g, 3 mmol) with trimethoxy benzoyl chloride (0.8 g, 4 mmol) according to the general procedure A. Compound 15a was obtained as a yellow amorphous solid product, m.p. 242–4 °C, yield 71%. IRʋmax/cm−1 3271, 3236, 3183 (NH), 3076, 3024 (aromatic C–H), 2967, 2941 (aliphatic C–H) 2211 (CN), 1672 (COs), 1609, 1578, 1556 (C═C, C═N), 1432, 1318, 1232 (C–N, C–O, C–C). 1H NMR (CDCl3, 500 MHz, δ ppm): 2.57–2.63 (m, 2H, pyrrolizine CH2-2), 3.09 (t, 2H, J= 7.5 Hz, pyrrolizine CH2-1), 3.97 (s, 6H, 3″-OCH3+5″-OCH3), 3.98 (s, 3H, 4″-OCH3), 4.58 (t, 2H, J= 7.1 Hz, pyrrolizine CH2-3), 7.13 (t, 1H, J= 7.3 Hz, CH-4′), 7.19 (s, 2H, Ph CH-2″+CH-6″), 7.35 (t, 2H, J= 7.6 Hz, Ph CH-3′+CH-5′), 7.68 (d, 2H, J= 7.8 Hz, Ph CH-2′+CH-6′), 9.11 (s, H, N═CH), 10.68 (s, H, CONH). 13C NMR (CDCl3, 125 MHz, δ ppm): 24.59 (pyrrolizine CH2-2), 25.46 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.35 (2C, 3″-OCH3+5″-OCH3), 61.09 (4″-OCH3), 84.71 (pyrrolizine C-7), 105.90 (2C, Ph CH-2″+CH-6″), 116.31 (pyrrolizine C-6), 117.71 (CN), 119.79 (2C, Ph CH-2′+CH-6′), 124.12 (Ph CH-4′), 129.04 (2C, Ph CH-3′+CH-5′), 130.75 (Ph C-1″), 138.23 (pyrrolizine C-5), 139.40 (pyrrolizine C-7a), 142.18 (Ph CH-1′), 148.19 (Ph C-4″), 153.82 (2C, Ph C-3″+C-5″), 158.48 (PhNHCO) 159.52 (N═CH). DEPT135 (CDCl3, 125 MHz, δ ppm): δ 24.59 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.35 (2C, 3″-OCH3+5″-OCH3), 61.09 (4″-OCH3), 105.90 (2C, Ph CH-2″+CH-6″), 119.79 (2C, Ph CH-2′+CH-6′), 124.12 (Ph CH-4′), 129.04 (2C, Ph CH-3′+CH-5′), 159.52 (N═CH). MS (EI): m/z (%) 445 ([M + 1]+, 4), 444 (M+, 12) 443 ([M–1]+, 2), 367 (4), 352 (36), 322 (5), 308 (6), 294 (4), 278 (12), 277 (45), 249 (13), 222 (7), 196 (13), 195 (100), 194 (7), 186 (10), 174 (13), 168 (15), 154 (5), 139 (11), 119 (5), 122 (2), 107 (10), 92 (6), 77 (10). Anal. Calcd. for C25H24N4O4 (444.48): C, 67.55; H, 5.44; N, 12.60. Found: C, 67.38; H, 4.98; N, 12.69.</p><!><p>The title compound was prepared from the reaction of compound 14b (0.84 g, 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol) according to the general procedure A. Compound 15b was obtained as a yellow amorphous solid product, m.p. 251–3 °C, yield 78%. IRʋmax/cm−1 3275, 3232, 3179 (NH), 3070 (aromatic C–H), 2962, 2926 (aliphatic C–H), 2211 (CN), 1668 (COs), 1611, 1576, 1552 (C═C, C═N), 1459, 1337, 1233 (C–N, C–O, C–C). 1H NMR (CDCl3, 500 MHz, δ ppm): 2.33 (s, 3H, 4′-CH3), 2.54–2.60 (m, 2H, pyrrolizine CH2-2), 3.06 (t, 2H, J= 7.4 Hz, pyrrolizine CH2-1), 3.94 (s, 6H, 3′′-OCH3+5′′-OCH3), 3.95 (s, 3H, 4′′-OCH3), 4.54 (t, 2H, J= 7.1 Hz, pyrrolizine CH2-3), 7.12 (d, 2H, J= 7.7 Hz, Ph CH-3′+CH-5′), 7.16 (s, 2H, Ph CH-2″+CH-6″), 7.54 (d, 2H, J= 7.6 Hz, Ph CH-2′+CH-6′), 9.08 (s, H, N═CH), 10.58 (s, H, CONH). 13C NMR (CDCl3, 125 MHz, δ ppm): δ 20.88 (CH3), 24.58 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1), 50.13 (pyrrolizine CH2-3), 56.35 (2C, 3″-OCH3+5″-OCH3), 61.09 (4″-OCH3), 105.87 (2C, Ph CH-2″+CH-6″), 116.36 (pyrrolizine C-7), 117.82 (CN), 119.75 (2C, Ph CH-2′+CH-6′), 129.52 (2C, Ph CH-3′+CH-5′), 130.79 (pyrrolizine C-6), 133.74 (Ph C-1″), 135.67 (pyrrolizine C-5), 139.23 (pyrrolizine C-7a), 142.13 (Ph C-1′), 142.69 (Ph C-4′), 148.08 (Ph C-4″), 153.80 (2C, Ph C-3″+C-5″), 158.39 (PhNHCO), 159.39 (N═CH). DEPT135 (CDCl3, 125 MHz, δ ppm): δ 20.88 (CH3), 24.58 (pyrrolizine CH2-2), 25.47 (pyrrolizine CH2-1), 50.13 (pyrrolizine CH2-3), 56.36 (2C, 3″-OCH3+5″-OCH3), 61.09 (4″-OCH3), 105.82 (2C, Ph CH-2″+CH-6″), 119.75 (2C, Ph CH-2′+CH-6′), 129.52 (2C, Ph CH-3′+CH-5′). MS (EI): m/z (%) 460 ([M + 2]+, 3), 459 ([M + 1]+, 19), 458 (M+, 72), 457 (7), 429 (4), 353 (21), 352 (100), 336 (4), 322 (7), 308 (7), 292 (11), 291 (54), 280 (2), 278 (3), 263 (8), 229 (4), 196 (3), 195 (21), 194 (2), 186 (9), 168 (9), 154 (3), 106 (2), 91 (2), 77 (4). Anal. Calcd. for C26H26N4O4 (458.51): C, 68.11; H, 5.72; N, 12.22. Found: C, 68.53; H, 5.32; N, 12.67.</p><!><p>The title compound was prepared from the reaction of compound 14c (0.9 g, 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol) according to the general procedure A. Compound 15c was obtained as a yellow amorphous solid product, m.p. 263–5 °C, yield 74%. IRʋmax/cm−1 3281, 3240, 3185 (NH), 3077, 3000 (aromatic C–H), 2942, 2841 (aliphatic C–H) 2208 (CN), 1674 (COs), 1605, 1578, 1511 (C═C, C═N), 1334, 1234 (C–N, C–O). 1H NMR (CDCl3, 500 MHz, δ ppm): δ 2.55–2.61 (m, 2H, pyrrolizine CH2-2), 3.06 (t, 2H, J= 7.4 Hz, pyrrolizine CH2-1), 3.82 (s, 3H, 4′-OCH3), 3.95 (s, 6H, 3″-OCH3+5″-OCH3), 3.99 (s, 3H, 4″-OCH3), 4.52 (t, 2H, J= 7.1 Hz, pyrrolizine CH2-3), 6.88 (d, 2H, J= 8.6 Hz, Ph CH-3′+CH-5′), 7.28 (s, 2H, Ph CH-2″+CH-6″), 7.61 (d, 2H, J= 8.3 Hz, Ph CH-2′+CH-6′), 8.96 (s, 1H, CONH), 10.70 (s, 1H, CONH). 13C NMR (CDCl3, 125 MHz, δ ppm): δ 24.62 (pyrrolizine CH2-2), 25.48 (pyrrolizine CH2-1), 50.10 (pyrrolizine CH2-3), 55.54 (4′-OCH3), 56.42 (2C, 3″-OCH3+5″-OCH3), 61.20 (4″-OCH3), 106.45 (2C, Ph CH-2″+CH-6″), 106.72 (pyrrolizine C-7), 114.16 (2C, Ph CH-3′+CH-5′), 114.77 (CN), 116.08 (pyrrolizine C-6), 118.05 (Ph C-1′), 121.49 (2C, Ph CH-2′+CH-6′), 129.60 (Ph C-1″), 131.29 (pyrrolizine C-7a), 148.00 (pyrrolizine C-5), 153.66 (Ph C-4″), 153.71 (2C, Ph C-3″+C-5″), 156.32 (Ph C-4′), 158.06 (CONH), 160.30 (N═CH). DEPT C135 (CDCl3, 125 MHz, δ ppm): δ 24.62 (pyrrolizine CH2-2), 25.48 (pyrrolizine CH2-1), 50.10 (pyrrolizine CH2-3), 55.54 (4′-OCH3), 56.41 (2C, 3″-OCH3+5″-OCH3), 61.20 (4″-OCH3), 106.42 (2C, Ph CH-2″+CH-6″), 114.16 (2C, Ph CH-3′+CH-5′), 121.48 (2C, Ph CH-2′+CH-6′). MS (EI): m/z (%) 474 (M+, 23), 449 (8), 422 (5), 396 (34), 383 (43), 361 (58), 332 (100), 320 (37), 261 (28), 190 (11), 120 (22), 93 (8), 69 (9). Anal. Calcd. for C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 65.44; H, 5.21; N, 12.15.</p><!><p>The title compound was prepared from the reaction of compound 14d (0.9 g, 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol) according to the general procedure A. Compound 15d was obtained as a yellow amorphous solid product, m.p. 271–3 °C, yield 64%. IRʋmax/cm−1 3277, 3234, 3177 (NH), 3065 (aromatic C–H), 2996, 2939 (aliphatic C–H) 2208 (CN), 1676 (COs), 1611, 1577, 1547 (C═C, C═N), 1417, 1398, 1291 (C–N, C–O). 1H NMR (CDCl3, 500 MHz, δ ppm): 2.55–2.61 (m, 2H, pyrrolizine CH2-2), 3.07 (t, 2H, J= 7.4 Hz, pyrrolizine CH2-1), 3.94 (s, 6H, 3″-OCH3+5″-OCH3), 3.96 (s, 3H, 4″-OCH3), 4.53 (t, 2H, J= 7.1 Hz, pyrrolizine CH2-3), 7.15 (s, 2H, Ph CH-2″+CH-6″), 7.28 (d, 2H, J= 7.8 Hz, Ph CH-3′+CH-5′), 7.60 (d, 2H, J= 7.8 Hz, Ph CH-2′+CH-6′), 9.08 (s, H, N═CH), 10.67 (s, H, CONH). 13C NMR (CDCl3, 125 MHz, δ ppm): δ 24.60 (pyrrolizine CH2-2), 25.45 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.37 (2C, 3″-OCH3+5″-OCH3), 61.11 (4″-OCH3), 105.95 (2C, Ph CH-2″+CH-6″), 116.18 (pyrrolizine C-7), 117.42 (CN), 120.90 (2C, Ph CH-2′+CH-6′), 126.45 (pyrrolizine C-6), 128.98 (Ph C-1″), 129.05 (2C, Ph CH-3′+CH-5′), 130.65 (Ph C-4′), 138.87 (pyrrolizine C-5), 139.60 (pyrrolizine C-7a), 142.34 (Ph C-1′), 148.37 (Ph C-4″), 153.85 (2C, Ph C-3″+C-5″), 158.46 (PhNHCO), 159.75 (N═CH). DEPT135 (CDCl3, 125 MHz, δ ppm): δ 24.61 (pyrrolizine CH2-2), 25.45 (pyrrolizine CH2-1), 50.14 (pyrrolizine CH2-3), 56.37 (2C, 3″-OCH3+5″-OCH3), 61.12 (4″-OCH3), 105.95 (2C, Ph CH-2″+CH-6″), 120.90 (2C, Ph CH-2′+CH-6′), 129.05 (2C, Ph CH-3′+CH-5′), 159.75 (N═CH). MS (EI): m/z (%) 480 ([M + 2]+, 1), 479 ([M + 1]+, 1), 478 (M+, 4), 458 (7), 353 (13), 352 (73), 323 (4), 322 (8), 311 (11), 308 (10), 292 (12), 291 (44), 278 (8), 263 (13), 252 (9), 229 (8), 222 (11), 196 (15), 195 (100), 186 (20), 168 (28), 154 (12), 147 (6), 111 (4), 106 (5), 91 (7), 77 (12). Anal. Calcd. for C25H23ClN4O4 (478.93): C, 62.70; H, 4.84; N, 11.70. Found: C, 63.14; H, 5.07; N, 11.22.</p><!><p>The title compound was prepared from the reaction of compound 14e (1.04 g, 3 mmol) with 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol) according to the general procedure A. Compound 15e was obtained as a yellow amorphous solid product, m.p. 277–80 °C, yield 68%. IRʋmax/cm−1 3172 (NH), 3063 (aromatic C–H), 2961, 2939, 2838 (aliphatic C–H) 2209 (CN), 1679 (COs), 1610, 1578, 1545 (C═C, C═N), 1418, 1334, 1232 (C–N, C–O, C–C). 1H NMR (CDCl3, 500 MHz, δ ppm): δ 2.57–2.62 (m, 2H, pyrrolizine CH2-2), 3.08 (t, 2H, J= 7.5 Hz, pyrrolizine CH2-1), 3.96 (s, 6H, 3″-OCH3+5″-OCH3), 3.99 (s, 3H, 4″-OCH3), 4.52 (t, 2H, J= 6.9 Hz, pyrrolizine CH2-3), 7.21 (s, 2H, Ph CH-2″+CH-6″), 7.44 (d, 2H, J= 7.7 Hz, Ph CH-3′+CH-5′), 7.59 (d, 2H, J= 8.2 Hz, Ph CH-2′+CH-6′), 9.01 (s, 1H, CONH), 10.77 (s, 1H, CONH). 13C NMR (CDCl3, 125 MHz, δ ppm): δ 24.64 (pyrrolizine CH2-2), 25.45 (pyrrolizine CH2-1), 50.15 (pyrrolizine CH2-3), 56.42 (2C, 3″-OCH3+5″-OCH3), 61.20 (4″-OCH3), 106.22 (2C, Ph CH-2″+CH-6″), 106.72 (pyrrolizine C-7), 116.55 (CN), 117.54 (pyrrolizine C-6), 121.28 (2C, Ph CH-2′+CH-6′), 130.46 (Ph C-4′), 131.73 (Ph C-1″), 131.97 (2C, Ph CH-3′+CH-5′), 132.62 (pyrrolizine C-5), 137.34 (pyrrolizine C-7a), 148.41 (Ph C-1′), 153.67 (Ph C-4″), 153.78 (2C, Ph C-3″+C-5″), 158.31 (PhNHCO), 160.29 (N═CH). DEPT C135 (CDCl3, 125 MHz, δ ppm): δ 24.64 (pyrrolizine CH2-2), 25.44 (pyrrolizine CH2-1), 50.15 (pyrrolizine CH2-3), 56.40 (2C, 3″-OCH3+5″-OCH3), 61.20 (4″-OCH3), 106.18 (2C, Ph CH-2″+CH-6″), 121.25 (2C, Ph CH-2′+CH-6′), 131.97 (2C, Ph CH-3′+CH-5′). MS (EI): m/z (%) 525 ([M + 3]+, 6), 524 ([M + 2]+, 21), 523 ([M + 1]+, 8), 522 (M+, 14), 368 (28), 352 (100), 313 (22), 236 (13), 195 (17), 109 (16), 97 (26), 69 (35). Anal. Calcd. for C25H23BrN4O4 (523.38): C, 57.37; H, 4.43; N, 10.70. Found: C, 56.93; H, 4.57; N, 11.16.</p><!><p>A mixture of pyrrolizine-5-carboxamides 14a–e (3 mmol) and 3,4,5-trimethoxybenzoyl chloride (1 g, 4.4 mmol) in dry benzene (30 mL) was stirred for 48 h at room temperature (Scheme 1). The reaction mixture was filtered, set a side. The formed precipitate was recrystallised from ethanol–acetone (1:1).</p><!><p>The title compound was prepared from the reaction of compound 14a (0.8 g, 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol) according to the general procedure B. Compound 16a was obtained as a white solid product, m.p. 253–5 °C, yield 63%. IRʋmax/cm−1 3190, 3131 (NHs), 3064, 3014 (aromatic C–H), 2968, 2909 (aliphatic C–H) 2224 (CN), 1656, 1639 (COs), 1584, 1567 (C═C, C═N), 1465, 1338, 1297 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, δ ppm): 2.47–2.51 (m, 2H, pyrrolizine CH2-2), 3.04 (t, 2H, J= 7.4 Hz, pyrrolizine CH2-1), 3.73 (s, 3H, 4″-OCH3), 3.84 (s, 6H, 3″-OCH3+5″-OCH3), 4.32 (t, 2H, J= 7.2 Hz, pyrrolizine CH2-3), 7.06 (t, H, J= 7.4 Hz, Ph CH-4′), 7.31 (t, 2H, J= 7.9 Hz, Ph CH-3′+CH-5′), 7.35 (s, 2H, Ph CH-2″+CH-6″), 7.58 (d, 2H, J= 7.7 Hz, Ph CH-2′+CH-6′), 9.78 (s, H, CONH), 10.34 (s, H, CONH). 13C NMR (DMSO-d6, 125 MHz, δ ppm): δ 24.88 (pyrrolizine CH2-2), 25.71 (pyrrolizine CH2-1), 49.64 (pyrrolizine CH2-3), 56.57 (2C, Ph 3″-OCH3+5″-OCH3), 60.63 (4″-OCH3), 84.98 (pyrrolizine C-7), 105.80 (2C, Ph CH-2″+CH-6″), 115.26 (CN), 119.06 (Ph C-1″), 119.75 (2C, Ph CH-2′+CH-6′), 124.16 (Ph CH-4′), 128.80 (pyrrolizine C-5), 128.84 (pyrrolizine C-7a), 129.34 (2C, Ph CH-3′+CH-5′), 139.00 (pyrrolizine C-6), 141.23 (Ph C-1′), 146.44 (Ph C-4″), 153.21 (2C, Ph C-3″+C-5″), 157.92 (PhNHCO), 166.38 (PhCONH). DEPT C135 (DMSO-d6, 125 MHz, δ ppm): δ 24.88 (pyrrolizine CH2-2), 25.71 (pyrrolizine CH2-1), 49.64 (pyrrolizine CH2-3), 56.56 (2C, Ph 3″-OCH3+5″-OCH3), 60.63 (4″-OCH3), 105.78 (2C, Ph CH-2″+CH-6″), 119.75 (2C, Ph CH-2′+CH-6′), 124.16 (Ph CH-4′), 129.34 (2C, Ph CH-3′+CH-5′). MS (EI): m/z (%) 460 (M+, 1), 373 (2), 368 (2), 367 (6), 344 (2), 266 (1), 169 (10), 195 (100), 173 (11), 167 (3), 147 (9), 145 (22), 122 (5), 117 (20), 106 (5), 105 (17), 93 (6), 91 (40), 78 (2). Anal. Calcd. for C25H24N4O5 (460.48): C, 65.21; H, 5.25; N, 12.17. Found: C, 65.54; H, 5.17; N, 12.64.</p><!><p>The title compound was prepared from the reaction of compound 14b (0.84 g, 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol) according to the general procedure B. Compound 16b was obtained as a white solid product, m.p. 265–7 °C, yield 66%. IRʋmax/cm−1 3187, 3112 (NHs), 3038 (aromatic C–H), 2968, 2870 (aliphatic C–H) 2221 (CN), 1656, 1638 (COs), 1586, 1549 (C═C, C═N), 1466, 1389, 1263 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, δ ppm): 2.23 (s, 3H, Ph-CH3), 2.46–2.54 (m, 2H, pyrrolizine CH2-2), 3.03 (t, 2H, J= 7.3 Hz, pyrrolizine CH2-1), 3.74 (s, 3H, 4″-OCH3), 3.85 (s, 6H, 3″-OCH3+5″-OCH3), 4.31 (t, 2H, J= 7.0 Hz, pyrrolizine CH2-3), 7.10 (d, 2H, J= 7.9 Hz, Ph CH-3′+CH-5′), 7.35 (s, 2H, Ph CH-2″+CH-6″), 7.47 (d, 2H, J= 7.8 Hz, Ph CH-2′+CH-6′), 9.65 (s, H, CONH), 10.32 (s, H, CONH). 13C NMR (DMSO-d6, 125 MHz, δ ppm): δ 20.87 (CH3), 24.88 (pyrrolizine CH2-2), 25.72 (pyrrolizine CH2-1), 49.63 (pyrrolizine CH2-3), 56.63 (2C, 3″-OCH3+5″-OCH3), 60.64 (4″-OCH3), 85.07 (pyrrolizine C-7), 105.89 (2C, Ph CH-2″+CH-6″), 115.21 (CN), 119.32 (Ph C-1″), 119.77 (2C, Ph CH-2′+CH-6′), 127.09 (pyrrolizine C-5), 128.70 (pyrrolizine C-7a), 129.69 (2C, Ph CH-3′+CH-5′), 133.23 (Ph C-1′), 136.47 (pyrrolizine C-6), 141.36 (Ph C-4′), 146.32 (Ph C-4″), 153.24 (2C, Ph C-3″+C-5″), 157.69 (PhNHCO), 166.40 (PhCONH). DEPT C135 (DMSO-d6, 125 MHz, δ ppm): δ 20.87 (CH3), 24.88 (pyrrolizine CH2-2), 25.72 (pyrrolizine CH2-1), 49.63 (pyrrolizine CH2-3), 56.63 (2C, 3″-OCH3+5″-OCH3), 60.64 (4″-OCH3), 105.89 (2C, Ph CH-2″+CH-6″), 119.77 (2C, Ph CH-2′+CH-6′), 129.69 (2C, Ph CH-3′+CH-5′). MS (EI): m/z (%) 475 ([M + 1]+, 2), 474 (M+, 9), 368 (34), 367 (37), 352 (6), 341 (4), 284 (6), 280 (1), 200 (11), 196 (11), 195 (100), 174 (2), 167 (6), 146 (2), 145 (1), 122 (4), 117 (2), 107 (30), 106 (5), 92 (3), 78 (2), 77 (7). Anal. Calcd. for C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 65.30; H, 5.71; N, 11.99.</p><!><p>The title compound was prepared from the reaction of compound 14c (0.90 g, 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol) according to the general procedure B. Compound 16c was obtained as a light buff solid product, m.p. 259–61 °C, yield 69%. IRʋmax/cm−1 3196 (NHs), 3068, 3003 (aromatic C–H), 2963, 2941, 2838 (aliphatic C–H) 2223 (CN), 1657, 1634 (COs), 1586, 1511 (C═C, C═N), 1464, 1339, 1234 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, δ ppm): δ 2.39–2.44 (m, 2H, pyrrolizine CH2-2), 2.88 (t, 2H, J= 7.0 Hz, pyrrolizine CH2-1), 3.71 (s, 3H, 4′-OCH3), 3.72 (s, 3H, 4″-OCH3), 3.81 (s, 6H, 3″-OCH3+5″-OCH3), 4.25 (t, 2H, J= 7.0 Hz, pyrrolizine CH2-3), 6.85 (d, 2H, J= 8.8 Hz, Ph CH-3′+CH-5′), 7.45 (s, 2H, Ph CH-2″+CH-6″), 7.52 (d, 2H, J= 8.4 Hz, Ph CH-2′+CH-6′), 10.69 (broad s, 2H, two CONHs). 13C NMR (DMSO, 125 MHz, δ ppm): δ 24.68 (pyrrolizine CH2-2), 25.56 (pyrrolizine CH2-1), 49.47 (pyrrolizine CH2-3), 55.66 (4′-OCH3), 56.39 (2C, 3″-OCH3+5″-OCH3), 60.58 (4″-OCH3), 84.33 (pyrrolizine C-7), 105.76 (2C, Ph CH-2″+CH-6″), 106.44 (CN), 114.35 (2C, Ph CH-3′+CH-5′), 116.20 (Ph C-1′), 120.89 (2C, Ph CH-2′+CH-6′), 121.46 (Ph C-1″), 132.77 (pyrrolizine C-5), 140.40 (pyrrolizine C-7a), 145.92 (pyrrolizine C-6), 152.32 (Ph C-4″), 152.96 (2C, Ph C-3″+C-5″), 155.57 (Ph C-4′), 158.33 (PhNHCO), 166.33 (PhCONH). DEPT C135 (DMSO, 125 MHz, δ ppm): δ 24.68 (pyrrolizine CH2-2), 25.56 (pyrrolizine CH2-1), 49.47 (pyrrolizine CH2-3), 55.66 (4′-OCH3), 56.39 (2C, 3″-OCH3+5″-OCH3), 60.58 (4″-OCH3), 105.75 (2C, Ph CH-2″+CH-6″), 114.35 (2C, Ph CH-3′+CH-5′), 120.89 (2C, Ph CH-2′+CH-6′). MS (EI): m/z (%) 492 ([M + 2]+, 3), 491 ([M + 1]+, 14), 490 (M+, 52), 382 (4), 368 (100), 341 (16), 267 (6), 195 (45), 123 (41), 77 (7). Anal. Calcd. for C26H26N4O6 (490.51): C, 63.66; H, 5.34; N, 11.42. Found: C, 63.78; H, 5.61; N, 11.69.</p><!><p>The title compound was prepared from the reaction of compound 14d (0.9 g, 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol) according to the general procedure B. Compound 16d was obtained as a white solid product, m.p. 281–3 °C, yield 61%. IRʋmax/cm−1 3193, 3108 (NHs), 3062, 3006 (aromatic C–H), 2969, 2937 (aliphatic C–H) 2220 (CN), 1644 (COs), 1586, 1524 (C═C, C═N), 1440, 1296, 1235 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, δ ppm): 2.47–2.51 (m, 2H, pyrrolizine CH2-2), 3.04 (t, 2H, J= 7.4 Hz, pyrrolizine CH2-1), 3.73 (s, 3H, 4″-OCH3), 3.84 (s, 6H, 3″-OCH3+5″-OCH3), 4.31 (t, 2H, J= 7.1 Hz, pyrrolizine CH2-3), 7.33 (s, 2H, Ph CH-2″+CH-6″), 7.37 (d, 2H, J= 8.8 Hz, Ph CH-3′+CH-5′), 7.63 (d, 2H, J= 8.8 Hz, Ph CH-2′+CH-6′), 9.94 (s, H, CONH), 10.31 (s, H, CONH). 13C NMR (DMSO-d6, 125 MHz, δ ppm): δ 24.90 (pyrrolizine CH2-2), 25.69 (pyrrolizine CH2-1), 49.62 (pyrrolizine CH2-3), 56.58 (2C, 3″-OCH3+5″-OCH3), 60.63 (4″-OCH3), 84.98 (pyrrolizine C-7), 105.82 (2C, Ph CH-2″+CH-6″), 115.23 (CN), 118.67 (Ph C-4′), 121.29 (Ph C-1″), 121.41 (2C, Ph CH-2′+CH-6′), 127.70 (pyrrolizine C-5), 128.87 (pyrrolizine C-7a), 129.21 (2C, Ph CH-3′+CH-5′), 138.03 (pyrrolizine C-6), 141.21 (Ph C-1′), 146.58 (Ph C-4″), 153.19 (2C, Ph C-3″+C-5″), 158.05 (PhNHCO), 166.24 (PhCONH). DEPT C135 (DMSO-d6, 125 MHz, δ ppm): δ 24.90 (pyrrolizine CH2-2), 25.70 (pyrrolizine CH2-1), 49.61 (pyrrolizine CH2-3), 56.57 (2C, 3″-OCH3+5″-OCH3), 60.63 (4″-OCH3), 105.80 (2C, Ph CH-2″+CH-6″), 121.40 (2C, Ph CH-2′+CH-6′), 129.21 (2C, Ph CH-3′+CH-5′). MS (EI): m/z (%) 496 ([M + 2]+, 1), 494 (M+, 1), 480 (1), 450 (6), 436 (4), 400 (7), 396 (5), 387 (7), 372 (6), 352 (20), 340 (14), 339 (11), 329 (17), 328 (7), 327 (9), 313 (13), 299 (12), 298 (15), 280 (4), 279 (11), 273 (21), 269 (15), 268 (48), 254 (20), 240 (26), 228 (30), 210 (40), 196 (23), 195 (31), 174 (100), 169 (31), 147 (29), 145 (17), 123 (31), 117 (29), 107 (44), 92 (36), 77 (14). Anal. Calcd. for C25H23ClN4O5 (494.93): C, 60.67; H, 4.68; N, 11.32. Found: C, 61.13; H, 4.87; N, 11.06.</p><!><p>The title compound was prepared from the reaction of compound 14e (1.04 g, 3 mmol) with 3,4,5-trimethoxy benzoyl chloride (1 g, 4.4 mmol) according to the general procedure B. Compound 16e was obtained as a light buff amorphous solid product, m.p. 286–8 °C, yield 64%. IRʋmax/cm−1 3222 (NHs), 3060 (aromatic C–H), 2972, 2938, 2835 (aliphatic C–H) 2220 (CN), 1644 (COs), 1587, 1524 (C═C, C═N), 1414, 1388, 1296 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, δ ppm): δ 2.46–2.51 (m, 2H, pyrrolizine CH2-2), 3.04 (t, 2H, J= 7.2 Hz, pyrrolizine CH2-1), 3.73 (s, 3H, 4″-OCH3), 3.83 (s, 6H, 3″-OCH3+5″-OCH3), 4.30 (t, 2H, J= 7.0 Hz, pyrrolizine CH2-3), 7.32 (s, 2H, Ph CH-2″+CH-6″), 7.50 (d, 2H, J= 8.4 Hz, Ph CH-3′+CH-5′), 7.57 (d, 2H, J= 8.6 Hz, Ph CH-2′+CH-6′), 9.91 (s, 1H, CONH), 10.29 (s, 1H, CONH). 13C NMR (DMSO, 125 MHz, δ ppm): δ 24.90 (pyrrolizine CH2-2), 25.69 (pyrrolizine CH2-1), 49.61 (pyrrolizine CH2-3), 56.56 (2C, 3″-OCH3+5″-OCH3), 60.63 (4″-OCH3), 84.86 (pyrrolizine C-7), 105.77 (2C, Ph CH-2″+CH-6″), 115.23 (CN), 115.73 (Ph C-4′), 118.67 (Ph C-1″), 121.76 (2C, Ph CH-2′+CH-6′), 127.62 (pyrrolizine C-5), 128.81 (pyrrolizine C-7a), 132.13 (2C, Ph CH-3′+CH-5′), 138.43 (pyrrolizine C-6), 141.17 (Ph C-1′), 146.61 (Ph C-4″), 153.19 (2C, Ph C-3″+C-5″), 158.04 (PhNHCO), 166.23 (PhCONH). DEPT C135 (DMSO, 125 MHz, δ ppm): δ 24.90 (pyrrolizine CH2-2), 25.69 (pyrrolizine CH2-1), 49.61 (pyrrolizine CH2-3), 56.55 (2C, 3″-OCH3+5″-OCH3), 60.63 (4″-OCH3), 105.75 (2C, Ph CH-2″+CH-6″), 121.75 (2C, Ph CH-2′+CH-6′), 132.13 (2C, Ph CH-3′+CH-5′). MS (EI): m/z (%) 543 ([M + 5]+, 2), 541 ([M + 3]+, 3), 538 (M+, 3), 490 (31), 457 (12), 422 (89), 368 (100), 341 (46), 318 (37), 296 (18), 200 (18), 175 (18), 121 (6), 100 (17), 73 (31). Anal. Calcd. for C25H23BrN4O5 (539.38): C, 55.67; H, 4.30; N, 10.39. Found: C, 55.83; H, 3.91; N, 10.62.</p><!><p>A mixture of indolizine-3-carboxamides 19 (0.84 g, 3 mmol) and 3,4,5-trimethoxybenzaldehyde (0.8 g, 4 mmol), 0.5 mL glacial acetic acid in absolute ethanol (30 mL) was stirred under reflux for 5 h (Scheme 2). The solvent was then evaporated under reduced pressure. The solid obtained was collected and recrystallised from acetone–chloroform (1:1). Compound 20 was obtained as a yellow amorphous solid product, m.p. 247–9 °C, yield 73%. IRʋmax/cm−1 3182 (NH), 3068 (aromatic C–H), 2938, 2875, 2824 (aliphatic C–H) 2209 (CN), 1677 (COs), 1609, 1594, 1575 (C═C, C═N), 1481, 1330, 1230 (C–N, C–O). 1H NMR (CDCl3, 500 MHz, δ ppm): δ 1.91–1.95 (m, 2H, indolizine CH2-7), 2.02–2.05 (m, 2H, indolizine CH2-6), 2.97 (t, 2H, J= 7.3 Hz, indolizine CH2-8), 3.95 (s, 6H, 3′-OCH3+5′-OCH3), 3.98 (s, 3H, 4′-OCH3), 4.54 (t, 2H, J= 6.5 Hz, indolizine CH2-5), 7.12 (t, 1H, J= 7.3 Hz, Ph CH-4″), 7.26 (m, 2H, Ph CH-3″+CH-5″), 7.32 (d, 2H, J= 7.6 Hz, Ph CH-2″+CH-6″), 7.69 (s, 2H, Ph CH-2′+CH-6′), 8.95 (s, 1H, N═CH), 10.96 (s, 1H, PhNHCO). 13C NMR (CDCl3, 125 MHz, δ ppm): δ 18.78 (indolizine CH2-7), 22.94 (indolizine CH2-8), 23.02 (indolizine CH2-6), 46.96 (indolizine CH2-5), 56.42 (2C, 3′-OCH3+5′-OCH3), 61.18 (4′-OCH3), 82.19 (indolizine C-1), 106.54 (2C, Ph CH-2′+CH-6′), 106.73 (CN), 115.79 (indolizine C-2), 119.63 (Ph C-1′), 120.06 (2C, Ph CH-2″+CH-6″), 124.10 (Ph C-4″), 128.72 (indolizine C-3), 128.97 (2C, Ph CH-3″+CH-5″), 129.42 (indolizine C-8a), 138.35 (Ph C-1″), 142.92 (Ph C-4′), 153.73 (2C, Ph C-3′+C-5′), 158.80 (N═CH), 161.09 (PhNHCO). DEPT C135 (CDCl3, 125 MHz, δ ppm): δ 18.78 (indolizine CH2-7), 22.94 (indolizine CH2-8), 23.03 (indolizine CH2-6), 46.97 (indolizine CH2-5), 56.41 (2C, 3′-OCH3+5′-OCH3), 61.19 (4′-OCH3), 106.50 (2C, Ph CH-2′+CH-6′), 120.05 (2C, Ph CH-2′+CH-6″), 124.02 (Ph C-4″), 128.98 (2C, Ph CH-3′+CH-5″). MS (EI): m/z (%) 460 ([M + 2]+, 5), 458 (M+, 8), 422 (83), 396 (30), 382 (30), 356 (47), 332 (100), 296 (15), 106 (13), 89 (26), 77 (12), 69 (31). Anal. Calcd. for C26H26N4O4 (458.51): C, 68.11; H, 5.72; N, 12.22. Found: C, 67.88; H, 5.46; N, 12.53.</p><!><p>Synthesis of compounds 20 and 21.</p><!><p>A mixture of indolizine-3-carboxamides 19 (0.84 g, 3 mmol) and 3,4,5-trimethoxybenzoyl chloride (1 g, 4.4 mmol) in dry benzene (30 mL) was stirred for 48 h at room temperature (Scheme 2). The reaction mixture was filtered, set aside. The precipitate obtained was recrystallised from ethanol–acetone (1:1). Compound 21 was obtained as a white solid product, m.p. 233–5 °C, yield 65%. IRʋmax/cm−1 3215, 3187 (NHs), 3061, 3040, 3015 (aromatic C–H), 2960, 2944, 2839 (aliphatic C–H), 2221 (CN), 1648 (COs), 1587, 1566 (C═C, C═N), 1317, 1235 (C–N, C–O). 1H NMR (DMSO-d6, 500 MHz, δ ppm): δ 1.72–1.76 (m, 2H, indolizine CH2-7), 1.85–1.89 (m, 2H, indolizine CH2-6), 2.71 (t, 2H, J= 5.7 Hz, indolizine CH2-8), 3.71 (s, 3H, 4′-OCH3), 3.76 (s, 6H, 3′-OCH3+5′-OCH3), 4.24 (t, 2H, J= 5.6 Hz, indolizine CH2-5), 6.97 (d, 2H, J= 7.3 Hz, Ph CH-4″), 7.23 (t, 2H, J= 7.7 Hz, Ph CH-3″+CH-5″), 7.37 (s, 1H, PhCONH), 7.44 (s, 2H, Ph CH-2′+CH-6′), 7.58 (d, 2H, J= 7.9 Hz, Ph CH-2″+CH-6″), 10.92 (broad s, 1H, PhNHCO). 13C NMR (DMSO, 125 MHz, δ ppm): δ 19.15 (indolizine CH2-7), 22.75 (indolizine CH2-8), 22.86 (indolizine CH2-6), 45.90 (indolizine CH2-5), 56.21 (2C, 3′-OCH3+5′-OCH3), 60.56 (4′-OCH3), 88.27 (indolizine C-1), 105.69 (2C, Ph CH-2′+CH-6′), 116.52 (CN), 119.25 (2C, Ph CH-2″+CH-6″), 123.00 (Ph C-1′), 125.71 (indolizine C-3), 128.80 (Ph CH-4″), 129.10 (2C, Ph CH-3″+CH-5″), 129.38 (indolizine C-8a), 139.84 (indolizine C-2), 140.12 (Ph C-1″), 140.20 (Ph C-4′), 152.77 (2C, Ph C-3′+C-5′), 159.62 (PhNHCO), 166.29 (PhCONH). DEPT C135 (DMSO, 125 MHz, δ ppm): δ 19.15 (indolizine CH2-7), 22.75 (indolizine CH2-8), 22.86 (indolizine CH2-6), 45.90 (indolizine CH2-5), 56.21 (2C, 3′-OCH3+5′-OCH3), 60.56 (4′-OCH3), 105.68 (2C, Ph CH-2′+CH-6′), 119.25 (2C, Ph CH-2″+CH-6″), 128.80 (Ph CH-4″), 129.10 (2C, Ph CH-3″+CH-5″). MS (EI): m/z (%) 475 ([M + 1]+, 3), 474 (M+, 10), 381 (82), 366 (6), 262 (4), 195 (64), 152 (6), 109 (20), 93 (57), 81 (71), 69 (98). Anal. Calcd. for C26H26N4O5 (474.51): C, 65.81; H, 5.52; N, 11.81. Found: C, 66.21; H, 5.78; N, 11.66.</p><!><p>MCF-7 (human breast adenocarcinoma), A2780 (human ovarian cancer) and HCT116 (human colorectal carcinoma), and MRC-5 (normal foetal lung fibroblast) cell lines were bought from the ATCC and were cultured in flasks which were incubated at 37 °C in an atmosphere of 5% CO2, 95% air, and 100% relative humidity, to maintain continuous logarithmic growth. MCF-7, A2780, and HCT116 were maintained in RPMI-1640 media (100 U/mL penicillin, 100 µg/mL streptomycin, and 10% foetal bovine serum, Gibco, Carlsbad, CA). MRC-5 cells were maintained in EMEM (100 U/mL penicillin, 100 µg/mL streptomycin, and 10% foetal bovine serum, Gibco, Carlsbad, CA). Doxorubicin (5 μg/mL, for three months) added for RPMI-1640 media containing MCF7 cells was used to develop doxorubicin-resistant MCF7/ADR cells. All cancer and normal cell lines were cultured according to our previous report13. Cells were used within 10–20 passages and were checked for mycoplasma every 6 months, by measuring the bioluminescence (Myco Alert sample detection kit; Lonza, Basel, Switzerland) using the multiplate reader (Synergy HT, BioTek, Winooski, VT).</p><!><p>The MTT assay was used to measure the cytotoxicity and growth inhibitory activities and cytotoxicity of the new compounds according to our previous report13,17. Cells from flasks of 70–80% confluency were separately seeded in 96-well flat-bottom microculture plates (Nalgene-Nunc, Thermo Fisher Scientific, Roskilde, Denmark) at a density of 3 × 103 cells (MCF-7, A2780, and MRC-5), 1 × 104 cells (HCT116), to a volume of 180 μL/well of culture medium. A Neubauer haemocytometer was used for cell counting. Cells were incubated at 37 °C overnight to allow attachment to the wells. The final concentrations of each compound in wells were: 0–50 μM in 200 μL of media (DMSO 0.1%). Lapatinib (Cayman, Ann Arbor, MI) was used as a positive control at the same final concentrations. Medium only (20 μL) was added to each control well, and each concentration was tested in triplicates (n = 3). Following incubation for 72 h, 50 μL MTT was added into each well. Plates were incubated for 3 h, the supernatant was aspirated, and 100 μL of DMSO was added to each well. The optical density (OD) of the purple formazan is proportional to the number of viable cells. When the amount of formazan produced by treated cells is compared with the amount of formazan produced by untreated control cells, the strength of the drug in causing growth inhibition can be determined, through plotting growth curves of absorbance against drug concentration, thus formulation concentration causing 50% inhibition (IC50) compared to control cell growth (100%) were determined. GraphPad Prism version 5.00 for Windows was used for analysis (La Jolla, CA). Cytotoxicity of the new compounds was calculated after treatment of cancer/normal cell lines for 72 h. The absorbance of the reduced MTT was determined using a microplate reader A570 nm.</p><!><p>The ability of compounds 16a,b,d to induce apoptotic changes in MCF-7 cells was determined by Annexin V-FITC/PI staining according to the previous report13,18,19. Additionally, the morphological changes in MCF-7 cells were examined by microscope. Following treatment with the test compounds at 0.1 µM for 24 h, the cells were stained with annexin V and PI. The determination of necrotic, early/late apoptotic changes were performed by flow cytometer (FC500, Beckman Coulter, Miami, FL).</p><!><p>Compounds 16a,b,d and imatinib were evaluated for their inhibitory activities against 20 kinases at a single concentration (10 μM). The test was performed using the radiolabeled ATP determination method (KINEXUS Bioinformatics Corporation, Vancouver, Canada). The test was performed following the previous reports13,20.</p><!><p>The ability of compounds 16a,b,d to inhibit the activity of CDK-2 was determined using ADP-Glo assay (Promega, Madison, WI). The assay was done according to the manufacturer's instructions and as described in the previous report21.</p><!><p>Cell cycle perturbations of MCF-7 cells treated with compounds 16a,b,d at 0.1 µM concentration for 24 h was determined using FC500 flow cytometer (FC500, Beckman Coulter, Miami, FL). The cell cycle analysis was performed according to our previous report13.</p><!><p>The effect of compounds 16a,b,d on tubulin polymerisation was measured in vitro using tubulin polymerisation fluorescence-based assay (cytoskeleton, cat. BK011P, https://www.cytoskeleton.com/bk011p). The assay was performed following the previous report22 and according to the manufacturer's instructions. Briefly, 2 mg/mL porcine tubulin was dissolved in buffer 1 (80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA pH 6.9, 10 µM fluorescent reporter, 1 mM GTP, 15% glycerol) to a final concentration of 10 mg/mL. Then tubulin solution was transferred to a pre-warmed 96-well plate that contained the test compounds 16a,b,d (5 μM), paclitaxel (3 μM), CaCl2 (0.5 μM), and control buffer. The polymerisation of tubulin was monitored as fluorescence at 37 °C for 60 min. Relative fluorescence units (RFU) were determined at 360 nm for excitation and at 460 nm for emission using the Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific, Roskilde, Denmark) at a reading speed of 1 cycle/min.</p><!><p>The MCF-7 cancer cells plated on coverslips on six-well plate (1 × 105/well) were treated with the indicated concentration of control, colchicine (3 μM), paclitaxel (3 μM), and the test compound 16b (5 μM) for 24 h as previously reported23. After treatment, cells were rinsed twice with PBS, fixed with 3.7% paraformaldehyde, and permeabilised with 0.1% Triton X-100. Cells then were blocked with 1% BSA in PBS for 1 h before further incubation with anti-β-tubulin mouse monoclonal antibody overnight at 4 °C (#86298, Cell Signaling, San Francisco, CA). Cells were incubated with Alexa Fluor® 488 secondary antibodies (Abcam, Cambridge, UK), after being washed with PBS for 1 h in a darkroom. Cellular microtubules were observed with the Nikon Eclipse Ti microscope (Minato-ku, Japan).</p><!><p>The molecular docking simulation of the new compounds was carried out by AutoDock 4.224. The crystal structure of CDK2 bound to CAN508 (pdb code: 3TNW)25, EGFR bound to erlotinib (pdb code: 1M17)26, Aurora A bound to VX6 (pdb code: 3E5A)27, and tubulin bound to C-A428, were obtained from protein data bank (http://www.rcsb.org/pdb). Ligand and protein files were prepared according to the previous report13,29. Docking and grid parameter files were prepared according to the previous reports30,31. The top 10 protein–ligand complexes were scored. The results including binding modes, affinities, and interactions of the best-fitting conformations of the new compounds were visualised using Discovery Studio Visualizer (v16.1.0.15350, Dassault Systems, San Diego, CA)32.</p><!><p>In this work, compound 11 was prepared from the reaction of pyrrolidin-2-one 10 and malononitrile as previously reported13. On the other hand, the acetanilides 13a–e were obtained from the reaction of (un)substituted anilines 13a–e with chloroacetyl chloride according to the reported procedures13 (Scheme 1). The starting materials 14a–e were prepared from the reaction of compounds 11 and 13a–e in acetone13.</p><p>The Schiff bases 15a–e were obtained on refluxing compounds 14a–e with 3,4,5-trimethoxybenzaldehyde in absolute ethanol in the presence of glacial acetic acid. On the other hand, the benzamide derivatives 16a–e were prepared from the reaction of compounds 14a–e with 3,4,5-trimethoxybenzoyl chloride in dry benzene (Scheme 1). Structural elucidation of the new compounds was performed using both spectral and elemental analyses. Copies of spectral data including IR, 1H NMR, 13C NMR, DEPT C135, and mass spectra are provided in supplementary data (Figs. S1–S141).</p><p>In addition, compound 18 (Scheme 2) was obtained on the treatment of piperidin-2-one 17 with dimethyl sulphate and malononitrile according to the reported procedures13. The starting material, indolizine 19 was synthesised using the same reaction conditions adopted for the synthesis of compound 14a with the replacement of 2-(pyrrolidin-2-ylidene)malononitrile 11 by 2-(piperidin-2-ylidene)malononitrile 18.</p><p>The Schiff base 20 was also obtained from the condensation of 3,4,5-trimethoxybenzaldehyde and 2-aminoindolizine-3-carboxamide 19 in ethanol. On the other hand, acylation of compound 19 with 3,4,5-trimethoxybenzoyl chloride yielded the benzamide derivative 21. Spectral (IR, H NMR, 13C NMR, DEPT C135, and mass spectra) and elemental analyses were performed to confirm the chemical structure of compounds 20 and 21. The spectra of the two compounds are also provided in Supplementary data (Figs. S6, S12, S49–S54, S96–S105, S125, and S141).</p><!><p>The cytotoxic activity of the new compounds (15a–e, 16a–e, 20, and 21) was evaluated in vitro using MTT assay13. The MTT assay depends on the reduction of the yellow tetrazole dye (MTT) to the purple formazan by the active mitochondrial dehydrogenase enzymes indicating the viability of the cells which can be measured as OD. The new compounds were evaluated against three non-resistant cancer cell lines: MCF-7, and A2780 cell lines were selected to compare cytotoxicity of the new compounds with the previously reported results of compounds 9a,b13. Cytotoxicity of the new compounds was also evaluated also against HCT116 cells which lacks COX-2 protein33,34. Selection of this cell line was based on the previous reports which revealed potent cytotoxic activity of the five membered heterocyclic derivatives against HCT116 cells35,36.</p><p>The results of the MTT assay (Table 1) revealed variable degrees of cytotoxic activities for the new compounds with IC50 values in the range of 0.030–36.880 μM against the three cancer cell lines. The results also revealed higher cytotoxic activities for the benzamide derivatives (16a–e and 21) compared to their corresponding benzylidene analogues (15a–e and 20).</p><!><p>Cytotoxicity (µM, IC50±SD) of compounds 15a–e, 16a–e, 20, and 21 against MCF-7, HCT116, and A2780 cancer cell lines in comparison to compounds 9a,b, lapatinib, and colchicine.</p><p>IC50: concentration of test compound which reduce cellular growth to 50% after 72 h treatment, results represent mean IC50 value ± SD (n = 3) of three independent experiments.</p><p>Average of the three IC50 values against the three cancer cell lines.</p><p>IC50 values quoted from our previous publication13.</p><p>IC50 values quoted from the previous publications37,38.</p><!><p>Compound 16b was the most potent in inhibiting the growth of MCF-7 cells, while compounds 16c,d exhibited the highest cytotoxic activity against HCT116 and A2780 cells, respectively. On the other hand, lapatinib exhibited cytotoxic activity against the three cancer cell lines with IC50 values in the range of 6.690–10.043 μM (Table 1).</p><p>The new compounds exhibited cytotoxic activities against MCF-7 cells with IC50 values in the range 0.068–36.880 μM. In addition, they inhibited the growth of A2780 cells with values in the range 0.030–33.360 μM. Interestingly, the new compounds also exhibited potent cytotoxic activities against HCT116 cells with IC50 values in the range of 00.032–19.835 μM. These results indicated that COXs inhibition may not be essential for the cytotoxicity of the compounds reported in this study.</p><!><p>Encouraged by the high cytotoxic activity of the new compounds against MCF-7 cells (Table 1), three of these derivatives (16a,b,d) were also selected to evaluate their cytotoxicity against doxorubicin-resistant MCF-7/ADR cells. The evaluation was performed using MTT assay13. The results are presented in Table 2. The results revealed cytotoxic activity for the three compounds against MCF-7/ADR cells with IC50 in the range of 0.52–6.26 μM. Among the three derivatives, compound 16d with the electron-withdrawing chloro atom exhibited the highest cytotoxic activity against MCF-7/ADR cells, while the methyl analogue 16b was the least active. These results suggested that electron-withdrawing groups on the phenyl ring of scaffold A could be used to enhance cytotoxic activity against MCF-7/ADR cells.</p><!><p>Cytotoxicity of compounds 16a, b, d against MCF-7/ADR cells.</p><p>IC50: concentration of test compound which reduce cellular growth of MCF-7/ADR cells to 50% after 72 h treatment, results represent mean IC50±SD, (n = 3) of three independent experiments.</p><!><p>Selectivity towards cancerous cells is one of the important issues which must be considered during the discovery and development of new anticancer agents. To assess the toxicity and selectivity of the new compounds (15a–e, 16a–e, 20, and 21), their growth inhibitory activities against normal human foetal lung fibroblast MRC-5 cells were evaluated using the MTT assay. The results expressed as IC50 values are presented in Table 3. The results revealed variable cytotoxic activity of the new compounds against the normal MRC-5 cells (IC50=0.155–17.080 μM), compared to 25.810 and 9.00 μM for compounds 9a,b, respectively13. However, compounds 15c,e, and 16a,b,d were more potent and/or more selective than compound 9a. The selectivity of the new compounds towards each of the three cancer cell lines was calculated by dividing their IC50 values against normal MRC-5 cells by their IC50 values against the corresponding cancer cell line (Table 3).</p><!><p>Cytotoxicity of compounds 9a,b, 15a–e, 16a–e, 20, 21, and lapatinib against normal MRC-5 cells.</p><p>IC50 against MRC-5 cells after 72 h treatment with the test compound, results represent mean IC50±SD (n = 3).</p><p>Selectively index (SI)=IC50 value against normal MRC-5 cells/IC50 value against cancer cell line.</p><p>IC50 value quoted from our previous publication13.</p><!><p>The five Schiff bases 15a–e exhibited lower selectivity (SI = 0.05–118.45) towards the three cancer cell lines than their benzamide analogues 16a–e (SI = 0.45–270). Among the new compounds, compound 16d was the most selective towards MCF-7 and A2780 cells, while compound 15e showed the highest selectivity to the HCT116 cancer cell line (Table 3).</p><p>Evaluation of cytotoxicity of the indolizine derivatives 20 and 21 against normal MRC-5 cells revealed IC50 values of 1.517 and 0.584 μM, respectively. The two compounds exhibited selectivity indices in the range of 0.27–6.14 towards the three cancer cell lines. Compound 20 was more cytotoxic towards the MRC-5 cells than its pyrrolizine analogue 15a. On the other hand, the indolizine 21 was less toxic to the normal cells compared to the pyrrolizine analogue 16a (Table 3).</p><!><p>The study of SAR of the new compounds is represented in Figure 4. The Schiff base 15a exhibited potent cytotoxic activity against HCT116 (IC50=0.353 μM) and very weak cytotoxicity against A2780 and MCF-7 cancer cell lines with IC50 values of 25.76 and 36.88 μM, respectively. On the other hand, replacement of the 3,4,5-trimethoxybenzylidene)amino in compound 15a with 3,4,5-trimethoxybenzamide moiety resulted in a significant increase in the cytotoxic activities against the three cancer cell lines.</p><!><p>SAR of cytotoxicity of the new compounds (15a–e, 16a–e, 20, and 21).</p><!><p>The substituents on the phenyl ring showed variable effects on cytotoxicity of the new compounds. In Schiff base derivatives 15a–e, substitution on para-position of the phenyl ring of compound 15a with electron-donating (OCH3) or electron-withdrawing (Cl/Br) atoms resulted in a significant increase (1.7–628.3-fold) in cytotoxic activity against MCF-7 and A2780 cells, while substitution with CH3 group decreased cytotoxicity against both HCT116 and A2780 cancer cell lines (Figure 4).</p><p>On the other hand, the study of SAR of the benzamide derivatives 16a–e showed that substitution on the phenyl ring of compound 16a with the electron-donating (OCH3) or electron-withdrawing (Br) groups resulted in a significant decrease (3.3–20.8-fold) in the cytotoxicity against MCF-7 and A2780, while substitution with CH3/Cl groups resulted in a very slight increase in cytotoxic activity against both MCF-7 and HCT116 cell lines (Figure 4).</p><p>Moreover, expansion of the pyrrolidine ring in compound 15a to the piperidine ring (compound 20) resulted in 1.4–27.5-fold increase in cytotoxicity against the three cancer cell lines (Figure 4).</p><p>Replacement of the 3,4,5-trimethoxybenzylidene-amino moiety in compound 15a with 3,4,5-trimethoxybenzamido moiety resulted in a remarkable increase (2.6–585.4-fold) in cytotoxic activities against the three cell lines. Expansion of the pyrrolidine ring in compound 16a to piperidine ring resulted in an increase in cytotoxic activity against the three cancer cell lines.</p><p>In conclusion, the 3,4,5-trimethoxybenzamido moiety and the electron-withdrawing atoms on the phenyl ring at C-5 on the pyrrolizine scaffold are favoured for high cytotoxic outcomes.</p><!><p>The cytotoxic activity of our previously reported pyrrolizine-5-carboxamide 9a was mediated by the induction of apoptosis in MCF-7 cells13,19. Accordingly, compounds 16a,b,d, the most active in the MTT assay (Table 1) were selected to investigate their apoptosis-inducing activities. The cancer cells were treated with the test compounds (0.1 μM) for 24 h. The induction of apoptosis in MCF-7 cells by the selected compounds was investigated using annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining assay. The assay procedures were the same as those applied in the previous report13. The results are presented in Table 4 and Figure 5. Compound 16a increased apoptosis (early and late events) in MCF-7 cells to 19% compared to 1% in the control. Moreover, compound 16b caused a significant increase of MCF-7 apoptotic cells which was similar to the effect of compound 16a. However, compound 16d was more potent in inducing apoptosis in MCF-7 cells (26%) compared to compounds 16a and 16b.</p><!><p>Evaluation of apoptosis-inducing activity of compounds 16a,b,d in MCF-7 cells using annexin V FITC/PI staining assay for 24 h (n= 3). x-axis: annexin V, y-axis: PI. (A) Vehicle control, (B) compound 16a (0.10 µM), (C) compound 16b (0.1 µM), (D) compound 16d (0.10 µM). Top left quarter: necrosis (PI+/annexin V–); top right quarter: late apoptosis (PI+/annexin V+); bottom left quarter: living cells (PI–/annexin V–); bottom right: early apoptosis (PI–/annexin V+).</p><p>Induction of apoptosis in MCF-7 cells treated with compounds 16a,b,d.</p><p>The results were obtained on treatment of MCF-7 cells by compounds 16a,b,d (0.1 μM) or vehicle control for 24 h (n= 3), values represent mean%±SD of three independent experiments.</p><!><p>The test compounds also caused variable degrees of necrosis in MCF-7 cells. Compounds 16a,d caused slight increases (3.50% and 2.51%, respectively) in the necrotic events compared to by the control (1.90%). On the other hand, compound 16b caused a 7.11% increase in necrotic events.</p><p>In conclusion, the new compounds 16a,b,d induced higher apoptotic events in MCF-7 cells at lower concentration compared to compound 9a (Table 4).</p><!><p>Compound 9a, the lead compound in this study, was previously evaluated in a kinase profiling test to identify its mechanism of action13. The results revealed the ability of compound 9a to inhibit the activity of five oncogenic kinases including ALK1, CDK-2/cyclin A1, DYRK3, GSK3 alpha, and NEK1 (inhibition%=3–25%) (Table 5).</p><!><p>Kinases inhibitory activity by compounds 9a, 16a,b,d and imatinib.</p><p>The negative values indicate inhibition, while the positive values indicate the activation in kinase activity.</p><p>Data quoted from our previous publication13.</p><!><p>In the current work, compounds 16a,b,d, the most active in MTT assay were also selected for the kinase profiling test to investigate their potential mechanism of action. The three compounds were tested at the same concentration (10 μM) against the same 20 kinases used in the previous report13. The test was performed by KINEXUS Bioinformatics Corporation (Vancouver, Canada), following previous reports13,20. The results are presented in Table 5 and Figure 6.</p><!><p>Inhibitory activity of compounds 9a, 16a,b,d, and imatinib against 20 protein kinases; negative values indicate inhibition in kinase activity, positive values indicate the activation of the enzyme.</p><!><p>The results revealed inhibition in the activities of 16 kinases by compounds 16a,b,d with inhibition% in the range of 1–38%. The activities of seven of these kinases were inhibited by the three compounds (2–28%) (Table 5 and Figure 6).</p><p>Treatment of CDK-2/cyclin A1 with compounds 16a,b,d resulted in 19–28% inhibition in the activity of the kinase compared to only 2% and 4% inhibition by imatinib and compound 9a, respectively. Compounds 16a,b also showed inhibitory activity against EGFR with inhibition% of 30 and 11%, respectively. Moreover, compound 16d exhibited 14% inhibition in the activity of Aurora A kinase compared to 38% inhibition for imatinib.</p><p>In addition, weak inhibition in the activities of ALK1 (11–19%), BLK (2–11%), CK1 Alpha 1 (7–15%), and DYRK3 (4–17%) was also observed following the treatment with compounds 16a,b,d. In addition, compounds 16a,b displayed inhibitory activity against EPHA1, MSK1, p38 Alpha, and SGK1 with inhibition% in the range of 5–22% (Table 5). Although these inhibitions in the activities of these kinases are weak, but they may also contribute to the overall cytotoxic activities of the three compounds.</p><p>The results of the kinase profiling test also showed significant improvement in the inhibitory activities of compounds 16a,b,d compared to compound 9a. This improvement was observed in the number of oncogenic kinases (8–13 kinases) inhibited by the new compared to only five kinases inhibited by compound 9a. In addition, compounds 16a,b,d exhibited higher inhibitory activities (1–38%) than those of compound 9a (3–25%) (Table 5 and Figure 6). These results suggested that the aromatic fragment in the side chain at C-6 of the pyrrolizine nucleus could have a better impact on the inhibitory activities of the new pyrrolizines compared to the aliphatic side chain in compound 9a.</p><!><p>The results of the kinase profiling test (Table 5 and Figure 6) revealed weak to moderate inhibitory activity against 7 kinases by compounds 16a,b,d. Among these kinases, CDK-2 was inhibited by the three compounds with inhibition% in the range 19–28%. To confirm the activity against CDK-2, the three compounds were also evaluated for their CDK-2 inhibitory activity using the ADP-Glo kinase assay kit (Promega, Madison, WI) according to the previous reports21. The results expressed in the IC50 values of the three compounds are presented in Table 6.</p><!><p>IC50 values of compounds 9a, 16a,b,d and staurosporine against CDK-2.</p><p>Average of three determinations; IC50, concentration which decrease CDK-2 activity to 50%.</p><p>Value quoted from previous publication13.</p><!><p>The results revealed higher inhibition in activity CDK-2 by compounds 16a,b,d compared to compound 9a, which was matched with the results of the kinase profiling test (Table 5). Among the three derivatives, compound 16a with the unsubstituted phenyl ring was the most potent as a CDK-2 inhibitor (IC50=0.086 μM). Moreover, compound 16d also exhibited potent inhibitory activity against CDK-2 (IC50=0.113 μM), while 16b with the electron-donating methyl group was the least active among the three benzamide derivatives (Table 6). These results suggested that for better inhibitory activity of CDK-2, the phenyl ring in scaffold A should be unsubstituted or substituted with electron-withdrawing group(s) rather than the electron-donating (4-CH3) group.</p><!><p>Based on the results of the MTT assay (Table 1), compounds 16a,b,d were selected to investigate their effect on cell cycle phases of MCF-7 cells. The cancer cells were treated with the test compounds at 0.1 μM and vehicle (control) for 24 h according to the previous report13. Cell cycle analysis was performed using FC500 flow cytometer (FC500, Beckman Coulter, Miami, FL). The revealed dramatic increases in the preG1 (11–14-fold) and G2/M (5–7-fold) cell cycle phases at the expense of 5–10-fold decrease of G1 events, all compared to control. On the other hand, the S phase was the least affected with less than a twofold decrease. Compound 16d showed the strongest preG1 (27.56%) and G2/M (45.62%) cell cycle effects in MCF-7 cells (Table 7, Figure 7).</p><!><p>Cell cycle analysis of MCF-7 cells treated with (A) vehicle (control), (B) compound 16a (0.1 µM), (C) compound 16b (0.1 µM), and (D) compound 16d (0.1 µM) for 24 h. x-axis: DNA content, y-axis: % cell number; (E) bar chart showing the effect of vehicle (control) and compounds 16a,b,d (0.1 µM) on cell cycle stages of MCF-7 cells after 24 h treatment. Data shown in mean %±SD (n = 2) of three independent experiments, statistical differences, compared with control cells, were assessed by one-way ANOVA with the Tukey's post hoc multiple comparison test (GraphPad Prism, La Jolla, CA). *p< 0.05 and **p< 0.01 were taken as significant.</p><p>Cell cycle effect of 16a,b,d on MCF-7 cells (24 h).</p><p>Data shown in mean %±SD (n = 2), treatment for 24 h at 0.1 μM, experiment was repeated 3×.</p><!><p>Cell cycle analysis of compound 9a (1 µM, 24 h) revealed blockade of MCF-7 cells at the S phase with more than a threefold increase compared to the control (17.3–55.6%), at the expense of decreases in other cell cycle phases13. However, in the current study, compounds 16a,b,d (0.1 µM) showed completely different effects on cell cycle phases of MCF-7 cells at a much lower concentration compared to compound 9a. The three compounds 16a,b,d increased the preG1 and G2/M cell cycle phases in MCF-7 cells at the same time point (24 h) (Table 7).</p><!><p>In the current study, the new compounds were designed bearing the trimethoxy phenyl moiety aiming to obtain new cytotoxic agents that can interfere with tubulin polymerisation in cancer cells. Herein, compounds 16a,b,d, the most active in MTT assay were selected to investigate their effects on the kinetics of the three phases of tubulin polymerisation (I: nucleation, II: growth, and III: steady-state equilibrium). In this assay, light scattering by microtubules which is directly proportional to the concentration of the microtubule polymer was used as a measure of tubulin polymerisation. The study was performed using a fluorescence-based tubulin polymerisation assay kit (Cytoskeleton Inc., Denver, CO; Cat # BK011P) following the manufacturer's instructions (https://www.cytoskeleton.com/bk011p) and according to the previous report22.</p><p>Paclitaxel (3 μM) was used as a tubulin polymerisation enhancer (positive control, enhancer), while calcium chloride (CaCl2, 0.5 μM) was used as a positive control (inhibitor) as per the manufacturer's instructions (https://www.cytoskeleton.com/pdf-storage/datasheets/bk011p.pdf). The vehicle was also used to evaluate the polymerisation when tubulin is incubated in the absence of the test compound. The effect of the selected compounds (5 μM) on tubulin polymerisation was evaluated and compared with those of the positive control. The results are represented in Figure 8.</p><!><p>Effect of compounds 16a,b,d on tubulin polymerisation: (A) tubulin polymerisation reactions of control, paclitaxel, CaCl2, and compounds 16a,b,d, I: nucleation, II: growth, and III: steady-state equilibrium phases; (B) semi-quantitative analysis of the inhibition in tubulin polymerisation showing the AUC on treatment with vehicle (control), paclitaxel, CaCl2, and compounds 16a,b,d.</p><!><p>The results revealed that the tubulin polymerisation enhancer, paclitaxel at 3 μM eliminated the nucleation phase I and enhanced the growth phase II. On the other hand, CaCl2 at 0.5 μM inhibited tubulin polymerisation and reduced the final polymer mass (Figure 8). The results also indicated moderate tubulin polymerisation inhibitory activity for compounds 16b,d compared to CaCl2. The inhibitory effect of compound 16b was higher than compound 16d. The effect of compound 16d on both nucleation and growth phases was similar to those of CaCl2, while its effect on the steady-state equilibrium phase was slightly lower than CaCl2 (Figure 8). On the other hand, compound 16a showed a similar effect to that of paclitaxel on the nucleation phase. However, this effect reached a plateau then started to decrease in the growth phase. The inhibitory effect of compound 16a on tubulin polymerisation was continued in the steady-state phase.</p><p>In the current work, the tubulin polymerisation assay was used as a qualitative assay to identify the potential mechanism of action of the new compounds 16a,b,d. However, area under the curve (AUC) indicates the total light scattered and corresponds to the total mass of the polymerised tubulin (Figure 8(A)). Accordingly, AUC was calculated for the tested compounds 16a,b,d and compared with those of the control, CaCl2, and paclitaxel (Figure 8(B)). The results showed a decrease in AUC of CaCl2 compared to the control indicating an inhibition in tubulin polymerisation and a decrease in the final polymer mass. On the other hand, an increase in AUC was observed by paclitaxel relative to control indicating a stabilisation in tubulin polymerisation. Moreover, compounds 16a,b,d exhibited similar effects on tubulin polymerisation like CaCl2. Among the three compounds, compound 16b exhibited the highest reduction in the final polymer mass. In addition, compound 16d showed a higher reduction in the final polymer mass than compound 16a. Considering the results of the three compounds, we can observe that compounds 16b,d have moderate inhibitory activity against tubulin polymerisation compared to the control and CaCl2, while the effect of compound 16a was weak.</p><!><p>To assess the effect of the new compounds on microtubule cytoskeleton in living cells, compound 16b, the most active in tubulin polymerisation assay (Figure 8) was selected. The effect of compound 16b was evaluated in an immune staining study using confocal immunofluorescent microscopy. The test was performed according to the previous report23. The cancer cells were incubated with the vehicle (control) and compound 16b (5 μM) for 24 h. The nuclei of the cancer cell stained blue with 4′,6-diamidino-2-phenylindole (DAPI), while microtubules were stained (green) with anti-β-tubulin mouse monoclonal antibody and Alexa Fluor® 488 secondary antibodies (Abcam, Cambridge, UK).</p><p>The immunofluorescence images of the untreated/treated cancer cells were taken to visualise the effect of the test compounds 16b on the microtubule cytoskeleton. The results are represented in Figure 9. The images of the control showed normal, clear slim, and fibrous microtubules which wrap around the nuclei and stretch through each cell. On the other hand, the cells treated with compound 16b showed shrank and partially disorganised microtubules which split partially into fragments. These results indicated the ability of compound 16b to induce a disruption in the formation of microtubules.</p><!><p>Immunofluorescence confocal microscopy images assessing cellular microtubule networks of MCF-7 cells after treatment with vehicle (A), compound 16b (B) for 24 h, nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI), microtubules stained (green) with anti-β-tubulin mouse monoclonal antibody and Alexa Fluor® 488 secondary antibodies (Abcam, Cambridge, UK).</p><!><p>In the current work, the molecular docking studies were performed into protein kinases (PKs) and tubulin to understand the mechanism of action of compounds 16a,b,d. The binding affinities, modes, orientations, and interactions of the new compounds into the relevant protein were investigated using AutoDock 4.224 and compared with those of the co-crystallised ligands. The results of the docking studies were visualised by the Discovery studio visualizer (Dassault Systems, San Diego, CA)32.</p><!><p>To rationalise the improvement of kinase inhibitory activity of the new compounds 16a,b,d compared to the lead compound 9a, a molecular docking study of the new compounds into the active sites of CDK-2, EGFR, and Aurora A were performed. The crystal structure of CDK-2 (pdb code: 3TNW)25, EGFR (pdb code: 1M17)26, and Aurora A (pdb code: 3E5A)27 was obtained from the protein data bank (https://www.rcsb.org/). AutoDock (v.4.2) was used to perform the docking study24. The study was performed following the previous reports29–31. Validation of the docking procedures into the selected kinases was also performed according to our previous report13. The results of the docking study of the new compounds and the co-crystallised ligands are presented in Table 8.</p><!><p>Docking results of compounds 16a,b,d into CDK-2 and EGFR in comparison to compounds 9a and the co-crystallised ligands.</p><p>Binding free energy (kcal/mol).</p><p>Inhibition constant.</p><p>HBs, number of hydrogen bonds.</p><p>Length in angstrom (Å).</p><p>CAN508, -4-[(E)-(3,5-diamino-1H-pyrazol-4-yl)diazenyl)phenol.</p><!><p>The results of the docking study of compounds 16a,b,d into the selected kinases revealed nice fitting into the active site of CDK-2 and EGFR. These results were confirmed by the absence of any steric clashes or unfavourable interactions with the amino acids in the active sites of the two kinases. Moreover, compounds 16a,b,d exhibited higher binding affinities towards CDK-2 and EGFR compared to CAN508, and erlotinib, respectively (Table 8). The three compounds 16a,b,d exhibited higher affinities (–7.91 to −9.21 kcal/mol) towards CDK-2 compared to −6.47 kcal/mol for CAN508 (IC50=3.5 μM against CDK-2)39. Compound 16a showed the highest affinity towards CDK-2 followed by the chloro analogue 16d. This order of binding affinities was in concordance with the inhibitory activities of the three compounds against CDK-2 (Table 6).</p><p>To understand the differences in the inhibitory activity of the three compounds against CDK-2 (Table 6), the binding modes and interactions of compounds 16a,b,d were also investigated. The results revealed that each of the two side chains in the three compounds can adopt an orientation that occupies the binding site of CAN508 in CDK-2.</p><p>Investigation of the binding mode of compound 16a revealed that the pyrrole ring together with the attached phenyl carboxamide side-chain adopted an orientation nearly superposing the position of CAN508 in CDK-2 (Figure 10). On the other hand, the pyrrole ring and the trimethoxyphenyl moiety in compounds 16b,d nearly superpose the positions of the imidazole and phenyl rings in CAN508, respectively. These differences in binding orientations of the three compounds can account even partially for the difference in their inhibitory activity against CDK-2.</p><!><p>Binding modes/interactions of compounds 16a,b,d (shown as sticks, coloured by element) and CAN508 (coloured in yellow) into CDK-2 (pdb code: 3TNW): (A) 3D binding mode of compound 16a overlaid with CAN508; (B) 3D binding mode of compound 16b overlaid with CAN508; (C) 3D binding mode of compound 16d overlaid with CAN508; (D) 2D binding mode of compound 16a; (E) 2D binding mode of compound 16b; (F) 2D binding mode of compound 16d, all showing H-bonding and hydrophobic interactions, hydrogen atoms were omitted for clarity.</p><!><p>Moreover, investigation of the binding interaction of compound 16a into the active site of CDK-2 revealed the formation of five conventional hydrogen bonds with LEU83 and LYS89. One carbon hydrogen bond was also observed between compound 16a and HIS84. In addition, compound 16a displayed several hydrophobic interactions with the key amino acids (ILE10, VAL18, ALA31, LYS33, VAL64, PHE80, LEU134, and ALA144) in the ATP binding site of CDK-2. These binding interactions were similar to those reported for several CDK-2 inhibitors40. On the other hand, each of compounds 16b,d displayed only three conventional hydrogen bonds with amino acids in CDK-2. The fewer number of conventional hydrogen bonds could also account for the lower inhibitory activities of compounds 16b,d compared to compound 16a (Figure 10).</p><p>Compounds 16a,b,d also displayed higher binding affinities towards EGFR compared to erlotinib (Table 8). Among the three derivatives, compound 16a showed the highest affinity for EGFR protein (ΔGb= −9.07 kcal/mol, Table 8). In addition, compound 16b displayed a binding affinity of −8.60 kcal/mol which was lower than the binding affinity of compound 16a. These results were matched with the inhibitory activities of the two compounds against EGFR (Table 5).</p><p>However, compound 16d displayed a good binding affinity (ΔGb= −8.53 kcal/mol) although it did not show in vitro inhibitory activity against EGFR (Table 5). To explain this difference, the binding interactions between the tested compounds (16a,b,d, and erlotinib) and the amino acids in EGFR were investigated. The results revealed that compound 16a exhibited three conventional hydrogen bonds with THR830 and ASP831 (Figure 11). Compound 16b also showed three conventional hydrogen bonds with LYS721 and MET769. However, investigation of the binding interactions of the best fit conformation of compound 16d into EGFR revealed only one conventional hydrogen bond formed with CYC773 (Table 8). These results indicated that the binding affinity of compound 16d towards EGFR is due mainly to the hydrophobic interactions with EGFR. The 2/3D binding modes of compounds 16b,d, and erlotinib are provided in the Supplementary data (Figs. S142–144).</p><!><p>Binding modes/interactions of compound 16a into EGFR (PDB code: 1M17): (A) 3D binding mode of compound 16a into the active site of EGFR, the co-crystallised erlotinib shown as orange line, receptor shown as hydrogen bond surface, hydrogen atoms were omitted for clarity; (B) 2D binding mode of compound 16a into EGFR showing different types of interactions with amino acids in the active site of the protein, hydrogen atoms were omitted for clarity.</p><!><p>In addition, among the three derivatives 16a,b,d, only compound 16d showed weak inhibitory activity against aurora A (14% inhibition) in the kinase profiling test. The docking study of compounds into the active site of aurora A (pdb code: 3E5A) revealed the highest affinity for compound 16d, Supplementary data (Table S3).</p><!><p>In this part, another molecular docking study was performed into the binding site of CA-4/colchicine in tubulin protein to investigate the binding mode/interactions of the new compounds correlate the obtained results with the inhibitory effects of these compounds on tubulin polymerisation (Figures 8 and 9). The crystal structure of tubulin protein (pdb code: 5LYJ) co-crystallised with CA-428 was obtained from the Protein Data Bank (https://www.rcsb.org/structure/5lyj). The protein and ligand files were prepared following the previous report29,41.</p><p>To validate the docking procedures, the co-crystallised ligand (CA-4) 1 was re-docked into the binding site of the co-crystallised ligand (7BA) in tubulin protein. The results revealed the superposition of the re-docked CA-4 with the co-crystallised ligand with RMSD of 0.9 Å. Moreover, the re-docked CA-4 showed similar binding mode, orientation, and exhibited the same binding interactions as those of the co-crystallised ligand (Figure 12).</p><!><p>Superposition of the re-docked/co-crystallised CA-4 1 into the binding site in tubulin (pdb code: 5LYJ): (A) 3D binding modes showing superposition of the re-docked CA-4 (shown as sticks, coloured by element) over the co-crystallised ligand (CA-4, shown as sticks, coloured in blue) into CA-4 1 binding site in tubulin, RMSD = 0.9 Å; (B) 2D binding mode of CA-4 showing H-bonding and hydrophobic interactions with amino acids in tubulin, hydrogen atoms were omitted for clarity.</p><!><p>Investigation of the binding interactions of CA-4 with the amino acids in tubulin protein revealed one conventional hydrogen bond with THR179, one pi-sulphur interaction with MET259, and three carbon hydrogen bonds with VAL238, VAL315, and ASN350. In addition, several hydrophobic interactions with CYS241, ALA250, LEU255, ALA316, and LYS352 were observed (Figure 12).</p><p>The results of the molecular docking study (Table 9) revealed a nice fitting of the new compounds into the binding site of CA-4 in tubulin, where no steric clashes or unfavourable interactions were observed. Moreover, the benzamide derivatives (16a–e and 21) displayed higher binding affinity (ΔGb= −8.80 to −12.16 kcal/mol) compared to −8.72 kcal/mol for CA-4, Supplementary data (Table S4). Moreover, the benzamide derivatives (16a–e and 21) also displayed higher binding free energies than their corresponding Schiff bases (15a–e and 20). These results were also matched with the results of the cytotoxic activities of the two series (Table 1).</p><!><p>Docking results of compounds 15a,b,d, 16a,b,d into tubulin protein (pdb code: 5LYJ) in comparison with the co-crystallised ligand 7BA (CA-4).</p><p>7BA, compound 1 (CA-4); values with asterisks indicate the carbon hydrogen bonds, the underlined atoms are the atoms involved in the H-bonding interactions.</p><p>Binding free energy (kcal/mol).</p><p>Inhibition constant.</p><p>HBs, number of hydrogen bonds.</p><p>Length in angstrom (Å).</p><!><p>To understand the reasons behind this noticeable difference in binding affinities of the two series, we have investigated their binding modes/orientations into tubulin protein. The best-fit conformations of the two series were overlaid with co-crystallised CA-4 in Figure 13. Investigation of their binding modes revealed that all the benzamide derivatives (16a–e and 21) exhibited binding orientations similar to that of CA-4. The two phenyl rings in each of the six benzamide derivatives adopted orientations superposing on the two phenyl rings of CA-4 with the trimethoxyphenyl moieties extended towards the α/β interface (Figure 13).</p><!><p>An overlay of the best fit conformation of the new compounds with the co-crystallised CA-4 shown as sticks, coloured by element (pdb code: 5LYJ): (A) compounds 15a–e and 20 (shown as blue lines) overlaid with the co-crystallised CA-4; (B) compounds 16a–e and 21 (shown as pink lines) overlaid with the co-crystallised CA-4, receptor in the two figs shown as hydrogen bond surface, hydrogen atoms were omitted for clarity.</p><!><p>On the other hand, the Schiff base derivatives (15a–e and 20) adopted completely different orientations from CA-4. The difference in the binding orientations of the two series could account even partially for the difference in their binding affinities towards tubulin and their cytotoxic activities.</p><p>Among the new benzamide derivatives, compound 16b displayed the highest binding affinity towards tubulin. Moreover, the chloro derivative 16d exhibited higher binding affinity than compound 16a. These results were quite matched with the results of tubulin polymerisation assay of the three compounds (Figure 8).</p><p>Furthermore, the binding orientations and interactions of the benzamide derivatives 16a,b,d into tubulin protein were deeply investigated. The results revealed that the three compounds adopted orientation like that of co-crystallised CA-4. In these orientations, the two phenyl rings of the three compounds superposed on the two phenyl rings of CA-4 (Figure 14). Like the co-crystallised CA-4, the trimethoxyphenyl moieties in the three compounds were located deeply into the β-subunit showing several interactions with important amino acids such as THR179, VAL238, and/or CYS241, through carbon–hydrogen bonding and hydrophobic interactions. Moreover, the pyrrolizine nucleus of the three derivatives was located towards the α/β interface with the double bond of the pyrrole ring (C2═C3) overlaid over the double bond in CA-4. In addition, the two phenyl rings in the three compounds were located on the same side of the pyrrole ring adopting a cis-like conformation similar to the cis-conformation of CA-4. These binding modes of compound 16a,b,d could account for their high binding affinities towards tubulin and even partially for the high cytotoxic activities of benzamide derivative.</p><!><p>Binding modes/interactions of compounds 16a,b,d (shown as sticks, coloured by element) into CA-4 (shown as sticks, coloured in yellow) binding site in tubulin (pdb code: 5LYJ): (A) 3D binding mode of compound 16a overlaid with the co-crystallised CA-4; (B) 3D binding mode of compound 16b overlaid with the co-crystallised CA-4; (C) 3D binding mode of compound 16d overlaid with the co-crystallised CA-4; (D) 2D binding mode of compound 16a; (E) 2D binding mode of compound 16b; (F) 2D binding mode of compound 16d, all showing H-bonding and hydrophobic interactions with amino acids in tubulin, hydrogen atoms were omitted for clarity.</p><!><p>The docking study into the binding site of CA-4 in tubulin protein was also performed for the remaining compounds (15c,e, 16c,e, 20, and 21). The results of this study are provided in the supplementary data (Table S4).</p><!><p>In the present study, two new series of pyrrolizine derivatives (15a–e and 16a–e) bearing 3,4,5-trimethoxyphenyl moiety were designed, synthesised, and evaluated for their cytotoxic activities. In addition, two new indolizines (20 and 21) were also synthesised and for their cytotoxic activities. Structural confirmation of these compounds was carried out by spectral (IR, 1H NMR, 13C NMR, DEPT C135, and mass) and quantitative elemental analyses. The results of the MTT assay revealed weak to potent cytotoxic activity for the new compounds against three (MCF-7, HCT116, and A2780) cancer cell lines with IC50 in the range of 0.03–36.88 μM. Among the new derivatives, compounds 16a,b,d exhibited the highest cytotoxicity against MCF-7 cells (IC50=0.068–0.082 μM), while compounds 16c,d were the most active against HCT116 and A2780 cells, respectively. On the other hand, the new compounds showed weak cytotoxicity against the normal MRC-5 cells (IC50=0.155–17.08 μM) compared to 13.66 μM for lapatinib. Compounds 16a,b,d exhibited also cytotoxic activities against doxorubicin-resistance MCF-7/ADR cells (IC50=0.52–6.26 μM). The study of SAR revealed higher cytotoxic activity and selectivity for the benzamide derivatives (16a–e and 21) compared to their corresponding Schiff bases (15a–e and 20). The kinase profiling test of compounds 16a,b,d revealed improvement in their inhibitory activities against several kinases (inhibition%=1–38%) compared to compound 9a. Moreover, the three compounds exhibited higher inhibitory activity against CDK-2 than compound 9a. Mechanistic studies of compounds 16a,b,d also revealed weak to moderate inhibitory activity against tubulin polymerisation. Cell cycle analyses of MCF-7 cells treated with compounds 16a,b,d revealed dramatic increases in the preG1 (11–14-fold) and G2/M (5–7-fold) cell cycle phases at the expense of 5–10-fold decrease of G1 events, all compared to the control. Compound 16d showed the strongest preG1 (27.56%) and G2/M (45.62%) cell cycle effects. The results of the docking study into tubulin/CDK-2/EGFR showed higher binding affinities for compounds 16a,b,d compared to the co-crystallised ligands (CAN508 and erlotinib). Taken together, compounds 16a,b,d could serve as promising lead compounds for the future development of new potent anticancer agents.</p><!><p>Click here for additional data file.</p>

PubMed Open Access

Investigating Endogenous Peptides and Peptidases using Peptidomics

Rather than simply being protein degradation products, peptides have proven to be important bioactive molecules. Bioactive peptides act as hormones, neurotransmitters and antimicrobial agents in vivo. The dysregulation of bioactive peptide signaling is also known to be involved in disease, and targeting peptide hormone pathways has been successful strategy in the development of novel therapeutics. The importance of bioactive peptides in biology has spurred research to elucidate the function and regulation of these molecules. Classical methods for peptide analysis have relied on targeted immunoassays, but certain scientific questions necessitated a broader and more detailed view of the peptidome\xe2\x80\x93all the peptides in a cell, tissue or organism. In this review we discuss how peptidomics has emerged to fill this need through the application of advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods that provide unique insights into peptide activity and regulation.

investigating_endogenous_peptides_and_peptidases_using_peptidomics

Bioactive Peptide Action, Production and Signaling<!>The Production and Signaling of Bioactive Peptides<!>Bioactive Peptide Discovery<!>Bioactive Peptide Function<!>Proteolytic Regulation of Bioactive Peptides<!>The Case for Peptidomics<!>Peptidomics Workflow, Instrumentation and Analysis<!>Using Peptidomics to Discover Bioactive Peptides<!>Quantitative Peptidomics<!>Application of Quantitative Peptidomics to Behavioral Studies<!>Quantitative Peptidomics to Elucidate Peptidases and Proteases Substrates<!>Prohormone Convertases<!>Prolyl Peptidases<!>A Peptidomics Strategy to Elucidate the Proteolytic Pathways that Process Bioacitve Peptides<!>Conclusions and Future Directions

<p>Organisms seamlessly integrate numerous classes of molecules, including bioactive peptides, into biochemical pathways that enable all the processes needed for life (1-3). Insulin and glucagon, for example, are two of several well-known pancreatic peptide hormones that partake in hormonal regulation of physiological glucose metabolism (4, 5). Neuropeptides such as substance P (6, 7) and the enkephalins (8, 9) signal within the central nervous system (CNS) and mediate behavioral processes. There are also more than 20 known antimicrobial peptides (AMPs) (10) that have important roles in the innate immune system. The defensin family of antimicrobial intestinal peptides thwart infection by serving as endogenous antibiotics (11) and more recent experiments have also shown that the defensins regulate the intestinal microbiome (12), all the bacteria present in the gut, to extend the physiological role of these peptides even further.</p><p>These examples highlight the broad influence of bioactive peptides and demonstrate why these pathways are of interest. Moreover, the discovery of these peptides has begun to impact medicine with the development of approved drug liraglutide (13), a glucagon like peptide 1 (GLP-1) mimetic, and AMP mimics that are promising antibiotic agents (14). Many more examples can be presented as bioactive peptides can be found in many biological niches and play many roles. Conotoxins, for example, are a group of neurotoxic peptides produced by the venomous marine cone snail that are potential therapeutics (15-18). Here, we primarily focus on the application of peptidomics to problems in mammalian biology, the exception being the work described on honeybee neuropeptidomics, but we recognize that peptidomics has the potential to impact many different areas of biology.</p><!><p>Though bioactive peptides have diverse functions and sequences, many of these peptides have analogous biochemical mechanisms that control their production, regulation and signaling (19) (Fig. 1). Bioactive peptide synthesis commences with the expression of a biologically inactive preprohormone that is typically 100-350 amino acids long (19). A signal peptide at the N-terminus (the "pre" in preprohormone) directs the peptide into the secretory pathway. The signal peptide is cleaved shortly after the peptide enters the lumen of the ER to afford the prohormone. Prohormones are then shuttled from the endoplasmic reticulum (ER) to the Golgi apparatus (Golgi). In the Golgi the prohormone begins its conversion into a mature bioactive peptide through proteolysis by a group of serine proteases called the prohormone convertases (PCs) (20, 21) (Fig. 1). This processing begins in the Golgi and continues into the secretory vesicles. Some peptides undergo additional proteolytic processing and post-translational modifications (acetylation, sulfation and amidation) to produce the active form (Fig. 2) (22, 23).</p><p>Peptides are released from cells into the extracellular milieu by fusion of secretory vesicles with the plasma membrane (2, 19) (Fig. 1). The secretion of a bioactive peptide is regulated by an external stimulus that causes the acute release of the peptide when needed (e.g. glucose release of insulin) (24, 25). After release, bioactive peptides travel to a target cell or tissue to bind their cognate membrane receptors, which initiates intracellular signal transduction and a biological response (26). The principle receptors involved in bioactive peptide signaling include seven transmembrane G-protein coupled receptors (GPCRs) (27) and receptor tyrosine kinases (RTKs) (28). Peptide signaling is terminated by removal of the peptide through renal clearance (29) or proteolyic inactivation of a peptide (30) (Fig. 1). By understanding these general pathways for a few peptides it has made it easier to discovery and characterization of new bioactive peptides, receptors and peptidases.</p><!><p>The methods used to investigate bioactive peptides have evolved as new techniques are introduced into biology. Classical methods for bioactive peptide discovery rely on bioassays that enable the biochemical purification of bioactive peptides from tissues by identifying fractions with a desired bioactivity (31). By performing multiple rounds of purification bioactive peptides could be purified and subsequently identified. Many important peptides were discovered through this type of approach including insulin (32), glucagon (33, 34), angiotensin II and the endorphins (35-37). Modern variants of this approach have replaced a cellular or physiological phenotypic bioassay with a targeted receptor-based assay to identify bioactive peptides and characterize the receptor too. The identification of GPCR ligands (38-40), for example, led to the discovery of sleep regulating orexins (41) and prolactin-releasing peptide (42) to demonstrate the effectiveness of this strategy in discovering new bioactive peptides.</p><p>To circumvent the need for bioassays, Tatemoto and Mutt developed an alternative approach bioactive peptide discovery that relies on the biochemical enrichment of peptides with a C-terminal amide (43-48), which are commonly found on bioactive peptides that are part of the secretory pathway (19). Oxidative cleavage of a C-terminal glycine by peptidyl alpha-amidating monooxygenase (PAM) (49-51) produces the C-terminal amide-containing peptides. C-terminal amides are thought to protect bioactive peptides from carboxypeptidase-mediated degradation and in some cases are required for bioactivity (52). Tatemoto and Mutt developed a biochemical method that enabled them to purify peptides with C-terminal amides from complex samples. This approach was highly successful and led to the discovery of neuropeptide Y (46), peptide histidine isoleucine (PHI(1-27)) (45) and galanin (48), which are now known to regulate a variety of physiological functions (3).</p><p>Lastly, bioactive peptides have also been discovered through messenger RNAs (mRNAs) that revealed a coding sequence containing PC-cleavage sites. Alternative splicing of the calcitonin gene affords two different mRNA products (53). Analysis of these splice forms revealed that one of the mRNAs encodes for the calcitonin peptide while the alternatively spliced form was predicted to encode a unknown peptide which was aptly named the calcitonin-gene related peptide (CGRP) (54). This predication was validated in subsequent experiments that demonstrated that CGRP was present in tissues (55, 56). Physiological experiments identified CGRP as a potent vasodilator (57). Most importantly, CGRP signaling was recently been linked the onset of migraines (58, 59) and this insight led to the development of CGRP-receptor antagonists a new class of anti-migraine drugs in clinical trials (60, 61). CGRP exemplifies the basic as well as biomedical impact of bioactive peptide discovery.</p><!><p>Pharmacology and genetics have been used to characterize the physiological functions for many peptides (9, 12). For instance, injection of glucagon like peptide 1 (GLP-1) reduces blood glucose levels to clearly define a role for GLP-1 in blood glucose homeostasis (62). Using pharmacology to ascertain peptide function is limited due to the poor bioavailability of most peptides (63). This is a particular problem in the study of neuropeptides since peptides do not readily cross the blood-brain barrier. Pharmacological studies with small-molecule receptor agonists (or antagonists) of neuropeptide receptors provide an alternative approach to understand the physiological function of a particular bioactive peptide (37, 61, 64). The analgesic natural product alkaloid morphine, for example, is an agonist for the opioid receptors to indicate that the endorphins, natural peptide agonists of the opioid receptors, are involved in pain sensation (37).</p><p>Genetic approaches have also proven to be a more general approach for studying bioactive peptide function in vivo (22, 65-67). For example, the role of the peptide YY (PYY), an intestinal hormone, in body mass regulation and feeding behavior was established by knocking this peptide out in a mouse model (68). Mice lacking PYY were obese and ate more than their wildtype counterparts, which demonstrates that PYY is a peripheral signal that is able to promote satiety (the feeling of fullness). These experiments also revealed that mice that ate protein, but not fat or carbohydrate, released more PYY and this finding provides a new model to explain why proteins are better appetite suppressants than fat or carbohydrate.</p><p>Knockout studies of peptides are often difficult, however, because a single gene sometimes produces more than one bioactive peptide (69-71). For example, the glucagon gene controls the production of glucagon, GLP-1 and GLP-2 (70, 71). As a result, knocking out the glucagon gene would result in loss of GLP-1 and GLP-2 as well. Glucagon and GLP-1 do have unique receptors though and knocking out the peptide receptors instead of the peptides enables the physiological functions of the bioactive peptide pathways to be assessed (27, 72, 73). To study glucagon signaling, for example, glucagon receptor knockout mice were generated and these mice showed improved glucose tolerance relative to their wildtype counterparts (74). The application of pharmacology and genetics has provided valuable information to further our understanding of the cellular and physiological roles of peptides and their receptors (Table 1).</p><!><p>An approach for controlling endogenous peptide signaling in medicine is to target the proteases and peptidases that regulate these pathways with small-molecule inhibitors (30, 75). A prototype example of the success of this approach is the inhibition of the angiotensin converting enzyme (ACE), a well studied exopeptidase, in the treatment of hypertension and cardiovascular disorders (75). ACE is responsible for the cleavage and activation of the angiotensin I peptide to generate angiotensin II, a ligand for the angiotensin receptor and a potent vasoconstrictor that causes hypertension. Understanding this mechanism led to the development of small-molecule ACE inhibitors as antihypertensive drugs that function by preventing the activation of angiotensin I. More recently, a similar strategy has successfully been employed in the development of anti-diabetic drugs that are inhibitors of dipeptidyl peptidase 4 (DPP4) activity.(30)</p><p>Bioactive peptides are often enzymatically regulated after release in ways that can regulate their activity. The insulinotropic hormone GLP-1 is released from the gut after a meal (30) and activates its receptors in the pancreas where it stimulates insulin biosynthesis and secretion (76). Detailed studies of GLP-1 signaling revealed that GLP-1 has a very short half-life in blood due to N-terminal processing of this peptide by a peptidase called DPP4 (77, 78). DPP4 removes an N-terminal dipeptide to inactivate GLP-1. Importantly, this pathway has been harnessed in the development of a new class of anti-diabetic drugs (30, 79) that inhibit DPP4. By blocking DPP4 activity endogenous GLP-1 levels are increased leading to higher levels of insulin and improved glucose tolerance (Fig. 3). The development of ACE and DPP4 inhibitors underscores the importance of bioactive peptides in basic research and medicine.</p><!><p>The importance of peptides supports the need for better methods to analyze these molecules in a biological setting. Classical methods for peptide analysis rely mostly on immunoassay approaches that require specific antibodies to recognize the peptide of interest (80, 81). Immunoassays are valuable because they provide a means to detect the peptides from a variety of biological samples (80, 82, 83). While effective, immunoassays do not provide a global analysis, require the time-consuming generation of antibodies, and can sometimes suffer from antibody cross-reactivity with other peptides (82). For example, Jankowski, et al. discovered a novel vasoconstrictive peptides angiotensin A (Ang A) by mass spectrometry and showed that this peptide cross-reacts with antibodies against angiotensin II (AngII) (82). More specifically, cross-reactivity is a particular problem when studying peptide proteolysis since shorter or longer peptides may still contain the antibody epitope making it difficult to measure the peptide of interest. Peptidomics approaches can overcome the challenges associated with immunoassays because liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides a specific and global analysis of peptidome. These attributes enable new types of experiments to elucidate important features of bioactive peptide regulation.</p><!><p>Peptidomics refers to any method that provides a broad view of the entire peptide pool in a biological sample, the peptidome (84, 85). Modern peptidomics approaches rely heavily on LC-MS/MS to provide sensitive detection and identification of peptides from biological samples. The two predominant types of mass spectrometers used in peptidomics studies are quadrupole time-of-flight (Q-TOF) and ion trap (IT) instruments (86). These instruments enable quantitation using several approaches and provide the tandem mass spectra that are necessary to identify the peptide sequence. The key steps in every peptidomics experiment are peptide isolation, peptide fractionation and processing, mass spectrometry, and data analysis (Fig. 4). Svensson and colleagues provided an early example of an effective peptidomics workflow during their discovery of neuropeptides (84). Protease inhibitors are not effective enough to prevent peptide degradation so Svensson and coworkers used focused microwave radiation of rodent brains (84, 87, 88) to ablate proteolytic activity by heat denaturing all proteins prior to peptide isolation. Heating tissues prior to isolation of the peptidome has become a standard process in peptidomics even though different heating methods are used (e.g. ex-vivo heating of tissues by microwave (89) or boiling (90-92)).</p><p>Following microwave irradiation the brain tissue was homogenized in slightly acidic buffer and centrifuged to remove any insoluble debris. Next, Svensson and colleagues fractionated their sample by passing it through a molecular weight cutoff (MWCO) filter (10 kilodalton) to separate the peptides from larger proteins (84). MWCO filters are used in almost every peptidomics experiment to enrich the peptidome. Other fractionation schemes can also be included to provide different views of the peptidome, including enzymatic digestion (e.g. trypsin), strong cation exchange (SCX) and isoelectric focusing (IEF) (92) (Fig. 4). The use of additional peptide fractionation improves the peptidome coverage. In one example a 10-fold increase in coverage was obtained through the addition of an SCX step into the workflow. The isolated neuropeptides were then subjected to reverse phase LC-MS/MS using an electrospray ionization (ESI) Q-TOF-MS. The identification of peptides is carried out using SEQUEST (93) or Mascot (94) software packages.</p><p>Using this workflow Svensson and colleagues identified greater than 500 neuropeptides (84). Some of these peptides are known bioactive neuropeptides, including substance P, beta-endorphin, and neurotensin, which validated the methodology. There were also a number of novel neuropeptides. Many of these novel peptides were derivatives of known neuropeptides, including longer or shorter peptide fragments and post-translationally modified forms. The common PTMs observed in this data set included acetylation, phosphorylation, and pyroglutamination (Fig. 2). The characterization of these novel neuropeptides highlights the value of peptidomics to obtain information that is inaccesible by other approaches. More generally, Svensson, et. al. demonstrated how to obtain quality peptidomics data and their approach has been emulated in all subsequent peptidomics studies.</p><!><p>Yamaguchi and colleagues developed a peptidomics strategy to identify new bioactive peptides by detecting peptides that are secreted by TT cells, a human thyroid cell line (95). By using peptidomics to detect amidated peptides in the conditioned media of TT cells they were able to detect known bioactive peptides, such as CGRP (53, 57). In addition, these experiments detected two novel C-terminally amidated peptides derived from the VGF gene called neuroendocrine regulatory peptides-1 and -2 (NERP-1 and -2). Subsequent histological studies revealed that NERP-1 and NERP-2 are present in vivo and that they co-localized in the rat brain (supraoptic nucleus and paraventricular nucleus) with vasopressin, a multifunctional peptide hormone (96-101). This led to the hypothesis that the NERPs and vasopressin may interact. This idea was validated when pharmacological delivery of the NERPs suppressed vasopressin release in a dose dependent fashion to demonstrate a physiological activity for these peptides (95) (Fig. 5). In this case, the detection of C-terminal amides using peptidomics led to the discovery of two novel bioactive peptides and provided a peptidomics version of the Tatemoto and Mutt approach (44). Sasaki, et. al. generalized this approach further to include peptides that lack C-terminal amides by performing cell based assays to determine which of the peptides they detected were able to induce intracellular calcium release, which is a hallmark of bioactive peptides (102).</p><p>In a more focused search Osaki and Minamino developed a peptidomics strategy to discover novel mammalian antimicrobial peptides (103). They took advantage of the fact that antimicrobial peptides are typically highly basic peptides—containing multiple lysines and arginines—and developed a cation exchange fractionation method that specifically enriched basic peptides. Treatment of QGP-1 cells, a human pancreatic endocrine cell line, with forskolin and carbachol promotes the secretion of intracellular peptides into the media. Secreted peptides were enriched by cation exchange, fractionated and desalted by HPLC, and analyzed by matrix assisted laser desorption ionization (MALDI)-MS to reveal a novel peptide fragment of insulin-like growth factor-binding protein 5 (IGFBP-5) (104, 105).</p><p>IGFBP-5 is a known secreted protein, but its proteolysis and antimicrobial activity had not been reported before. The peptide fragment of IGFBP-5 identified in this study was referred to as antimicrobial peptide-IGFBP-5 (AMP-IFGBP-5) (103). Synthesis of the AMP-IGFBP-5 enabled the antimicrobial activity of this peptide to be tested. AMP-IGFBP-5 had an IC50 of ~ 1 μM with E.coli K12 and was on par or better than the established antimicrobial peptides cathelicidin (106) and β-defensin-2 (107). Additional experiments revealed that this peptide was active against a number of Gram-negative and Gram-positive bacteria, as well as the fungus Pichia pastoris GS115. In addition, AMP-IGFBP-5 is present in the brain and gut of rats, suggesting a potential physiological role for this peptide in the innate immune system (Fig. 6).</p><p>These examples highlight the utility of peptidomics to accelerate the discovery of novel bioactive peptides by enabling the detection and identification of these peptides. Most of these peptides would undetectable using standard molecular biology techniques used to identify proteins (e.g. silver stained SDS page gels or proteomics) (108, 109). In addition, immunoassays are not a discovery based approach because they require structural knowledge of the peptides first so that antibodies can be generated (110). Characterizing these new peptides these studies also indicate new functions for precursor proteins that generate these peptides, such as VGF (111) and IGFBP-5 (104, 105). As additional assays are integrated with peptidomics approaches the number of bioactive peptides discovered is bound to increase.</p><!><p>The above examples take advantage of the ability of peptidomics to identify peptides within complex mixtures (112). In some cases, however, it is important to quantify peptides between two samples to correlate changes in peptide levels with changes in a phenotype or genotype (89-92, 113-115). Quantitative peptidomics methods enable differences in relative peptide concentrations to be determined and can be carried out using isotopic labeling methods (113, 114, 116, 117) or a label-free approach (90-92, 118-121) (Fig. 7).</p><p>In one example of an isotope labeling approach a light and heavy form (e.g. deuterium or carbon-13) of a chemical labeling reagent, such as succinic anhydride, is used to differentially label two peptidome samples. Succinic anhydride and the deuterated version of succinic anhydride, d4-succinic anhydride, are readily available. Labeling of two different peptidomes with succinic anhydride and d4-succinic anhydride results in a mass difference of 4 daltons between the same peptide in each sample (113) (Fig. 7). After separate labeling reactions the two samples are mixed and analyzed during in same LC-MS/MS experiment. Differences in relative peptide concentrations are measured by comparing the ratio of the light labeled peptide to the heavy labeled peptide in the mass spectrum (Fig. 7). This approach enables the relative abundance of peptides between different samples to be determined, and additional labels can be created if more samples are to be included. By contrast label-free approaches simply rely on the ion intensity, absolute signal at the MS detector, between two samples to provide relative changes in peptide levels between two samples (92).</p><!><p>Using an isotopic labeling strategy, Sweedler and colleagues investigated changes in neuropeptides in the honeybee brain during behavioral assays (113). Honeybees were chosen as the model system because they show amazingly sophisticated behavior (122, 123). They have approximately 100 neuropeptides, and in this report they were able to quantitatively measure about 50 of those peptides. Quantitation was accomplished using a chemical labeling approach with succinic anhydride and d4-succinic anhydride to differentially label the peptidomes in a series of pair-wise comparisons. Changes in neuropeptide levels were measured as honeybees were foraging for food (124). Four categories were created based on whether the honeybees preferred pollen (P) or nectar (N) as a food source, or whether the honeybees were isolated when they arrived (A) or departed (D) from the food (Fig. 8).</p><p>Combinations of these categories led to four different sample sets for peptidomics: nectar arriving (NA), nectar departing (ND), pollen arriving (PA), and pollen departing (PD) samples (Fig. 8). Peptidomics analysis compared NA to ND, NA to PA, ND to PD, and PA to PD. Of the 50 peptides measured in these studies, 8 were significantly different in one of the four pairwise comparisons. The changes were distributed between nectar (N) and pollen (P) foragers, as well as differences between arriving (A) and departing (D) honeybees. Together these changes were distinct enough to categorize the four different groups using the peptide signatures and canonical discriminant analysis (125). Brockmann and colleagues pushed the frontiers of peptidomics by making measurements with small amounts of tissue and demonstrating that the honeybee neuropeptidome could be used to correlate neuropeptides with behavior. This work sets the stage to subsequently study these peptides an their functions in honeybees or other related organisms.</p><!><p>One major question for every enzyme is the identification of natural substrates (126). For most peptidases and proteases, the identification of candidate substrates is a two-step process. Traditionally, the substrate specificity of the peptidase or protease is determined in vitro using synthetic peptide libraries (127, 128). Based on the enzyme specificity, the next step is to look for candidate endogenous substrates and then test these natural substrates in vitro (129). Finally, in the best case scenario, the activity of the enzyme is perturbed via genetics or chemical inhibition and the endogenous levels of the substrate are evaluated to determine the physiological relevance of the prediction (77).</p><p>While occasionally successful, this strategy often leads to many false positives because in vitro experiments are poor predictors of in vivo substrate utilization due to a number of factors that cannot be recapitulated in vitro (91, 130). First, in vitro experiments cannot account for the distribution of enzymes and substrates within cells and tissues. Therefore, enzyme-substrate relationships that are determined in vitro might not occur in vivo because the two molecules do not have the opportunity to interact. Next, assays with pure enzymes do not factor in the relative contributions of other enzymes in the proteome. As a consequence, even if an enzyme and substrate interact in vitro, other enzymes may be responsible for processing the peptide in vivo (126). Lastly, the presence of competitive substrates in vivo can prevent an enzyme from cleaving an in vitro substrate in vivo (131). Together these cases highlight the difficulty in trying to predict endogenous peptidase-substrate relationships from in vitro experiments (91).</p><p>Peptidomics offers an elegant solution to this problem by enabling the substrates of peptidases to be measured directly in cells and tissues (90-92, 114, 115, 132, 133). In doing so, substrate discovery using peptidomics takes into account all of the aforementioned challenges presented by in vitro experiments. These peptidomics experiments rely on the fact that perturbation of the activity of a peptidase–either by using genetics to knock it out or pharmacological inhibition–results in different amounts of substrates and products in cells and tissues. Comparison of samples with differences in peptidase activity using quantitative peptidomics can identify these endogenous substrates and products (Fig. 9). The identification of peptidase-regulated peptides can help explain the biochemical, cellular, and physiological functions of the peptidase. We provide several examples below that attempt to target particular enzymes involved in peptide biosynthesis, degradation, and signaling.</p><!><p>Fricker and colleagues pioneered efforts to identify substrates and products of the PCs using peptidomics (134). As mentioned, PCs are the enzymes responsible for making the primary cut in the prohormone peptide in the secretory pathway that eventually leads to the production of the mature peptide (19). The PCs cleave prohormones at dibasic sites (e.g. KK, KR, RR), a very distinctive sequence that has been used to predict the presence of peptide hormones within longer genes. The PCs are a family of subtilsin-like serine proteases with seven members in mammals (135). PC1/3 and PC2 represent the majority of the PC activity in neuroendocrine cells and other tissues (135). For example, PC2 is involved in the production of a number of neuropeptides as well as insulin and glucagon in the pancreas.</p><p>Mice lacking PC2 show impaired neuropeptide processing by radioimmunoassasys (RIAs) that measured the levels of full-length peptide (136, 137). Like all immunoassays, RIAs suffer from issues with cross reactivity (82), and this problem may be exacerbated in peptide processing studies where incompletely processed peptides are very similar in structure, which would favor cross reactivity. As a result, Fricker and colleagues decided to employ a quantitative peptidomics approach to study PC2. In these experiments hypothalamic peptidomes from wildtype (PC2+/+) and PC2-null (PC2−/−) mice were compared by peptidomics to identify specific substrates of PC2 using an isotopic labeling method (134). The substrates would be expected to be elevated in the knockout mice, while the products would be elevated in the wildtype mice.</p><p>A number of neuropeptides were identified in this study including fragments of cocaine and amphetamine regulated transcript, chromogranins, preprotachykinins, provassopressin, and secretogranin (Table 2). The levels of the peptides detected in the PC2−/− sample were greatly reduced in comparison to the PC2+/+ sample. This data supports a general role for PC2 in the production of many neuropeptides in the hypothalamus. In addition, a number of protein fragments, most likely from the breakdown of proteins within the cell, were also detected in these samples. These fragments are not part of the secretory pathway and the levels of these fragments did not change, demonstrating the specificity of PC2 for secreted hormones. Finally, this specificity supports the notion that the methodology is capturing changes that are occurring in vivo, since any processing that occurred during the sample preparation would target secretory and non-secretory peptides equally. Fricker and colleagues also utilized their data to determine that aromatic residues or proline are preferred by PC2 at the P1' and P2' positions. In total, this work provided a physiological view of PC2 biochemistry and a template for comparative peptidomics studies that identify peptides that are regulated by peptidases and proteases.</p><!><p>One of the benefits of peptidomics is that it is unbiased and can be used to discover novel endogenous substrates for peptidases, which can help define the cellular and/or physiological function of peptidases (77). Examples of this are found in the analysis of members of the prolyl peptidase family. The prolyl peptidases are a family of serine peptidases that cleave peptides preferentially on the C-terminal side of proline residues (i.e. proline is strongly preferred at the P1 position). The prolyl peptidase family is composed of prolyl endopeptidases and dipeptidyl peptidases, which preferentially cut at proline residues at the penultimate position of the N-terminus (i.e. H2N-XP-peptide) (78).</p><p>Prolyl endopeptidase (PREP) is the founding member of the prolyl peptidase family (138, 139). Interest in this protein was derived from its unique selectivity for proline. Initial in vitro assays with PREP identified candidate substrates for the enzyme, such as vasopressin (139, 140), which led to new hypotheses about PREP function. Vasopressin was previously linked to memory performance and led to the development of selective PREP inhibitors, such as S17092 (141, 142), as potential anti-amnesic compounds. Interestingly, while S17092 inhibitors showed improved cognitive function in monkeys (143) and humans (144), PREP inhibition did not regulate physiological levels of vasopressin highlighting the difficulty in using in vitro experiments to predict endogenous substrates (140, 145).</p><p>To gain a deeper insight in the biochemistry of PREP in vivo, two groups utilized comparative peptidomics to investigate PREP substrates in the nervous system using selective pharmacological inhibition of PREP. The first study utilized the commercially available inhibitor, Z-ProProlinal, to study PREP activity in a rat model (133). In these experiments quantitation was performed using an isotopic labeling strategy termed iTRAQ (146). Using this approach, Tenorio-Laranga and colleagues identified a number of novel Prep substrates in the brains of inhibitor-treated rats. These were proline-containing peptides elevated in the inhibitor-treated samples versus the untreated samples. These peptides were not explicitly tested as Prep substrates, but fit into the known substrate profile for the enzyme. On the basis of their peptidomics data, Tenorio-Laranga and coworkers concluded that Prep is involved in the catabolism of peptides in the CNS (133).</p><p>Nolte and colleagues utilized the PREP inhibitor S17092 to study the impact of PREP on the CNS peptidome of mice (90). In this case, quantitation was performed using a label-free approach. The substrates identified in this study raised important questions about the PREP substrate specificity that were then studied in greater detail. Specifically, peptidomics was able to provide a possible explanation of why PREP had evolved a preference for cutting shorter peptides. One of the PREP substrates identified was a fragment of the bioactive peptide CGRP, CGRP(20-37), which was elevated in the S17092-treated sample (Fig. 10). The full length CGRP(1-37) was not a substrate, even though it contains the very same cut site. These data demonstrate that Prep uses sequence and length specificity to cleave a subset of proline-containing peptides in the nervous system.</p><p>Tagore and colleagues (91, 92) extended peptidomics to study another prolyl peptidase, DPP4, in the kidney. While the role of DPP4 in plasma GLP-1 regulation was established (77) the function of this enzyme in other tissues, including the kidney, was less clear. Peptidomics comparison of wildtype (DPP4+/+) and DPP4 null (DPP4−/−) samples identified a number of DPP4 substrates elevated in the DPP4−/− samples. One interesting question that emerged from these studies was an attempt to explain how the penultimate proline containing substrates of DPP4 are generated in vivo. The data suggested that peptides are cleaved first by aminopeptidases until a penultimate proline is encountered. Penultimate proline peptides are not aminopeptidase substrates and are then released and cleaved by DPP4. In this model, aminopeptidase and DPP4 activities form a biochemical pathway that is responsible for the N-terminal degradation of proline-containing peptides (Fig. 11). This model was tested by adding peptides with internal prolines to tissue lysates with and without aminopeptidase activity. In the absence of aminopeptidase activity the production of penultimate proline containing peptides was reduced, which demonstrated a role for kidney aminopeptidase activity in the production of DPP4 substrates (91, 92). In total, these studies highlight the value of peptidomics in providing detailed information about peptidases in vivo.</p><!><p>As mentioned, the regulation of bioactive peptides by peptidases is of basic interest and can also be used to develop novel therapeutics (e.g. DPP4 and ACE inhibitors). Despite the interest in these pathways we currently lack information about the proteolytic regulation of most bioactive peptides due to the lack of a general approach for elucidating these pathways in vivo. Specifically, any approach that can identify physiologically relevant peptide fragments can reveal the proteolytic pathways that process bioactive peptides and accelerate the discovery of the peptidases responsible for peptide processing. Tinoco, Kim, and colleagues developed a general peptidomics-based strategy to elucidate the endogenous pathways that regulate bioactive peptides coupling in vitro assays and in vivo peptidomics measurements of endogenous peptides (131) (Fig. 12).</p><p>This peptidomics-based approach was applied to investigate the proteolysis of PHI(1-27) (147), an intestinal peptide hormone which has been linked to a number of biological functions, including prolactin secretion (148) and glucose stimulated insulin secretion (GSIS) (149). First, PHI(1-27) was incubated with intestinal lysates and this revealed the production of PHI(3-27) and PHI(1-22) as the predominant fragments suggesting potential N- and C-terminus specific cut sites. In vitro experiments using chemical inhibitors demonstrated that the N-terminal processing is regulated by DPP4. Peptidomics experiments revealed the existence of PHI(1-22) but not PHI(3-27) in the intestine (150). The absence of PHI(3-27) in vivo was explained by the presence of competitive DPP4 substrates which can attenuate DPP4 processing of PHI(1-27). This hypothesis was supported by in vitro experiments with recombinant DPP4 where the presence of additional DPP4 substrates greatly slowed PHI(1-27) processing.</p><p>Lastly, the functional impact of the C-terminal proteolysis of PHI(1-27) was assessed using a GSIS assay (151) with mouse pancreatic β islets. In these experiments PHI(1-27) was active and able to promote insulin secretion, as reported, but PHI(1-22) was inactive to indicate that proteolysis of PHI(1-27) in vivo ablates the bioactivity of this peptide. Together this data demonstrates the utility of peptidomics in characterizing the endogenous proteolytic pathways that regulate bioactive peptide hormones. More generally, by coupling these experiments to improved strategies for peptidase discovery and bioassays, such as GSIS, additional enzymes and pathways that regulate important physiological pathways will be discovered.</p><!><p>As a whole, the application of LC-MS peptidomics has had a major impact on redefining the outlook of the peptide field. Advancements in peptidomics workflows and analytical tools have enabled improved strategies for identifying bioactive peptides and characterizing their functions (92). Peptidomics also helps to study peptidases and molecular pathways that regulate peptides in general (89-92, 114, 115, 131, 132). Because peptides are such powerful regulators of physiology, this research will impact our basic understanding of peptide and enzyme function in vivo, and may eventually provide novel insights that will be of benefit in the development of therapeutics like protease/peptidase inhibitors. Moreover, these same approaches can be extended from proteases to other classes of enzymes, such as kinases, that may modify bioactive peptides as well.</p><p>Looking forward there appears to be a number of opportunities for peptidomics to make an impact in biology and medicine. In the last decade, biologically active peptides encoded in short open reading frames (sORFs) have been serendipitously identified in mammals (152, 153), insects (154) and bacteria (155). These peptides encoded in sORFs, which lack signaling sequences and are released directly into the cytoplasm (156). Humanin was the first sORF derived peptide to be identified through a screen to identify genes that inhibit apoptosis and it exhibits neuroprotective properties (152, 157) by direct binding to proapoptotic proteins in the mitochondria. More recently, peptides encoded by the polished rice (pri) sORF gene have been shown to function as transcription factors that affect epidermal differentiation in Drosophila (158), suggesting that such peptides may be more ubiquitous than suspected. Improvements in the endogenous identification of sORF encoded peptides can be made by integrating genomic approaches with peptidomics (159-161).</p><p>Furthermore, as we improve our ability to deliver peptides in vivo the search for natural bioactive peptides is likely to ramp up. Natural product discovery was based on the fact that small-molecule drugs could be found by scouring nature's stockpile of biologically active chemicals (162). Similar approaches for peptides have been hindered because peptides were traditionally considered to be poor drugs (163). The development of biologics aims to overcome many of these issues (164-166). Drugs such as liraglutide (13) and pramlintide (167) are peptides that are derived from GLP-1 and amylin, respectively. The emergence of these peptide drugs and improved methods for intracellular delivery of bioactive peptides (168, 169) and proteins (170) suggests a bright future for bioactive peptide medicines. Much of this will be predicated on the discovery of new bioactive peptides and will be accelerated by the inclusion of peptidomics.</p>

PubMed Author Manuscript

Mice lacking the galanin gene show decreased sensitivity to nicotine conditioned place preference

Previous work has indicated that the neuropeptide galanin decreases sensitivity to the rewarding effects of morphine and cocaine, but increases alcohol drinking. The aim of the current study was to examine the role of galanin signaling in nicotine reward by testing the effects of nicotine in mice lacking galanin peptide (Gal \xe2\x88\x92/\xe2\x88\x92) as compared to wild-type (Gal +/+) controls. Using an unbiased, three-chamber conditioned place preference (CPP) paradigm the dose-response function for nicotine CPP was tested in Gal \xe2\x88\x92/\xe2\x88\x92 and Gal +/+ mice. Since activation of extracellular signal-related kinase (ERK2) is involved in the rewarding effects of several classes of drugs of abuse, we then measured the level of ERK2 phosphorylation in the nucleus accumbens shell (NACsh) and core (NACco) of Gal \xe2\x88\x92/\xe2\x88\x92 and Gal +/+ mice following re-exposure to the CPP chamber previously paired with nicotine as a marker of mesolimbic system activation. Finally, we examined whether acute nicotine administration affects ERK2 activity in Gal \xe2\x88\x92/\xe2\x88\x92 and Gal +/+ mice. Gal \xe2\x88\x92/\xe2\x88\x92 mice required a higher dose of nicotine to induce a significant CPP compared to Gal +/+ mice. Upon re-exposure to the CPP apparatus, only Gal +/+ mice showed activation of ERK2 in the NACsh after training with the optimal dose for nicotine CPP, suggesting that the CPP observed following training with a higher dose of nicotine in Gal \xe2\x88\x92/\xe2\x88\x92 mice resulted in differential recruitment of ERK signaling in the NACsh. In addition, no activation of ERK2 was observed following acute nicotine administration in either genotype. These data, along with prior results, suggest that galanin alters sensitivity to drugs of abuse differentially, with morphine, cocaine and amphetamine place preference suppressed, and nicotine and alcohol preference increased, by galanin signaling.

mice_lacking_the_galanin_gene_show_decreased_sensitivity_to_nicotine_conditioned_place_preference

1. Introduction<!>2.1 Subjects<!>2.2 Drugs<!>2.3 Experiment 1: Nicotine Conditioned Place Preference<!>2.4 Experiment 2: Acute Nicotine Administration<!>2.5 Western Blot Analysis for ERK Phosphorylation<!>3.1 Experiment 1: Nicotine Conditioned Place Preference<!>3.2 Experiment 2: Acute Nicotine Administration<!>4. Discussion<!>

<p>There has been increasing interest in determining the mechanisms by which orexigenic neuropeptides modulate the neurochemical and behavioral responses to drugs of abuse. There is considerable evidence suggesting that neuropeptides involved in feeding can alter activity within the mesolimbic dopamine system, thereby affecting a number of motivated behaviors including those associated with the rewarding effects of drugs of abuse (Chung et al. 2009; Ericson and Ahlenius 1999; Espana et al. 2010; LeSage et al. 2010; Maric et al. 2009; Sears et al. 2010). Among these neuropeptides, galanin is of interest because of its ability to modulate the rewarding effects of a number of classes of abused drugs, including opiates, psychostimulants and alcohol (reviewed in Picciotto et al. 2010).</p><p>Previous research has suggested that endogenous galanin inhibits the rewarding effects of morphine and cocaine as transgenic mice lacking the galanin peptide (Gal −/−) show increased sensitivity to the rewarding effects of these drugs in the conditioned place preference (CPP) paradigm compared to wild-type (Gal +/+) controls (Hawes et al. 2008; Narasimhaiah et al., 2009). Similarly, exogenous galanin administration (i.c.v.) decreases morphine-induced CPP in C57BL/6J mice (Zachariou et al. 2006). In contrast, galanin does not greatly alter cocaine-induced hyperactivity and does not change the dose-response function for cocaine self-administration, although Gal −/− mice are significantly more likely to be low responders for cocaine than Gal +/+ mice (Brabant et al, 2010). While the effect of galanin on amphetamine and alcohol reward have not been assessed directly, amphetamine-induced locomotor hyperactivity is decreased in transgenic mice over-expressing the galanin peptide (Kuteeva, et al. 2005) and GAL −/− mice voluntarily consume less ethanol compared to GAL +/+ or galanin over-expressing mice (Karatayev et al. 2009; Karatayev et al. 2010).</p><p>The mesolimbic dopamine system has been implicated in the motivation to obtain drugs of abuse and natural rewards (for review see Baldo and Kelley 2007; Everitt et al. 2008; Vucetic and Reyes 2010) and there is evidence that galanin modulates activity of this pathway, possibly via indirect mechanisms (for review see Robinson and Brewer 2008). Interestingly, it has recently been proposed that galanin regulates appetitive (i.e. food/drug-seeking) versus consummatory (i.e. food/drug-taking) aspects of reward, potentially through inhibition of the ascending mesolimbic dopamine system (McNamara and Robinson 2010), suggesting that this may be a mechanism underlying galanin's effects on behaviors related to drug addiction.</p><p>Galanin exerts its action through G-protein coupled receptors (GalR1, GalR2, and GalR3), which stimulate several intracellular signaling pathways implicated in synaptic plasticity underlying reward-learning and addiction (Lang, et al. 2007; Beninger and Gerdjikov 2004; Russo et al. 2010). For example, activation of GalR1 or GalR2 results in Gi-mediated inhibition of cyclic AMP-responsive element binding (CREB) protein, which is critical for conditioned place preference to nicotine (Badie-Mahdavi et al. 2005; Brunzell et al. 2009). In addition, activation of GalR1 (through Gi/o-type G-proteins via Gβγ-subunits) or GalR2 (through Gq/11-type G-proteins) can activate extracellular signal-related kinase (ERK; Lang, et al. 2007). ERK activity, which can be evaluated by its phosphorylated state (P-ERK), is crucial for various aspects of learning and memory (Thomas and Huganir 2004). This signaling pathway has also been implicated in the long-lasting neurobiological changes associated with administration of drugs of abuse (Girault A et al. 2007). It has been shown previously that preference for a cocaine-paired environment is dependent on the ERK signaling pathway (Valjent et al. 2006). In addition, ERK activation is altered in mice administered nicotine through drinking water (Brunzell, et al 2003). Interestingly, galanin and the galanin receptor agonist galnon have been shown to decrease morphine- and cocaine-induced increases in ERK signaling (Picciotto 2008).</p><p>The aim of the current study was to assess whether mice lacking the galanin peptide show an altered response to nicotine in the CPP paradigm. In addition, ERK activation in the nucleus accumbens shell and core (NACsh and NACco, respectively) was assessed in a subset of these mice following re-exposure to the CPP apparatus as a marker of plasticity in the mesolimbic system following repeated nicotine administration. In a separate group of mice, the effects of acute nicotine on ERK signaling in the nucleus accumbens were assessed to determine whether the altered ERK response was a direct effect of galanin signaling or was a response to the conditioned environment.</p><!><p>Congenic male galanin knockout (−/−) and wild type (+/+) mice on the 129 Ola/Hsd background were used for all experiments (Wynick et al. 1997). Mice used in these studies were generated using the following strategy to prevent any contribution of genetic drift to differences between congenic Gal +/+ and Gal −/− mice: Gal +/− mating pairs were used to generate Gal +/+ or Gal −/− mice and several breeding pairs of these homozygous mice were subsequently crossed to obtain experimental animals of the appropriate genotype (Narasimhaiah et al., 2009). Mice of the same genotype were housed together (2–5 per cage) in standard plastic mouse cages (Allentown Inc, Allentown, NJ USA) and had ad libitum access to chow (Harlan Teklad #2018) and water. Mice were allowed to habituate to the colony for at least 2 weeks before testing and were handled a minimum of 2 times a day for 2 days prior to experimental manipulation. All studies were conducted in accordance with the guidelines provided by the National Institutes of Health and were approved by the Yale Animal Care and Use Committee.</p><!><p>Nicotine biatartrate salt was obtained from Sigma-Aldrich (Saint Louis, MO, USA) and was dissolved in 0.9% NaCl (saline). The pH was adjusted to 7.4 with NaOH. All injections were administered IP in a volume of 0.01 ml/g body weight. All doses are referred to with respect to the freebase.</p><!><p>Behavioral experiments were conducted using modified three-chamber CPP boxes from Med Associates (ENV-256C Med Associates, Inc, St. Albans, VT, USA). Two conditioning chambers with retractable doors were separated by a smaller, grey neutral chamber with a grey Plexiglas floor. Both conditioning chambers had black walls. One conditioning chamber had a wire mesh floor and the other had a metal grid floor. Movement of each animal was recorded by photocell beam breaks and time spent in each chamber was recorded with Med-PC IV software.</p><p>Mice were transported to the testing room and allowed to habituate for at least 30 min prior to behavioral testing. Nicotine CPP was assessed in a similar manner to previous studies of morphine and cocaine CPP (Hawes et al., 2008; Narasimhaiah et al., 2009). On Day 1 (pre-conditioning test), mice were placed in the center chamber and allowed to explore all 3 chambers for 15 min to determine baseline preference for each of the chambers. Mice that showed greater than 70% preference for any chamber were excluded from further testing (n=13 Gal +/+ and n=10 Gal −/−). Two conditioning sessions per day were conducted on Days 2, 3, and 4. During the AM session (beginning at approximately 0900 h), mice were confined to one conditioning chamber for 30-min following saline injection. During the PM session (beginning at approximately 1300 h), mice were confined to the opposite conditioning chamber for 30-min following nicotine administration (0.05, 0.09, 0.18 or 0.36 mg/kg; n=10–12/group). On Day 5, the post-conditioning test was conducted. Mice were initially placed in the center chamber and allowed free access to all three chambers for a 15-min post-conditioning test. Animals were counterbalanced for drug-paired chamber based on genotype and baseline preference. The pre- and post-conditioning tests were conducted at an intermediate time between the AM and PM conditioning sessions (at approximately 1100 h). Data were collected as time spent in each chamber (sec) during the pre- and post-conditioning test. In Figure 1, the time spent in each of the chambers during the pre- (baseline) and post-conditioning test for each genotype is presented. These raw data were then used to calculate difference scores that reflect a change from baseline (post- minus pre-conditioning chamber), with positive numbers indicating an increase in time spent from baseline and negative numbers indicating a decrease in time spent from baseline for each genotype (Figure 2). All data were analyzed using two-tailed, paired samples t-tests and level of significance was set at alpha < 0.05. Within 5 min following completion of the post-conditioning test, mice were sacrificed and brains were immediately frozen on dry ice and stored at −80C until western blot analysis.</p><!><p>The effect of acute nicotine administration on ERK phosphorylation was assessed. In order to habituate mice to the injection procedure, they were injected once a day for 3 days with saline. Mice received an injection of nicotine (0, 0.18 or 0.36 mg/kg, IP; n=8/group) and brains were harvested 20 min later by rapid decapitation, immediately frozen using dry ice and stored at −80ºC until western blot analysis. Previous reports indicate the highest level of ERK activation following nicotine occurs at this time point in CD-1 mice (Valjent et al., 2004).</p><!><p>A random subset of the brains harvested following the post-conditioning test in Experiment 1 (n = 6–9/group) were used to determine levels of phosphorylated ERK (pERK) by western blotting as has been described (Hawes et al., 2008). Briefly, bilateral tissue punches (18 gauge, 1 mm thick slices) were taken from NACsh and NACco and cell lysis buffer (50 mM Tris, 1 mM EDTA, 1 mM EGTA, 1% SDS, and 1 mM PMSF) was added and immediately pulse sonicated for 5 sec. Lowry reagents (Bio-Rad, Hercules, CA, USA) were used to determine protein concentrations according to manufacturer's instructions. For pERK and ERK immunoblots, 6 μg of protein for each sample was separated on a 10% poly-acrylamide gel and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Blots were blocked in 5% Milk/Tris-buffered saline Tween-20 (TBST) for 1 h, washed with TBST, and incubated overnight in primary antibodies (ERK and pERK) diluted in TBST. Polyclonal antisera specific for p42/p44- MAPK (ERK 1/2) and the phosphorylated form of ERK 1/2 (Cell Signaling, Beverley, MA, USA) were used at a dilution of 1:1000 for both antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoreactivity was used as an internal standard to control for protein loading. Following overnight incubation, blots were rinsed and incubated for 30 min at room temperature in anti-GAPDH monoclonal primary antibody (Advanced ImmunoChemical Inc., Long Beach, CA, USA) diluted 1:5000 in TBST. After washing three times for 5 min with TBST, blots were incubated with secondary antibodies for 1 h at room temp. The blots were incubated in IR Dye 800-conjugated anti-rabbit IgG (Rockland Inc., Gilbertsville, PA, USA) and Alexa fluor 680-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. Bands were visualized and quantified using the LI-COR Odyssey imager (LI-COR Biosciences, Lincoln, NE, USA) with Odyssey imaging software. The levels of ERK phosphorylation were determined by calculating the ratio of phosphorylated band intensity to total band intensity for ERK 2 (p42ERK2). Data were normalized to the respective saline control to allow comparison across multiple blots by dividing each value by the saline group average and multiplying by 100 and were analyzed using two-tailed, independent samples t-tests. The level of significance was set at alpha<0.05.</p><!><p>No differences in chamber preference during the pre-conditioning test (baseline) were observed across genotypes in any of the treatment groups (Figure 1A and 1B). There was a significant difference between time spent in the saline- and nicotine-paired chambers observed in Gal +/+ mice that were conditioned with the 0.18 mg/kg dose of nicotine (t13=2.32; p=0.04). Interestingly, the dose response function is shifted to the left in Gal −/− mice as indicated by a trend towards significance following conditioning with the 0.18 mg/kg dose of nicotine (t11=1.99; p=0.07), as well as the higher dose (0.36 mg/kg; t11=2.00; p=0.07; Figure 1C and 1D). Similarly, analysis of the difference scores in Figure 2 indicated significant CPP in the GAL +/+ mice following conditioning with the 0.18 mg/kg dose of nicotine (t13=3.17; p=0.01) and in the GAL −/− mice following conditioning with the 0.36 mg/kg dose of nicotine (t11=2.46; p=0.03). These results suggest that GAL −/− mice are less sensitive to the rewarding effects of nicotine compared to GAL +/+ mice. The results of measurements of ERK phosophorylation in the NACsh and NACco following re-exposure to the conditioning compartment are shown in Figure 3. A statistically significant (t14=2.64; p<0.05) increase in ERK phosphorylation in the nucleus accumbens shell following the post-conditioning test was observed in GAL +/+ mice conditioned with the 0.18 mg/kg dose of nicotine compared to saline control.</p><!><p>In order to determine whether the P-ERK signal was related to the rewarding effects of nicotine administered in the place preference context, or whether the pharmacological effects of nicotine were sufficient to induce ERK phosphorylation in these mice, the acute effects of nicotine on ERK phosphorylation were also assessed in GAL +/+ and GAL −/− mice. Acute nicotine administration (0.18, 0.36 mg/kg; IP) did not induce ERK phosphorylation in GAL +/+ or GAL −/− mice in either subregion of the NAC (Fig. 4).</p><!><p>Several hypothalamic neuropeptides, including galanin, modulate the neurochemical and behavioral responses to drugs of abuse. Previous research has indicated that mice with constitutive knockout of the galanin peptide display increased sensitivity to the rewarding effects of morphine and cocaine, suggesting that endogenous galanin signaling opposes or inhibits the neurochemical actions of morphine and cocaine (Hawes et al., 2008; Narasimhaiah et al., 2009). In contrast, results from the current study indicate that mice lacking the galanin peptide display decreased sensitivity to nicotine in the CPP paradigm, suggesting that galanin does not modulate drug-induced neurochemical changes similalry across all classes of abused drugs.</p><p>Although each drug of abuse has different initial effects on neurotransmission through interactions with specific receptors and subsequent sigaling pathways, these initally different mechanisms of action converge to increase mesolimbic and nigrostriatal dopamine transmission; however, it is important to keep in mind that each of these drug classes has unique effects in parts of the mesocorticolimbic circuitry that can modulate reward-related behavior (Pierce and Kumaresan 2006). For example, systemic administration of nicotinic acetylcholine receptor antagonists and α-noradrenergic receptor decrease nicotine reward, but have little or no effect on cocaine reward (Sershen et al., 2010). In addition, it has been shown that antagonism of hippocampal nicotinic receptors and ablation of prefrontal cortical noradrenergic afferents inhibits morphine CPP (Rezayof et al., 2006; Ventura et al., 2005). Since galanin can modulate both acetylcholine and noradrenergic release in the hippocampus, it is possible that galanin differentially modulates the effects of drugs of abuse dependent on function of these neurotransmitters (Kehr et al. 2001; Elvander and Ogren 2005).</p><p>An imbalance between cholinergic and dopaminergic signaling in the nucleus accumbens has been implicated in a variety of neurological disorders, likely because alterations in these neurotransmitter systems can impair the functioning of cortico-basal ganglia-thalamocortical loops (for review see Aosaki et al., 2010; Livingstone & Wonnacott, 2009). Cholinergic innervation of the ventral tegmental area and substantia nigra can influence the firing of dopaminergic neurons via nicotinic receptors on dopaminergic cell bodies, presynaptic glutamatergic terminals and GABAergic interneurons, resulting in modulation of dopamine release in target nuclei, such as the striatum (Mansvelder et al., 2002; Mena-Segovia et al., 2008). In addition, presynaptic nicotinic receptors on dopaminergic terminals and neurotransmission from striatal cholinergic interneurons in the striatum can affect dopamine release (Exley and Cragg 2008). It has also been suggested that the cholinergic system may be involved in galanin-mediated modulation of dopamine signaling (Picciotto, 2008). Galanin administration into the paraventricular nucleus of the hypothalamus can decrease extracellular levels of acetylcholine and increase extracellular dopamine levels in NAC (Rada et al., 1998). Given the ability of acetylcholine release to alter dopamine dynamics and the important role of dopamine in both feeding and drug-related behaviors, it seems possible that galanin-induced alterations in cholinergic signaling could modulate drug-abuse related behaviors. Further research is needed to assess whether the basal levels of acetylcholine and dopamine are changed in Gal −/− mice, an effect that may result in differential responses to cocaine, morphine and nicotine reward. In addition, it will be important to determine the role of galanin in the reinforcing effects of morphine, alcohol, and nicotine using the self-administration paradigm as previous research has indicated galanin may modulate cocaine reward, but not reinforcement (Brabant et al., 2010; Narasimhaiah et al., 2009).</p><p>Exposure to a previously nicotine-paired chamber results in activation of CREB in several brain regions, including the VTA, NACsh, cingulate cortex and pedunculopontine tegmental nucleus and CREB activation is required in the NACsh for expression of nicotine CPP (Brunzell et al., 2009; Walters et al., 2003). One prominent regulator of CREB activity in neurons is ERK. Activation of the ERK signaling pathway in the NAC, which plays an important role in associative learning, is necessary for the expression of cocaine and morphine reward (Valjent et al., 2006). While acute nicotine (0.4 mg/kg) has been reported to induce ERK activation in the NAC, similar to cocaine and morphine, it is unknown if ERK activation in the NAC is necessary for the expression of nicotine CPP (Valjent et al., 2004).</p><p>In contrast to previous studies using the CD-1 strain of mouse, acute nicotine administration did not alter ERK activation in the NAC. This may be due to strain differences since the Gal +/+ and Gal −/− mice were developed on the 129/OlaHsd background. Despite the lack of an acute increase in ERK phosphorylation following nicotine administration, both Gal +/+ and Gal −/− mice showed a significant nicotine CPP, raising the possibility that ERK activation occurs at a different time-point post nicotine administration in these mice compared to CD-1 mice (Valjent et al., 2004). In the current study, levels of ERK activation in the NACsh and NACco were determined in a random subset of mice from each conditioning group following behavioral assessment of nicotine CPP. Gal +/+ mice that exhibited significant nicotine (0.18 mg/kg) CPP showed increased ERK activation in the NACsh. However, no ERK activation was observed in the NAC of Gal −/− mice that exhibited significant nicotine (0.36 mg/kg) CPP, suggesting that ERK activation in NACsh may not be required for nicotine CPP in these mice or that the time-course of ERK activation was altered in these animals. While speculative, it is possible that the CREB activity in the NACsh, which is critical for nicotine CPP, is not regulated by ERK, but instead by another signaling cascade such as PKA or CaMKIV (Selcher et al., 2002). It is also possible that the ERK/CREB signaling pathway in other brain regions (not assessed in the current study) are critical for acquisition of nicotine CPP, such as the hippocampus.</p><p>In conclusion, the current results demonstrate that constitutive loss of the galanin peptide does not modulate the conditioned reinforcing effects of all classes of abused drugs uniformly, as assessed using the CPP paradigm. It is possible that compensatory mechanisms in these mice underlie the current results and further studies are needed to fully elucidate the role of galaninergic receptor signaling in drug-induced behaviors. Given the role of galanin, as well as other orexigenic neuropeptides, in motivated behavior for both food and drug-reward, galanin signaling remains a potential target for development of therapeutics to combat drug abuse.</p><!><p>Mice lacking the galanin peptide showed decreased sensitivity to nicotine in the conditioned place preference paradigm compared to wild-type mice.</p><p>Only Gal+/+ mice showed an increase in ERK2 activity following re-exposure to the CPP apparatus.</p><p>No activation of ERK2 was observed following acute nicotine administration in either genotype.</p><p>Total time spent in the saline- and nicotine-paired compartment by each group of animals before and after conditioning. Data represent total time spent (sec) in saline-paired or nicotine-paired chamber (mean ± SEM). Panel A and B: Total time spent in each chamber during the pre-conditioning test (baseline). Panel C and D: Total time (sec) spent in each chamber during the post-conditioning test. *p<0.05, #p<0.07</p><p>Mice lacking the galanin peptide (GAL −/−) are less sensitive to the rewarding effects of nicotine. Data represent change from baseline expressed as the difference in time spent (sec) between the pre- and post-conditioning test for each chamber (mean ± SEM). *p<0.05</p><p>Change in ERK activation following re-exposure to the CPP apparatus in NACsh and NACco. Data are expressed as mean ± SEM, relative to the respective saline control. *p<0.05</p><p>Change in ERK activation following acute nicotine exposure in NACsh and NACco. Data are expressed as mean ± SEM, relative to the respective saline control. *p<0.05</p>

PubMed Author Manuscript

A facile, rapid, one-pot regio/stereoselective synthesis of 2-iminothiazolidin-4-ones under solvent/scavenger-free conditions

A rapid and efficient one pot solvent/scavenger-free protocol for the synthesis of 2-iminothiazolidin-4-ones has been developed. Interestingly, the regio/stereoselective synthesis affords the regioisomeric (Z)-3-alkyl/aryl-2-(2-phenylcyclohex-2-enylimino)thiazolidin-4-one as the sole product in good yield. The selectivities observed have been rationalized based on the relative magnitude of the allylic strains developed during the course of the reaction. This is the first report wherein the impact of allylic strains in directing the regiocyclization has been noted.

a_facile,_rapid,_one-pot_regio/stereoselective_synthesis_of_2-iminothiazolidin-4-ones_under_solvent/

Introduction<!>Results and Discussion<!>Conclusion

<p>Thiazolidin-4-one derivatives are well known for their bioactivities such as antidiabetic [1], anticancer [2], calcium-channel blocker [3,4], platelet activating factor (PAF) antagonist [5] and anti-HIV [6] activity. In addition, 2-iminothiazolidin-4-ones exhibit remarkable hypnotic [7,8], antitubercular [9], cardiovascular [10] and cyclooxygenase (COX) inhibitory [11] activities (Figure 1).</p><p>A common strategy involved in the prevailing synthetic protocols for 2-iminothiazolidin-4-ones [12][13][14] is the cyclization of thioureas with α-halocarboxylic acids [15] or acyl halides [16,17] or carboxylic esters [18]. These protocols are generally solution phase methods using organic solvents and acid scavengers. In the present scenario, such protocols may not be recommended by the principles of green chemistry. Consequently, the search for simple and efficient environmentally friendly methodologies for the synthesis of 2-iminothiazolidin-4-ones is worth attempting.</p><p>In this regard, and in continuation of our recent reports on the solvent-free synthesis of amides [19,20], thioamides [21], cyclic imides [22], thiazolidin-4-ones [23], spirothiazolidin-4-ones [24], 1,2,3-triazoles [25], 1,2,3-triazolylchalcones [26], and 1,2,3-triazolyldihydropyrimidine-2-thiones [27], we herein present a one-pot solvent/scavenger-free synthetic protocol for 2-iminothiazolidin-4-ones. This environmentally benign method avoids toxic organic solvents and acid scavengers, the details of which are presented below.</p><!><p>At the outset, optimization of the one-pot reaction was attempted by varying the solvents and using triethylamine as the acid scavenger (Table 1). The reaction was also attempted under solvent-free conditions. The latter was more promising in the sense that the reaction was very rapid affording the product 4f in 15-20 min (Table 1, entries 7 and 8) compared to 2-6 h (Table 1, entries 1-6) in solvents. The structure of the product 4f was assigned as (Z)-2-(2-phenylcyclohex-2-enylimino)-3-ptolylthiazolidin-4-one based on the single-crystal XRD data [28,29] of its analogues (4c and 4j).</p><p>Though the rate of reaction rate could be accelerated, the yield of 2-iminothiazolidin-4-one 4f was not good (41-66%) under both solution-phase and solvent-free conditions. Hence, as an attempt to optimize the yield, the solvent-free protocol was screened with and without the acid scavenger. Hereto, the yield of the product was poor (Table 2). Thus, the screening indicated that the scavengers had no positive, but rather an impeding effect. To develop an insight in this regard, a plausible mechanism of the reaction in the presence of the acid scavenger was proposed (Scheme 1).</p><p>From the mechanism, it can be envisaged that the acid scavenger may neutralize the HCl (that is generated during the course of the reaction) or the iminium ions by deprotonation. Also, another possibility is that the use of base as the scavenger may lead to the acid-base reaction resulting in the formation of the carboxylate anion of one the starting materials viz. chloroacetic acid or the thiourea-chloroacetic acid coupled product. This may retard the direct amine-carboxylic acid coupling, thus decreasing the yield of the product. In view of the above perception, the solvent-free protocol was screened with one equivalent or in the absence of acid scavenger and varying equivalents of chloroacetic acid at 100 °C (Table 2). The optimum conditions were found to be with 3 equivalents of chloroacetic acid in the absence of acid scavenger affording a good yield of 2-iminothiazolidin-4-one 4f (Table 2, entry 6).</p><p>In this context, it is pertinent to mention that while the prevailing solution-phase protocol [30][31][32] uses an acid scavenger, such as sodium hydroxide, triethylamine, pyridine or sodium acetate, the solvent-free methodology involved in the present investigation does not require any acid scavenger.</p><p>The scope of the new synthetic protocol was proved through the synthesis of a library of 2-iminothiazolidin-4-ones (Table 3). However, its limitations were realized when the synthesis of ortho-tolyl/1-napthyl analogues and the para-substituted (NO 2 and COOH) phenyl analogues failed. Apparently, the reason for this can be attributed to the retardation of the nucleophilic attack of the amines on the isothiocyanate due to the steric effect (Figure 2) in the former, and decrease of the nucleo- philicity of the amines by the electron-withdrawing group in the latter, thus not affording the expected thiourea.</p><p>Having established the new protocol for the synthesis of 2-iminothiazolidin-4-ones, the method was extended to the rapid synthesis of a library of thiazolidinone derivatives (Table 4).</p><p>Further, it is pertinent to mention here the interesting regio/ stereoselectivity noted in the synthesis. Though the formation of a Yield of isolated product, b regioisomeric mixtures obtained. the four regio/stereoisomeric 2-iminothiazolidin-4-ones 4a, 4b, 5a and 5b is possible, it is novel to note that only one of them, viz. 4b, is formed exclusively (Figure 3).</p><p>The high regio/stereoselectivity of the reaction can be rationalized based on the relative magnitudes of allylic strains (A 1,2 and A 1,3 ) developed during the course of the regiocyclization (Scheme 2).</p><p>In this context, it is relevant to recall the literature reports on the factors directing the regioselectivity in the synthesis of 2-iminothiazolidin-4-ones. Only a couple of reports in this regard are available in the literature. While one of these reports suggests that the pK a [17] of amines directs the regioselectivity, another investigation indicates that the chelating effect [18] of the substituent directs the regiochemical outcome. In both the reports, two regioisomeric 2-iminothiazolidin-4-ones are obtained. Thus, the present investigation affording a single regioisomeric product exclusively is the first report wherein the allylic strains are noted to direct the high regioselectivity.</p><p>Finally, the stereoselective formation of the (Z)-stereoisomer is also explicable based on allylic strain, which is summarized in Figure 4.</p><!><p>In conclusion, a new solvent/scavenger-free synthetic protocol for 2-iminothiazolidin-4-ones has been reported. Unlike the prevailing solution-phase protocols employing organic solvents and acid scavengers, the present study avoids solvents and scavengers. The rate of the reaction is prominently enhanced under solvent-free conditions compared to that in the solution phase. Apparently, the intimacy of the highly polar reactants in the fused state in the absence of solvent may be responsible for the rate enhancement.</p>

Beilstein

Light-regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP

We developed a new system for light-induced protein dimerization in living cells using a novel photocaged analog of rapamycin (pRap) together with an engineered rapamycin binding domain (iFKBP). Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.

light-regulation_of_protein_dimerization_and_kinase_activity_in_living_cells_using_photocaged_rapamy

<p>Rapamycin (Rap, Figure 1a), also known as sirolimus, is a complex macrolide natural product isolated from the bacterium Streptomyces hygroscopicus, found in a soil sample on Easter Island in 1975.1–4 Rapamycin mediates heterodimerization of the proteins FKBP12 (FK506 binding protein 12) and FRB (FKBP12 rapamycin binding domain).1 Due to rapamycin's excellent physiological properties, including good pharmacokinetics, permeability across the blood-brain barrier, and oral bioavailability,2 it has been used as a small molecule dimerizer for a wide range of applications in mammalian cells and organisms.3 Furthermore, the FRB-FKBP12 interaction has proven valuable in a broad range of basic research applications, where it has been engineered to control gene function through rapamycin-induced transcription,4, 5 protein localization,4 protein degradation,6 and DNA recombination.7 We recently showed that rapamycin can control kinase activity when an engineered version of FKBP is inserted at a conserved position in the kinase active site8. Thus, a photo-activatable analog of rapamycin represents a unique and important biological research tool, enabling the regulation of heterodimerization and kinase activity using light as a noninvasive regulatory element that can be controlled with high spatial and temporal resolution.</p><p>Photo-activatable derivatives of small molecules are typically generated through the installation of a light-removable protecting group, a so called "caging group", at a site crucial for biological activity of the small molecule.9 This renders the molecule inactive, until the caging group is removed through light-irradiation, typically with UV light of 365–405 nm.10 The feasibility of this approach has been demonstrated through successful photochemical regulation of numerous small molecules in cellular environments and multicellular organisms.9, 11 Here, we report the synthesis of a photocaged rapamycin analog (pRap, Figure 1a) which, together with an engineered FKBP (iFKBP), enables successful photo-control of the FKBP-FRB interaction (Figure 1b). The caged rapamycin is applied to regulate both protein dimerization and kinase activity in live cells. Interestingly, caging is not effective with unaltered FKBP, but requires the FKBP mutations described here. An analysis of the chemically accessible sites of rapamycin (Rap) revealed that the methoxy group on C-16 can undergo nucleophilic substitution13–17 and β-elimination.12 The hydroxyl groups at C-28 and C-40 can be protected with silyl groups,13,14 and a trifluoromethylsulfonyl group.15 The lactone at C-34 can be hydrolyzed and eliminated22–24 and, importantly, the hydroxyl group at C-40 can be converted into a carbonate group15 and be esterified.15, 20, 21 Thus, C-40 represents the most suitable site for chemical modification with a carbonate-linked caging group that can provide facile installation and quick photolysis. Importantly, based on the crystal structure of the ternary complex of rapamycin, FRB, and FKBP12, the hydroxyl group at C-40 undergoes hydrogen bond formation with the glutamine 53 of FKBP12 (Figure 1c).16 Thus, we hypothesized that disruption of that hydrogen bond through installation of a sterically demanding α-methyl-6-nitropiperonyloxycarbonyl (MeNPOC) group would prevent protein dimerization.</p><p>The caged rapamycin pRap was synthesized in one step from rapamycin (Rap) via chemoselective acylation with the mixed carbonate of 6-nitro-piperonyl alcohol and N-hydroxysuccinimide (NPOC-NHS, Figure 1a). The identity of purified pRap was confirmed by 1H NMR and HRMS analysis (see Supporting Information).</p><p>We first tested whether pRap could induce dimerization of FKBP12 and FRB. For this we created a GFP-FRB protein fusion and wild-type FKBP12 fused to the N-terminus of focal adhesion kinase (FAK). FAK localizes prominently to focal adhesions in living cells,17 allowing us to test dimerization in vivo by observing rapamycin-mediated translocation of GFP-FRB into focal adhesions. Prior to live cell co-localization studies, the constructs were tested in pull-down assays, comparing the ability of pRap and Rap to mediate the intracellular dimerization of FKBP-FAK and FRB. Cells expressing both FKBP-FAK and FRB were treated with rapamycin or pRap for one hour, with or without irradiation. Complex formation was assayed by pulling down myc-FKBP-FAK from cell lysates and blotting for GFP-FRB (Supporting Figure 1a, b). Surprisingly, both small molecules generated dimerization with similar effectiveness, with or without UV irradiation (Supporting Figure 1a, b). These results were further confirmed by an mTOR activity assay (Supporting Figure 2). This data showed that the FKBP12-rapamycin-FRB complex was not sufficiently sensitive to rapamycin modification for a successful light-activation approach through photocaging.</p><p>We hypothesized that alteration of FKBP12 could be used to render the ternary rapamycin-FKBP-FRB interaction more sensitive to the functional groups of pRap affected by photolysis. We tested a recently developed modified FKBP, named iFKBP, that is proposed to have increased structural mobility of the Lys52-Glu54 loop positioned next to the C-40 hydroxyl group of rapamycin (Figure 1c).8 Using both an N-terminal iFKBP-FAK fusion (as tested above for FKBP) and a fusion of iFKBP internally, at position 413 of FAK (Figure 2a), we examined whether pRap could mediate heterodimerization of iFKBP and FRB in a light dependent manner. Indeed, pRap (at concentrations of up to 20 µM) failed to mediate interaction between iFKBP-FAK and GFP-FRB, while irradiation of pRap-treated cells with 365 nm UV light successfully removed the caging group and induced iFKBP-FRB dimerization (Figure 2b–d). Uncaging kinetics were dependent on both light dosage and pRap concentration. Importantly, in the presence of pRap, translocation of FRB into focal adhesions was observed only upon decaging, indicating successful protein dimerization between FAK-iFKBP and FRB in live cells (Figure 2e–f). These studies demonstrated that pRap can effectively mediate light-dependent protein heterodimerization when used with iFKBP rather than FKBP12.</p><p>Recently, we developed a new method for the regulation of protein kinases in live cells.8 Insertion of iFKBP at a structurally conserved position within the catalytic domain of several kinases, including FAK, rendered the kinases inactive. Our previous studies indicate that insertion of the iFKBP increases the mobility of the critical G-loop in the catalytic domain of FAK, resulting in the inhibition of catalytic activity.8 Formation of iFKBP-rapamycin-FRB complex through addition of rapamycin significantly restricts iFKBP dynamics, thus stabilizing the G-loop and rescuing the kinase activity.8 This was used to achieve specific control of kinases in living cells with high temporal resolution.8</p><p>Here we tested the light-mediated regulation of FAK activity with pRap using RapR-FAK (rapamycin-regulated FAK) as a model (Figure 3a). Myc-tagged RapR-FAK was co-expressed with GFP-FRB in HEK293T cells. As in the published validation of RapR-FAK, we examined rapamycin's ability to induce RapR-FAK phosphorylation of the N-terminal fragment of paxillin,18 a signal transduction adaptor protein and natural FAK substrate. Unlike rapamycin, pRap was completely inactive at concentrations of up to 20 µM, producing no detectable paxillin Tyr31 phosphorylation. UV irradiation alone did not activate RapR-FAK in the absence of pRap, but irradiation in the presence of pRap (1–20 µM) induced activation of RapR-FAK through protein complex formation with FRB (Figure 3b), leading to robust phosphorylation of paxillin. Light-mediated interaction between RapR-FAK and GFP-FRB was further confirmed by co-immunoprecipitation of the two proteins (Figure 3b) and by translocation of FRB to focal adhesions (Figure 2f) upon pRap irradiation.</p><p>Finally, we examined whether light induced activation of pRap could be used to control cell behavior. FAK activation has been shown to produce large dorsal membrane ruffles8. We therefore examined effects of RapR-FAK activation on the membrane dynamics of HeLa cells. In the presence of pRap, but without UV irradiation, transfected HeLa cells displayed normal, small peripheral ruffles around the border of the cell (Figure 3c), consistent with inactive RapR-FAK. In contrast, UV irradiation (365 nm) produced very large and dynamic ruffles across the dorsal cell surface (Figure 3d and Supporting Movie 1). This UV-induced phenotype was displayed in 40% of analyzed cells (9 of 22 cells), in excellent agreement with effects of regular rapamycin on RapR-FAK (56% positive cells).8</p><p>To explore how modification of FKBP to iFKBP was able to render pRap inactive until irradiated with UV light, we performed discrete molecular dynamics (DMD) simulations.19 Within the sampled conformations we identified the dominant ensemble and compared the localization of residues that form the iFKBP-FRB and FKBP12-FRB interfaces. Surprisingly, our simulations demonstrated that binding of the caged rapamycin to iFKBP is at least as strong as that to FKBP12, but the interaction of the added piperonyloxycarbonyl moiety with iFKBP distorts the protein's binding-competent conformations and prevents binding of FRB. The most notable difference between the iFKBP and FKBP complexes is a significant distortion of the FRB-binding interface formed by the segment Asp41-Leu49 (Figure 4a–b; Supporting Movies 2–5). The interface area formed between pRap and iFKBP is larger than that between pRap and FKBP12. Due to its higher structural plasticity, the iFKBP protein is able to deform and create additional contacts with pRap, which is unachievable by the more rigid and stable FKBP12. Based on these simulations, we suggest that strong binding between iFKBP and pRap distorts the FRB-binding interface in FKBP12, and thus prevents further binding to FRB.</p><p>In summary, we have developed a new photocaged analog of rapamycin, pRap, which can be used together with iFKBP, an engineered version of FKBP12, for the light-mediated regulation of protein dimerization. We demonstrated successful use of this new dimerization system by modulating protein interactions for two different FAK-iFKBP fusions in living cells. Furthermore, we achieved light-mediated activation of an engineered protein kinase, FAK, and demonstrated light-induced changes in cell behavior characteristic of this kinase. Rapamycin-mediated protein dimerization and regulation of kinases have lacked the precise spatial and temporal control of light-mediated processes. Our new caged rapamycin approach will significantly enhance the many existing methodologies that use rapamycin for the regulation of protein activity in cells and multicellular organisms. We are currently exploring the synthesis of caged, orthogonal rapalogs and two-photon activated caged rapamycin.</p><p>Photocaging of rapamycin. (a) Structure of rapamycin (Rap) and its synthetic transformation into caged rapamycin (pRap) through selective acylation of the C-40 hydroxyl group with nitro-piperonyloxycarbonyl N-hydroxysuccinimide carbonate (NPOC-NHS). (b) Schematic of the light-induced heterodimerization of the proteins FRB and iFKBP using pRap. (c) Crystal structure of the ternary complex between rapamycin, FKBP12 (green), and FRB (blue). The 2.65 Å hydrogen bond (possibly mediated through a water molecule) between Gln53 of FKBP12 and the C-40 hydroxyl group of rapamycin is indicated. PDB 2FAP.</p><p>Light-regulated dimerization of iFKBP and FRB. (a) Positions of iFKBP insertions into FAK. (b–d) HEK293T cells co-transfected with GFP-FRB and either myc-iFKBP-FAK (b, c) or myc-FAK-iFKBP413 (d) were treated with either rapamycin (0.5 µM) or the indicated concentrations of pRap. Ten minutes after addition of pRap or Rap, cells were irradiated with 365 nm UV light for 1 min (b, d) or 5 min (c), and incubated for 1 hour. Control cells were not irradiated. Myc-iFKBP-FAK was immunoprecipitated from cell lysates using anti-myc antibody and co-immunoprecipitation of GFP-FRB was detected by Western Blot using anti-GFP antibody. (e) HeLa cells co-transfected with GFP-FAK-iFKBP413 and mCherry-FRB were treated with pRap (20 µM) for 30 min, followed by UV irradiation (365 nm, 2 min). TIRF images were taken before and after irradiation. (f) HeLa cells co-transfected with GFP-RapR-FAK and mCherry-FRB were treated with pRap (5 µM) for 30 min, followed by UV irradiation (365 nm, 2 min). TIRF images were taken before and after irradiation.</p><p>Light-mediated activation of a protein kinase. (a) Schematic of RapR-FAK regulation by pRap. (b) Myc-RapR-FAK kinase was co-expressed with GFP-FRB in HEK293T cells. Cells were treated with the indicated amount of rapamycin (Rap), caged rapamycin (pRap), or DMSO (control). The indicated samples were exposed to UV light (365 nm, 1 min). All cells were incubated at 37 °C for 1 hour after treatment. Myc-RapR-FAK was immunoprecipitated using an anti-myc antibody and tested in a kinase assay using an N-terminal fragment of paxillin as a substrate. The level of phosphorylation of paxillin on Tyr31 (probed with anti-phospho-Tyr31 paxillin antibody) indicates the kinase activity. (c, d) HeLa cells co-transfected with GFP-RapR-FAK and Cherry-FRB were treated with pRap (1 µM) and imaged before (c) and after (d) UV irradiation (365 nm, 1 min). Arrows indicate formation of large dorsal ruffles stimulated by activated RapR-FAK.</p><p>Molecular dynamics simulations of the FKBP12-pRap-iFKBP interaction. (a) Tube representations of FKBP12 (left) and iFKBP (right) with pRap in the binding pocket. Warmer colors and thicker backbones correspond to fluctuations within the structure. Note the greater mobility of iFKBP. (b) Binding interfaces of the pRap, FRB, and FKBP12 or iFKBP complexes. For iFKBP (right) the interface area is reduced due to loss of contacts between the Asp41-Leu49 segment and FRB. In the iFKBP complex, residues that were in contact for the FKBP12 complex are marked in light brown. The segment deleted from FKBP12 to produce iFKBP is marked with dark color in FKBP12.</p>

PubMed Author Manuscript

Seconds-resolved pharmacokinetic measurements of the chemotherapeutic irinotecan <i>in situ</i> in the living body

The ability to measure drugs in the body rapidly and in real time would advance both our understanding of pharmacokinetics and our ability to optimally dose and deliver pharmacological therapies. To this end, we are developing electrochemical aptamer-based (E-AB) sensors, a seconds-resolved platform technology that, as critical for performing measurements in vivo, is reagentless, reversible, and selective enough to work when placed directly in bodily fluids. Here we describe the development of an E-AB sensor against irinotecan, a member of the camptothecin family of cancer chemotherapeutics, and its adaptation to in vivo sensing. To achieve this we first re-engineered (via truncation) a previously reported DNA aptamer against the camptothecins to support high-gain E-AB signaling. We then co-deposited the modified aptamer with an unstructured, redox-reporter-modified DNA sequence whose output was independent of target concentration, rendering the sensor's signal gain a sufficiently strong function of square-wave frequency to support kinetic-differential-measurement drift correction. The resultant, 200 mm-diameter, 3 mm-long sensor achieves 20 s-resolved, multi-hour measurements of plasma irinotecan when emplaced in the jugular veins of live rats, thus providing an unprecedentedly high-precision view into the pharmacokinetics of this class of chemotherapeutics.

seconds-resolved_pharmacokinetic_measurements_of_the_chemotherapeutic_irinotecan_<i>in_situ</i>_in_t

Introduction<!>Results and discussion<!>Conclusions<!>Conflicts of interest

<p>The goal of personalized medicine is to precisely tailor treatment to the individual. 1,2 To this end, an ability to measure drugs in the living body with seconds resolution would allow clinicians to dene drug dosing based on high-precision, patient-specic pharmacokinetic measurements rather than on indirect predictors of drug metabolism such as age, body mass, or pharmacogenetics. 3,4 Ultimately, the ability to measure drugs in the body in real-time would enable closed-loop feedback-controlled delivery, 5 vastly improving dosing precision by actively responding to minute-to-minute uctuations in a patient's metabolism. 4,6 The development of such technology, however, faces signicant hurdles. 7,8 First, an in vivo sensor must be small enough to be placed in the body without causing undue damage. Second, it cannot require the addition of exogenous reagents or the use of batch processing, such as washing or separations. Third, it must make measurements at a frequency that is rapid relative to the drug's pharmacokinetics. Finally, it must be selective and stable enough to work for prolonged periods in the complex, uctuating environments found in vivo. To this end we are developing electrochemical aptamer-based (E-AB) sensors, a technology that, by achieving these goals, supports the high frequency, real-time measurement of specic molecules directly in the living body. 9 E-AB sensors employ an electrode-bound, redox-reporter-modied aptamer as their recognition element (Fig. 1A). Binding of the target molecule to this aptamer induces a conformational change that produces an easily measured electrochemical output (here we employ square wave voltammetry) without needing reagent additions or wash steps. Because E-AB signaling is generated by a binding-induced conformational change and not, as is the case for most other reagentless biosensor architectures, by the adsorption of target to the sensor surface, 7 E-AB sensors are largely insensitive to non-specic adsorption and support multi-hour measurements in biological uids not only in vitro 10 but also in vivo. 9 Finally, because their signaling arises due to target binding alone, and not, as is the case, for example, of the continuous glucose monitor, 11 from the chemical reactivity of the target, E-AB sensors are a platform technology generalizable to a wide variety of analytes, including two of which, the aminoglycosides and doxorubicin, have been measured in vivo. 9 Building on this foundation we describe here the fabrication and characterization of an E-AB sensor adapted to measurements in situ in the body, one directed against the camptothecin family of anticancer drugs, an important class of chemotherapeutic agents used in the treatment of a range of human cancers. 12,13</p><!><p>As the recognition element in our sensor we employ a DNA aptamer that binds to the camptothecins. 14,15 Specically, a 40base version of this aptamer, termed CA40, which folds into a target-recognizing G-quadruplex anked by a 12-base-pair stem (Fig. 2A), binds the camptothecin, irinotecan, with a dissociation constant of 475 AE 10 nM when the unmodied aptamer is free in solution (Fig. 2B and S1 †). To adapt this into an E-AB sensor modied its 3 0 end with a methylene blue redox reporter and deposited it onto a gold electrode via a six-carbon thiol at its 5 0 end (Fig. 1A). Electrochemically interrogating the resulting sensor in buffer we observe the expected Langmuir isotherm binding with an estimated K D of 126 AE 24 mM (error bars here and, unless otherwise noted, reect the standard deviation derived using at least three independently fabricated sensors), signal gain (the relative change in signal upon the addition of saturating target) of 84 AE 4% (Fig. 2C, blue curve) and association and dissociation kinetics too rapid to measure (time constants < 5 s at clinically relevant concentrations; Fig. 2D). We presume that the poorer affinity the aptamer exhibits in the context of the sensor arises due to interactions with the electrode surface, as this is known to destabilize the folding (thus hindering binding) surface-attached oligonucleotide. 16 Despite this, when challenged in buffer the sensor supports the detection of irinotecan over the clinically relevant 1 to 15 mM range. 17,18 When challenged in whole blood, however, the (apparent) affinity of the surface-bound aptamer is poorer still (K D ¼ 291 AE 15 mM), presumably because the concentration of the free drug is reduced due to protein binding. 17 Worse, under these conditions the gain falls to 15 AE 1%, pushing its useful dynamic range out of the clinically relevant concentration window (Fig. 2C, red curve).</p><p>To improve sensor performance in whole blood we reengineered the camptothecin-binding aptamer to better populate its "unfolded" state in the absence of target, thus increasing the sensor's gain. 19,20 To do so we destabilized the aptamer's doublestranded stem (Fig. 3A) via either truncation (CA36, CA32, CA28, CA16) or the introduction of one (CA40_2MM) or two (CA40_2MM) G-T mismatches. As estimated using the nucleic acid folding predictor NUPACK 21 these strategies should decrease the stability of folded aptamer from the À31.8 kJ mol À1 of the C40 parent to as low as À0.3 kJ mol À1 for CA28 (Fig. S2 †). Characterizing sensors fabricated using these variants (Fig. 3A) we obtain dissociation constants ranging from 38 AE 11 mM to 254 AE 48 mM (Table 1 ESI †) and signal gain of up to 755% (Fig. 3B and Table S1 †) when challenged in working buffer. Testing in whole blood (Fig. 3C) once again reduces both gain and apparent affinities (Table 1 ESI †). Even under these more challenging conditions, however, sensors employing the CA32, CA28, and CA16 variants support high-gain E-AB sensing.</p><p>Having achieved good in vitro performance with a sensor employing the CA32 variant we next set out to adapt this to use in situ in the veins of live rats. Under such conditions E-AB sensors oen exhibit signicant baseline dri. 9,22 We have previously corrected this using "Kinetic Differential Measurements" (KDM) an approach that exploits the generally strong square-wave frequency dependence of E-AB signal gain. 9,22 Specically, the signal gain of the E-AB sensors we have previously described is so great that they exhibit a "signal-on" (target binding increases the signaling current) response at some square-wave frequencies no observable gain or even "signal-off" behavior at others. 9,22 Conveniently, the signals obtained under these different regimes dri in concert such that taking their difference via KDM removes the dri seen in vivo. 9 And since the two signaling currents respond in opposition in the presence of their target, taking their difference also improves signal gain. Uniquely in our experience, however, the gain of the camptothecin-detecting E-AB sensors (CA32, CA28, and CA16) is only a weak function of square-wave frequency (Fig. S3 †), necessitating the development of a new approach to performing KDM.</p><p>To enhance the frequency-dependence of the sensor's gain in support of KDM dri correction we added a second reporter-modied DNA strand to the sensor that: (1) transfers electrons more rapidly than the aptamer does and (2) does not respond to the presence of the target (Fig. S4A †). Our rational for doing so was that, at frequencies at which this "non-responsive" DNA dominates the signal (i.e., at higher frequencies) the gain of the resultant sensor will be low, and at frequencies at which, instead, the aptamer dominates the signal gain will be higher (Fig. S5B †).</p><p>To achieve this we co-deposited the CA32 aptamer variant and an unstructured 10-base strand comprised of a random sequence of adenines and thymines (Fig. 4A) that is known to transfer electrons at a rate of 80 s À1 (Fig. S4 †). 23 Per our expectations, sensors employing a 1 : 1 mixture of this sequence and the aptamer achieve sufficiently frequency-dependence gain to enable KDM (Fig. 4B). To our surprise, however, the resultant frequency dependence is so strong that the sensor's gain becomes slightly negative at low frequencies, an observation inconsistent with the expectations described above (if the currents are additive, the gain cannot go below zero). We presume this occurs due to interactions between the two sequences on the surface that alter their electron transfer kinetics. Irrespective of its origins, however, the effect supports accurate KDM dri correction. Specically, using the signals obtained at 10 Hz (signal-off) and 120 Hz (signal-on) to perform KDM (Fig. S5 †) we can easily monitor irinotecan in both buffer and whole blood (Fig. 4C) over the entire 0.5 to 15 mM (0.06 to 10 mg mL À1 ) human therapeutic range. 17,18 KDM-corrected indwelling E-AB sensors readily support the real-time, high frequency irinotecan measurements in situ in the bodies of live rats. To demonstrate this we fabricated sensors using 75 mm-diameter gold, platinum and silver wires as the working, counter and reference electrodes, respectively (Fig. 1B). We inserted the resulting sensor in the jugular vein of anesthetized Sprague-Dawley rats via a previously emplaced 22gauge catheter (Fig. 1C). Testing this with a single intravenous injection (20 mg kg À1 ) of irinotecan we nd that the signal observed at both 10 and 120 Hz respond to the drug, but these are also accompanied by the expected 9 signal dri (Fig. 5A). And the gain observed at 10 Hz becomes, under these conditions, slightly positive. We are nevertheless still able to use KDM to correct the sensor's dri and recover stable baselines, thus enabling continuous, real-time measurements of the drug at therapeutically relevant concentrations (Fig. 5B).</p><p>To further characterize the performance of the camptothecin sensor we used it to monitor sequential intravenous injections of irinotecan (at 10 and 20 mg kg; Fig. 6A). The resultant maximum concentrations (C MAX ¼ 39.8 AE 3.2 mM and 20.9 AE 2.0 mM, respectively; here and below the condence intervals reect standard errors calculated from the ts) and distribution rates (a ¼ 0.58 AE 0.07 min and 0.48 min (xed value, see ESI Table 2 †)) are comparable to those seen in previous studies that employed ex-vivo drug-level measurements (Fig. S6A and Table S2 †). [24][25][26][27][28] In contrast, the elimination rates (b ¼ 9.2 AE 1.4 min and 8.4 AE 2.2 min) we observe are more rapid than those Fig. 3 We reengineered the parent aptamer to produce higher-gain E-AB signaling. (A) We did so by destabilizing the aptamer's stem-loop (thus increasing the population of unfolded molecules poised to respond to target) via either introduction of one (CA40_1MM) or two (CA40_2MM) mismatches or via truncation (CA36, CA32, CA28, CA16) of the stem. (B) When challenged in a simple buffered solution all of the re-engineered variants exhibited higher gain than that of the parent aptamer (see ESI Table 1 †), with the most destabilized (CA40_2MM, CA32, CA28, CA16) producing the greatest signal gain. (C) When tested in whole blood their gain and affinity are reduced, but the best performing nevertheless still support high-gain E-AB sensing.</p><p>reported previously and, thus, the resulting "areas under the curve" for the drug are reduced (Fig. S7 and Table S2 †). [24][25][26][27][28] We believe this discrepancy (Fig. S7 †) occurs because the prior work used chromatographic and mass spectrometric methods to Fig. 4 To correct the drift of seen during in vivo deployment we modified the E-AB sensor so that it better supports "Kinetic Differential Measurements" (KDM). (A) KDM requires that the gain of an E-AB sensor be a strong function of square wave frequency. 22 To induce this we codeposited the aptamer with a redox-reporter-modified linear DNA sequence that does not respond to target. (B) The signal gain (relative signal change between no target and saturating target -i.e., 100 mM) of the original E-AB sensor (100 : 0 black curve) is a relatively minor function of square-wave frequency. Upon co-deposition with increasing amounts of the linear-strand (to a maximum ratio of 50 : 50, red curve) we observe increasingly strong frequency dependence, albeit with a corresponding reduction in the maximum gain. (C) A sensor fabricated using a 50 : 50 mixture of the two strands and employing KDM drift correction (here the difference in the relative signals seen at 10 and 120 Hz) responds to target over the clinically-relevant range (0.5 mM to 15 mM; 0.06 and 10 mg mL À1 ) 17,18 in both buffer and in undiluted whole blood.</p><p>Fig. 5 The KDM-corrected E-AB sensor supports real time, secondsresolved measurements of plasma irinotecan levels. 9,22 (A) In the absence of KDM signals collected at high (120 Hz) and low (10 Hz) frequencies both drift significantly, but because they drift in concert (B) taking their difference (KDM) produces a stable baseline. (C) As expected, control injections of either a saline "blank" or a second chemotherapeutic (5-fluorouracil, which is often co-administered with irinotecan) 31,32 do not produce any measurable sensor response.</p><p>This journal is © The Royal Society of Chemistry 2019 Chem. Sci., 2019, 10, 8164-8170 | 8167 measure total drug levels (which requires removal of blood samples from the animal's body and the extraction of the total drug into buffer). 24-28 E-AB sensors, in contrast, measure the free drug, which is the fraction of the drug that is pharmacologically active. 29 And, in general, the elimination and clearance of free drug are more rapid than those of total drug as drugs that interact strongly with plasma proteins tend to clear more slowly than those that do not. 30 E-AB-derived measurements of irinotecan pharmacokinetics represent a signicant advance over prior pharmacokinetic studies of the camptothecins. [24][25][26][27][28] For example, the 20 s temporal resolution of our measurements (dened by the time required to take the two square wave scans necessary to perform KDM) is at least an order of magnitude better than that of the most highly time resolved prior study. [24][25][26][27][28] Moreover, all prior studies reported plasma level measurements averaged over multiple animals, thus eliminating their ability to explore subject-to-subject pharmacokinetic variability. The present E-AB-derived measurement parameters, in contrast, provide 300 time points per hour in each animal, and thus determine the pharmacokinetics of individuals with exceptional precision. Because the excretion phase of irinotecan exhibits signicant inter-patient variability (due to drug-drug interactions, variations in health status, and pharmacogenetics), 17,18,33,34 this latter Fig. 7 (A) To determine irinotecan's pharmacokinetics with more precision we performed an intravenous injection at a much higher dose (60 mg kg À1 ). (B) The higher peak concentrations reached in this experiment lead to longer measurement runs, in turn improving the precision of our estimates of the relevant pharmacokinetic parameters. point is likely of clinical signicance. To illustrate our ability to measure such variability we performed sequential 10 and 20 mg kg À1 irinotecan injections in three rats (Fig. 6). The resulting measurements reveal only small ($10 to 20%) variation in either C MAX or the rate of the distribution phase (Fig. S6B and C, and Table S2 †). In contrast, however, the rate of drug excretion and its clearance values vary many fold from individual to individual. As all of the animals we employed in these experiments were healthy male rats these pharmacokinetic differences arose solely due to metabolic variability between the animals.</p><p>The elimination rate and clearance of irinotecan are the pharmacokinetic parameters used to determine its optimized, personalized dosing during chemotherapy. 17,18,33,34 To measure these parameters with greater precision we administered a large dose (60 mg kg À1 ) of the drug over a longer period. The higher plasma concentrations this produces lead, in turn, to a longer measurement period (aer delivery ceases) before the sensor's limit of detection is reached (Fig. 7). The $150 plasma drug measurements we thus achieve in a single pharmacokinetic prole produces estimates of the drug's elimination half-life (10.4 AE 0.4 min) and clearance (18.6 AE 1.4 mL min À1 ) that are far more precise than those produced in prior, ex vivo studies, which typically achieve less than a dozen measurements per prole, [24][25][26][27][28] much less the two measurements used in typical "peaks-and-troughs" clinical measurements.</p><!><p>Here we describe an indwelling E-AB sensor supporting the seconds-resolved measurement of the anticancer drug irinotecan in situ in the living body over the course of hours. Design of the sensor required the reengineering of a parent aptamer to support high-gain E-AB signaling and the development of a novel method to ensure sufficient frequency-dependent signal gain to support KDM-based dri correction. Using the resulting sensors we measured plasma irinotecan levels with micromolar concentration resolution and seconds temporal resolution, with the latter representing an orders of magnitude improvement over that of prior studies. The resulting measurements dene the pharmacokinetics of irinotecan of individual animals, providing an unprecedented high precision view of the drug's inter-subject pharmacokinetic variability.</p><p>E-AB sensors are a platform technology that supports the high frequency, real-time measurement of specic molecules (irrespective of their chemical reactivity) in situ in the living body. When coupled with the platform's convenience and precision this versatility provides signicant opportunities to improve drug dosing. As noted above, for example, irinotecan suffers from signicant inter-patient metabolic variability, 35,36 leading to toxicity and increasing side effects. 17,34 But because current methods for measuring plasma drug levels are slow and cumbersome, 37 the FDA has invoked pharmacogenetic estimates of metabolism as the primary means of reducing the risk associated with this variation. 35 In this light, the ease with which E-AB sensors provide high precision, patient-specic measurements of drug elimination (as opposed to indirect estimates), suggests the platform could provide a valuable adjunct to chemotherapeutic treatment.</p><p>In addition to improving the precision and accuracy of personalized dose determination, E-AB-derived measurements may also support a new paradigm for personalized drug delivery. Specically, we have recently used the real-time concentration information provided by E-AB sensors to inform closed-loop feedback controlled drug delivery. 5 In this the rate of drug administered is optimized multiple times a minute, enabling the maintenance of plasma drug concentrations at a pre-dened value with precision of better than 20% despite $3-fold hour-to-hour changes in drug pharmacokinetics. This approach to drug delivery provides an unprecedented means of overcoming pharmacokinetic variability, improving the overall efficacy and safety of treatment. Given this, drug-detecting E-AB sensors could prove a powerful new tool in the clinician's arsenal.</p><!><p>One author (K. W. P.) has a nancial interest in and serves on the scientic advisory boards of two companies attempting to commercialize E-AB sensors. A. I., N. A. C. and K. W. P. have led a provisional patent based on the work presented in this paper.</p>

Royal Society of Chemistry (RSC)

Central carbon metabolism of Plasmodium parasites

The central role of metabolic perturbation to the pathology of malaria, the promise of antimetabolites as antimalarial drugs and a basic scientific interest in understanding this fascinating example of highly divergent microbial metabolism has spurred a major and concerted research effort towards elucidating the metabolic network of the Plasmodium parasites. Central carbon metabolism, broadly comprising the flow of carbon from nutrients into biomass, has been a particular focus due to clear and early indications that it plays an essential role in this network. Decades of painstaking efforts have significantly clarified our understanding of these pathways of carbon flux, and this foundational knowledge, coupled with the advent of advanced analytical technologies, have set the stage for the development of a holistic, network-level model of plasmodial carbon metabolism. In this review we summarize the current state of knowledge regarding central carbon metabolism and suggest future avenues of research. We focus primarily on the blood stages of Plasmodium falciparum, the most lethal of the human malaria parasites, but also integrate results from simian, avian and rodent models of malaria that were a major focus of early investigations into plasmodial metabolism.

central_carbon_metabolism_of_plasmodium_parasites

Introduction<!>Glycolysis<!>Other carbon sources<!>Pentose phosphate pathway<!>Glycosylation and aminosugars<!>Lipid biogenesis<!>Tricarboxylic acid metabolism<!>Systems level studies<!>Suggested research avenues and concluding remarks<!>

<p>While the process of evolving into a parasitic niche seems to have resulted in a `paring down' of many Plasmodium metabolic pathways, including the wholesale loss of de novo amino acid and purine biosynthesis, pioneering early work suggested that most of the core conserved components of carbon metabolism – glycolysis [1, 2], the pentose phosphate pathway [3], lipid biogenesis [4, 5], glycosylation [6, 7] and at least some components of citric acid metabolism [8, 9] – were present in some form. However, efforts to precisely map the flow of carbon through the metabolic network of the malaria parasite were often stymied by difficulties pertaining to the isolation and culturing of parasites, complications arising from the interconnected nature of parasite and host cell metabolism, technical limitations of metabolic tracing using classical methodologies and in some cases marked divergence between pathways in Plasmodium spp. and the model organisms in which they were first elucidated (reviewed in [10–12]). Dedicated efforts by a host of researchers, however, have resolved most of these technical problems and filled in many of the blank spots in the metabolic map.</p><p>The picture that arises from these studies is of a network that is both more streamlined and more modular than that found in free-living protozoa. This seems to be a consequence of dispensing with the flexibility that a free-living microbe must maintain in order to dynamically regulate its metabolic network to consume any of a wide variety of possible combinations of nutrients. Within the nutrient-rich and homeostatic environment of the blood stages, the parasite appears to consume a defined set of nutrients (glucose, glutamine, free fatty acids, etc.) via several discrete pathways (glycolysis/pentose phosphate pathway, carboxylic acid metabolism, fatty acid elongation and modification) with a low degree of interconnectivity. A fine-grained understanding of plasmodial metabolism will hopefully aid in exploiting this metabolic rigidity when selecting drug targets and designing antimalarials.</p><p>We expect the broad outline of central carbon metabolism to be conserved between the Plasmodium spp. due to the markedly similar life cycles, comparable drug sensitivities and low levels of divergence in metabolic enzymes at the genome level. However, we caution that the metabolism of the rodent and avian malaria parasites in particular may well be more complicated given their proclivity to invade immature, nucleated erythrocytes (reticulocytes) that are more metabolically complex than the mature erythrocytes favored by simian parasites.</p><!><p>Based on numerous classical experiments, carbon metabolism of the malaria parasites has been considered largely synonymous with carbohydrate metabolism, principally the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis. It has long been clear from in vitro studies of blood-stage Plasmodium parasites that they are voracious consumers of glucose (blood sugar). The host cell, at least in the human parasites, is the mature erythrocyte (red blood cell, or RBC), whose metabolism has been exhaustively studied and is comparatively simple enough to be simulated by comprehensive kinetic models [13, 14]. The RBC lacks a mitochondrion and therefore is entirely dependent on glucose fermentation. As a nonproliferative cell with modest energetic needs, RBC glucose consumption is relatively low, on the order of 5 μmol glucose / 24 hours / 109 RBCs [15]. Upon invasion by a Plasmodium parasite, however, the glucose consumption rate is estimated to increase up to 100-fold at the most metabolically active trophozoite and schizont stages [12].</p><p>In vitro culture experiments using several other sugars have established that, besides glucose, only fructose can support continuous growth (albeit at a reduced rate) [16, 17]. Most of the glucose consumed (60–70%) by P. falciparum is incompletely oxidized to lactic acid and excreted [15], although the exact percentage varies between different Plasmodium species and the atmospheric culture conditions used. This glucose consumption contrasts with the >90% glucose-to-lactate conversion observed in uninfected RBCs and reflects the increased flux of glucose carbon into biomass (nucleic acids, lipids, glycosylated proteins) required for proliferating parasites. Only a very small fraction of the total glucose is completely oxidized to carbon dioxide, at least in mammalian malaria parasites [1, 18, 19], which has generally been taken to indicate the absence of a functional citric acid cycle contributing to energy generation. This is in keeping with the observation that in vitro cultures of P. falciparum exhibit only minimal oxygen consumption [20] (and in fact prefer microaerophilic conditions of ~5% oxygen, being growth-inhibited by normal atmospheric oxygen concentrations [21].) These results strongly suggest that blood-stage Plasmodium rely primarily upon glucose fermentation for their energetic needs. Accordingly, inhibitors of mitochondrial respiration have only a small effect on parasite ATP pools [22].</p><p>The parasite accommodates this vastly increased need for glucose by expressing at least one essential hexose transporter to the surface of the infected cell [23], increasing the hexose permeability of the erythrocyte membrane [24]. Such modifications of the host cell raise the question of precisely how the host glycolytic machinery interacts with that of the parasite. All the enzymes required for a complete EMP pathway are 1) encoded in the parasite genome [25], 2) expressed during the blood stages [26], and 3) detected in infected cells, in some cases substantially increasing the activity normally observed in RBCs (reviewed in [27]). Free glucose should be quickly phosphorylated to glucose-6-phosphate by the host cell hexokinase upon entry into the RBC cytosol, rendering it membrane-impermeant and effectively trapping it within the host compartment. Its import into the parasite may depend on dephosphorylation by an acid phosphatase (PFI0880c) that is trafficked to the erythrocyte and cleaves phosphate from a diversity of small molecules [28]. This nonspecific cleavage of high-energy phosphate bonds might have the effect of draining energy from the host cytosol, despite the fact that the parasite seems to require the host cell to remain "viable" (maintaining its redox state, etc.) until lysis occurs in order to successfully complete its developmental cycle. Malaria parasites may circumvent this difficulty by actively supplying the host with metabolically useful molecules such as ATP [29] and glutathione [30]. This secreted phosphatase may also help explain the decline observed in infected cells in the levels of 2,3-diphosphoglycerate [31–33], an allosteric regulator of hemoglobin oxygen affinity produced by an enzyme (diphosphoglycerate mutase) present in the erythrocyte but not the parasite [25, 34].</p><!><p>Many microbes that prefer sugars as a carbon source maintain the ability to metabolize other compounds such as acetate, pyruvate, ethanol or amino acids depending on their availability. Generating the 5- and 6-carbon sugars necessary for growth alternatively depends on gluconeogenesis, in which the catabolic reactions of glycolysis are essentially reversed through the use of different enzymes at the regulated thermodynamic control steps (reviewed in [35]). However, the complete sequencing of the P. falciparum genome revealed no homologs of fructose bisphosphatase, an enzyme normally required for gluconeogenesis [25]. Strangely, the parasite does possess phosphoenolpyruvate carboxykinase (PEPCK, PF13_0234) [36], an enzyme converting oxaloacetate and ATP to PEP and carbon dioxide that usually functions in supplying citric acid cycle or amino acid-derived carbon to gluconeogenesis. The parasites also encode a PEP carboxylase (PEPC, PF14_0246), which has almost the opposite gene expression profile (peak expression at 18 and 47 hours post invasion for PEPCK and PEPC, respectively) [26]. PEPC has been purified from P. berghei [37], and essentially runs the reverse reaction: PEP and carbon dioxide converted to oxaloacetate and inorganic phosphate. The role of this enzyme remains unclear, but would suggest a regulated carbon fixation step.</p><p>It is well-established through 14CO2 incorporation experiments that all studied Plasmodium spp. possess the ability to fix carbon, although the nature of the end products differs between species [11]. Our own metabolomic investigation of carbon flux in P. falciparum showed clear evidence of PEPC activity, as parasites fed uniformly 13C-labeled glucose generated oxaloacetate/malate labeled at three of the four carbons (indicative of the carboxylation of PEP) [38] (Fig. 1). The purpose of this side pathway is unclear since we have found that the majority of the malate produced in this fashion is excreted from the cell. This results in an energetic cost of one ATP molecule for every molecule of PEP that does not complete glycolysis. Perhaps PEPC acts in vivo to supply oxaloacetate as a carbon skeleton for the generation of aspartate by aspartate transaminase (PFB0200c) when aspartate concentrations are limiting. However, in cell culture, aspartate levels are relatively high and this flux may be redirected to malate, which is then excreted due to overflow metabolism. In our studies, aspartate was clearly interconverting with cytosolic oxaloacetate/malate pools, although we were unable to detect any gluconeogenic PEPCK activity in the asexual stages, since 13C-labeled aspartate failed to label PEP, pyruvate or lactate [38]. Interestingly, the up-regulation of PEPCK in P. falciparum gametocytes and zygotes has prompted the hypothesis that there is a switch to gluconeogenic metabolism at these stages [36]. How this can be achieved in the absence of fructose bisphosphatase remains unclear.</p><p>Several early reports suggested that various Plasmodium spp. possess at least a limited ability to metabolize a number of other substrates, such as glycolytic end-products or tricarboxylic acid cycle intermediates [39–42]. However, most of these experiments rely on the stimulation of oxygen uptake as a measure of nutrient consumption, which can be problematic given the unclear relationship between this metric and parasite growth. Possible contaminating sources for this activity have been extensively discussed elsewhere [10–12]. Other experiments using simian and avian malaria parasites directly demonstrated the conversion of radioactively labeled lactate and pyruvate to glycolytic intermediates, organic acids and volatiles such as formate and acetate [1, 9, 39]. However, we caution that since these experiments used non-physiological glucose-free conditions and/or involved erythrocyte-free parasite preparations, special care should be taken in weighing their relationship to in vivo parasite metabolism, where 1) the parasite resides within a selectively permeable host erythrocyte, 2) serum glucose levels are robustly maintained by the host, and 3) lactate is rapidly excreted.</p><p>Plasmodium spp. also lack the ability to generate carbohydrate stores in the form of polysaccharides such as glycogen [18, 19, 43]. Thus the blood-stage parasites are obligately dependent on the fermentation of a constant and abundant supply of glucose. This adaptation is sensible given the parasite's adaptation to a peculiar evolutionary niche: glucose is the most abundant nutrient in human serum and its homeostasis is maintained by a powerful regulatory system. The availability of carbohydrates or other potential carbon sources in the other stages of the life cycle is difficult to study given the general intractability of culturing these stages, but it has been shown that the hemolymph of Anopheles stephensi is rich in glucose and the storage carbohydrate trehalose [44] and the essential P. berghei hexose transporter (PB000562.01.0) is expressed throughout development in the mosquito [23].</p><!><p>The pentose phosphate pathway (PPP, also known as the hexose monophosphate shunt), a critical conserved pathway in virtually all cells capable of metabolizing carbohydrates as a carbon source, is composed of two interconnected branches. The oxidative arm, in which glucose-6-phosphate is ultimately oxidized to ribose-5-phosphate, generates both the riboses needed for nucleic acid synthesis, as well as NADPH, which is used for redox control and as a cofactor for biosynthetic reactions. The non-oxidative arm, comprising a series of reversible reactions interconverting 3, 4, 5, 6 and 7-carbon sugar phosphates can either recycle ribose-5-phosphate generated by the oxidative arm back into glycolytic intermediates (when NADPH is required but nucleotide synthesis is not) or else converts glycolytic intermediates into ribose-5-phosphate without concomitant NADPH production (Fig. 1). It is one of the major metabolic pathways in human erythrocytes, consuming 3–11% of the glucose metabolized under normal conditions [45], as it is the only source of the NADPH required to reduce glutathione in response to oxidative stress. Though counterintuitive given its indispensable nature in proliferating cells, the existence of a complete PPP in the malaria parasites was for decades a point of controversy due to difficulties in detecting the necessary enzymes in parasite extracts [11] and early reports of very slight increases in pathway activity in RBCs infected with simian, avian and rodent malaria parasites [3, 9, 19]. However, the presence of the first and rate-limiting enzyme in the pathway, glucose-6-phosphate dehydrogenase (G6PDH, PF14_0511), was eventually established [46, 47]. The remaining enzymes are also encoded in the genome [25] and expressed [26], with the singular exception of transaldolase, for which no homolog has yet been identified [25].</p><p>The infected RBC commits roughly the same fraction of glucose to the oxidative PPP as does a normal RBC [48]. Since glucose consumption is massively increased during infection [12], the absolute flux through the PPP is likewise increased. The most recent investigation of this pathway in P. falciparum determined that the activity of the oxidative branch increased 78-fold by the trophozoite stages [48]. 82% of this activity could be recapitulated in free parasites, indicating that the parasite is responsible for the majority of the flux but also that the erythrocyte PPP is up-regulated 24-fold, to levels roughly similar to those observed when subjecting uninfected RBCs to oxidative insult. Two reports, one using P. falciparum [48] and the other in the simian parasite P. knowlesi [3], have found that significant amounts of radioactivity are incorporated into parasite nucleic acids from both 1- and 6- 14C-glucose. Since the carbon at position 6 is lost to decarboxylation in the oxidative PPP and so cannot be incorporated into ribose, this implies that the non-oxidative PPP (in which glycolytic intermediates can be converted to riboses) contributes significantly to the total pool of ribose-5-phosphate.</p><p>Despite the observation that the parasite PPP predominates in terms of flux, clear evidence for the importance of the host pathway is found in the fact that G6PDH deficiency, the most common human enzymopathy, is associated with resistance to clinical malaria (reviewed in [49]). The mechanistic basis for this protection remains somewhat controversial; in some cases slowed growth in G6PDH-deficient erythrocytes has been directly observed in vitro [50], while another group disputes this claim and suggests that the protection is mediated instead by an increase in oxidative damage to the host cell membrane, marking it for early phagocytosis by macrophages [51]. Nevertheless, the parasite requires a certain degree of viability from its host cell during the progression through blood-stage development, which includes maintenance of an adequate glutathione pool, with a sufficiently high ratio of reduced (GSH) to oxidized glutathione (GSSG, glutathione disulfide), within the host cell cytosol [52]. The regeneration of GSH from GSSG is fueled by NADPH from the RBC's pentose phosphate metabolism, whose flux is limited by G6PDH. Intriguingly, it has been reported that the parasite also actively supplies GSH to the host compartment while inducing the excretion of GSSG [30], suggesting that a combined effort encompassing both recycling by the host and biosynthesis by the parasite may be required to sustain the infected cell.</p><!><p>The malaria parasite expresses a variety of glycoconjugated proteins (circumsporozoite protein, the merozoite surface proteins), several of which are essential for invasion and virulence [53]. However, the range of glycoconjugates produced, and the corresponding biosynthetic enzymes, appears to be severely restricted, limited almost entirely to the production of glycophosphatidylinositol (GPI) anchors required for protein-membrane association (reviewed in [54]). These moieties are assembled stepwise in the lumen of the endoplasmic reticulum on a phosphatidylinositol core through the sequential addition of glucosamine (as its activated form, UDP-N-acetyl-glucosamine, subsequently deacetylated) and four mannose molecules. The inositol is acylated, typically by myristic acid, at the 2-O position prior to mannosylation; the third mannose in the chain serves as the site for addition of ethanolamine phosphate, through which the GPI anchor is then attached to a protein.</p><p>This pathway thus draws carbon compounds from a variety of sources: hexose sugars (mannose, the glucose moiety of glucosamine), inositol biosynthesis, lower glycolysis (glycerol phosphate from the reduction of dihydroxy-acetone-phosphate, acetyl-CoA from the oxidation of pyruvate), ethanolamine from the host, and lipids from scavenging and modification (Fig. 1). We have recently shown that the acetyl moieties utilized for the synthesis of UDP-N-acetyl-glucosamine ultimately derive from glucose, presumably through the action of the pyruvate dehydrogenase (PDH) complex on glycolytic pyruvate [38]. Thus it is surprising that the PDH E1-alpha and E3 subunit genes of P. yoelii can be disrupted with no obvious growth phenotype until the liver stages [55], given that GPI biosynthesis is essential for blood-stage survival [56]. It is possible that in the event of PDH inactivation a separate acetyl-CoA-generating mechanism (discussed below) might compensate, indicating a degree of metabolic flexibility within this pathway. Nonetheless, the importance of GPI-anchored proteins to parasite development and the unique nature of certain of the enzymatic steps has suggested this pathway as a potential drug target [54].</p><!><p>The highly proliferative growth of the blood stage parasite depends on manufacturing significant quantities of new membrane. However, early experiments tracking the incorporation of radio-labeled glucose revealed that the amount of carbon from this nutrient incorporated into the lipid fraction of P. knowlesi biomass was low and occurred mainly in the glycerol backbone instead of the acyl chains [5]. This suggests that de novo fatty acid biosynthesis, or at least that using carbohydrates as a carbon source, is not a significant component of parasite metabolism. Interest in fatty acid metabolism has recently resurged with the discovery that the parasite genome encodes the complete suite of enzymes necessary for Type II (bacterial) fatty acid synthesis (FAS). These enzymes are all targeted to the apicoplast, a nonphotosynthetic plastid-like organelle derived from a secondary endosymbiotic event that is suspected to play a role in a number of plant-derived metabolic pathways [25, 57]. Type II FAS is an attractive drug target and considerable excitement has been generated by the discovery that triclosan, an antibiotic targeting bacterial enoyl-acyl carrier protein reductase (ENR), efficiently inhibits both the incorporation of 14C-acetate into parasite fatty acids and P. falciparum growth in culture [58]. However, the transcripts for various enzymes in the Type II FAS pathway show very low to undetectable blood-stage expression levels in microarray analyses [26], and subsequent investigations determined that several of these, including ENR, could be genetically disrupted in both P. berghei and P. falciparum without any discernible affect on blood-stage growth [59, 60]. However, the FAS pathway is essential during the liver stages, as these mutants exhibit a block in liver cyst development. Therefore, these studies conclusively demonstrate that ENR is not the blood-stage target of triclosan. That triclosan remains lethal to these ENR knockout parasites, and in fact still inhibits acetate incorporation [58], imply that it targets another aspect of parasite metabolism, perhaps acyl chain extension.</p><p>A more complete understanding of the structure of parasite fatty acid metabolism derives from a series of experiments involving metabolic tracers and different lipid precursor supplementations [61]. P. falciparum is typically cultured in serum-free medium supplemented with lipid-rich albumin, which is required for growth due to the complex mixture of fatty acids it provides. By systematically testing different combinations of free fatty acids for their ability to support parasite growth, Mi-ichi et al. were able to determine six combinations of C14, C16, C18 fatty acids and several desaturation products thereof that enable continuous culture of P. falciparum. No single fatty acid was required in every combination because the parasite possesses a limited ability to modify exogenously supplied fatty acids as needed. Specifically, metabolic labeling experiments using radioactive fatty acid species determined that the parasite can elongate *****C14 to C16 and C16 to C18, as well as desaturate acyl chains (primarily at the ω–9 position).</p><p>A model that arises from the data above is of a lipid metabolism in which preformed fatty acids are 1) scavenged from the host cell and serum, 2) subjected to a limited set of modifications by parasitic elongases and desaturases, and 3) incorporated into membrane glycerides with a glycerol backbone derived ultimately from glucose through the EMP pathway (Fig. 1). The two-carbon units (in the form of acetyl-CoA) necessary for elongation might be produced from glycolytic pyruvate in the apicoplast by the PDH complex localized to that compartment [62]. However, this enzyme is not essential during the blood stages [55]. It is possible that a glutamine-driven pathway (discussed below) could supply acetyl-CoA to lipid elongation, either under normal circumstances or else solely in the PDH mutant. This remains to be demonstrated however, since the few metabolic labeling experiments to robustly label acyl chains in parasite cultures have used very high levels of free 14C-acetate (P. knowlesi [5], P. falciparum [58]). In these experiments, only a small fraction (~15%) of the total radioactivity incorporated into lipids from 14C-glucose in P. knowlesi was detected in the acyl chain [5]. In addition, we have recently used gas chromatography-mass spectrometry profiling of lipids in parasites fed either 13C-glucose or 13C-glutamine, but were unable to measure any 13C incorporation into fatty acids [38]. Fatty acids acquired through scavenging from the extracellular environment evidently cannot serve as carbon sources themselves due to the absence from the genome of any of the β-oxidation enzymes necessary for the breakdown of acyl chains. Whole, preformed lipids might in theory be scavenged directly from the host cell membrane or host serum, but there is no evidence of lipid (as opposed to fatty acid) scavenging from the extracellular environment. Moreover, there is a net 6-fold increase in the phospholipid content of the infected erythrocyte [63], indicating significant amounts of phospholipid biosynthesis on the part of the parasite. The `bewildering' series of plasmodial pathways responsible for assembling complex phospholipids, and the open questions pertaining to their study, have been very well reviewed elsewhere [64].</p><!><p>Mitochondrial tricarboxylic acid (TCA) metabolism has long been considered a "black box" within the rest of the malaria parasite's metabolism, unclear both in terms of its function as well as its basic architecture. In most free-living microbes this pathway acts as a versatile central hub of carbon metabolism in which carbon derived from glycolysis or other nutrients (ethanol, acetate, amino acids, etc.) is fully oxidized to carbon dioxide while generating energy (by oxidative phosphorylation), reducing equivalents and biosynthetic precursors (such as for amino acid synthesis). However, from the earliest reports in the malaria literature onwards a more confusing and contradictory picture emerges. Electron micrographic observations of the human malaria parasites' morphology revealed the single mitochondrion to be only minimally cristate [65], calling into question its role as a site of active metabolism (although cristae are observed in the mitochondria of avian parasites [65, 66]). The low levels of oxygen consumption observed in culture and the centrality of anaerobic glycolysis to energy metabolism discussed above further implied that the primary mitochondrial function of oxidative phosphorylation was absent or of minimal importance. In fact, the absence of obvious homologs to the F0 a and b subunits of the F1F0 ATP synthase in the parasite genome led to speculation that this enzyme complex may be incapable of generating ATP [25], although candidate homologs have recently been identified bioinformatically [67]. Malaria parasites have also dispensed with de novo amino acid biosynthesis, instead acquiring them through scavenging from the host serum or hemoglobin catabolism [68], which generates such an excess of amino acids that most are excreted from the infected cell as waste [69]. Biochemical and informatic studies suggest that the parasite malate dehydrogenase is actually cytosolic [70, 71], and so cannot contribute to a mitochondrial TCA cycle. Furthermore, as mentioned above the absence of the PDH enzyme complex from the mitochondrion [62], where it generally serves as the fundamental link between carbohydrate and mitochondrial carboxylic acid metabolism, converting glycolytic pyruvate to mitochondrial acetyl-CoA for citrate synthesis, raised the question of how, if at all, carbon enters the TCA cycle. This coincides with early reports that only very minimal amounts of 14CO2 and keto acids are produced from 14C –glucose during intraerythrocytic growth of both simian and avian parasites [1, 2, 9].</p><p>However, other lines of evidence indicate that some form of mitochondrial carboxylate metabolism occurs during the blood stage. The simian malaria parasite P. knowlesi and avian parasite P. lophurae fix 14CO2 into metabolites including citrate, malate and succinate [8, 72]. With the advent of the genome sequence it was realized that the parasite encodes homologs to all of the enzymes necessary for a complete TCA cycle [25], which are all are roughly coexpressed during the intraerythrocytic stage [26] and possess putative mitochondrial signal sequences save for malate dehydrogenase. In place of malate dehydrogenase, a second enzyme capable of essentially running the same reaction (malate:quinone oxidoreductase, PFF0815w) is expressed and appears to be mitochondrially targeted. In addition, the P. falciparum citrate synthase homolog (PF10_0218) [73], aconitase (PF13_0229) [74] and isocitrate dehydrogenase (IDH, PF13_0242) [38] have been localized entirely or partially to the mitochondrion. Intriguingly, while most eukaryotes possess at least three isoforms of IDH (mitochondrial NADP-dependent, mitochondrial NAD-dependent and cytosolic NADP-dependent), the parasite encodes only the mitochondrial NADP-dependent enzyme. Enzymatic activity for the TCA cycle enzymes succinate dehydrogenase (PFL0630w and PF10_0334) [75], aconitase [74] and IDH (in P. knowlesi) [76] have been detected in parasite extracts, and IDH has been cloned and characterized [77, 78]. Furthermore, the Plasmodium spp. possess an essential de novo heme biosynthetic pathway which presumably requires succinyl-CoA to function [79]. As the only known source of this precursor in the parasite's metabolic network, at least a subset of the TCA cycle enzymes (2-oxoglutarate dehydrogenase or succinyl-CoA synthetase) must be active. Finally, a metabolomic survey of the P. falciparum blood-stage developmental cycle discovered that the levels of several TCA cycle intermediates (ketoglutarate, iso/citrate, aconitate) oscillate periodically over the intraerythrocytic developmental cycle roughly in phase with the TCA cycle enzymes, suggesting that they are actively synthesized [80]. In order to explain these disparate strands of data, several models have been put forward. One possibility is that the full TCA pathway is active during different parasite life cycle stages, with the parasite's branched-chain alpha-keto-acid dehydrogenase complex possibly supplying mitochondrial acetyl-CoA through amino acid catabolism [81], or perhaps by acting as a surrogate pyruvate dehydrogenase [82].</p><p>Efforts to address this enigma in our laboratory have taken advantage of recent advances in mass spectrometry-based high throughput chemimetric technologies [83–85]. These metabolomic platforms have the advantage of permitting rapid and simultaneous quantification of a large panel of cellular metabolites, and, using mass spectrometry for detection, allows for relatively simple metabolic tracing experiments using stable isotope-labeled nutrients in which the number and position of labeled atoms can be determined. By feeding P. falciparum cultures 13C-glucose and analyzing isotopic distributions we have confirmed that carbohydrate metabolism does not contribute significantly to the TCA cycle, though there is clear evidence of cytosolic CO2 fixation through PEP carboxylase to oxaloacetate and malate [38]. 13C-glutamine, by contrast robustly labels all TCA cycle intermediates measured. It seems that glutamine is efficiently taken up by the infected cell [86] and converted to glutamate (likely through glutamate synthase, PF14_0334), which is itself converted to 2-oxoglutarate by glutamate dehydrogenase (GDH). P. falciparum encodes 3 distinct GDH genes: an NADP-dependent enzyme presumably targeted to the apicoplast (PF14_0286) [87], which may supply NADPH for biosynthetic reactions in that compartment; another NADP-dependent GDH lacking a cleavable signal sequence, and so presumably cytosolic (PF14_0164) [88]; and a large, fungal-type NAD-dependent GDH (PF08_0132) [87] with no predicted signal sequences. While we can only speculate at this point which of these predominates in terms of flux, one hypothesis that has been put forward has it that the cytosolic NADP-GDH might be a major source of NADPH for redox control [12], which would concomitantly generate cytosolic 2-oxoglutarate. Studies in our lab have determined that a large amount of 2-oxoglutarate is effluxed from infected cells as an apparent waste product, but some fraction appears to enter the mitochondrion, possibly through a malate:oxoglutarate antiporter homolog (PF08_0031), and serves as the carbon source driving TCA metabolism [38].</p><p>Surprisingly, the observed isotope labeling patterns when cells are fed 13C-glutamine indicated that this metabolism comprises two largely independent pathways: an oxidative branch running successively from 2-oxoglutarate to succinyl-CoA, succinate, fumarate and malate (essentially half of a canonical TCA cycle), and a reductive branch running from 2-oxoglutarate to isocitrate, citrate, oxaloacetate and malate (Fig. 1). We find that the resulting malate is excreted along with malate produced during the cytosolic carbon fixation process. The oxidative branch is unlikely to carry a very high flux but should generate reducing power (NADH) for the electron transport chain, GTP (via substrate-level phosphorylation) and, critically, the succinyl-CoA required for heme biosynthesis. The reductive branch, by contrast, produces two-carbon units during the citrate cleavage step rather than consuming two-carbon units as acetyl-CoA.</p><p>These results help to clarify a long-standing mystery in the field of Plasmodium metabolism by revealing a significantly diverged architecture for this fundamental pathway. Several other puzzling observations become explicable in the context of this model. For example, losing the canonical TCA cycle's NAD-dependent IDH while retaining the NADP-dependent isoform may be explained by noting that the reaction catalyzed by the NAD-dependent isoform is typically irreversible in the oxidative direction [89] while the NADP-dependent reaction is freely reversible in a wide variety of organisms [89–92]. Thus this permits the pathway to run in the reductive direction. Also, the presence of the putative mitochondrial malate:oxoglutarate antiporter has been difficult to interpret given the absence of any identifiable glutamate:aspartate antiporter, as the pair normally function together to complete the metabolic circuit known as the malate-aspartate shuttle [81]. However, if the mitochondrion is consuming 2-oxoglutarate and excreting malate then this transporter's role would simply be to exchange substrate for product. Other questions remain open: our data suggest that the parasite has evolved (at least) two independent pathways for the production of acetyl-CoA, and that these two pathways therefore might play different metabolic roles. However, it is not completely clear at this point whether this reductive pathway localizes entirely to the mitochondrion, or how essential it is for parasite growth. Further study is required to elucidate these and other puzzles.</p><!><p>A variety of recent research efforts have aimed to elevate our understanding of plasmodial biology and metabolism to a systems level through the use of high-throughput "-omics" approaches, both in in vitro and in vivo settings. Though generally more difficult to interpret than classical approaches directed at one to a few nodes in a biological pathway, such datasets offer the potential to capture behaviors of the entire network. A study of the transcriptional profiles of P. falciparum samples harvested directly from clinical malaria patients for example, found that the parasites may exist in three broad clusters that seem to reflect a difference in metabolism [93]. Based on the gene expression of glycolytic and TCA cycle genes, the authors concluded that one cluster resembled normal anaerobic fermentative metabolism commonly seen in culture, while another seemed to show evidence of a starvation response and a switch to oxidative energy generation. Our biochemical evidence described above renders it doubtful that oxidative phosphorylation plays a significant energetic role, although it is possible that these data indicate an up-regulation of mitochondrial metabolism to generate two-carbon units in under conditions of reduced glucose availability in starved parasites (although other investigators have challenged these results as artifacts of analysis [94]). Several groups have reported that TCA cycle enzymes are present during all life cycle stages of the parasite analyzed, and may be particularly up-regulated in gametocytes, ookinetes and sporozoites of P. falciparum and P. yoelii [95–99]. The functional implications of this remain mysterious, given both the unlikely nature that this pathway plays an energetic role and the unclear metabolic requirements at the stages of development when the parasite is not actively proliferating.</p><p>Of course, metabolomic methodologies will be critical in the push towards dissecting the interlocking carbon metabolic pathways of the Plasmodium parasites and their host cells (reviewed in [64, 100]). Such techniques have been used to probe metabolic perturbations induced by malarial infection in in vitroP. falciparum cultures [80] and in vivo rodent malaria models [101, 102]. More directly, the ability of analytic technologies such as mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy to trace the flow of heavy isotope-labeled nutrients through pathways is proving to be exceptionally valuable in this regard. Our own pathway analysis of carbon flow in P. falciparum, discussed in detail above, resulted in a new model architecture for mitochondrial carboxylic acid metabolism [38]. An investigation of labeling from 13C-glucose by NMR has suggested the intriguing possibility that, in addition to severely increasing the glucose consumption of the infected cell, P. falciparum is capable of somehow inhibiting glucose utilization by uninfected erythrocytes (seemingly through the inhibition of the host phosphofructokinase and pyruvate kinase by an excreted soluble factor) [33, 103]. If this finding can be confirmed and the inhibitory molecule identified it would be a fascinating insight into the parasitic modulation of host metabolism. Another group has reported, again using NMR-based measurements of 13C-labeled glucose metabolism, that in vitro cultures of P. falciparum produce glycerol as a major metabolite of glucose [104]. This finding is surprising given the rarity of glycerol production among eukaryotes and the absence of any enzyme in the current P. falciparum genome annotation capable of generating glycerol, such as a glycerol-3-phosphatase or a reversible glycerol kinase, although this activity is found in other parasitic protozoa such as the Trypanosomes [105–107]. These authors hypothesize that a functional glycerol-3-phosphate shuttle is therefore present in Plasmodium spp. and that a reappraisal of parasite redox metabolism may be necessary. However, since no quenching or deproteination step was used during metabolite extraction, it is possible that the observed glycerol may be the product of a nonspecific phosphatase acting on glycerol-3-phosphate produced by the parasite (for example, the excreted acid phosphatase discussed above was found to cleave phosphate from a wide variety of small-molecule substrates, including glycerol-2-phosphate [28]). Nevertheless, these recent observations suggest that the parasite may have some further metabolic tricks up its sleeve and that our current views of redox control and carbon metabolism should be interpreted with an open mind.</p><!><p>The preceding decades of malaria research have vastly clarified our understanding of the biology of the parasite and filled in many of the blank spaces of its metabolic map. The challenge now facing the malaria community is to build on the foundation of these results a comprehensive understanding of Plasmodium physiology, metabolism and host-parasite interactions both at the cellular and organismal level. One major stumbling block on the way toward this goal is our poor understanding of the metabolic interconnection between the RBC and its invader. The infected cell is highly compartmentalized and in one sense the essence of intracellular parasitism is the exchange of nutrients and wastes between parasite, host cell and environment. This is due largely to the significant difficulty inherent to compartmental analysis of metabolite concentrations and fluxes and a paucity of information about the localization and even identity of plasmodial proteins (most of which are unannotated). One clear starting point to unraveling this web of interactions is a concerted effort to identify and localize the many putative transporters of unknown function expressed during the blood stage [108]. A more complete understanding of the transport capabilities of the infected cell will significantly aid in efforts to understand how Plasmodium spp. mediate their exchange with the environment through the host cytoplasm.</p><p>Another fruitful area of inquiry is the genetic control of metabolism. Many regulatory interactions are difficult to study in malarial systems due to the lack of genetic tools. However, the progeny of phenotypically distinct strains have been used in conjunction with quantitative trait loci (QTL) analysis to uncover the loci governing traits such as drug resistance [109] and mRNA expression level [110]. This tool has also been used with metabolomics to probe metabolic regulation in Arabidopsis thaliana [111, 112]. Such an approach has enormous potential in malaria research to reveal interactions not accessible by other methods.</p><p>A more fine-scaled understanding of not just the architecture of the metabolic pathways of the infected cell but their dynamics is achievable through the use of fluxomics to map out the fluxes through the metabolic network. Such studies entail using a variation on classical pulse-chase techniques, rather using stable isotope-labeled nutrients and analytical platforms (MS or NMR-based) capable of measuring changes in the isotopic labeling pattern of downstream intermediates. Such studies measure both steady-state fluxes and the transient change in fluxes induced by a perturbation, both of which provide significant insight into the dynamic regulation and enzyme kinetics at play in the system [113–115]. Again, several technical challenges must be resolved before such studies can be carried out with current in vitro malaria culture techniques, which are complicated by the presence of host-cell compartments, enzymes, the presence of uninfected cells and other, more subtle peculiarities of the system. For example, our initial investigations into the TCA cycle were complicated by the observation that citrate concentrations in our RBC extracts were far in excess of the negligible erythrocyte citrate content reported in the literature [116]. We determined that this was due to citrate entering the RBCs during storage in the high citrate (>10 mM final concentration) anticoagulant solutions routinely used during blood collection. This issue was easily resolved using blood collected in sodium heparin solutions [38] but points to the special care that must be taken to minimize the effect of metabolic perturbations resulting from the nutrient environment during in vitro tissue culturing.</p><p>The avenues above will ultimately prove their worth by contributing to a systems level model for Plasmodium metabolism. Flux-balanced (stoichiometric) in silico metabolic networks have been developed for well-studied model organisms such as yeast [117] and E. coli [118] as well as several pathogens [119–121], and recent efforts by several groups have focused on constructing such an informatic tool for P. falciparum [122–124]. At present these are limited by our lack of understanding about the structure, dynamics and compartmentalization of the infected cell's metabolism, and such models must be refined by experimental evidence such as described above. A sufficiently accurate model will permit immediate in silico experiments to determine vulnerable drug targets (enzymes and transporters) and multi-drug dosing strategies, potentially significantly speeding the process of rational drug design. A fundamental understanding of central carbon metabolism in Plasmodium spp., and the unique adaptations differentiating the parasite and its host provide exciting new avenues for future therapeutic intervention strategies.</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><p>Embden-Meyerhof-Parnas</p><p>red blood cell</p><p>phosphoenolpyruvate</p><p>PEP carboxykinase</p><p>PEP carboxylase</p><p>pentose phosphate pathway</p><p>glucose-6-phosphate dehydrogenase</p><p>glycophosphatidylinositol</p><p>enoyl-acyl carrier protein reductase</p><p>pyruvate dehydrogenase</p><p>tricarboxylic acid</p><p>isocitrate dehydrogenase</p><p>nuclear magnetic resonance</p><p>reduced glutathione</p><p>oxidized glutathione (glutathione disulfide)</p><p>An integrated map of carbon flow through the metabolic network of Plasmodium falciparum. Arrows show the proposed direction of flux through the corresponding enzymatic reaction as suggested by experimental evidence; note that this is only intended to indicate net flux, and that the reaction in question might be reversible. Cofactors (ATP, NADH, etc.) are not shown for the sake of clarity. Text in circles represent major biomass components; the circled question mark indicates uncertainty about the existence of the enzyme transaldolase. Abbreviations: Glc, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GADP, glyceraldehyde-3-phosphate; 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Lac, lactate; Ac-CoA, acetyl-CoA; Ac-R, either acetate or acetyl-CoA; GlycP, glycerol-3-phosphate; Glyc, glycerol; Man6P, mannose-6-phosphate; Man1P, mannose-1-phosphate; GDP-Man, GDP-mannose; GlcN6P, glucosamine-6-phosphate; GlcNAc6P, N-acetyl-glucosamine-6-phosphate; GlcNAc1P, N-acetylglucosamine-1-phosphate; UDP-GlcNAc, UDP-N-acetyl-glucosamine; 6PGL, 6-phosphoglucono-δ-lactone; 6PGa, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; X5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; Asp, aspartate; Gln, glutamine; Glu, glutamate; 2OG, 2-oxoglutarate; ICT, isocitrate; Cit, citrate; OAA, oxaloacetate; Mal, malate; Suc-CoA, succinyl-CoA; Suc, succinate; Fum, fumarate; GPI, glycophosphatidylinositol.</p>

PubMed Author Manuscript

NaGd(MoO4)2 nanocrystals with diverse morphologies: controlled synthesis, growth mechanism, photoluminescence and thermometric properties

Pure tetragonal phase, uniform and well-crystallized sodium gadolinium molybdate (NaGd(MoO 4 ) 2 ) nanocrystals with diverse morphologies, e.g. nanocylinders, nanocubes and square nanoplates have been selectively synthesized via oleic acid-mediated hydrothermal method. The phase, structure, morphology and composition of the as-synthesized products are studied. Contents of both sodium molybdate and oleic acid of the precursor solutions are found to affect the morphologies of the products significantly, and oleic acid plays a key role in the morphology-controlled synthesis of NaGd(MoO 4 ) 2 nanocrystals with diverse morphologies. Growth mechanism of NaGd(MoO 4 ) 2 nanocrystals is proposed based on timedependent morphology evolution and X-ray diffraction analysis. Morphology-dependent down-shifting photoluminescence properties of NaGd(MoO 4 ) 2 : Eu 3+ nanocrystals, and upconversion photoluminescence properties of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ and Yb 3+ /Tm 3+ nanoplates are investigated in detail. Charge transfer band in the down-shifting excitation spectra shows a slight blue-shift, and the luminescence intensities and lifetimes of Eu 3+ are decreased gradually with the morphology of the nanocrystals varying from nanocubes to thin square nanoplates. Upconversion energy transfer mechanisms of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ , Yb 3+ /Tm 3+ nanoplates are proposed based on the energy level scheme and power dependence of upconversion emissions. Thermometric properties of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ nanoplates are investigated, and the maximum sensitivity is determined to be 0.01333 K −1 at 285 K.

nagd(moo4)2_nanocrystals_with_diverse_morphologies:_controlled_synthesis,_growth_mechanism,_photolum

<!>Results and Discussion<!>Conclusion<!>Chemicals.

<p>Nowadays, lanthanide-doped nanocrystals, especially upconversion nanocrystals, have become the current focus of intensive researches due to their unique photoluminescence properties and consequently numerous applications, such as bio-imaging and bio-probe, photodynamic/chemo-therapy, drug delivery, temperature sensing, solar cells, optoelectronics and photocatalysis [1][2][3][4][5][6][7][8][9][10] . Compared with the luminescence from conventional organic dyes or quantum dots, lanthanide luminescence from nanocrystals exhibits many advantages, including high photostability (high resistance to optical blinking and photobleaching), large Stokes/anti-Stokes shifts, sharp emission bandwidths, abundant emission channels, long excited-state lifetime, low cytotoxicity, and low synthesis expenditure [11][12][13][14][15] . All these merits endow lanthanide-doped nanocrystals with efficient luminescence, high detection sensitivity and signal-to-noise ratio, and ease of use in aforementioned applications. The morphology (size and shape) of nanocrystals will affect their physicochemical properties, and the synthesis of nanocrystals with tunable morphologies is particularly significant for the applications in biological and biomedical fields [16][17][18] . So morphology-controlled synthesis of nanocrystals has attracted much attention from researchers.</p><p>Double alkaline rare-earth molybdates ARe(MoO 4 ) 2 (A = alkali metal cation, Re = trivalent rare-earth metal cation) have been demonstrated to be promising candidates as luminescent host materials for numerous applications, due to their favorable chemical and physical stability, large lanthanide admittance, and relatively low phonon energy [19][20][21][22][23] . Many researches have been devoted to the synthesis and luminescence properties of lanthanide-doped molybdate microcrystals or phosphors [24][25][26][27][28][29] . Nevertheless, the synthesis or luminescence properties of double alkaline rare-earth molybdate nanocrystals are rarely reported, which result from the faster crystallization and growth rate and difficulty in controlling the growth process of double molybdates 30,31 . Bipyramid-like NaLa(MoO 4 ) 2 : Eu 3+ nanocrystals were synthesized hydrothermally using oleic acid/oleylamine as surfactant 32 . NaLa(MoO 4 ) 2 : Eu 3+ , Eu 3+ /Tb 3+ shuttle-like nanorods composed of nanoparticles were prepared hydrothermally using ethylene glycol as ligand and their luminescent properties were discussed 33,34 . However, most of these works focused on either nanocrystals with only a single morphology or poor-crystallized composite nanoparticles, and controlled synthesis of double molybdates nanocrystals with diverse morphologies has not been reported so far.</p><p>Solution-based wet chemical methods, which allow a fine control of size, shape and chemical homogeneity of the products by fine tuning of experimental conditions, are universally employed to synthesize nanocrystals 35,36 . Some organic additives with functional groups or long hydrocarbon chains (e.g. oleic acid, citrate acid, oleylamine, ethylenediamine tetraacetic acid, and cetyltrimethyl ammonium bromide) can act as complexing agents and shape modifier by adjusting the growth rate of different facets under hydrothermal conditions 37,38 . In this work, we present a novel template-free morphology-controlled hydrothermal synthesis of NaGd(MoO 4 ) 2 nanocrystals. Pure tetragonal phase, uniform and well-crystallized NaGd(MoO 4 ) 2 nanocrystals with several distinct morphologies, including nanocubes and square nanoplates, can be selectively synthesized by a modified hydrothermal method using oleic acid as complexing agent. The morphology of the synthesized NaGd(MoO 4 ) 2 nanocrystals can be controlled by simply tuning the contents of oleic acid in the precursor solution. Effects of oleic acid and sodium molybdate (Na 2 MoO 4 ) on the formation of NaGd(MoO 4 ) 2 nanocrystals and growth mechanism of NaGd(MoO 4 ) 2 nanoplates are discussed. Meanwhile, morphology-dependent down-shifting photoluminescence properties of NaGd(MoO 4 ) 2 : Eu 3+ nanocrystals, upconversion photoluminescence properties of Yb 3+ /Er 3+ and Yb 3+ /Tm 3+ square nanoplates, and thermometric properties of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ square nanoplates are investigated in detail.</p><!><p>Crystal structures, morphologies and compositions. NaGd(MoO 4 ) 2 nanocrystals with specific uniform morphologies are synthesized hydrothermally at 180 °C for 12 h with different contents of oleic acid (0.25, 0.75, 1.25 and 1.5 ml) and 10 mmol Na 2 MoO 4 in the initial precursor solutions. Figure 1a shows the XRD patterns of NaGd(MoO 4 ) 2 nanocrystals samples with four typical morphologies, including nanocubes (pattern i) and square nanoplates with different aspect ratios (patterns ii-iv), in the 2 theta range of 10-80°. All the diffraction peaks can be readily indexed to a tetragonal phase NaGd(MoO 25-0828). The average crystallite sizes calculated using Scherrer's formula from the broadening of the diffraction peak (112) in the four patterns are 58, 51, 48 and 49 nm, respectively. What's more, compared with the standard diffraction data, quite intense (004) diffraction peak is found in the pattern iii of Fig. 1a, indicating a preferentially oriented crystallization might exist along the (001) planes of NaGd(MoO 4 ) 2 nanocrystal. Due to the morphological characters of the samples and discrepancy in sample preparation procedure for the XRD measurements, the enhancement of (004) peaks in patterns ii and iv is not evident.</p><p>The SEM images of the NaGd(MoO 4 ) 2 nanocrystals with four typical morphologies were shown in Fig. 1b-e. Obviously, all the samples exhibit uniform, regular and well-crystallized nanocrystals. In Fig. 1b, the sample consists of monodisperse and uniform nanocubes with side length of ~150 nm. In Fig. 1c-e, the samples are composed of monodisperse and uniform square nanoplates. The thicknesses are about 85, 70, 50 nm and the side lengths are about 250, 400 and 500 nm for the samples in Fig. 1c-e, respectively. The chemical composition of the NaGd(MoO 4 ) 2 nanocrystals with the morphology of square nanoplates (shown in Fig. 1d) was analyzed by the EDS spectrum shown in Fig. 1f. The sample is confirmed to be composed of Na, Gd, Mo and O. The measured atomic ratio of Na, Gd, Mo and O is close to the stoichiometric proportion of NaGd(MoO 4 ) 2 . C peak and excessive proportion of O come from a little oleic acid adsorbed on the surface of the sample. The Si and Pt peaks arise from silicon substrate and conductive coating.</p><p>More details about the morphological and structural features were further investigated by employing TEM, HRTEM and SAED. The morphologies of the samples shown in the TEM images (Fig. 2a-d) are consistent with that in SEM images. The HRTEM images taken at the edge of the nanocube/nanoplates (Fig. 2e-h) reveal perfect crystalline surfaces. The interplanar distances between adjacent lattice fringes of the four samples are all about 0.26 nm, which correspond to the d spacing of (200) or (020) planes of tetragonal NaGd(MoO 4 ) 2 structure. These lattice fringes indicate that the nanoplates grow along [100] and [010] directions, namely (001) planes, which agrees well with the speculation from the XRD analysis. Taking the morphology of the nanocrystals (square plate) into account, it is also inferred that the normal direction of the upper surface of the square nanoplates is the zone axis ([001] orientation). Thus the upper surface of the square nanoplates belongs to (001) planes of tetragonal NaGd(MoO 4 ) 2. The SAED patterns of all the samples show highly ordered sharp spots, which indicate the single crystalline nature of the samples. The diffraction dots are indexed to (200) and (020) planes of tetragonal NaGd(MoO 4 ) 2 .</p><p>FTIR analysis was performed to investigate the surface properties of the samples. Supplementary Fig. S1 presents the FTIR spectra of the NaGd(MoO 4 ) 2 nanocrystals with four typical morphologies (Fig. 1b-e). The spectra are similar in shape. A broad band at about 3399 cm −1 corresponds to the O-H stretching vibrations is observed, arising from surface-adsorbed ambient water. Small peaks at about 2927 and 2856 cm −1 are attributed to the stretching vibration of -CH 2 in skeletal chain of oleic acid. The peaks at about 1635 and 1460 cm −1 are ascribed to the vibrations of the C = O groups of oleic acid 39 . The strong absorption bands at 796 and 704 cm −1 are assigned to the F 2 (ν 3 ) antisymmetric stretch and the peak at 434 cm −1 is ascribed to F 2 (ν 4 ) bending mode vibrations related to the O-Mo-O stretching vibrations in the MoO 4 tetrahedron 40 . These results show the existence of residual complexing ligand on the surface of the samples.</p><p>Formation of the NaGd(MoO 4 ) 2 nanocrystals. Effects of Na 2 MoO 4 on the morphology of the NaGd(MoO 4 ) 2 nanocrystals. Generally speaking, the molar ratio of the starting source reagents in the precursor solutions would affect the morphology and/or phase of the products in the hydrothermal synthesis procedure. To investigate the effects of the Na 2 MoO 4 on the morphology of the synthesized NaGd(MoO 4 ) 2 nanocrystals, NaGd(MoO 4 ) 2 samples are synthesized with different amounts of Na 2 MoO 4 varying from 2 to 12 mmol, and the fixed amount of oleic acid (1.25 ml) in the precursor solution. Supplementary Figs S2 and S3 present the XRD patterns and SEM images of the as-synthesized samples. From the XRD patterns, it is observed that all the samples are pure tetragonal phase NaGd(MoO 4 ) 2 (ICDD No. 25-0828). Similarly, intense diffraction peak (004) is also observed in pattern c of Supplementary Fig. S2. As can be seen from the SEM images, the samples are comprised of nanoparticles and bipyramids when a small amount of Na 2 MoO 4 is added. With the increasing amount of Na 2 MoO 4 , the samples evolve toward square nanoplates. And both the side length and thickness of the square nanoplates are reduced with the increase of Na 2 MoO 4 content (Supplementary Fig. S3c-e). The sample exhibits irregular nanoflakes with nearly round shape at further increasing Na 2 MoO 4 content (12 mmol, Supplementary Fig. S3f).</p><p>According to Bravais-Friedel-Donnay-Harker theory, high-index facets with high surface free energy have a large growth rate and will not be expressed in the equilibrium morphology of the resulting crystal 41 . Based on crystal structure models of tetragonal NaGd(MoO 4 ) 2 shown in Supplementary Fig. S4, packing density of Gd 3+ /Na + for some low-index facets are calculated to be 0.0364 Å −2 for {001} facets, 0.0332 Å −2 for {010}/{100} facets and 0.0227 Å −2 for {101} facets. The packing densities of Gd 3+ /Na + on the {001} facets are higher than that on other facets. Na 2 MoO 4 will ionize and provide (MoO 4 ) 2− anions in the hydrothermal solution. When excess (MoO 4 ) 2− exist in the solution, it will be preferentially adsorbed on {001} facets of tetragonal NaGd(MoO 4 ) 2 nanocrystal nuclei due to the strong electrostatic interaction between (MoO 4 ) 2− and Gd 3+ /Na + 42 . This preferential adsorption of (MoO 4 ) 2− on {001} facets will reduce the growth rate along [001] directions and cause preferentially oriented crystallization along the (001) plane in the NaGd(MoO 4 ) 2 nanocrystals. So the samples exhibit thinner square nanoplates at higher Na 2 MoO 4 contents. A slightly lower adsorption energy of (MoO 4 ) 2− for the other facets than that for {001} facets will also cause the adsorption of (MoO 4 ) 2− on the other non-{001} facets, but the restriction of the growth rate is weaker than that for {001} facets. Thus the side length is slightly reduced with increasing Na 2 MoO 4 content. More excess amounts of Na 2 MoO 4 will provide more (MoO 4 ) 2− anions, and break the equilibrium of growth dynamics for the nanocrystals and make the products more irregular.</p><p>Effects of oleic acid on the formation of NaGd(MoO 4 ) 2 nanocrystal. As is previously discussed, appropriate amount of Na 2 MoO 4 favor the formation of the synthesized NaGd(MoO 4 ) 2 nanocrystals with regular morphology. So the amount of Na 2 MoO 4 is further fixed at 10 mmol and NaGd(MoO 4 ) 2 nanocrystal samples synthesized with different contents of oleic acid (0-1.75 ml) in the initial precursor solution. As can be seen from the XRD patterns shown in Supplementary Fig. S5, all the diffraction peaks of the as-synthesized samples can be easily indexed as pure tetragonal phase NaGd(MoO 4 ) 2 (ICDD No. 25-0828). No peak from impurities or other phases can be found in these patterns, indicating high purity of the samples. A relatively intense diffraction peak (004) is observed in pattern f (Supplementary Fig. S5). Figure 3 shows the SEM images of the synthesized NaGd(MoO 4 ) 2 nanocrystal samples. When no oleic acid is added, the products exhibit barrel-like nanocylinders with the height of ~300 nm and diameter of ~200 nm (Fig. 3a). Whereas when oleic acid was added into the reaction system, new morphologies appear. Nanocubes were obtained when 0.25 ml oleic acid was added (Fig. 3b). When the content of oleic acid is increased to 0.5 ml, the morphology of the products varies from nanocubes to square nanoplates (Fig. 3c). With the increase of oleic acid content ranging from 0.5 to 1.5 ml, the morphologies of the products are all square nanoplates, whereas the thickness of the nanoplates decreases and the side length increases gradually (Fig. 3c-g). The square nanoplates become increasingly thinner and flatter with the increase of oleic acid contents, and seem to be squashed gradually. When 1.75 ml oleic acid is added, the products become irregular thinner nanoflakes with near-circular shape and thickness less than 50 nm (Fig. 3h). It is noted that oleic acid acts as complexing agent and shape modifier, and plays a key role in the morphology-controlled synthesis of NaGd(MoO 4 ) 2 nanocrystals. When lanthanide nitrates solution is added into the mixed solution of oleic acid and ethanol, lanthanide oleate complexes ((RCOO) 3 Ln) are formed through strong coordination interaction and ion exchange process. The oleate complexes could control the concentration of free Ln 3+ in solution and thus help to control the growth of the nanocrystals in a dynamical view 43 . In addition, the oleic acid in the hydrothermal solution will limit the growth rate of specific planes of the nanocrystals through interactions with lanthanide ions 44 . In our case, oleate ions are supposed to be selectively adsorbed on the {001} facets of square NaGd(MoO 4 ) 2 nanocrystals. The adsorbed oleate ions will reduce the reactivity of the {001} facets and limit the growth rate along the [001] direction (perpendicular to the {001} planes) of NaGd(MoO 4 ) 2 nanocrystals. Therefore, the more amount oleic acid is added, the more (001) and (001) facets are expressed in the eventual equilibrium morphologies of nanocrystals, which will result in the formation of NaGd(MoO 4 ) 2 square nanoplates. When excess amounts of oleic acid is added (1.75 ml or more in our case), the growth kinetics will be changed. A more reduced crystal growth rate along the [001] directions means a relatively faster growth rate along (001) planes (i.e. along [100] and [010] directions). A faster and faster growth rate along (001) planes will make the growth behavior out of kinetic control and lead to the formation of irregular near-circular nanoflakes. Therefore, the morphology of the as-synthesized NaGd(MoO 4 ) 2 nanocrystals evolves in the sequence of nanocylinders, nanocubes, square nanoplates and irregular nanoflakes with the increase in the oleic acid content.</p><p>Growth mechanism of NaGd(MoO 4 ) 2 nanocrystals. It is hard to observe the crystallization process in the hydrothermal apparatus directly and the growth mechanism of hydrothermally synthesized nanocrystals is generally inferred from the morphology observation and XRD analysis of the products obtained at different reaction time intervals. Taking NaGd(MoO 4 ) 2 square nanoplates as an example, time-dependent morphology evolution and XRD analysis are carried out to disclose the growth mechanism of NaGd(MoO 4 ) 2 nanocrystals. The SEM images and XRD patterns of the products obtained for different reaction time (0, 0.5, 1, 3 and 6 h) with 1.25 ml oleic acid and 10 mmol Na 2 MoO 4 in the initial precursor solutions are presented in Supplementary Fig. S6. Amorphous poor-crystalline precursors were formed in the initial stage before hydrothermal reaction, which can be confirmed by the corresponding SEM images and XRD patterns of the products obtained at 0 h. When the reaction time is prolonged to 0.5 h, some small particles appear in the SEM image (Supplementary Fig. S6b) and a small peak emerges in the XRD pattern, which means crystal nuclei are gradually formed as the hydrothermal reaction proceed. Square nanoplates are found when the reaction time is prolonged to 1 h and some small ones are also found in the SEM image (Supplementary Fig. S6c). Meanwhile the diffraction peaks in the XRD patterns of the products become sharper and stronger, and the peaks fit well with the pure tetragonal phase NaGd(MoO 4 ) 2 . This indicates that the nuclei grow bigger first to form rudiments of NaGd(MoO 4 ) 2 nanocrystals with the morphology of square nanoplates under hydrothermal conditions in the presence of oleic acid. Then some of the rudimental crystals grow even bigger. By and large, the nanocrystals in this stage are not well developed, since some square nanoplates with smaller size are always observed in the SEM image (Supplementary Fig. S6c). With further increasing reaction time, the rudimental NaGd(MoO 4 ) 2 square plates grow bigger and bigger, and the smaller ones disappear gradually in the meantime (Supplementary Fig. S6c-e). This can be deemed as the ripening process of the NaGd(MoO 4 ) 2 nanocrystals. In this stage, large square nanoplates develop even bigger at the expense of smaller ones, driven by the thermodynamic minimization of the surface energies of the nanocrystals. This phenomenon is often observed in the synthesis process of nanocrystals, and universally known as Ostwald ripening 45 . Eventually, uniform and well-crystallized NaGd(MoO 4 ) 2 nanocrystals with regular morphology are formed at the end of the ripening process. A possible growth mechanism is proposed based on the morphology evolution as follows. First the precursor is converted to NaGd(MoO 4 ) 2 nuclei in the nucleation stage under hydrothermal conditions. Subsequently, the NaGd(MoO 4 ) 2 nuclei grow to rudimental NaGd(MoO 4 ) 2 nanocrystals, followed by the Ostwald ripening process until well-crystallized NaGd(MoO 4 ) 2 nanocrystals are formed. In brief, NaGd(MoO 4 ) 2 nanocrystals are formed through "Nucleation → Crystallization → Ostwald ripening" growth process.</p><p>From the above analysis, it can be seen that the amounts of Na 2 MoO 4 and oleic acid have significant effects on the formation of the NaGd(MoO 4 ) 2 nanocrystals, and the NaGd(MoO 4 ) 2 nanocrystals are formed step by step with increasing reaction time under hydrothermal conditions in the growth process. The effects of Na 2 MoO 4 and oleic acid on the formation of NaGd(MoO 4 ) 2 nanocrystals with diverse morphologies, and the growth mechanism of NaGd(MoO 4 ) 2 square nanoplates is summarized and illustrated schematically in Fig. 4.</p><p>Photoluminescence and thermometric properties of NaGd(MoO 4 ) 2 : nanocrystals upon lanthanide (Eu 3+ , Yb 3+ /Er 3+ and Yb 3+ /Tm 3+ ) doping. Morphology-dependent down-shifting photoluminescence of NaGd(MoO 4 ) 2 : Eu 3+ nanocrystals. NaGd(MoO 4 ) 2 : 5% Eu 3+ nanocrystals with the morphologies of nanocubes and square nanoplates with different thicknesses (corresponding to the morphologies shown in Fig. 3b-g) are synthesized, and their morphology-dependent down-shifting photoluminescence properties are investigated in detail. Figure 5a shows the photoluminescence excitation spectra of NaGd(MoO 4 ) 2 : 5% Eu 3+ nanocrystals monitoring at 616 nm. Broad and intense excitation bands lie in the range from 235 to 350 nm, which are referred to charge transfer (C-T) absorption corresponding to the electron transfer from 2p orbit of O 2− to 5d orbit of Mo 6+ within the MoO 4 2− group in the host molybdate. The full widths at half-maximum (FWHM) of the C-T bands are all about 55 nm. These broad and intense C-T excitation bands indicate that the dopant Eu 3+ in NaGd(MoO 4 ) 2 nanocrystals can be excited efficiently by ultraviolet light radiation around 280 nm. Sharp peaks centered at 362, 395 and 465 nm are observed for all spectra shown in Fig. 5a. These peaks are attributed to the characteristic 4f → 4f transitions ( 7 F 0 → 5 D 4 , 7 F 0 → 5 L 6 and 7 F 0 → 5 D 2 ) of Eu 3+ . The asymmetric C-T band can be fitted by two Gaussian peaks, and the fitting curves for spectrum (i) and (vi) in Fig. 5a are depicted in Supplementary Figs S7 and S8. A blue-shift of more than 2 nm in the C-T bands from spectrum (i) to (vi) is observed in the fitting curves. The nanocrystals with a smaller thickness will have a larger energy gap due to quantum confinement effect. The charge transfer band, which is related to the bandgap of NaGd(MoO 4 ) 2 host, is thus shifted towards the higher energy side. On the other hand, the blue-shift of the C-T bands indicate that the bonding energy between the central Mo 6+ and the ligand O 2− becomes stronger with the morphology varying from nanocubes to thin nanoplates. The intensities of C-T bands and other intrinsic peaks of Eu 3+ in excitation spectrum decrease gradually, which result from luminescent quenching effect.</p><p>Figure 5b shows the photoluminescence emission spectra of NaGd(MoO 4 ) 2 : 5% Eu 3+ nanocrystals under excitation of C-T band at 280 nm. The spectra for different morphologies are similar in shape, in which four emission peaks at 592, 616, 655, and 703 nm are associated with 5 D 0 → 7 F J (J = 1, 2, 3, 4) transitions of Eu 3+ . The sharp and intense red emission lines at 616 nm ( 5 D 0 → 7 F 2 transition) suggest that the Eu 3+ dopant ions occupy the sites without inversion symmetry in the host NaGd(MoO 4 ) 2 nanocrystals. What's more, the emission intensity decreases slightly from spectrum (i) to (vi) due to the decreased radiative transition probability caused by surface quenching effect. Compared with the nanocubes, the thin nanoplates are smaller in size and have a larger surface-to-volume ratio. The energy of activator (Eu 3+ ) may be trapped by surface defects, ligands and other quenchers which leads to enhanced surface quenching effect 46 . The luminescence dynamics of NaGd(MoO 4 ) 2 : 5% Eu 3+ nanocrystals at 616 nm for different morphologies are investigated, as shown in Fig. 5c. All the curves show single-exponential decay and can be well-fitted by a single-exponential function I = I 0 exp(− t/τ), where I 0 is the luminescence intensity at t = 0, τ is the lifetime. The lifetimes of Eu 3+ are determined to be 0.672, 0.620, 0.603, 0.592, 0.586 and 0.571 ms for different morphologies from nanocubes to thin square nanoplates, respectively. The lifetime of an excited state depends on the depopulation (radiative or nonradiative transitions) probability of electrons from this excited state. Due to the surface quenching effect, the energy in the upper excited state of Er 3+ easily migrates to the surface and is trapped by the surface defects, ligands or other quenchers, which increases the nonradiative transition probability and therefore reduces the lifetime of the excited state. So the thin square nanoplates with the smallest size and the largest surface-to-volume ratio have the lowest lifetime.</p><p>Upconversion photoluminescence of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ and Yb 3+ /Tm 3+ thin square nanoplates. Upconversion luminescence properties of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ , Yb 3+ /Tm 3+ thin nanoplates are investigated. Figure 6a presents the upconversion luminescence spectra of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ nanoplates with fixed Yb 3+ concentration (10%) and different Er 3+ concentrations (0.5%, 1% and 2%) under 980 nm excitation. Intense green luminescence peaks centered at 530 and 553 nm, and red emission peaks at 657 and 670 nm are observed, which are both characteristic intra-configurational 4f → 4f transitions of Er 3+ . The two green emission peaks at 530 and 553 nm are ascribed to the transitions 2 H 11/2 → 4 I 15/2 and 4 S 3/2 → 4 I 15/2 of Er 3+ , respectively. The red emission peaks correspond to the transition 4 F 9/2 → 4 I 15/2 of Er 3+ . Both the green and red upconversion emissions are split into several subpeaks due to Stark splitting of the upper energy levels. The integral emission intensities of the three samples are depicted in the inset of Fig. 6a. The sample with Er 3+ doping concentration of 1% possesses the highest integral emission intensity, indicating the optimal Er 3+ doping concentration is 1%. At higher Er 3+ doping concentration the upconversion luminescence becomes less efficient, owing to the concentration quenching effect and the cross relaxation between Er 3+ .</p><p>To ascertain the upconversion energy transfer mechanism, investigation of power dependence of upconversion emissions is performed. The double logarithmic plots of green and red upconversion emission intensities versus pump powers for the NaGd(MoO 4 ) 2 : 10% Yb 3+ /1% Er 3+ nanoplates are depicted in Fig. 6b, together with the linear fitting curves. Generally, the number of photons involved in the upconversion process may be inferred from the slopes of the plots 47 . The slope value of the fitting curves for green and red upconversion emissions is 1.96 and 1.86 respectively, revealing two photons are involved in both green and red upconversion processes.</p><p>The proposed upconversion mechanism based on the energy level scheme and power dependence of upconversion luminescence is schematically shown in Fig. 6c. Yb 3+ ions act as sensitizer to absorb energy of 980 nm excitation light and transfer it to activator Er 3+ ions. Electrons in the ground state ( 4 I 15/2 ) of Er 3+ can be excited to 4 I 11/2 state through energy transfer (ET) process from Yb 3+ , and subsequently excited to 4 F 7/2 state through energy transfer upconversion (ETU) process. The states 2 H 11/2 and 4 S 3/2 can be populated by means of nonradiative multiphonon relaxation (MPR) form the state 4 F 7/2 . Radiative transitions from 2 H 11/2 / 4 S 3/2 to the ground state of Er 3+ generate green upconversion emission. For red upconversion emission, there are two ways to populate the upper excited state 4 F 9/2 : MPR process from state 4 S 3/2 and ETU process from state 4 I 13/2 . Then red upconversion emission can be expected by radiative transition from the populated state 4 F 9/2 to the ground state.</p><p>Figure 7a presents the upconversion luminescence spectra of NaGd(MoO 4 ) 2 : Yb 3+ /Tm 3+ nanoplates with fixed Yb 3+ concentration (10%) and different Tm 3+ concentrations (0.5%, 1% and 2%) under 980 nm excitation. Intense emission peaks at 796 nm in the infrared wave range corresponding to the transition 3 H 4 → 3 H 6 of Tm 3+ are observed in all the three spectra. All the three samples exhibit nearly pure near-infrared upconversion luminescence. The upconversion emission intensity of the NaGd(MoO 4 ) 2 : Yb 3+ /Tm 3+ samples decreases with increasing Tm 3+ concentration, which is caused by the concentration quenching effect. The sample doped with 10% Yb 3+ /0.5% Tm 3+ possesses the most intense emission intensity. Some relatively weak emission peaks can also be observed in the visible region of the magnified spectra for the samples doped with 0.5% and 1% Tm 3+ (shown in inset of Fig. 7a). Blue emission peaks at 477 nm and red emission peaks at 649 nm are ascribed to transitions 1 G 4 → 3 H 6 and 1 G 4 → 3 F 4 of Tm 3+ . Since the emission peaks at 796 nm locate in the invisible wave range, the upconversion luminescence appears blue in color to the naked eye.</p><p>Figure 7b depicts the double logarithmic plots of near-infrared upconversion emission intensities versus pump powers for the NaGd(MoO 4 ) 2 : 10% Yb 3+ /0.5% Tm 3+ nanoplates. The slope value of the fitting curves is 1.93 for near-infrared upconversion emissions and 2.30 for near-infrared emission, suggesting that the near-infrared upconversion luminescence belongs to two-photon process, while the blue one involve three-photon absorption. Figure 7c shows the proposed upconversion mechanism and the energy level scheme of NaGd(MoO 4 ) 2 : Yb 3+ /Tm 3+ . There are energy mismatches between the transitions within Yb 3+ ( 2 F 5/2 → 2 F 7/2 ) and Tm 3+ ( 3 H 6 → 3 H 5 , 3 F 4 → 3 F 2 , and 3 H 4 → 1 G 4 ). So the energy transfer between Yb 3+ and Tm 3+ needs the assistance of phonons of the host. State 3 H 5 is populated from 3 H 6 by phonon assisted ET process. Electrons in state 3 H 5 can relax to state 3 F 4 through the MPR process. The phonon assisted ETU process will populate the state 3 F 2, 3 from state 3 F 4 . Electrons in the state 3 F 2, 3 can relax to state 3 H 4 , from which electrons relax radiatively to the ground state generating dominated near-infrared upconversion emission (796 nm). State 1 G 4 might be populated by another phonon assisted ETU process from 3 H 4 to 1 G 4 . Electrons in 1 G 4 state can decay radiatively to either state 3 F 4 or state 3 H 6 , which will cause the blue (477 nm) or red (649 nm) upconversion emissions. For the ET and ETU processes 3 H 6 → 3 H 5 , 3 F 4 → 3 F 2 , and 3 H 4 → 1 G 4 , the number of phonons needed in a similar host NaGd(WO 4 ) 2 is about 2, 3 and 5, respectively 48 . The more the number of phonons is, the lower the probability of energy transfer is. Therefore, it is speculated that the ETU process 3 H 4 → 1 G 4 hardly occurs and the electrons in the 3 H 4 state are likely to relax radiatively to the ground state rather than to be excited to 1 G 4 state through phonon assisted ETU process in our case. This explains why the infrared upconversion luminescence is quite intense compared with the visible upconversion luminescence.</p><p>Thermometric properties of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ thin square nanoplates. Since the emission intensity ratio from two thermally coupled energy levels of lanthanide ions is sensitive to ambient temperature, optical thermometry can be realized in Ln 3+ doped upconversion nanocrystals based on the temperature-dependent upconversion luminescence 49 . Adjacent thermally coupled energy levels 2 H 11/2 and 4 S 3/2 of Er 3+ follow a Boltzmann-type population distribution, and are employed to investigate thermometric properties of NaGd(MoO 4 ) 2 : 10% Yb 3+ /1% Er 3+ nanoplate crystals. Figure 8a shows the temperature-dependent upconversion luminescence spectra (normalized to 1 at the maximum emission value) from 85 to 285 K under excitation of 980 nm. The upconversion luminescence intensity ratio R (I 525nm /I 550nm ) of the two green emission bands increases evidently with increasing ambient temperature.</p><p>The intensity ratio R can be expressed as: , where g, σ, ω are degeneracy, emission cross-section and angular frequency of radiative transitions from the 2 H 11/2 and 4 S 3/2 levels to the ground level 4 I 15/2 ), Δ Ε is the energy gap between the 2 H 11/2 and 4 S 3/2 levels, k B is the Boltzmann constant, and T is the absolute temperature. The dependence of upconversion luminescence intensity ratio R with the temperature is plotted in Fig. 8b. The intensity ratio R varies from 0.0006 to 0.9752 with the temperature increasing from 85 to 285 K. The fitting curve is also presented according to the above equation, which matches well with the experimental data. Δ Ε can be further calculated to be 777.45 cm −1 from the fitting results, which is very close to the experimental energy gap between the two levels.</p><p>The thermometric sensitivity S is defined as the rate of change of R(T) as follows,</p><p>The calculated sensitivity is plotted as a function of absolute temperature in Fig. 8c. The thermometric sensitivity of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ nanocrystals increases with increasing temperature in the temperature range of measurement. Compared with the square plate NaGd(MoO ) 2 : 10% Yb 3+ /1% Er 3+ microcrystals synthesized as previously reported by us (temperature-dependent upconversion luminescence and thermometric sensitivity are shown in Supplementary Figs S9 and S10), the NaGd(MoO 4 ) 2 : 10% Yb 3+ /1% Er 3+ nanocrystals possess a more sensitive thermometric property. The maximum value of sensitivity of the NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ nanocrystals is 0.01333 K −1 at 285 K, which is higher than reported values of many other Yb 3+ /Er 3+ doped materials in a similar temperature range (around 300 K) 23,[49][50][51][52][53][54][55] .</p><p>From the fitted equations for luminescence intensity ratio (R)-Temperature curves for nanocrystals and microcrystals (shown in Fig. 8b and Supplementary Fig. S10a), it is found that, compared with that for microcrystals, both Δ E and the proportionality constant C increase in Equation ( 1 increases, which leads to the enhanced sensitivity as defined in Equation ( 2). This is why nanocrystals have a more sensitive response to temperature, compared with microcrystals.</p><!><p>Pure tetragonal phase, uniform and well-crystallized NaGd(MoO 4 ) 2 nanocrystals with diverse regular morphologies can be selectively synthesized via oleic acid-mediated hydrothermal synthesis method by simply tuning the contents of oleic acid in the precursor solution. (MoO 4 ) 2− ions will be preferentially adsorbed on the {001} facets of tetragonal NaGd(MoO 4 ) 2 , which have a higher packing density of Gd 3+ /Na + ions (0.0364 Å −2 ). Thus, appropriate amount of Na 2 MoO 4 in the precursor solution favor the formation of NaGd(MoO 4 ) 2 nanocrystals with regular morphology. Since oleic acid in the hydrothermal solution helps to control the growth rate of the nanocrystals, especially along [001] directions of tetragonal NaGd(MoO 4 ) 2 , the amount of oleic acid plays a key role in the morphology-controlled synthesis of NaGd(MoO 4 ) 2 nanocrystals. The morphology of the as-synthesized NaGd(MoO 4 ) 2 nanocrystals evolves in the sequence from nanocylinders, nanocubes, square nanoplates to irregular nanoflakes with increasing oleic acid content. Time-dependent morphology evolution and XRD analysis of the products suggest that the growth of NaGd(MoO 4 ) 2 nanocrystals is governed by a "Nucleation → Crystall ization → Ostwald ripening" growth mechanism. Investigation of down-shifting photoluminescence properties confirm that lanthanide dopant in NaGd(MoO 4 ) 2 host nanocrystals can be excited efficiently by broad band ultraviolet light through charge transfer absorption (around 280 nm). Due to quantum confinement effect and stronger bonding energy between the Mo 6+ and ligand O 2− , the charge transfer band has a slight blue-shift, and the intensity is decreased with the morphology of the nanocrystals varying from nanocubes to thin nanoplates. As a result of surface quenching effect, both the down-shifting emission intensity and lifetime of the Eu 3+ doped nanocrystals decrease gradually from nanocubes to thin square nanoplates. On the basis of energy level scheme and pump power dependence of upconversion emissions, the mechanisms for upconversion photoluminescence of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ , Yb 3+ /Tm 3+ nanocrystals are proposed. Two-photon process accounts for both the visible (green and red) upconversion emissions of NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ nanocrystals and the near-infrared upconversion emission of NaGd(MoO 4 ) 2 : Yb 3+ /Tm 3+ nanocrystals. While the blue upconversion emission of NaGd(MoO 4 ) 2 : Yb 3+ /Tm 3+ nanocrystals involves three-photon absorption. Lower probability of phonon assisted ETU process 3 H 4 → 1 G 4 of Tm 3+ lead to nearly pure near-infrared upconversion luminescence of NaGd(MoO 4 ) 2 : Yb 3+ /Tm 3+ nanocrystals. NaGd(MoO 4 ) 2 : Yb 3+ /Er 3+ nanocrystals exhibit excellent thermometric properties with a relatively high sensitivity (0.01333 K −1 at 285 K). NaGd(MoO 4 ) 2 nanocrystals have a more sensitive response to temperature compared with microcrystals. Investigations of photoluminescence and thermometric properties manifest that NaGd(MoO 4 ) 2 nanocrystals are promising candidates for luminescent hosts in luminescent imaging, temperature sensing, color display and other tremendous down-shifting/upconversion applications. Synthesis of NaGd(MoO 4 ) 2 and NaGd(MoO 4 ) 2 : Eu 3+ , Yb 3+ /Er 3+ and Yb 3+ /Tm 3+ nanocrystals. A predetermined amount of oleic acid was added into 10 ml ethanol. After vigorous stirring for 30 min, 0.5 ml Gd(NO 3 ) 3 solution (0.5 mmol) and a certain amount of Na 2 MoO 4 solution were added into the above solution under continuous stirring. After additional agitation for 1 h, the as-obtained translucent precursor solution (total volume 41 ml) was transferred into a 60 ml Teflon-lined stainless steel autoclave, which was then sealed and maintained at 180 °C for 12 h. The final precipitate products were collected by centrifugation, washed several times with deionized water and ethanol, and dried at 50 °C for 5 h in air.</p><!><p>NaGd(MoO 4 ) 2 : Eu 3+ , Yb 3+ /Er 3+ , Yb 3+ /Tm 3+ nanocrystals were synthesized following a similar procedure except for introducing the proper amount of corresponding lanthanide nitrates to the precursor solution as described above.</p><p>Characterization. Powder X-ray diffraction (XRD) was performed on a Rigaku Smartlab diffractometer with Cu Kα radiation at a scanning rate of 10° min −1 . Scanning electron microscope (SEM, FEI Quanta 400F) and transmission electron microscope (TEM, FEI Tecnai G2 F30) were employed for the observation of the morphology. Energy dispersive X-ray spectroscopy (EDS) data were obtained using the SEM equipped with the energy dispersive X-ray spectrometer. TEM images, high-resolution TEM (HRTEM) images and selected-area electron diffraction (SAED) patterns were performed at an accelerating voltage of 300 kV. Fourier transform infrared (FTIR) spectra were obtained in transmission mode on a Bruker Equinox 55 FTIR spectrometer with the samples sandwiched between two KBr plates. Photoluminescence excitation and emission spectra were recorded on an Edinburgh FLSP920 spectrometer equipped with a 980 nm diode laser, a 450 W continuous xenon lamp and a 60 W microsecond flash lamp as excitation sources and a R928 red-sensitive photomultiplier tube as detector. The samples were annealed at 500 °C for 1 h prior to upconversion luminescence measurements. All the measurements were performed at room temperature except for the thermometric upconversion photoluminescence.</p>

Scientific Reports - Nature

Efficient 18F-Labeling of Synthetic Exendin-4 Analogues for Imaging Beta Cells

A number of exendin derivatives have been developed to target glucagon-like peptide 1 (GLP-1) receptors on beta cells in vivo. Modifications of exendin analogues have been shown to have significant effects on pharmacokinetics and, as such, have been used to develop a variety of therapeutic compounds. Here, we show that an exendin-4, modified at position 12 with a cysteine conjugated to a tetrazine, can be labeled with 18F-trans-cyclooctene and converted into a PET imaging agent at high yields and with good selectivity. The agent accumulates in beta cells in vivo and has sufficiently high accumulation in mouse models of insulinomas to enable in vivo imaging.

efficient_18f-labeling_of_synthetic_exendin-4_analogues_for_imaging_beta_cells

Introduction<!>Results and Discussion<!>General<!>3-maleimido propanamide-tetrazine (maleimido-Tz) 3<!>E4Tz12 5<!>18F-E4Tz12\n7<!>Cell lines<!>Western Blot<!>Mice<!>Whole pancreas islet imaging<!>18F-E4Tz12\n7 biodistribution studies<!>MicroPET-CT imaging<!>Modeling

<p>The ability to visualize beta cells noninvasively could have far reaching implications for both biomedical research and clinical practice. Progressive loss of functional beta cell mass (BCM) is the underlying cause of autoimmune type 1 diabetes mellitus, and is also responsible for the secondary failure of clinical drugs in type 2 diabetes. It is widely believed that noninvasive imaging of beta cells could ultimately facilitate not only our understanding of the natural history of islet formation but also the pathophysiology of diabetes. In turn, we would have the capability to diagnose diabetes earlier, monitor the efficacy of widely used drugs, as well as advance the discovery of new therapies. Furthermore, beta cell-specific imaging approaches could be used to diagnose and localize insulinomas and aid the assessment of transplanted islets or pancreata.</p><p>In a previous report, we described the development and validation of near infrared fluorescent exendin-4 analogues for imaging beta cells at single cell resolutions,[1[ and for fiberoptic, endoscopic or intraoperative imaging.[2[ We showed that one lead agent, derived from exendin-4 (E4K12-FL), had sub-nanomolar EC50 binding concentrations and high specificity. In addition, its binding could be inhibited by glucagon-like peptide 1 (GLP-1) receptor agonists. Following intravenous administration to mice, pancreatic islets could be readily distinguished from exocrine pancreas, achieving target-to-background ratios of 6:1. Serial imaging subsequently revealed rapid accumulation kinetics (with initial signal in the islets detectable within 3 min and peak fluorescence occurring within 20 min of injection). Such properties make this an ideal agent for in vivo imaging. Together with other reports of various exendins labeled with chelates,[3–9] we hypothesized that 18F-labeled exendin-4 analogues could be used for noninvasive imaging with positron emission tomography-computed tomography (PET–CT). While two approaches of 18F-labeling have been recently reported,[10, 11[ the 18F-conjugation methods used in these studies do not appear to have been used in concert with removal of unreacted material via bioorthogonal scavenging resins.[12[ In this study, we started with a cysteine (C12) version of our previously validated exendin-4 (E4K12), by exchanging the lysine at position 12 with a cysteine. Using bioorthogonal labeling strategies employing 18F-trans-cyclooctene (18F-TCO) and tetrazine (Tz) modified molecules,[13–15] we report the facile synthesis and purification of 18F-labeled exendin-4. The described reaction demonstrated fast reaction times (20 min), high purity as well as specific activity. Given that the ultimate goal is to translate this technology to the clinic, a lead 18F-labeled compound was subsequently applied to PET–CT imaging of insulinoma in a mouse model. Pharmacokinetic modeling, the plasma clearance and tracer-uptake data obtained from these experiments were subsequently used for extrapolation to humans.</p><!><p>We previously demonstrated that modification of the exendin-4 amino acid sequence at position 12 does not result in perturbation of the molecule's intrapancreatic binding, selectivity or specificity for the GLP-1 receptor. In order to translate this finding into a noninvasive 18F-PET probe, we designed the cysteine-tetrazine (Tz) cross-linker, maleimide-Tz 3 (Scheme 1). The compound was synthesized from the literature-known Tz amine 1[16[ and the maleimide-NHS ester 2[17[ in 68% isolated yield. This crosslinker readily reacted with E4C12, an exendin-4-related peptide in which the natural lysine at position 12 (K12) was exchanged for a cysteine (C12) yielding the bioorthogonally reactive Tz-labeled peptide E4Tz12 5. Figure 1 shows liquid chromatography-mass spectrometry (LC–MS) traces of both maleimide-Tz 3 (Figure 1A) and E4Tz12 5 (Figure 1B), which confirm the identities of the cold precursors.</p><p>Similar to the techniques used for small-molecule radiolabeling, we subsequently incubated E4Tz12 5 with 18F-trans-cyclooctene (18F-TCO) 6.[12[ The radiolabeled bioorthogonally reactive prosthetic group 18F-TCO 6 was synthesized in 46% decay-corrected radiochemical yield (dcRCY) by nucleophilic substitution of the tosylate precursor with 18F-fluoride in the presence of tetrabutylammonium bicarbonate (TBAB), as previously described.[13[ 18F-TCO 6 (>94% pure after HPLC purification) and E4Tz12 5 were then combined in dimethyl sulfoxide (DMSO; 1000 μCi [37 MBq] and 5.5 nmol, respectively) and stirred vigorously for 20 min before yielding a mixture of 18F-E4Tz12 7 and unlabeled 5. Removal of 5 with TCO-modified scavenger resin[12[ followed by centrifugal filtration, provided desired 18F-E4Tz12 7. Simple dilution with phosphate buffered saline (1 × PBS) afforded the material ready for injection. The octanol/water and octanol/1 × PBS partition coefficients (logP) were determined and found to be −1.56 ± 0.06 and −1.75 ± 0.07, respectively, indicating good water solubility.</p><p>The blood half-life of 18F-E4Tz12 7 was determined through serial retro-orbital bleeds, and the individual data points were then fitted using a biexponential decay curve. This resulted in a weighted half-life (t1/2) for 18F-E4Tz12 7 of 6.8 min [t1/2(slow) = 26.8 min (20%); t1/2(fast) = 1.9 min (80%); R2 of 0.991; Figure 2A]. Biodistribution of 18F-E4Tz12 7 showed dominant renal and hepatobiliary excretion of the compound, with the majority accumulating in the kidneys (17.8 ± 0.6% injected dose per milligram [% IDg−1]), urine, and bowel. Data from dynamic microPET scans generated time–activity curves (Figure 2C) which support the ex vivo excretion profiles. Tissue levels of the compound were highest in the lungs (4.1 ± 1.5% IDg−1) and pancreas (1.2 ± 0.1% IDg−1); although, uptake of 18F-E4Tz12 7 was found to be significantly lower in the pancreata of mice that had been preinjected with cold exenatide (Byetta®, 0.36 ± 0.05% IDg−1). Accumulation in the bone was low (0.6 ± 0.1% IDg−1), indicating minimal defluorination of 18F-E4Tz12 7.</p><p>To determine the intra-pancreatic distribution of the compound (islets of Langerhans comprise only 1–2% of the pancreatic mass), we performed autoradiography. We injected 18F-E4Tz12 7 (92 ± 12μCi [3.40 ± 0.44 MBq]) via tail vein into transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of the mouse insulin promoter [mouse insulin promoter (MIP)-green fluorescent protein (GFP)].[18[ After 3 h, the mice were euthanized, and their pancreata excised. The pancreata were then imaged using surface reflectance imaging (to show the islet distribution) before being exposed for autoradiography (to show the distribution of 18F-E4Tz12 7). Figure 3 shows good colocalization between the fluorescence of the GFP islet and the autoradiographic signal from 18F-E4Tz12 7 with a Pearson's coefficient of 0.83 ± 0.04 (Rcoloc.). Based on micro-dissected specimens and target-to-background ratios, we calculated a concentration of approximately 40% IDg−1) in the islets.</p><p>To determine the utility of 18F-E4Tz12 7 for insulinoma detection, we tested it in different murine models: NIT-1, 916-1 or WTRT2 mouse insulinoma xenografts. These cell lines were chosen for their elevated GLP1R expression as verified by Western blot (Figure 4C). For tumors, uptake values of 2.5 % IDg−1 (916-1), 2.0 % IDg−1 (WTRT2) and 0.7 % IDg−1 (NIT-1) were obtained, which allowed them to be detected by whole body PET imaging (Figure 4A and B). Tumor-to-muscle ratios from ex vivo scintillation counting data were 13.4, 10.5, and 14.6 for 916-1, WTRT2 and NIT-1, respectively. In all cases, preinjection of cold exenatide (250 μL, 60 μM) resulted in a significant reduction of the standard uptake values (916-1: 82% reduction; WTRT-2: 54% reduction; NIT-1: 62% reduction). In contrast, muscle standard uptake values were not affected by preinjection with cold exenatide (0.11% IDg−1) This confirms the applicability and selective uptake of 18F-E4Tz12 7 as a targeted probe for GLP-1 receptor-rich tissues.</p><p>Ultimately, these agents are being developed for their clinical application. While their clearance is very rapid in mice (80% with a 1.9 min half-life and 20% with a 26.8 min half-life for 18F-E4Tz12 7), we were interested in determining the optimal clearance kinetics in humans. A compartmental pharmacokinetic model was thus developed to extrapolate our results from mice. The advantage of this model is that some of the parameters (e.g., plasma clearance) that vary between species can be scaled up, while others (e.g., the binding rate constants and radioactive decay half-life) are kept constant.</p><p>Using clinical data available for exenatide, the plasma concentration after continuous infusion[19[ was fit to a two-compartmental model, in order to predict the percent clearance of a bolus imaging dose. The results indicated that 73% of the imaging agent dose redistributes to peripheral tissues with a rapid 1 min half-life, while the remaining 27% clears with a 63 min half-life. This is close to the percent clearance observed with inulin in humans following an intravenous bolus injection (76% with a 10 min half-life and 24% with a 86 min half-life[20[); the model therefore provides a reasonable estimate of clearance.</p><p>The exchange rate of the compound between the plasma and extracellular space was subsequently estimated from literature values[21[ and adjusted to fit our experimental results in mice (Figure 5A). The results in Figure 5B show estimates of human uptake and clearance, based on clinical data, and using mechanistic rate constants from mice. In both cases, the specific uptake of the compound in islets is significantly higher than in the exocrine pancreas due to its specific target binding.</p><p>The GLP-1 receptor is highly expressed in beta cells within the islets of Langerhans as well as in functioning beta cell islet tumors (insulinomas). GLP-1 analogues are a new class of peptide-based drugs used for the treatment of diabetes. Exenatide, the first FDA approved GLP-1 analogue, is a synthetic version of exendin-4. It is a 39-amino acid peptide isolated from the saliva of the Gila monster (Heloderma suspectum) and contains 53% sequence homology with GLP-1. A recent crystal structure of the extracellular domain of the GLP-1 receptor showed the binding mode of exendin-4 (amino acids 9-39).[22[ From this crystal structure, it was clear that lysine 12 (K12) is not involved in binding to the GLP-1 receptor domain. Moreover, it explains why K12-modified exendins retain high affinity for the receptor.[1,2] Our results further demonstrate that K12 modification with tetrazines are not only stable but allow rapid site-specific and high-yield fluorinations. Tetrazine functionalization of the peptide also allows removal of unreacted starting material with the complimentary trans-cyclooctene beads, an option not available to other current 18F or metal chelation labeling strategies. The resulting compounds exhibit appropriate pharmacokinetics for PET imaging of beta cells in a mouse model.</p><p>In an effort to predict the compound's kinetics in humans, we applied pharmacokinetic modeling and allometric scaling.[23,24] In the mouse, the synthesized compound had a weighted half-life (t1/2) of 6.8 min. Using our modeling and scaling approach, we predicted a t1/2 value of 18 min in human. Importantly, we found that this molecule size has a beta phase clearance half-life of 63 min. Agents with clearance rates that are much slower than the radioactive half-life could have a background that is too high during the imaging window. Conversely, agents that clear much faster than the radioactive half-life could have inefficient accumulation within the target tissue. Given that the decay of 18F is 109.8 min, our modeling indicates that this compound would have close to ideal clearance for human imaging. The pharmacokinetic modeling also indicates that further improvements in linker modification could reduce exocrine uptake and improve detection sensitivity. For example, by using bioorthogonal chemistry, which allows facile modulation of the linkers, further improvements in the reaction kinetics, stability and biocompatibility of the compound could be achieved.[25,26]</p><!><p>Unless otherwise noted, all reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification. Exendin-4 (exenatide, Byetta®) was obtained from Amylin/Eli Lilly (San Diego, CA, USA). E4C12 (4163 g mol−1; HGEGTFTSDLSCQMEEEAVRLFIEWLKNGGPSSGAPPPS) was obtained from Genscript (Piscataway, NJ, USA). [18F]-Fluoride (n.c.a.) was purchased from PETNET Solutions (Woburn, MA, USA). 3-maleimido-propanoic acid succinimidyl ester 1, tetrazine (Tz) amine 2 and 18F-trans-cyclooctene (18F-TCO) 4 were synthesized as described elsewhere.[13,16,17,27] High performance liquid chromatography–electro-spray ionization mass spectrometry (HPLC–ESI-MS) analyses and HPLC purifications were performed on a Waters LC-MS system (Milford, MA, USA). For LC–ESI-MS analyses, a Waters XTerra® C18 5 μm column was used. For preparative runs, an Atlantis® Prep T3 OBD™ 5 μm column was used. High-resolution ESI mass spectra were obtained on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) in the Department of Chemistry Instrumentation Facility at the Massachusetts Institute of Technology. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian AS-400 (400 MHz) spectrometer. Chemical shifts for protons are reported in parts per million (ppm) and are referenced against the [D6]acetone lock signal (1H, 2.05 ppm). NMR data are reported as follows: chemical shift, multiplicity (s=singlet, d = doublet, t = triplet, m = multiplet), coupling constants (Hz) and integration.</p><!><p>A solution of 3-maleimido-propanoic acid succinimidyl ester 1 (5 mg, 19 μmol, 20 mg mL−1, 250 μL in dimethylformamide (DMF) was added to a solution of Tz amine 2 (3.5 mg, 19 μmol) and Et3N (5.3 μL) in MeCN (1 mL), and the resulting reaction mixture stirred at RT for 1 h. Volatiles were removed in vacuo and the crude product purified using HPLC to give compound 3 as a pink solid (4.4 mg, 13 μmol, 68%): 1H NMR (400 MHz, [D6]acetone): δ = 10.43 (s, 1 H), 8.52 (d, 3JHH = 8.3, 2H), 7.78 (m, 1 H), 8.58 (d, 3JHH = 8.2, 2H), 6.86 (s, 2H), 4.52 (d, 3JHH = 6.0, 2H), 3.80 (t, 3JHH = 7.4, 2H), 2.59 ppm (t, 3JHH = 7.4, 2 H); LC–ESI-MS(+): m/z (%): 339.2 (100) [M + H]+, 677.4 (29) [2M + H]+; LC–ESI-MS(−): m/z (%): 337.1 (29) [M − H]−, 383.1 (100) [M + HCOO]−, 721.3 (27) [2M + HCOO]−; HRMS-ESI: m/z [M − H]+ calcd for [C16H14N6O3Na]+ 361.1020, found 361.1013 [M + Na]+.</p><!><p>A solution of maleimido-Tz 3 (50 μL 10 mM) in DMF was added to a solution of E4C12 4 (3.0 mg, 0.7 μmol) in 1 × PBS (1000 μL), and the resulting solution was stirred at RT for 3 h. The reaction mixture was purified using an Amicon® Ultra 3 kDa centrifugal filter (Millipore, Carrigtwohill, Ireland) before being subjected to HPLC purification, yielding compound 5 as a rose-colored solid (0.8 mg, 0.2 μmol, 29%): LC–ESI-MS(+): m/z (%): 1125.9 (100) [M + 4H]4+, 1501.3 (51) [M + 3H]3+; LC–ESI-MS(−): m/z (%): 1498.7 (100) [M − 3H]3−.</p><!><p>2-[18F]-(E)-5-(2-Fluoroethoxy)cyclooct-1-ene (18F-TCO) was prepared in a similar manner to previously described procedures[13[ employing a Synthra RN Plus automated synthesizer (Synthra GmbH, Hamburg, Germany) operated by SynthraView software in an average time of 102 min. The synthesizer reagent vials were filled with the following: A2 with MeCN (350 μL), A3 with (E)-2-(cyclooct-4-enyloxy)ethyl 4-methylbenzenesulfonate (2.0 mg, 12.3 μmol) in DMSO (400 μL), A5 with MeCN (150 μL), and B2 with H2O (800 μL). The starting activity well was filled with [18F]-F− (n.c.a.) (2072 MBq, 56 ± 15 mCi) in H218O (500–1000 μL), tetrabutylammonium bicarbonate (TBAB, 250 μL, 75 mM in H2O), and MeCN (200 μL). The [18F]-F−/TBAB solution was transferred to the reaction vessel and dried by azeotropic distillation with MeCN. After drying, TCO-tosylate (2 mg, 15 mM) in DMSO was added and heated to 90°C for 10 min. After cooling to 30°C, the mixture was filtered through an Alumina-N cartridge (100 mg, 1 mL, Waters) into reaction vessel 2. The Alumina-N cartridge was washed with MeCN (150 μL) and the combined filtrates were then diluted with H2O (800 μL). This solution was subsequently subjected to preparative HPLC purification (MeCN/H2O, 50:50). 18F-TCO was collected (tR=13.5 min) in 5–6 mL of solvent and isolated by manual C18 solid phase extraction. It was then eluted with DMSO (2 × 450 μL) to give 10.1 ± 5.9 mCi of 18F-TCO in 46.1 ± 12.2% (n = 4) decay-corrected radiochemical yield (dcRCY) in an average time of 102 min (once drying of [18F]-F− (n.c.a.) had ended). Analytical HPLC demonstrated >94% radiochemical purity of 18F-TCO.</p><p>E4Tz12 5 (5.5 nmol, 1 mM in DMSO) was added to the 18F-TCO 6 in DMSO. After stirring at RT for 20 min, TCO-beads (150 uL suspension of 10 mg mL−1; TCO loading: 13 nmol mg−1) were added to the mixture and stirred for 20 min. The reaction mixture was filtered using an Amicon® Ultra 3 kDa centrifugal filter (Millipore, Carrigtwohill, Ireland) to give 18F-E4Tz12 7 (1.8 ± 0.9 mCi, 46.7 ± 17.3% (n = 4) dcRCY).</p><p>18F-E4Tz12 7 (approx. 14 μCi [0.52 MBq]) in DMSO/1 × PBS (4:1, 5 μL) was added to octanol (500 μL) and H2O (MilliQ, 500 μL) in a 1.5-mL microcentrifuge tube. The mixture was vortexed for 1 min at RT and centrifuged (15 000 rpm, 5 min). After centrifugation, 100-μL aliquots of both layers were measured using a γ-counter. The experiment was carried out in quintuplicate. This experiment was repeated with octanol/1 × PBS (1:1, 1000 μL).</p><!><p>We chose three different insulinoma tumor cell lines (NIT-1, WTRT2, 916-1), to correlate imaging findings and to elucidate how 18F-E4Tz12 behaves in different insulinoma tumor environments. Both WTRT2 and 916-1 were generously provided by Johanna Joyce (Memorial Sloan–Kettering Cancer Center, New York City, USA). NIT-1 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). WTRT2 and 916-1 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum (10%), L-glutamine, penicillin (100 I.U.), and streptomycin (100 μg mL−1). NIT-1 were cultured in F-12K medium (Kaighn's Modification of Ham's F-12 Medium, ATCC, Manassas, VA) supplemented with fetal bovine serum (10%), sodium bicarbonate (2%), L-glutamine, penicillin (100 I.U.), and streptomycin (100 μg L−1). All cell lines were cultured at 37°C and 5% CO2.</p><!><p>916-1, WTRT2, and NIT-1 cells seeded into six-well plates were washed twice with ice-cold 1 × PBS and lysed on ice for 10 min with ice-cold RIPA lysis buffer (100 μL) supplemented with a 100-fold dilution of protease inhibitor cocktail for mammalian cells (Sigma–Aldrich). The lysate was centrifuged (10 min, 10000 rcf) and the supernatant collected. Protein concentrations were determined using bicinchoninic acid (BCA) protein assays (Pierce, Rockford, IL, USA). Cell lysates (10 μg) were subjected to SDS-PAGE, followed by immunoblotting using anti-GLP-1R antibody (#39072, Abcam, Cambridge, UK), goat-anti-rabbit secondary (Jackson ImmunoResearch, West Grove, PA, USA), and detection with chemiluminescence (Picowestern ECL substrate, Pierce). Blots were stripped using Restore Stripping Buffer (Thermo Scientific), labeled with anti-GAPDH antibody (AF 5718, R&D Systems) followed by detection with chemiluminescence.</p><!><p>Experiments were performed in Nu/Nu mice (from Massachusetts General Hospital, Boston, MA; for tumor implantations and imaging; n = 6), C57BL/6 (B6) mice (from The Jackson Laboratory, Bar Harbor, ME; for biodistribution and pharmacokinetics; n = 8), or B6.Cg-Tg(Ins1-EGFP)1Hara/J mice (from The Jackson Laboratory, Bar Harbor, ME; for autoradiography/surface reflectance imaging; n = 3).[18[ B6.Cg-Tg(Ins1-EGFP)1Hara/J mice express the enhanced green fluorescent protein (eGFP) in the islets under the control of the mouse insulin 1 promoter (MIP-GFP). For all surgical procedures and imaging experiments, mice were anesthetized with 2.0% isoflurane in O2 at 2.0 L min−1. For imaging experiments lasting longer than 1 h, the isoflurane flow rate was reduced to ~1.0% isoflurane in O2 at 2.0 L min−1. Surgeries were conducted under sterile conditions with a zoom stereomicroscope (Olympus SZ61). All procedures and animal protocols were approved by the Massachusetts General Hospital subcommittee on research animal care.</p><!><p>B6.Cg-Tg(Ins1-EGFP)1Hara/J (MIP-GFP) mice[18[ were administered 18F-E4Tz12 7 (92 ± 12 μCi [3.40 ± 0.44 MBq]) via intravenous tail-vein injection, and the GPL-1 receptor-specific probe was allowed to accumulate and clear for 3 h. Mice were then euthanized, their organs perfused using 1 × PBS (30 mL) and the pancreata harvested. They were subsequently weighed and placed between two glass cover slides using a 1 mm rubber gasket, maintaining a constant thickness. Initially, fluorescence reflectance was recorded by imaging the entire pancreas on an OV110 epifluorescence imager (Olympus America, Center Valley, PA, USA). The pancreata were then transferred to an autoradiography phosphor imaging plate (SI, Molecular Dynamics) and exposed at −20°C for 12 h before the plate was analyzed using a Typhoon scanner (GE Healthcare). Image analysis was conducted using ImageJA 1.45 software.</p><!><p>C57BL/6 (B6) mice were used for blood half-life determinations. Mice were administered 18F-E4Tz12 7 (68 ± 12 μCi [2.52 ± 0.44 MBq]) by intravenous tail-vein injection. Blood sampling was performed by retro-orbital puncture using tared, heparinized capillary tubes. Samples were subsequently weighed and activity measured using a Wallac Wizard 3″ 1480 Automatic Gamma Counter (PerkinElmer). Blood half-life data were fitted to a biexponential model using Graphpad Prism 4.0c software (GraphPad Software Inc., San Diego, CA), and results were reported as the weighted average of the distribution and clearance phases. For biodistributions, (B6) mice were intravenously injected via tail vein with 18F-E4Tz12 7 (131 ± 18 μCi [4.85 ± 0.67 MBq]). Animals were euthanized at 3 h and their organs perfused using 1 × PBS (30 mL). Tissues were subsequently harvested, weighed and their radioactivity counted using a Wallac Wizard 3″ 1480 Automatic Gamma Counter. Statistical analysis was performed using Graphpad Prism 4.0c.</p><!><p>Mice were imaged by PET-CT using an Inveon small animal microPET scanner (Siemens Medical Solutions). Mice were injected with 18F-E4Tz12 7 (557 ± 38 μCi [20.61 ± 1.41 MBq]) via tail-vein injection under isoflurane anesthesia (see above). Acquisition for static microPET images started 2 h post injection and acquisition took approximately 30 min. For dynamic microPET imaging, mice were injected approximately 30 s after the start of microPET acquisition, and data was collected for 2 h. The radioactivity concentration for a tissue was determined by measuring within regions of interest (ROIs) for a given tissue with the units of Bq mL−1 min−1. A tissue density of 1 g mL−1 was assumed and ROIs were converted to Bq g−1 min−1 and divided by the injected activity to obtain an imaging ROI-derived % IDg−1. For GLP-1 receptor blocking experiments, unlabeled exenatide (250 μL, 60 μM) was preinjected 45 min prior to injection of 18F-E4Tz12 7. A high-resolution Fourier rebinning algorithm was used, followed by a filtered back-projection algorithm using a ramp filter, to reconstruct 3D images without attenuation correction. The image voxel size was 0.796 × 0.861 × 0.861 mm, for a total of 128 × 128 × 159 voxels. Peak sensitivity of the Inveon accounts for 11.1 % of positron emission, with a mean resolution of 1.65 mm. The total counts acquired was 600 million per PET scan. Calibration of the PET signal with a cylindrical phantom containing 18F was performed before all scans. CT images were reconstructed using a modified Feldkamp reconstruction algorithm (COBRA) from 360 cone-beam X-ray projections (80 kVp and 500 μA X-ray tube). The isotropic voxel size of the CT images was 60 μm. The reconstruction of data sets, PET-CT fusion, and image analysis were performed using Inveon Research Workplace (IRW) software (Siemens). 3D visualizations were produced using a digital imaging and communications in medicine (DICOM) viewer (OsiriX Foundation, Geneva, Switzerland).</p><!><p>A compartmental model was used to extrapolate results from mouse-imaging studies to humans. The model includes biexponential loss from the plasma compartment (due to redistribution and clearance), and separate compartments for the endocrine and exocrine pancreas. Exchange with the endocrine tissue (islets) was estimated as a function of the vascular surface area-to-volume ratio (measured at 505 ± 146 cm−1 using CD31 stained histology slides),[28[ and permeability was estimated at 30 μm s−1 (for this sized molecule in the fenestrated capillary bed).[21[ Exocrine pancreas was modeled in a similar manner, while the exchange parameters were adjusted to fit experimental data. Within the compartments, the imaging agent is able to bind the target, dissociate, internalize, and be degraded and washed out.[24[ These rate constants were assumed constant between species. For plasma clearance in humans, the rate constants for exchange and clearance from a two-compartmental model were fit to experimental data taken from patients undergoing an intravenous infusion of exenatide[19[ using a least-squares fitting algorithm in Matlab (Mathworks, Natick, MA, USA). Estimates for humans were obtained by entering the plasma clearance values from human clinical data into the model together with the microscopic transport rates obtained from mouse experiments.</p>

PubMed Author Manuscript

Evaluation of a commercial liquid-chromatography high-resolution mass-spectrometry method for the determination of hepcidin-25

IntroductionReliable determination of hepcidin-25, a key regulator of iron metabolism, is important. This study aimed at evaluating the performance of the Hepcidin-25 Liquid Chromatography-Tandem Mass-Spectrometry (LC-MS/MS) Kit (Immundiagnostik AG, Bensheim, Germany) for quantification of the hepcidin-25 protein.Materials and methodsPrecision, accuracy, linearity, and preanalytical requirements of the liquid-chromatography high-resolution mass-spectrometry (LC-HR-MS) method were evaluated. The imprecision and bias acceptance criteria were defined ≤ 15%. We investigated sample stability at room temperature (RT) and after repeated freeze and thaw cycles. Additionally, we assessed serum hepcidin-25 concentrations of 165 healthy adults referred for a medical check-up.ResultsThe hepcidin-25 LC-MS/MS assay was linear over the concentration range of 3 – 200 ng/mL. Within- and between-run precision ranged between 1.9 – 8.6% and 5.1 – 12.4%, respectively. The mean bias of the low and high control material was - 2.7% and 2.1%, respectively. At RT, serum samples were stable for 3 h (mean bias + 0.3%). After two and three freeze and thaw cycles, hepcidin-25 concentrations showed a bias of + 8.0 and + 20%, respectively. Of 165 healthy adults, 109 females had a significantly lower median of 8.42 (range: 1.00 – 60.10) ng/mL compared to 56 males with 15.76 (range: 1.50 – 60.50) ng/mL (P = 0.002).ConclusionsThe hepcidin-25 LC-MS/MS kit shows a broad analytical range and meets the imprecision and bias acceptance criteria of ≤ 15%. Serum samples can be stored at RT for 3 h and resist up to two freeze and thaw cycles.

evaluation_of_a_commercial_liquid-chromatography_high-resolution_mass-spectrometry_method_for_the_de

Introduction<!>Subjects<!>Chemicals<!>Instrumentation and conditions<!><!>Preparation of calibration<!>Sample preparation<!>Assay evaluation<!>Analyte stability measurements<!>Statistical analysis<!>Analytical performance of the hepcidin-25 assay<!><!>Analytical performance of the hepcidin-25 assay<!>Pre-analytical analyses<!><!>Hepcidin-25 serum concentrations<!>Discussion<!>

<p>In recent years, hepcidin-25, an essential key regulator of human iron homeostasis, has gained substantial attention. This cysteine-rich acute-phase protein, which consists of 25 amino acids, is synthesized in the liver and excreted by the kidneys (1, 2). The hepatic synthesis of hepcidin-25 is induced by iron loading or inflammation and inhibited by erythropoiesis (3, 4).</p><p>Hepcidin-25 lowers circulating iron in the bloodstream by binding to and downregulating the cellular iron efflux channel ferroportin, which is highly expressed in duodenal enterocytes and macrophages of the reticuloendothelial system (5, 6). Increased serum hepcidin-25 concentrations decrease the enteral iron absorption and the release of stored iron from macrophages and hepatocytes (4). Conversely, suppressed hepcidin-25 production enhances intestinal iron absorption and the ability of the reticuloendothelial system to export recycled iron from senescent erythrocytes (4, 7).</p><p>As hepcidin-25 is a promising biomarker in the assessment of the human iron status, the quantitative analysis of this parameter is of great interest. In clinical practice, the establishment of various diagnostic tools (i.e. immunoassays, liquid chromatography-tandem mass-spectrometry (LC-MS/MS)) showed substantial differences in absolute hepcidin-25 concentrations and reproducibility of results between routine laboratories (8). Although no gold standard procedure for hepcidin-25 measurements has been defined yet, LC-MS/MS has been proposed to be more specific and sensitive compared to immunoassays (8-10).</p><p>The LC-MS/MS method is a powerful and valuable tool, which has become a widely used technique for quantitative determination of small molecules (i.e. steroid hormones) (11). High specificity, precision and flexibility, together with the potential of simultaneous determination of many different target compounds are the main advantages of this method (12). However, at present clinical laboratories rarely use LC-MS/MS for the quantitation of proteins in daily practice. Large molecule size and the complexity of the matrix are challenges for accurate quantification (10).</p><p>Beside the triple-quadrupole mass-spectrometry (QQQ-MS) with low resolution, there are also high-resolution (HR) instruments with ion trap MS available, which may include q-Orbitrap-MS (Q-Orbi-MS) and q-time-of-flight-MS (Q-TOF-MS). High-resolution MS is more applicable for analysis of intact peptides and proteins compared to quadrupole-instruments working in unit resolution (13, 14). Furthermore, HR-MS instruments have the advantage that data can be acquired also in full-scan mode, allowing retrospective search for compounds, not initially targeted.</p><p>Currently, the Hepcidin-25 LC-MS/MS Kit (Immundiagnostik AG, Bensheim, Germany) and the Hepcidin-25 LC-MS/MS Kit (Li StarFish, Cernusco, Italy) are commercially available to facilitate the quantification of this protein biomarker via LC-MS/MS in clinical research. However, a thorough evaluation of the analytical performance by the use of an HR-MS instrumentation is recommended before these assays can be used in clinical routine. The aim of the present study was to evaluate the precision, accuracy, linearity, the limit of detection (LoD), and the limit of quantification (LoQ) of the Hepcidin-25 LC-MS/MS Kit from Immundiagnostik AG with a HR-MS method. Additionally, we performed a stability study evaluating sample storage at room temperature (RT), repeated freeze and thaw cycles, and the auto-sampler stability.</p><!><p>For the performance evaluation of the Hepcidin-25 LC-MS/MS Kit (Immundiagnostik AG, Bensheim, Germany), remaining blood samples (serum) from routine analysis of 165 ambulatory healthy adults, who were referred to the outpatient clinic of the Institute of Clinical Chemistry and Laboratory Medicine of the General Hospital Steyr (Steyr, Austria) for a medical check-up of the iron status, were used. The study period was from January to June 2017. A total number of 109 individuals (66%) were females and 56 (34%) were males. The median age was 43 (range: 15 – 90) years. All participants provided their written informed consent. They underwent blood sampling after an overnight fasting state (12h) in the morning between 08:00 and 10:00 a.m. Four mL VACUETTE® Z Serum Clot Activator tubes (Greiner Bio-one International GmbH, Kremsmünster, Austria) were used for blood draw from a peripheral vein. Serum samples were centrifuged at 1800xg for 10 minutes at RT and immediately analysed after blood draw. All 165 serum samples were used to evaluate and compare hepcidin-25 medians between females and males.</p><p>The study was approved by the Ethical Committee of the Johannes Kepler University Linz (Linz, Austria) and carried out in accordance with the current version of the Declaration of Helsinki.</p><!><p>The Hepcidin-25 LC-MS/MS Tuning Kit (Immundiagnostik AG, Bensheim, Germany) was used for optimization of LC-HR-MS ionization settings. This Tuning Kit consists of a highly pure hepcidin-25 and the internal standard (IS), Calcitonin Gene related Peptide human, each with a concentration of 1 µg/mL. For serum hepcidin-25 measurements, the Hepcidin-25 LC-MS/MS assay was purchased from Immundiagnostik AG. All further reagents and solvents of the kits are described in the manuals of the manufacturer in detail. Oasis® hydrophilic-lipophilic-balanced (HLB), 1cc (10 mg) cartridges (Waters, Eschborn, Germany) and foetal bovine serum (Sigma-Aldrich, Vienna, Austria) were used.</p><!><p>An ultra-high-pressure liquid-chromatography (UHPLC) Accela 1250 pump, a column oven (MayLab, Vienna, Austria) and an auto-sampler Accela Open AS were coupled on a Q Exactive hybrid Q-Orbitrap-MS (ThermoFisher Scientific, San Jose, California). The LC-MS instrument control was performed using XcaliburTM software version 2.2. (ThermoFisher Scientific, San Jose, California). For chromatography, a XSelect charged surface hybrid (CSH) C18 column (130Å, 3.5 µm, 2.1 mm x 50 mm; Waters, Eschborn, Germany) and a gradient of mobile phase A and B (Immunodiagnostics AG, Bensheim Germany) were used for separation and elution. Gradient settings for eluents A/B (in %, v/v) were, 90/10 (0 min), 90/10 (1.50 min), 5/95 (7 min), 5/95 (8 min), 90/10 (9 min), 90/10 (10 min; re-equilibration start). Flow rate was 400 μL/min. Ionization in positive mode was performed with a heated electrospray ionization (ESI) ion source. In brief: sheath gas flow rate 35 mL/min, auxiliary gas flow rate 10 mL/min, sweep gas flow rate 0 mL/min, spray voltage 4.00 kV, capillary temperature 350 °C, S-lens radio frequency (RF) level 90, and auxiliary gas heater temperature 150 °C. Positive ion full scan mode was set between mass to charge ratio (m/z) = 740 to m/z = 950, resolution was 70,000 (specified at m/z = 200).</p><p>The isotopic abundance and the accurate mass extraction of hepcidin-25 and the IS are shown in Figure 1 (A and B). For isotopic distribution of hepcidin-25, the most abundant peak was the threefold charged ion [M+3H]3+, revealed as the highest intensity (A). This and the next three isotopic peaks were used for post-acquisition data processing of hepcidin-25. For the IS determination, the fivefold charged ion [M+5H]5+, which showed the highest intensity, together with the area of two further isotopic peaks were used (B). The deviations of the m/z of target ions from their theoretical masses were within the region of 6 ppm. Peak area ratios from hepcidin-25 versus the IS were calculated and used to construct the calibration curves (1/X˄2 weighting). Mass calibration of the instrument was carried out at least every third day.</p><!><p>Isotopic distribution and accurate mass extraction of the molecular mass from hepcidin-25 and the internal standard, Calcitonin Gene related Peptide human. (A) Relative isotopic distribution of hepcidin-25. The threefold charged ion [M+3H]3+ revealed highest intensity. This and the next three isotopic peaks were used for quantification. (B) Relative isotopic distribution of the internal standard. Most intense ion was the fivefold charged ion [M+5H]5+. This and the next three isotopic peaks were used for quantification. m/z – mass to charge ratio.</p><!><p>Instead of the two-point calibration, as recommended by the manufacturer, an in-house calibration curve was prepared with analyte-free foetal bovine serum, which was spiked with hepcidin-25 (from the Tunning Kit), obtaining seven different concentrations (3.13, 6.25, 12.50, 25, 50, 100 and 200 ng/mL). Two- and seven-point calibration results were compared to prove that seven-point calibration yields comparable results.</p><!><p>Oasis® HLB 1cc (10 mg) cartridges (Waters, Eschborn, Germany) were conditioned by consecutive rinsing with methanol (200 μL) and deionized water (200 μL). 200 µL serum, together with 100 µL IS-solution, were loaded on the cartridges under vacuum followed by three washing steps using the wash solution (each with 200 µL), provided by the manufacturer. Analytes were eluted with the elution solution (100µL) from the kit and diluted equally (v/v, 60µL/60µL) with the wash solution 1. Subsequently 50 µL were injected onto the LC-HR-MS system.</p><!><p>Evaluation of the Hepcidin-25 LC-MS/MS Kit was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (15-17).</p><p>Precision measurements and recovery tests were assessed with seven calibrators with the expected hepcidin-25 concentrations of 3.13, 6.25, 12.50, 25, 50, 100 and 200 ng/mL (Table 1). Until analysis, the calibrators were stored at -20 °C. The within- and between-run precisions were assessed by replicate analyses (N = 5) of seven hepcidin-25 concentrations (3.13, 6.25, 12.50, 25, 50, 100 and 200 ng/mL) on the same day and on five consecutive days (15). According to the literature, the precision goal for each concentration was not to exceed 15% of the coefficient of variation (CV) (18).</p><p>The accuracy was assessed as the difference between the result of the mean value of six measurements of the low and high control material provided by the manufacturer (Immundiagnostik AG, Bensheim, Germany) compared to its "true" value (17). According to the literature, the acceptance criteria for the accuracy were defined ≤ 15% (18).</p><p>The LoD was defined as the lowest concentration, which showed a signal of at least three times higher than the average background noise of an unspiked blank (19). For the determination of LoD, the lowest calibrator (3.13 ng/mL) was added by decreasing concentrations to the blank matrix.</p><p>The LoQ was defined as the lowest concentration that can be determined with an acceptable level of repeatability precision (< 10%) (19). The LoQ was performed by measuring the calibrator with lowest concentration (3.13 ng/mL) at five consecutive days.</p><p>The manufacturer's claim for within- and between-run imprecisions were 2.6 and 3.8 – 7.3%, respectively. The LoD was quoted 1 ng/mL (20).</p><!><p>To investigate analyte stability, three serum samples were used to prepare a serum pool of 5 mL. This serum pool was divided in 20 aliquots (250 µL each), which were stored at RT. Three aliquots were measured at 0 and 3 h, and after 4 and 7 days. The RT in the laboratory is constant 25 °C and monitored by continuous record of the air conditioner.</p><p>A second serum pool of 5 mL was prepared with three other serum samples. This pool was also divided in 20 aliquots (250 µL each), which were stored at -20 °C. On days 1, 2 and 7, all aliquots were thawed, three of them were assayed and the rest again deep-frozen at -20 °C. A Kirsch MED-340 freezer (Kirsch, Offenburg, Germany) was used for -20 °C sample storage. The continuous record of the temperature ensures a high-quality monitoring. The specific concentrations for each time point of the three measured aliquots of both serum pools were calculated as arithmetic means.</p><p>The three aliquots of serum analyte stability on the auto-sampler tray (4 °C) was investigated with calibrator 3 and 4. Both calibrators were measured on days 1 – 4.</p><!><p>Descriptive statistics was used to summarize and present the study results. The distribution of the hepcidin-25 measurements was calculated with the Kolmogorov-Smirnov test. The exact Mann-Whitney U-test was used for subgroup comparison. A P-value < 0.05 was considered statistically significant. Statistical tests were performed with the Analyse-it® software version 4.92 (Analyse-it Software, Ltd., Leeds, United Kingdom). The formulas for bias calculations were as follows: absolute bias (ng/mL) = measured concentration – expected concentration and measured concentration – initial concentration (in terms of stability measurements); mean bias (%) = measured value – expected value/expected value x 100.</p><!><p>Figure 2 (A-C) shows a representative chromatogram of the high-resolution technique of hepcidin-25 and of IS. The commercial Hepcidin-25 LC-MS/MS Kit was linear over the concentration range of 3 – 200 ng/mL. The coefficient of determination (r2) was 0.9898. LoD was 1 ng/mL and LoQ was 3 ng/mL respectively.</p><!><p>Representative chromatogram of high-resolution mass-spectrometry for the determination of hepcidin-25 concentration. (A) Total ion chromatogram. (B) Hepcidin-25 detected with a retention time at 4.54 min. (C) Internal standard (Calcitonin Gene related Peptide human) with a retention time at 4.58 min. m/z – mass to charge ratio.</p><!><p>The results of the precision studies and the recovery of the LC-HR-MS method are shown in Table 1. Within-run CVs varied between 1.9 - 8.6% and between-run CVs ranged between 5.1 - 12.4% and were within the acceptance criteria of ≤ 15%. Observed recovery was between 88 – 107%.</p><p>The accuracy studies were within the acceptance criteria of ≤ 15% and showed an absolute and mean bias of - 1.25 ng/mL and - 2.7%, respectively, for the low control material ("true" vs. measured value: 45.50 vs. 44.25 ng/mL) and an absolute and mean bias of 2.28 ng/mL and 2.1%, respectively, for the high control material ("true" vs. measured value: 110.50 vs. 112.78 ng/mL).</p><p>Results of compared two-point and seven-point calibrations are shown in Table 2. The mean absolute and relative bias were - 0.8 ng/mL and - 11.3%, respectively.</p><!><p>As shown in Table 3, serum samples were stable at RT for at least 3 h. The mean difference of repeated measurements within three aliquots of serum pool 1 after 3 h was + 0.3%. Mean hepcidin-25 concentrations decreased with - 49, - 68 and - 79% after 24 h, 4 days, and 7 days, respectively. Freeze and thaw cycle experiments demonstrated a mean hepcidin-25 concentration increase of + 1.4, + 8.0 and + 20% after specimens were thawed, analysed and again deep-frozen on day 1, day 2 and day 7, respectively.</p><p>Auto-sampler stability measurements (4 °C) with calibrator 3 (12.50 ng/mL) and 4 (25.0 ng/mL) are shown in Figure 3. After 4 days, the hepcidin-25 measurements were stable. The CVs for calibrator 3 and 4 were 8.6% (10.80 ± 0.92 ng/mL) and 2.6% (23.80 ± 0.62 ng/mL), respectively.</p><!><p>Auto-sampler stability measurements (4 °C). After 4 days, the hepcidin-25 measurements with calibrator 3 (12.5 ng/mL) and 4 (25 ng/mL) were stable. The coefficients of variation (CVs) for calibrator 3 and 4 were 8.6 and 2.6% (acceptance criteria ≤ 15%), respectively.</p><!><p>Hepcidin-25 measurements of all included individuals (N = 165) showed a median value of 10.80 (range: 1.0 – 60.50) ng/mL. Females (N = 109) had a significantly lower median of 8.42 (range: 1.0 – 60.10) ng/mL compared to males (N = 56) with 15.76 (range: 1.50 – 60.50) ng/mL (P = 0.002), respectively. Sex-related hepcidin-25 serum medians stratified by 10-year groups were lower in females than males in all decades of life with the exception of the age groups 60 – 69 years and < 20 years, in which women showed higher medians compared to men (data not shown).</p><!><p>In the present study, the within- and between-run imprecisions of the Hepcidin-25 LC-MS/MS Kit from Immundiagnostik AG applied on a HR-MS instrumentation varied between 1.9 - 8.6% and 5.1 - 12.4%. The LoD was 1 ng/mL. These results were in line with the manufacturer's specifications quoted for within- and between-run imprecisions and LoD (20). The accuracy (bias) studies were ≤ 15%, which conform with the acceptance criteria of the Food and Drug Administration (FDA) Guidelines for Bioanalytical Method Validation (18).</p><p>Here, higher CVs were observed for higher and lower CVs for lower analyte concentrations. The authors themselves cannot fully explain this phenomenon but to our experience, the performance of LC-MS/MS methods is often so that at higher analyte concentrations, the ionisation leads to more imprecision. A second point may be the fact that we did not use an isotopically labelled IS in our study.</p><p>Beside the two-point calibration curve proposed by the manufacturer, we additionally fitted a calibration curve with seven in-house calibration standards. Such a multi-point calibration is proposed in the validation recommendations for LC-MS/MS methods (18). Present data show the equivalence of the two calibrations (mean bias ≤ 15%). Our laboratory prefers the seven-point calibration curve because the lowest calibrator of the manufacturer was 22.1 ng/mL and the determined LoQ was 3.1 ng/mL.</p><p>LC-HR-MS has been shown as an accurate and reliable technique for the quantitative determination of small molecules in clinical routine, for example in determination of bile acids (21). Performing Q Exactive MS instruments with Orbitrap-technology, proteins up to 30 kDa can be isotopically resolved by the use of high resolution. Accurate quantification with significantly increased sensitivity can be achieved by summarizing the area of all resolved isotope-peaks of a particular ionization status. In the current literature, only two publications describe the determination of hepcidin-25 performed with LC-HR-MS (22, 23). To the best of our knowledge, this is the first report evaluating the commercially available Hepcidin-25 LC-MS/MS Kit with this high-resolution technique.</p><p>Within the last years, the diagnostic use of LC-MS/MS methods and immunoassays for human serum hepcidin-25 measurements increased rapidly (2, 24-26). Earlier studies, which report the development of quantitative in-house hepcidin-25 LC-MS/MS methods, used synthetic human hepcidin-25 from Peptide Institute (Osaka, Japan) for standard curve calculations (24-26). In comparison, we used the hepcidin-25 of the Tunning Kit from Immundiagnostik AG (Bensheim, Germany). Various sources of synthetic hepcidin-25 and different protocols for sample preparation and chromatographic separation could be possible reasons for differences observed between LC-MS/MS methods (26). In addition, pre-analytical factors, sample storage and analyte stability must be considered in order to obtain reproducible and comparable results (27). Moreover, circulating hepcidin-25 concentrations underlie a circadian rhythm with lowest levels in the morning and highest values in the afternoon (28).</p><p>Herein, we performed blood sampling in a fasting state in the early morning and studied preanalytical stability measurements. At ambient RT, serum samples were stable for up to 3 h. The mean decrease of hepcidin-25 concentration after 24 h was - 49%. These data indicate that delays in transportation, aliquoting or measuring hepcidin-25 blood samples at RT should be avoided (29). In comparison, a previous study reported hepcidin-25 serum concentrations to be stable at RT for one day (27). Recently, Handley et al. showed serum hepcidin-25 measurements to be stable for at least up to three weeks (23). The authors presumed that protein LoBind tubes, which are especially designed to minimize protein absorption, had contributed to this extraordinary hepcidin-25 stability at RT (23).</p><p>In this work, hepcidin-25 measurements were stable after two freeze and thaw cycles. Previous reports showed analyte stabilities for at least three and five repeated analyses after freezing (- 20°C) and thawing (RT) of hepcidin-25 serum samples (23, 27). All these data are indicative for the prevention of repeated freeze and thaw cycles in laboratories, which handle hepcidin-25 measurements in a patch workflow.</p><p>Herein, females showed significantly lower hepcidin-25 serum concentrations compared to males (P = 0.002). This finding is in line with previous published studies, which reported that serum hepcidin-25 concentrations are substantially higher in men than in women (27, 30). It is believed that iron loss during menstruation explains this gender differences (27, 30).</p><p>Several limitations of this study must be mentioned. For precision and analyte stability testing calibration samples were used because it was difficult to get samples of patients. Reference ranges were not calculated because data on subjects' disorders or therapy, which are necessary for this determination, were not included. Hepcidin-25 stability measurements were not performed with pathological analyte concentration and determinations at RT were performed within the first 24 h, only.</p><p>In conclusion, the Hepcidin-25 LC-MS/MS Kit from Immundiagnostik AG shows a broad analytical range and meets the imprecision and bias acceptance criteria of ≤ 15%. Serum samples can be stored at RT for 3 h and resist up to two freeze and thaw cycles. These data are indicative for a reliable and robust diagnostic method for clinical practice.</p><!><p>Potential conflict of interest: Data from this paper were presented as poster at the 5th Annual Congress of the Association for Mass Spectrometry to the Clinical Lab (MSACL) EU 2018, in Salzburg, Austria.</p>

PubMed Open Access

Total Synthesis and Chemoproteomics Connect Curcusone Diterpenes with Oncogenic Protein BRAT1

Natural products are an indispensable source of lifesaving medicine, but natural product-based drug discovery often suffers from scarce natural supply and unknown mode of action. The study and development of anticancer curcusone diterpenes fall into such a dilemma. Meanwhile, many biologicallyvalidated disease targets are considered "undruggable" due to the lack of enzymatic activity and/or predicted small molecule binding sites. The oncogenic BRCA1-associated ATM activator 1 (BRAT1) belongs to such an "undruggable" category. Here, we report our synthetic and chemoproteomics studies of the curcusone diterpenes that culminate in an efficient total synthesis and the identification of BRAT1 as a cellular target. We demonstrate for the first time that BRAT1 can be inhibited by a small molecule (curcusone D), resulting in impaired DNA damage response, reduced cancer cell migration, potentiated activity of the DNA damaging drug etoposide, and other phenotypes similar to BRAT1 knockdown.

total_synthesis_and_chemoproteomics_connect_curcusone_diterpenes_with_oncogenic_protein_brat1

2<!>Experimental Procedures and Data

<p>Natural products have been valuable sources and inspirations of lifesaving drug molecules 1 . Their accumulated evolutionary wisdom together with their structural novelty and diversity makes them unparalleled for novel therapeutic development. However, their natural scarcity, structural complexity, and unknown mode of action often hamper their further development in the drug discovery pipeline. Total synthesis is frequently employed to solve the material supply and chemical probe synthesis for comprehensive biological profiling and target identification 2 . Meanwhile, many biologically validated disease targets are considered as "undruggable" from a chemical standpoint due to the lack of enzymatic activity and/or small molecule binding sites 3 . The BRCA1-associated ATM activator 1 (BRAT1) protein has been validated as an oncogenic protein involved in various cancers but belongs to the "undruggable" category with no known small molecule inhibitors to date. Herein, we report a collaborative effort in the total synthesis and chemoproteomics profiling of curcusone natural products which reveals BRAT1 as a key cellular target and validated curcusone D as the first BRAT1 inhibitor.</p><p>The curcusone diterpenes (Fig. 1A) were isolated from Jatropha curcas, a widely used ingredient in traditional remedies for a variety of ailments including cancer. Structurally, they share a characteristic [6-7-5] tricyclic skeleton with the daphnane and tigliane diterpenes 4 . Curcusones A-D (1a-d), isolated by Clardy and co-workers in 1986, were unambiguously identified as two epimeric pairs at the C2 position 5 .</p><p>Since then, around thirty curcusone molecules have been isolated including curcusones F-J 6 , which lack the dienone moiety in the seven-membered ring. Structurally rearranged analogs like spirocurcasone (3) and dimeric analogs such as dimericursone A (2a) and dimericursone B (2b) were discovered recently 7,8 . Among them, 1a-1d exhibited low micromolar IC50 values against a broad spectrum of human cancer cell lines 6 . However, their mode of action remained unknown and no total syntheses of 1a-1d were reported prior to this study. While the closely related daphnane and tigliane diterpenes have attracted a significant amount of synthetic interest [9][10][11][12][13] , the curcusone molecules have surprisingly received little attention despite their therapeutic potential. In 2017, we reported the first total syntheses of the putative structures of 1i and 1j in 21 steps (Fig. 1B), ultimately leading to the conclusion that the originally proposed structures of both 1i and 1j were incorrect 14 . Our synthesis involves a gold-catalyzed tandem furan formation and furan-allene</p><p>[4+3] cycloaddition to build the 5,7-fused ring system with an oxa bridge and a Diels-Alder reaction to construct the 6-membered ring. In 2019, Stoltz and co-workers reported their studies toward synthesizing 1a-1d (Fig. 1C) [15][16][17] . Their approach features a divinylcyclopropane-cycloheptadiene rearrangement to forge the 7-membered ring and reached advanced intermediate 14 after 12 steps from 8.</p><p>Total Synthesis and Probe Synthesis. Our ongoing interest in natural products that can covalently modify cellular proteins 18 prompted us to continue pursuing the total synthesis and target identification of 1a-1d with an electrophilic cycloheptadienone moiety. This unique structural feature could allow them to form a covalent bond with nucleophilic residues of certain cellular proteins 19 . Previous cytotoxicity studies found that reduction and/or oxidation of the C6-C7 double bond greatly reduced their anticancer activity 6 .</p><p>As such, an approach allowing variation of the C6 and C7 substituents would be highly desirable. We envisioned 15 as an advanced intermediate (Fig. 1D). α-Halogenation followed by two methylation reactions would lead to 1a and 1b, which could be oxidized to 1c and 1d via a-hydroxylation. A ring closing metathesis (RCM) or an intramolecular aldol condensation was planned to form the 7-membered dienone Our synthesis started with preparing 23 (Fig. 2), a known compound synthesized from 8 in three steps -extended silyl enol ether formation, vinylogous Mukaiyama aldol reaction, and NaBH4 reduction 20 .</p><p>We combined the first two steps into a one-pot reaction; crude 22 was then subjected to NaBH4 reduction directly to produce multi-decagram scale of 23 in one batch. We next needed to prepare 24 for the Claisen rearrangement. NaH-promoted addition-elimination between 23 and 20b afforded 24, albeit in low yield (35%). We then used a Mitsunobu reaction between 23 and 20a to prepare 24, but the hydrazine byproduct derived from diethyl azodicarboxylate could not be separated from 24. The recently reported redox-neutral organocatalytic Mitsunobu conditions were also explored but failed to provide 24 21 . Fortunately, the hydrazine byproduct could be tolerated in the Claisen rearrangement. After 24 was heated at 140-150 °C in DMF for 18 h, the Claisen rearrangement did occur, but the rearranged product 25 further cyclized to provide tricyclic compound 26 (X-ray, Fig. S2) as a single diastereomer in 48% yield from 23.</p><p>Without getting 25 to prepare triflate 18, we decided to continue with 26 and explore the hidden cyclopentane-1,3-dione symmetry to synthesize 17. We started with investigating 1,2-addition of lithiated ethyl vinyl ether (27) to 26 theorizing that a global hydrolysis would release the methyl ketone and the aldehyde at once to form 17 for the aldol condensation. This 1,2-addition turned out to be nontrivial. When two equiv. of 27 was used, only less than 10% yield of 28 was obtained. Owing to their oxophilicity, cerium chloride and lanthanum chloride have been used to promote 1,2-additions 22 . Unfortunately, both failed in our case. Eventually, the 1,2-addition was improved by increasing the amount of 27 to 10 equiv. 23 , and 28 was prepared in 57% yield. 28 was then subjected to hydrolysis upon the treatment with p-toluenesulfonic acid and 17 was obtained in 51% yield from 26. Meanwhile, we were delight to observe the formation of 15 in the same reaction, albeit in very poor yield (<5%). We were encouraged to achieve a global hydrolysis/aldol condensation cascade to synthesize 15 from 28 in one step and identified FeCl3 24 in combination with TMSCl as the optimal conditions. When crude 28 was treated with a premixed FeCl3 (0.2 M in 2-methyltetrahydrofuran) and TMSCl in toluene at room temperature, global hydrolysis occurred to generate 17 in situ, which further underwent FeCl3-promoted intramolecular aldol condensation to afford 15 in 39% yield from 26.</p><p>With the [6-7-5] tricyclic carbon skeleton quickly assembled in only six steps, we next needed to introduce the two methyl groups. Johnson iodination converted 15 to iodoenone 31, which was surprisingly unstable. Therefore, after a quick workup, crude 31 was immediately subjected to the next Stille cross coupling with tetramethylstannane to provide 32 in 45% yield over two steps. Finally, α-methylation of enone 32 at C2 position delivered a 1:1 mixture of separable (-)-1a and (-)-1b in 41% yield (88% brsm; 9 steps total). α-Hydroxylation of 1a with KHDMS and MoOPH gave separable (-)-1c and (-)-1d in 63% yield (d.r. 1:1; 84% brsm; 10 steps total). Additionally, in order to obtain analogs for biological activity comparison, we converted (-)-1b to (+)-3 and (-)-33 (a synthetic derivative named as pyracurcasone) by following a reported one-step procedure 7 . The 1 H, 13 C NMR, and other analytic data of our synthetic samples matched well with the reported ones, which also conclude that the absolute configuration of 1a-1d assigned by Clardy et al. in 1986 is opposite of the actual ones.</p><p>We then set out to synthesize 2a from 1a-1d via a biomimetic dimerization. The proposed biosynthesis of 2a consists of a sequence of oxidative dehydrogenation of 1a/1b or dehydration of 1c/1d to form a reactive cyclopentadienone intermediate followed by Diels-Alder dimerization and cheletropic extrusion of carbon monoxide 8 . From there, 2a could be converted to 2b via another oxidative dehydrogenation and double bond isomerization. We started with 1c and 1d. After an unfruitful attempt to synthesize 2a by heating them directly at elevated temperatures, a 1:1 mixture of them was first converted to their mesylates (34). After extensive exploration, we identified that using triethylamine as a base in 1,4dichlorobenzene at 150 °C produced (-)-2a in 18% yield over two steps, which provides a direct evidence to support the proposed biosynthetic pathway. Under our conditions, the formation of 2b was not observed.</p><p>To elucidate the anticancer mechanism and identify potential cellular targets of curcusones, an alkyne-tagged probe molecule 37 was designed for chemoproteomics studies. Since the dienone is likely protein-reactive and is critical for the observed activity, we decided to minimize structural perturbation of this part and used the tertiary alcohol as a handle to link with a terminal alkyne. 37 was synthesized in 59% yield from (−)-1d via a DCC-promoted coupling with 36.</p><p>Cytotoxicity and Target Identification. We evaluated the cytotoxicity of curcusones and their analogs in breast cancer MCF-7 cells using the WST-1 assay (Fig. 3A). Synthetic 1a-1d, natural 1b and 1d, and intermediates 15 and 32 exhibited micromolar EC50 values against MCF-7 cells with 1d being the most potent curcusone. Importantly, the cytotoxicity values for synthetic 1b and 1d were virtually identical to the values of their naturally isolated counterparts. Analog 33 showed slightly better antiproliferation activity indicating the feasibility of finely tuning the cycloheptadienone moiety to improve potency, but 2a was not active even at 100 µM. Likely due to the full confluency of the tested MCF-7 cells, the EC50 values we obtained were about one order of magnitude higher than previously reported (1.6-3.1 µM EC50 values for 1a-1d) 6 . Gratifyingly, 37 retained similar anticancer properties of 1d, thus warranting its use in competitive chemoproteomic studies.</p><p>We then identified the cellular targets of curcusones by competitive chemoproteomics using probe 37. MCF-7 cells were treated with 1d for 4 hours followed by lysis, treatment with 37, CuAAC with biotin azide, enrichment, digestion, and LC-MS/MS analysis using label-free quantification (Fig. 3B and Table S1). The best competed target was BRAT1, which acts as a master regulator of the DNA damage response (DDR) and DNA repair by binding to BRCA1 and by activating DDR kinases such as ATM and PRKDC (DNA-PKcs) following DNA damage [25][26][27] . Knockdown of BRAT1 increased the constitutive level of apoptosis in human osteosarcoma cells 25 and decreased cancer cell proliferation and tumorigenicity in vitro and in mouse tumor xenografts 27 . BRAT1 is also an unfavorable prognostic marker in kidney and liver cancers 28 . Therefore, targeting BRAT1 is a promising strategy for cancer treatment. 9 We next characterized the physical interaction between 1d and BRAT1. We overexpressed FLAG-BRAT1 in HEK-293T cells and performed a thermal shift assay by treating lysates with 1d, heating as indicated, and probing the remaining soluble FLAG-BRAT1 by Western blotting (Fig. 3C). We observed 10 thermal destabilization of 1d-treated BRAT1 indicating a direct interaction. To validate endogenous BRAT1 as a target of 1d in live cells, we employed a competitive pulldown experiment. MCF-7 cells were treated with 1d for 4 hours before lysis, treatment with probe 37, CuAAC with biotin azide, streptavidin enrichment, elution, and Western blot visualization. Indeed, native BRAT1 was enriched by 37 and was competed by 1d (Fig. 3D). Additionally, 1d competed the enrichment of BRAT1 from cervical cancer HeLa and triple negative breast cancer MDA-MB-231 cells, thus validating native BRAT1 as a cellular target of 1d across these cell lines. In situ treatment of 1d in live HeLa cells competed BRAT1 enrichment by 37 at low micromolar concentrations (EC50 = 2.7 µM; Fig. 3E). Assuming that 1d binds to BRAT1 irreversibly, we determined the binding constants Ki (3.5 µM) and kinact (0.0079 min -1 ; Fig. 3F). These results demonstrate that 1d is the first small-molecule binder of BRAT1. BRAT1 Modulation. To determine whether 1d inhibits BRAT1 in cells, we generated stable BRAT1 KD HeLa cells via shRNA retroviral transduction (Fig. S1A). We then compared the protein expression profiles of BRAT1 KD cells versus 1d-treated cells (3 µM, 24 h) by global proteomics analysis (Fig. 4A-C and Table S2). Among 3347 quantified proteins in compound-treated cells, we found only 36 up-and 42 down-regulated proteins. Importantly, 31 of the 78 dysregulated proteins were also dysregulated in BRAT1 KD cells, thus indicating that 1d functionally inhibits BRAT1 in cells. Notably, several wellknown cancer migration and progression drivers were downregulated (Fig. 4B), including TRIM47 which mediates cancer migration 29 , the bona fide oncoprotein and potential biomarker WBP2 30 , and frequently highly amplified oncogene FNDC3B 31 . None of these proteins have previously been functionally linked to BRAT1. We then investigated the effect of 1d treatment and BRAT1 KD on cancer cell migration in WT and BRAT1 KD HeLa cells, as well as WT MCF-7 and MDA-MB-231 cells (Fig. 4D-G). As expected, BRAT1 knockdown greatly diminished migration of HeLa cells, and treatment with 1d at 1 µM concentration also reduced migration of all cell lines by ~4-fold. Our global proteomics experiment also revealed several commonly downregulated key DNA repair proteins (Fig. 4C) such as (i) POLD1 which synthesizes DNA during repair 32 , (ii) USP47 which facilitates base-excision repair 33 , (iii) FANCI which mediates the repair of DNA double strand breaks and interstrand crosslinks 34 , and (iv) BRCC3 which stabilizes the accumulation of BRCA1 at DNA breaks 35 . These proteins have not been previously linked to BRAT1 either. Most notably, 1d treatment (24 hours) significantly downregulated the actual physical target, BRAT1 (ratio 0.18), as confirmed by Western blotting (Fig. S1B).</p><p>Collectively, these findings demonstrate the importance of BRAT1 as a master regulator of the DDR and that 1d inhibits BRAT1 in cells.</p><p>We then investigated whether 1d would potentiate the DNA damaging effect of the clinical drug and topoisomerase inhibitor etoposide via BRAT1 inhibition. WT or BRAT1 KD HeLa cells were treated with DMSO, etoposide, 1d, or etoposide and 1d combined. Subsequent DNA damage was then measured by fluorescence microscopy using γH2AX staining (Fig. 4H, I and Fig. S1C). Treatment with 1d (3 µM) or KD of BRAT1 alone did not increase γH2AX signal. However, co-treatment of 1d with etoposide led to a 2-fold increase. Similarly, etoposide treatment significantly increased γH2AX signal in BRAT1 KD cells, recapitulating the 1d/etoposide co-treatment results. Importantly, 1d treatment did not increase γH2AX signal in etoposide-treated BRAT1 KD cells, confirming that the 1d-etoposide synergism is linked to BRAT1 inactivation. Furthermore, co-treatment of 1d with etoposide also increased cytotoxicity in HeLa, MCF-7, and MDA-MB-231 cells (Fig. 4J). Likewise, there was increased cell death in BRAT1 KD HeLa cells following etoposide treatment relative to WT cells (Fig. 4K). Altogether, these results demonstrate that targeting BRAT1 with 1d is a promising anticancer strategy for chemosensitization to DNA damaging drugs.</p><p>In summary, we completed the first asymmetric total synthesis and target identification of the curcusone natural products. Our convergent synthesis builds upon a cheap and abundant chiral pool molecule (8) and features a thermal [3,3]-sigmatropic rearrangement and an FeCl3-promoted global hydrolysis/aldol condensation cascade to rapidly construct the critical cycloheptadienone core. This efficient synthetic route yielded 1a and 1b in 9 steps, 1c and 1d in 10 steps, and 2a in 12 steps from (S)-(−)-8. The successful synthesis of 2a from 1c/1d experimentally supports the proposed Diels-Alder dimerization and cheletropic extrusion biosynthesis. By performing chemoproteomics with the alkyne probe 37, we identified the previously "undruggable" oncogenic protein BRAT1 as a key cellular target of 1d. Furthermore, 1d inhibits BRAT1 in cancer cells, thereby reducing cancer cell migration, increasing susceptibility to DNA damage, and inducing chemosensitization to the approved drug etoposide. To our knowledge, 1d is the first known small-molecule inhibitor of BRAT1, a master regulator of the DDR and DNA repair. Many promising clinical trials are underway targeting DDR proteins such as PARP, ATR, ATM, CHK, and DNA-PK as monotherapies or in combination with other treatments [36][37] . Olaparib, a PARP inhibitor, was approved by FDA in 2014 as a monotherapy to treat germline BRCA1/2-mutant ovarian cancer 36 . Further structure-activity optimizations of the curcusones may thus yield novel BRTAT1 inhibitors as potential lead medicines for monotherapies or combination therapies.</p><!><p>For detailed experimental procedures and compound characterization data, see the Supplementary Information.</p>

ChemRxiv

pH-Mediated Single Molecule Conductance of Cucurbit[7]uril

Recognition tunneling technique owns the capability for investigating and characterizing molecules at single molecule level. Here, we investigated the conductance value of cucurbit[7]uril (CB[7]) and melphalan@CB[7] (Mel@CB[7]) complex molecular junctions by using recognition tunneling technique. The conductances of CB[7] and Mel@CB[7] with different pH values were studied in aqueous media as well as organic solvent. Both pH value and guest molecule have an impact on the conductance of CB[7] molecular junction. The conductances of CB[7] and Mel@CB[7] both showed slightly difference on the conductance under different measurement systems. This work extends the molecular conductance measurement to aqueous media and provides new insights of pH-responsive host-guest system for single molecule detection through electrical measurements.

ph-mediated_single_molecule_conductance_of_cucurbit[7]uril

Introduction<!>Results and Discussion<!><!>Single Molecule Conductance Measurements of CB[7]<!><!>Single Molecule Conductance Measurements of CB[7]<!>Single Molecule Conductance Measurements of Mel@CB[7]<!><!>Single Molecule Conductance Measurements of Mel@CB[7]<!><!>Single Molecule Conductance Measurements of Mel@CB[7]<!>Chemicals<!>STM Probes Fabrication<!>Gold Substrates Preparation<!>Recognition Tunneling Measurements<!>UV-Vis Measurements<!>Contact Angle of SAMs<!>Conclusions<!>Data Availability Statement<!>Author Contributions<!>Conflict of Interest<!><!>Supplementary Material<!>

<p>Molecular electronics, which focuses on the single molecules in the electronic junctions used to make electronic devices, has been extensively studied. Molecular electronics aims at investigating single molecules at electrical junctions for fabricating electronic devices, which has been extensively investigated (Cui, 2001; Choi and Mody, 2009; Herrer et al., 2018). A variety of methods have been developed to measure the electrical properties of single molecules by constructing metal-molecule-metal junctions, including scanning probes techniques such as scanning tunneling microscopy (STM) techniques (Xu and Tao, 2003; Haiss et al., 2006; Venkataraman et al., 2006; Chen et al., 2020; Yu et al., 2020), conducting atomic force microscopy (AFM) (Cui, 2001), mechanically controlled break junctions (MCBJ) (Reed et al., 1997; Smit et al., 2002), and nanoparticle dimers (Dadosh et al., 2005; Fernandez et al., 2014).</p><p>Considering possible contaminations in the air and electrochemical leakage current in aqueous media, STM measurements are normally performed in organic solvent system. This limited the applications in biomedical field to a certain extent. The local molecular environment should be taken into account when measuring single-molecule electrical properties in the design of single molecule sensing devices (Zhang et al., 2016).</p><p>Molecular interactions included some dynamic behaviors in biological systems can be greatly influenced by various stimuli, such as pH, temperature, light, and so on (Angelos et al., 2008; Mendes and Paula, 2008; Yu et al., 2015). Stimuli-responsive host-guest interactions has been established in solution or on surfaces, and normally studied by traditional analytical methods like nuclear magnetic resonance (NMR), fluorescence, and isothermal titration calorimetry (ITC), etc. (Cabane et al., 2012; Yang et al., 2014a,b; Sinn and Biedermann, 2018; Xiao et al., 2019). However, electrical properties of pH-responsive supramolecular systems are rarely investigated by recognition tunneling technique.</p><p>Belong to a family of macrocyclic host molecule, barrel-shaped cucurbit[n]uril (CB[n]s) has gained much interest for applications including sensors, nanoreactors and drug delivery due to its good biocompatibility, high thermal stability and remarkable recognition properties (Macartney, 2011; Ma and Zhao, 2015; Kuok et al., 2017; Gao et al., 2018; Yin and Wang, 2018; Zhang et al., 2018; Braegelman and Webber, 2019; Cheng et al., 2020). CB molecule can be immobilized on the gold surface through the collective interactions between multiple carbonyl groups of CB and gold (An et al., 2008). CB[n] retains its recognition properties when bonded to the gold surface, making it a promising candidate for molecular sensors. In addition, the introduction of CB into the conductance measurement system provides a platform to investigate the molecules that cannot tether with gold. Here, we chose CB[7] as the host molecule to explore the electrical properties via STM fixed junction technique. CB[7] is known with good water solubility and high binding affinity with various guest molecules, and only one guest molecule can be confined inside its cavity at a given time.</p><p>CB[7] can allow the inclusion of melphalan (Mel) in its cavity. As an antineoplasic drug, Mel is indicated for the treatment of multiple myeloma and other types of cancer (Samuels and Bitran, 1995; Falco et al., 2007). While the usability of Mel is limited to its poor water solubility at neutral pH as well as rapid hydrolysis in physiological conditions. However, the solubility and stability of Mel can be efficiently improved upon introducing CB molecule, which promotes further application for therapy (Zhang and Isaacs, 2014; Villarroel-Lecourt et al., 2018). Notably, the binding affinity between Mel and CB[7] differs as the pH changes (Villarroel-Lecourt et al., 2018). This makes Mel@CB[7] complex a potential pH-responsive sensor.</p><p>We investigated the conductances of CB[7] and its host-guest complex (Mel@CB[7]) with different pH values by using STM fixed junction technique. CB[7] and Mel@CB[7] functionalized gold substrates were prepared by immersing gold substrates into various pH phosphate buffer solution (PB) containing CB[7] and Mel@CB[7], respectively. Both CB[7] and Mel@CB[7] showed differences on the conductance when pH value changed. In addition, we compared the conductance measurements performed in PB and organic solvent 1, 2, 4-trichlorobenzene (TCB). Slight difference on the conductance was observed under different measurement systems. This work extends the single molecule conductance measurements from normal organic system to aqueous media for investigating dynamic molecular interactions and aims at exploring potential application for fabricating pH-responsive supramolecular system for single molecule detection as well as the potential application for drug delivery.</p><!><p>The schematic setup of the conductance measurement of CB[7] is shown in Figure 1A. The conductance measurement method is based on the STM fixed-junction technique. Generally, a gold probe is placed in close proximity to gold surface functionalized with the target molecule where the distance between the probe and the substrate is fixed at a few tens of nanometers controlled by the piezoelectric transducer (PZT) of a STM. The target molecules are not disturbed by electrodes moving or crashing into each other, which makes it suitable for the study of noncovalent interactions between single molecules (Chen et al., 2020). No signal was observed on clean gold substrate (black curve of Figure 1B) or on gold surface modified with CB[7] where no effective molecular junctions form ("off" state). The target molecule on the substrate contacts the probe to form a molecular bridge, giving rise to the current jumps ("on" state) (Haiss et al., 2004, 2006). The current fluctuations represent the fast formation and breaking of molecular bridge. With the carbonyl group of CB[7] molecule on the surface binding to both electrodes, current spikes occurred (red curve of Figure 1B). These current spikes indicated the transient formation of Au-CB[7]-Au molecular junction. The time-dependent STM image of CB[7] functionalized gold substrate was shown in Figure S1A. The appearance of pits in the STM image suggested that CB[7] can interact with the gold substrate to relocate the surface gold atoms (Xiao et al., 2018), which indicated the successful modification of CB[7]. This was further proven by the decrease of contact angle after modifying CB[7] (Figure S1B).</p><!><p>Experimental setup for conductance measurement. (A) Schematic diagram of the conductance measurement of CB[7] (top), and the spontaneous formation of a molecular bridge between the tip and the substrate (bottom). (B) Typical current traces for the substrate before (black) and after (red) modified with CB[7] for overnight. Measurements were carried out with a sample bias of 0.1 V and a set-point current of 20 nA.</p><!><p>We first investigated the single-molecule conductance and bonding lifetime of CB[7] molecular junction where measurements were carried out in PB solution. The gold probes used under this condition were insulated to rule out the electrochemical leakage current. Freshly cleaned gold substrates were immersed in PB buffer containing CB[7] molecules with various pH values for overnight prior to the conductance measurements. The pH values tried here were 1, 4, and 7.</p><p>The single molecule conductance of CB[7] can be determined by transient current changes (Haiss et al., 2004; Xiao et al., 2018).</p><p>Where I is current, I0 is the tunneling current before the observation of the current jump, V is the applied bias. In this report, the baseline current I0 is 20 nA, and the bias V is 0.1 V, which follows the previous report (Xiao et al., 2018). The histograms of conductance G vs. G0 were plotted in semi-log scale, where G0 is quantum of conductance 77.4 μS, as shown in Figure 2A (in PB) and C (in TCB). The corresponding results are displayed in Table 1.</p><!><p>Single molecule conductance and bonding lifetime of CB[7] molecular junction. The molecule conductance histograms (A,C) and lifetime histograms (B,D) of switching events for CB[7] were plotted in logarithm scale. Gold substrates were modified with CB[7] in PB buffer solution with the pH values at 1, 4, and 7. STM measurements were performed in PB (A,B) and TCB (C,D) at a constant bias of 0.1 V with the setpoint of 20 nA.</p><p>Single molecule conductance and lifetime of switching events for CB[7].</p><!><p>Based on the Gaussian fit, the mean conductance of CB[7] measured in PB were 81.61 nS, 73.75 nS, and 70.26 nS for pH 1, 4, and 7, respectively. The conductance gradually decreased as the pH value increased. More protons would interact with carbonyl group under acid condition, which may contribute to the electron transfer of CB[7] molecular junction. Meanwhile, we also checked the width of the switching spike, giving the information of bonding stability between CB[7] molecule and gold electrodes. The histogram and Gaussian fit of the switching lifetime Δt, plotted in log scale was shown in Figure 2B. The mean value of switching events for CB[7] was between 7 and 8 ms, which indicated the short-time stability of the binding geometry associated with the collective interaction between single or few carbonyl groups of CB[7] and gold electrode.</p><p>We also studied single-molecule conductance measurements of CB[7] performed in TCB. The histograms and Gaussian fit of the switching events for CB[7] measured in TCB are shown in Figures 2C,D. Based on the results in Table 1, the mean conductance of CB[7] measured in TCB were 73.58 nS, 71.08 nS, and 70.27 nS for pH 1, 4, and 7, respectively. Just like the conductance measured in PB, the conductance here showed slightly decrease as the pH value increased. To confirm the pH effect, we further investigated the conductance of CB[7] where the pH was 9 (Figure S2). The mean conductance of CB[7] measured in PB and TCB were 64.52 nS and 63.2 nS, respectively (Table S1). The conductance of CB[7] at pH 9 was smaller than that at pH 7, which further indicated the interactions between protons and carbonyl group affected the electron transfer process of the CB[7] molecular junction.</p><p>In contrast to the lifetime and the conductance of CB[7] measured in PB, the values measured in TCB were smaller under the same pH value, especially for pH 1. This suggested the interactions between protons and carbonyl group under aqueous condition may promote better electron transfer to a certain extent.</p><!><p>We further explored the conductance of CB[7] with melphalan encapsulated inside its cavity. The chemical structure of CB[7] and Mel are shown in Figure 3A. Mel has been reported to form 1:1 host-guest complex with CB[7], with the equilibrium binding affinity log K = 6 at pH 1 (Villarroel-Lecourt et al., 2018). The protonation of Mel is essential for binding to the macrocycle due to the cation-dipole interactions. We first studied the pH value effect on the interactions between Mel and CB[7] through absorption spectroscopy.</p><!><p>Spectroscopic characterization for Mel@CB[7] interaction. (A) Chemical structure for CB[7] and Mel. (B–D) UV-Vis absorption of Mel (20 μM) upon addition of CB[7] (0–40 μM) in 10 mM phosphate buffer (B, pH = 1, C, pH = 4, D, pH = 7).</p><!><p>UV/vis spectra of Mel showed a remarkable decrease in the absorption band at 260 nm at pH 1 in the presence of increasing concentrations of CB[7] (Figure 3B), which attributed to the encapsulation of the drug. Slightly decrease for the absorption spectra of Mel was observed at pH 4 with the increasing of CB[7] (Figure 3C). Interestingly, this change was not observed at pH 7 (Figure 3D) because of the weak host-guest interaction of the complex at this pH, which is also consistent with the simulation of Mel@CB[7] complex in the docking study (Villarroel-Lecourt et al., 2018). Considering the rapid hydrolysis of Mel as well as the lack of obvious binding between Mel and CB[7] under alkaline conditions, subsequent conductance measurements focused on pH values at 1, 4, and 7.</p><p>Similar to CB[7]'s immobilization, Mel@CB[7] complexed molecules were deposited onto the gold substrate prior to conductance measurements. CB[7] here served as a nanocontainer to immobilize the guest molecule for STM measurements. No single molecule conductance event was observed for Mel due to no effective molecular junction forming.</p><p>The histograms and Gaussian fit of G vs. G0 and the lifetime Δt in semi-log scale for Mel@CB[7] complex are shown in Figure 4, where measurements were performed in PB (A, B) buffer and TCB (C, D), respectively. The responding results are summarized in Table 2.</p><!><p>Single molecule conductance and bonding lifetime of Mel@CB[7] molecular junction. The molecule conductance histograms (A,C) and lifetime histograms (B,D) of switching events for Mel@CB[7]. Gold substrates were modified with Mel@CB[7] PB buffer solution where the pH values were 1, 4, and 7. STM measurements were performed in PB buffer (A,B) and TCB (C,D) at a bias of 0.1 V with the setpoint 20 nA.</p><p>Single molecule conductance and lifetime of switching events for Mel@CB[7].</p><!><p>As shown in Table 2, the mean conductance of Mel@CB[7] complex measured in PB were 71.41 nS, 69.3 nS, and 68.35 nS for pH 1, 4, and 7, respectively. The conductances of Mel@CB[7] measured in PB gradually decreased with the increase of pH value, which is similar to the change trend of CB[7]. However, it is worth noting that compared with CB[7], the Mel@CB[7] complex showed a smaller conductance at the same pH. Guest molecule in the cavity of CB[7] had an impact on electronic transport process, which is consistent with the previous report (Xiao et al., 2018). Furthermore, the mean lifetime of the switching events for Mel@CB[7] was between 6 and 7 ms, which was smaller than that for CB[7]. This suggested that the inclusion of Mel inside CB[7]'s cavity weakened the bonding stability between CB[7] molecule and gold electrodes.</p><p>The mean conductance of Mel@CB[7] measured in TCB were 69.08 nS, 66.64 nS, and 64.82 nS for pH 1, 4, and 7, respectively. The conductance here also slightly decreased as the pH value increased. As for the measurements performed in TCB, both the mean conductance and the mean lifetime of Mel@CB[7] complex were smaller than that of CB[7] under the same pH value. This further indicated the guest molecule in the cavity of CB[7] disturbed the electron transport process, as well as the bonding stability between CB[7] molecule and gold electrodes.</p><p>However, compared with the conductance measured in PB, only a slight decrease of the conductance at pH 1 was observed for Mel@CB[7] complex when the measurement was performed in TCB. This may further indicate that the guest molecule in the cavity play a dominant role in disturbing the electron transfer in the molecular junction.</p><!><p>Cucurbit[7]uril (CB[7]) was synthesized following a procedure published by Day's group (Day et al., 2001). Gold wire, sodium chloride, sodium dihydrogen phosphate, dibasic sodium phosphate, sodium hydroxide, hydrochloric acid and absolute ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. Melphalan was purchased from aladdin. 1, 2, 4-trichlorobenzene was purchased from Sigma-Aldrich. All the purchased chemicals were used directly without further purification. Deionized water from a Milli-Q water purifying system was used to prepare all of the solutions (18.2 MΩ).</p><!><p>STM probes were fabricated by following previous report (Tuchband et al., 2012). A gold wire was electrochemically etched and cleaned by dipping into piranha solution (H2SO4:H2O2 = 3:1, by volume), rinsed with copious amounts of deionized water, and dried with argon. This gold tip was directly used in the STM measurements carried out in organic solvent. For the measurements performed in PB buffer, the gold tip was further insulated with high-density polyethylene (HDPE) to expose only the tip apex.</p><!><p>10 mM phosphate buffer solution with pH 7 was prepared by mixing equal concentration of dibasic sodium phosphate solution and sodium dihydrogen phosphate solution (10 mM). PB buffer with pH 1, 4 and 9 were prepared by dropping hydrochloric acid and sodium hydroxide solution into pH 7 PB buffer, respectively. A 1 mM CB[7] solution was prepared by dissolving CB[7] powders in PB buffer with sonication for 5 min. A 1 mM Mel@CB[7] complex solution was prepared by mixing Mel powders in the CB[7] solution, followed by ~15 min sonication. Freshly cleaned gold substrates were immersed in molecular solution for overnight at room temperature. The modified gold substrates were washed with copious amounts of ethanol and deionized water, dried with argon gas, and used immediately.</p><!><p>Recognition Tunneling measurements were performed on a STM (Keysight 5,500) with the sample and probe submerged in a liquid cell containing 1, 2, 4-trichlorobenzene or PB buffer at a bias of 0.1 V with the setpoint 20 nA. Current-time traces were recorded with a Picoview software. For each sample, 2~3 gold probes were used to collect hundreds of experimental runs over different sample positions. The PZT servo response time was about 30 ms, as described previously (Xiao et al., 2018). Current spikes were analyzed by home-built Labview programs.</p><!><p>10 mM PB buffer with pH 1, 4, 7 were prepared. A 20 μM Mel solution was prepared by dissolving Mel powders in PB buffer, following with sonication for 10 min. The association of Mel to CB[7] (0-40 μM) was measured by absorption spectroscopy (UV-3600, Japan).</p><!><p>Water contact angles were measured using a goniometer (OCA15EC, Germany) immediately after the addition of 5.0 μL of water droplets on gold substrate.</p><!><p>In summary, we investigated the statistical conductance value of CB[7] and its host-guest complex molecular junctions by using STM fixed junction technique. The conductances of CB[7] and Mel@CB[7] complex with different pH value were determined in PB buffer and organic solvent TCB. Both CB[7] and Mel@CB[7] complex showed decreased conductance as the pH value increased. This suggested that the pH affected the electron transfer process of the molecular junction. The mean conductance and the mean lifetime of Mel@CB[7] complex were smaller than that of CB[7] under the same pH value, which indicated that the encapsulation of Mel molecule inside CB[7]'s cavity disturbed the electron process and weakened the electronic coupling between host molecule and gold electrodes. In addition, the values of the lifetime and the conductance for both CB[7] and Mel@CB[7] measured in PB were greater than that in TCB under the same pH value, suggesting the interactions between protons and carbonyl group under aqueous condition promoting better electron transfer to a certain extent. We believed this work can contribute to exploring multi-functional stimuli-responsive supramolecular system for single molecule detection.</p><!><p>All datasets generated for this study are included in the article/Supplementary Material.</p><!><p>The project was conceptually designed by FL. The majority of the experiments and data analysis were carried out by QA. The manuscript was prepared and revised by QA, QF, and FL. All authors discussed the results and implications and commented on the 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><!><p>Funding. The project was supported by the National Natural Science Foundation of China (21372183, 21807083) and the Program for Innovative Teams of Outstanding Young and Middle-aged Researchers in the Higher Education Institutions of Hubei Province (T201702).</p><!><p>The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2020.00736/full#supplementary-material</p><!><p>Click here for additional data file.</p>

PubMed Open Access

INHIBITION OF THE VESICULAR GLUTAMATE TRANSPORTER (VGLUT) WITH CONGO RED ANALOGS: NEW BINDING INSIGHTS.

The vesicular glutamate transporter (VGLUT) facilitates the uptake of glutamate (Glu) into neuronal vesicles. VGLUT has not yet been fully characterized pharmacologically but a body of work established that certain azo-dyes bearing two Glu isosteres via a linker were potent inhibitors. However, the distance between the isostere groups that convey potent inhibition has not been delineated. This report describes the synthesis and pharmacologic assessment of Congo Red analogs that contain one or two glutamate isostere or mimic groups; the latter varied in the interatomic distance and spacer properties to probe strategic binding interactions within VGLUT. The more potent inhibitors had two glutamate isosteres symmetrically linked to a central aromatic group and showed IC50 values ~ 0.3 \xe2\x80\x93 2.0 \xce\xbcM at VGLUT. These compounds contained phenyl, diphenyl ether (PhOPh) or 1,2-diphenylethane as the linker connecting 4-aminonaphthalene sulfonic acid groups. A homology model for VGLUT2 using D-galactonate transporter (DgoT) to dock and identify R88, H199 and F219 as key protein interactions with Trypan Blue, Congo Red and selected potent analogs prepared and tested in this report.

inhibition_of_the_vesicular_glutamate_transporter_(vglut)_with_congo_red_analogs:_new_binding_insigh

Introduction<!>Materials and Methods.<!>Synthesis of the compounds.<!>Glutamate Vesicular Uptake Assay.<!>Software and methods used for the homology modeling and docking studies.<!>VGLUT2 Homology Model.<!>Congo Red Analog Design and Syntheses.<!>Inhibition of VGLUT by the Congo Red Analogs.<!>Docking of Congo Red Analog 2a (Table 2).<!>Conclusions.

<p>As the primary excitatory neurotransmitter in the mammalian CNS, L-glutamate (Glu) is produced in the cytosol of neurons from glutamine or α-ketoglutarate.[1] Synaptic vesicles are then loaded with Glu via ATP/H+-dependent vesicular glutamate transporters (VGLUTs 1–3; SLC17A6, SLC17A8 and SLC17A7) [2] via Cl- stimulation that can occur from either side of the vesicle membrane [3–5] (Scheme 1). Vesicles that fuse with the plasma membrane during synaptic transmission release Glu into the synaptic cleft where it can activate ionotropic (iGluR) and metabotropic receptors (mGluR). To terminate the transmission, high-affinity, Na+-dependent protein transporters (EAATs 1–5) remove Glu from the synapse by sequestering it in glia and neurons (Scheme 1).[6–11] High affinity, selective ligands have been essential to the pharmacological characterization of the glutamate receptors and transporters,[2, 14] but by comparison far fewer have been identified that act at VGLUTs, where there is an unexplored potential to control vesicle filling, regulate pre-synaptic quantal size (Fig. 1)[15, 16] and cause a corresponding change in Glu-mediated signaling.[17] Although numerous advances have been made toward understanding VGLUT function, selective ligands for routine pharmacologic and/or physiologic manipulation of the transporter have been limited. Considering VGLUTs differ in function, transport criteria, substrate specificity and cellular location from EAATs and GluRs suggest they can be selectively targeted and pharmacologically-manipulated by small molecules without disrupting other transport phenomena. VGLUTs are believed to transport substrate using an alternating-access (rocker) mechanism[18] with a substrate binding pocket midway in the protein channel.[19–25] Phosphate serves as an alternative substrate but binds at a second, undefined binding domain.[3, 26] Using a homology model for VGLUT1 based on GlpT, a possible second substrate site for glutamate was proposed[19]; a notion supported in part by computational and experimental studies at other transporters.[27–31] The presence of a second substrate binding site on VGLUT also appears plausible because Trypan Blue (TB) and related azo-dye structures possessing two symmetric glutamate or amino acid mimics (Fig. 1) are potent inhibitors, whereas a ligand containing only one amino acid mimic is much less active. In this study, we first evaluated the possibility of dual binding sites by conducting preliminary docking of VGLUT2 to the known inhibitors Trypan Blue and Congo Red (CR) to determine the interatomic distance between glutamate isostere groups. Using docking information, analogs of CR were designed and prepared with glutamate isosteres attached to linker groups that varied in interatomic length to afford the optimal binding interactions to effectively inhibit VGLUT.</p><!><p>Solutions were concentrated by rotary evaporation (10 to 160 torr) or on a high-vacuum manifold (ca. 0.1 to 0.25 torr) at 25 – 60 °C. Flash column chromatography was performed using silica gel (60 Å, particle size 0.043 – 0.060 mm, EMD Chemicals). Protocols calling for the cooling of reaction mixtures to 0 °C were done so in a brine/ice bath. All reagents and solvents were used as received from commercial suppliers. NMR spectra were recorded on a Bruker Avance III (1H, 400 MHz; 13C 100 MHz) spectrometer. 1H and 13C NMR chemical shifts (δ) are reported in parts per million (ppm) and referenced to the solvent (13C) or the residual proton resonance of the solvent (1H) as reported in literature.23 UV-Vis absorption and fluorescent spectra were recorded on a Synergy Mx microplate reader. High-resolution mass spectra were obtained by the Mass Spectrometry Facility of the Core Laboratory for Neuromolecular Production at the University of Montana. Melting / decomposition points are uncorrected and were recorded on a Melt Temp II apparatus (Laboratory Devices Inc.).</p><!><p>The synthetic procedures and full characterizations are provided in the Online Resources.</p><!><p>Whole rat brains were purchased from Innovative Research (Novi, MI USA), the synaptic vesicles isolated, and the glutamate uptake assay conducted as reported previously [32–34] in which the control studies were done in the absence of ATP. Intact rat brain vesicles contain more than one VGLUT isoform. All efforts were made to minimize animal suffering and to reduce the number of animals used in this study.</p><!><p>Homology model and molecular docking were conducted using YASARA (Version 18.10.11)[35]. A homology model for VGLUT2 was built using D-galactonate transporter (DgoT)[36] and the model subsequently employed to conduct docking experiments. VGLUT2 secondary structure was predicted using PSI-Pred algorithm [37, 38] and produced 21 homology models that were further refined to a model with a quality Z-core of −1.14. Molecular docking in the VGLUT2 homology model was performed using YASARA using customized versions of AutoDock and VINA [39, 40]. Inhibitors were built using standard bond lengths and angles and minimized at pH 7.4 in the YAMBER force field. Clusters of poses were obtained that were scored in binding energy inclusive of typical binding interactions (hydrogen bonding, hydrophobic, polar, etc.) and repulsive forces. Substrate binding domains were initially identified by comparison to reported models [3, 5, 19, 26, 41, 42]. The simulation cell coordinates spanned two-thirds the length of VGLUT2 (Y-axis ~ 50 Å) with X-axis = 30 Å and Z-axis = 40 Å inclusive of each of the following ten residues: R88, H128, R184, E191, H199, R211, R322, Y327, H356 and T454. The docking results represent the top energy binding pose some of which were exported into 2D protein ligand map programs to identify interactions, and visualized in PyMol ver 2.1 (Schrodinger) or YASARA. Some binding orientations, docked molecules and structure overlays for VGLUT2-ligand displays had occluding protein domains made transparent to assist with clarity. Additional details on the procedures and methods used for the computational studies are provided in the Online Resources.</p><!><p>To investigate the possibility of multiple binding domains for glutamate, a homology model for VGLUT2 based on the D-galactonate transporter (DgoT)[36] was constructed and docking performed with TB and Congo Red (CR) (Figure 2) [41]. The VGLUT2-DgoT homology structure is similar to that previously reported from GlpT [19, 26] and the outward-facing, open, substrate-bound conformer (6E9O) reveals a long channel lined with ionizable residues. In the computational studies, residues considered important to docking were derived from prior models and reports that VGLUTs contain two anion binding sites and one cation binding site that permit vesicle filling to adjust to changing ionic conditions.[5] It should be noted that during the submission of this work, a structure for rat VGLUT2 obtained by cryo-electron microscopy was reported at 3.8-angstrom resolution.[43] Future studies will utilize the cryo-EM data and compare with the DgoT model. It was also understood that the modeling takes into account that VGLUTs have been shown to transport glutamate and inorganic phosphate (Pi) using a single, substrate binding site although coupling to cation gradients differ [44]. Although the possibility of a second binding domain would not alter the Glu and Pi transport mechanisms, VGLUT occupancy by an inhibitor bearing dual Glu mimics would also be likely block Pi transport.</p><p>Docking of TB led to a high binding energy pose (~ 16 kcal/mol) with the inward pointing naphthalene disulfonate (glutamate mimic) interacting with R88 and T454, and a second naphthalene disulfonate glutamate mimic forming interactions near the cytosolic entry or vestibule binding site with R211 and other residues (Fig. 2).[41] Likewise, the high energy binding pose for CR reveals interactions between the sulfonate and R88 and also a second ionic between the lower sulfonate with R211. The initial docking experiments support aryl disulfonates as glutamate mimics binding these residues. Whereas TB contains four substituents on each naphthalene moiety, CR has only two, and the biphenyl group is unsubstituted in CR.</p><p>Symmetric structures capable of engaging a second binding site may be advantageous to enhance the selectivity at VGLUTs. If two distinct binding domains were present, ligands constructed with a matched inter-domain distance may display improved binding over other known inhibitor types. In this study, a panel of new structures were designed containing dual carboxylate mimics separated by a linker moiety to vary the interatomic distance and potentially capitalize on simultaneously binding two domains in VGLUT.</p><!><p>Due to structure simplicity and known VGLUT inhibition (Ki ~ 550 μM), CR was selected as a scaffold to develop the new analogs (Fig. 3). The two 4-aminonaphthylsulfonic acid (ANS) groups present in CR were considered as glutamate isosteres to probe dual binding sites, and the intramolecular distance between these isosteres could be manipulated by replacement of the biphenyl linker with spacer groups including phenyl, diphenyl methyl, diphenyl ether, diphenyl thioether, diphenyl sulfone, diphenyldisulfide, 1,2-diphenylethane, 1,2-diphenylethylene and 1,2-diphenylethyne (Fig. 3). The diazo moiety was retained for structure-activity comparisons to CR and due to the known reliable chemistry connecting the linkers to substituted naphthylenes.</p><p>Altering the linker properties could also help characterize favorable interactions within the interdomain region. As controls, select structures were also prepared with only one aminonaphthylene sulfonic acid attached to a linker. The syntheses, in vitro inhibition of VGLUT, preliminary structure-activity relationships, and molecular modeling studies were conducted to support these binding hypotheses. Considering these analogs may possess emission spectra that could serve as reporters of VGLUT activity and ligand binding, we briefly investigated the UV-Vis spectral properties.</p><p>The syntheses of the mono- and bis-containing aminonaphthyl sulfonic acids were conducted as shown in Scheme 2. Diamine 1 was diazotized with NaNO2, the unreacted HNO2 quenched with urea, and the mixture added to a basic solution of ANS. The crude bis-ANS products 2 precipitated for most reactions, and could be collected by filtration. In other cases, precipitation was induced by addition of solid NaCl. For some products, silica gel column chromatography using CH2Cl2/CH3OH (9:1 to 4:1) was used to purify the analogs. The diphenylether analog 2d was isolated cleanly without additional purification. The side product from the coupling reactions was the mono-addition product 3. In the preparation of 2c and 2g, sufficient quantity of the mono-addition adduct was isolated for characterization and VGLUT screening.</p><p>In general, isolated yields for bis-linked analogs 2 were low (< 25%) due to incomplete reaction, competing reduction, diazotization decomposition, and chromatographic loss. However, these reactions were amenable to scale-up and the desired compounds were easily isolated. Compounds 2a and 2c-j representing the bis-linked analogs were characterized by 1H and 13C-NMR, mass spectrometry, and UV-Vis spectroscopy (see: Online Resources). As noted, compounds containing one ANS group, i.e., analogs 3a-d and 3i represent important controls lacking the second isotere. In order to expand this analog set, three additional unsymmetric analogs were synthesized using the same reaction conditions (Scheme 2) with the monoamine coupling partners aniline, 4-phenyl aniline, and 4-amino-biphenylether as diazotization substrates. The resultant mono-substituted analogs bearing azophenyl (3a), azobiphenyl (3b) and azo diphenyl ether (3d) were isolated in 30–40% yield. Compound 3b represents the truncated mono-substituted analog of Congo Red. The synthetic analogs gave UV-Vis absorption spectra with extinction coefficients ~ 104 M−1·cm−1, and most exhibited emission bands between 415 – 440 nm when excited at the maximum UV absorbance wavelength (c.f., Congo Red λmax = 340, 498 nm with an λex at 417 nm [45]), however, none were suitable for spectrometric assay development.</p><!><p>CR analogs were screened as VGLUT inhibitors anticipating that analogs with two ANS isosteres would show greater potency if the proper interatomic positions were obtained. The VGLUT assay [32, 46, 47] quantifies 3H-L-glutamate transported into synaptic vesicles, with the amount of radioactivity correspondingly reduced by inhibitors. All values have been corrected for non-specific uptake as determined by the accumulation of 3H-L-glutamate in the absence of ATP. The activities of the analogs were first screened at a concentration of 10 μM against 250 μM L-glutamate and reported as percent of control (i.e., 100% glutamate uptake = no inhibition and 0% uptake = complete blockade of glutamate uptake into vesicles). A more detailed IC50 evaluation was conducted on select analogs that reduced glutamate uptake to less than 10% of control (Table 1). Previously reported values for Congo Red (2b) and ANS were included for comparison [48, 49].</p><p>Prior work characterized the activity of 4-ANS and CR (2b) as inhibitors of VGLUT-mediated uptake [38]. Thus, while 4-ANS proved to be only a moderate to weak inhibitor itself (≈ 50% inhibition at 100 μM vs 250 μM 3H-L-Glu), the presence of two ANS groups linked via a biphenyl moiety (i.e., CR, 2b) resulted in a marked increase in inhibitory activity (IC50 = 0.6 ± 0.2 μM with 250 μM 3H-L-Glu) in agreement with more recent reports [49]. All of the compounds tested showed some inhibition of VGLUT with some preliminary differences observed between the compounds containing two ANS moieties 2a-2j (symmetric) or those with one 3a-d, 3i (asymmetric). Compounds 2a, 2c-2j reduced VGLUT uptake from 63–100% inhibition. Based on the VGLUT2 model, the bis-aryl or longer linkers were anticipated to be more potent owing to similarity to azo dyes such as Trypan Blue, however, compound 2a bearing a single phenyl linker was the most effective inhibitor exhibiting an IC50 ≈ 300 nM. To this point, compounds 2d (-PhOPh-), 2g (-PhCH2CH2Ph-) and 2j (-PhC≡CPh-) with slightly longer linkers were also among the more potent inhibitors in this new series. While compound 2c with a diphenylmethylene linker and compounds 2ef (thioether and sulfone) were only tested at a single concentration, the resulting levels of inhibiton demonstrate all are less potent than 2d, indicating the importance of a possible interaction between VGLUT residues and the linker oxygen atom. Among ligands with two atom spacers between phenyl groups, compound 2g (-CH2CH2-), which exhibited an IC50 ≈ 2.3 μM was a more effective inhibitor than the trans-stibene 2h, disulfide 2i or alkyne 2j. Although 2g would be expected to have greater conformational flexibility owing to its ethane bridge, than the other three, this flexibility alone cannot account for the favorable interaction with VGLUT.</p><p>In general, the asymmetric mono-ANS analogs exhibited less than or similar levels of VGLUT inhibition when compared with the corresponding bis-ANS analogs, although generalization such as these should be interpreted cautiously, as comparisons of % uptake (or % inhibition) are based on activity determinations at single concentrations. The diphenylmethylene and diphenyldisulfide were equivalent in activity indicating, at least with these derivatives, that no additional favorable interactions occurred with the inclusion of a second ANS group. ANS, which lacked any diazo linkers, showed modest inhibition of VGLUT (~ 50%), while the mono-substituted structures with phenyl 3a and biphenyl 3b groups did not appear to enhance binding. Extending the linker length in compound 3cdi improved inhibition likely through additional hydrophobic interactions. Still, the finding that the shortest symmetric analog 2a was the most potent blocker of VGLUT was highly unexpected and may help advance the development of future inhibitor structures. Further investigation was conducted to identify the activity of compounds 2a, 2d and 2g that appear comparable if not greater than Congo Red as VGLUT inhibitors. Detailed dose/response analyses for 2a (Fig. 4; IC50 plot shown), 2d and 2g afforded IC50 values of 300 nM, 1500 nM and 2300 nM, respectively, with plots consistent with competitive inhibition. Only 2a was more potent than Congo Red (ki ~ 490 nM; IC50 = 550 nM)[48] (Table 1). Attempts were made to assess compound 2j but poor solubility limited this experiment.</p><!><p>Since compound 2a proved to be a more potent inhibitor than CR, despite being shorter, docking experiments in YASARA[35] were conducted to assess possible interactions. The docking revealed a relatively high energy binding orientation for 2a (12.6 kcal/mol) with ionic interactions identified between sulfonates and R88 and H199 (Table 2; Entries 1 and 2) but not R211. A 2D protein-ligand interaction profile (PLIP)[50] revealed additional interactions and distances for ionic interactions His199 (3.8 Å) and R88 (4.3 Å) and hydrophobic, hydrogen bonding and a key pi-stacking interaction with Phe 219 (Table 2). Overall, the docking uncovered a number of interactions between 2a and VGLUT that support the unexpected inhibitory potency of this analog although additional analogs will need to be prepared to fully assess the validate these interactions.</p><p>The pi-stacking interaction between 2a and Phe219 (Entry 5) may explain, in part, why 3a, which lacks a second ANS moiety, retained some ability to block VGLUT activity. To investigate this, five additional ANS-diazophenyl analogs were prepared and tested based on the structure of 3a. When assayed at VGLUT, the p-methyl (51% ± 11), p-hydroxy (42% ± 4), p-methoxy (63% ± 6), p-bromo (65% ± 5), and p-iodo (56% ± 3) 3a analogs reduced uptake levels similarly. Docking did not reveal any deeper insights for these analogs but the p-hydroxy analog of 3a can form an additional hydrogen bonding interaction with VGLUT.</p><p>The role of the sulfonate group in CR as a glutamate mimic was also briefly investigated. Analogs in which the sulfonic acid group of ANS and compound 3a were substituted for a sulfonamide were prepared and tested. The reasoning for this exchange was to alter the pKa and degree of ionization, and consequently to attempt to change the interaction with R88 from ionic to hydrogen bonding and/or assess the role of Tyr195 and Tyr327. However, neither sulfonamide showed any activity as an inhibitor at the concentrations tested possibly suggesting the importance of charge interactions. Also, no analogs were prepared with substituents on the linker because benzopurpurin 4B, the 2,2'-bis-methyl diphenyl analog of CR is less active IC50 ~ 680 uM[51] potentially due to reduced pi-stacking interactions.</p><!><p>Acher and co-workers[52] reported a similar approach with modification of the linker but using the naphthalene scaffold of Trypan Blue rather than Congo Red. They were able to show that bi-aryl derivatives and 8-hydroxylated substituents are key to inhibitory activity - a useful and crucial finding as it aligns with our prior work using lipophilic groups improve the activity quinoline-2,4-dicarboxylates as inhibitors.[53, 54]</p><p>Brilliant Yellow (BY) is among the most potent VGLUT inhibitors yet identified and possesses sulfonic acid groups on the linker portion. Ueda and co-workers used a clever substitution of sulfonate to carboxylate and showed these BY analogs retained excellent inhibitory activity.[55] Molecular modeling indicated conserved distances between the azenyl nitrogen and sulfonate oxygens for the more potent inhibitors. As speculated by the authors [55], this arrangement might engage Tyr195, which was an insightful prediction that is supported by our docking study showing clear involvement of Tyr195 at both azo and sulfonates (Table 2). We are currently investigating new chemical entities that will utilize these collective findings to identify increasingly potent and selective inhibtors of VGLUT.</p>

PubMed Author Manuscript

Computational Screening of All Stoichiometric Inorganic Materials

The compositional space for inorganic materials remains vastly unexplored. Walsh and colleagues have designed procedures that use well-established chemical knowledge to quantify the number of possible multi-component compounds. They show how chemical filters can be applied to quickly and effectively narrow down the number of results and focus on those with target functionality.

computational_screening_of_all_stoichiometric_inorganic_materials

INTRODUCTION<!>The Bigger Picture<!>Elemental Combinations<!>Compositional Descriptors<!>Descriptor 1: Electronic Chemical Potential<!>Electronic Structure: Photoelectrodes<!>Conclusions<!>EXPERIMENTAL PROCEDURES

<p>Currently, over 184,000 entries in the Inorganic Crystal Structure Database (ICSD) involve 9,141 structure types; 1 66,814 of these materials have also been subject to quantum mechanical calculations, and information on their basic electronic structures and thermodynamics is included in the Materials Project 2 (powered by the PYMATGEN infrastructure 3 ).</p><p>The configurational phase space for new materials is immense, but blind exploration of the periodic table is a daunting task. Fortunately, over a century of research in the physical sciences has provided us with myriad rules for assessing the feasibility of a given stoichiometry and the likelihood of particular crystal arrangements. Examples of chemical phenomenology include the radius ratio rules 4 and Pettifor maps 5 for structure prediction, as well as electronegativity and chemical hardness for predicting reactivity. 6 Pauling's rules 7 provide predictive power for ionic or heteropolar crystals. A wealth of knowledge exists for understanding the physical properties of tetrahedral semiconductors. 8 Recent examples of searches for new materials that draw from existing chemical knowledge include 18-electron ABX compounds, 9 hyperferroelectric superlattices, 10 and organic-inorganic perovskites. 11,12 The reliability and predictive power of atomistic material simulations is increasing. 13,14 Many approximations are being removed as high-performance supercomputers reach petaflop scale. This includes more accurate quantum</p><!><p>The discovery of functional materials is critical for technological advancements that will play a role in addressing global challenges, ranging from catalysis for sanitation, semiconductors for harvesting solar energy, and biomimetic materials for health. There is a concerted global effort to reduce the time it takes to realize new materials via databases, highthroughput screening, informatics, and mapping out the ''materials genome.'' Here, we show how the compositional space for stoichiometric, inorganic materials can be quantified by simple rules and how the vast space can be explored quickly and cheaply with the use of key chemical concepts and element properties in the search for candidate materials with target properties. We exemplify the application of this approach by identifying a chalcohalide material with potential for watersplitting applications and carrying out a comprehensive search for new compositions that could adopt the widely studied perovskite crystal structure. mechanical treatment of electron-electron interactions in the solid state, 15 as well as more realistic models of chemical disorder. 16 However, because of the computational cost, high-throughput screening with first-principle techniques is usually limited to hundreds or thousands of materials-a small fraction of the overall phase space.</p><p>We report a procedure for navigating the materials landscape with low computational effort, and it can be achieved with simple chemical descriptors. We first explore the magnitude of the task at hand by enumerating combinations of elements and ions for binary, ternary, and quaternary compositions. We demonstrate that chemical constraints can narrow the search space drastically. Examples of how deeper insights can be gained are illustrated for electronic (photoelectrodes for water splitting) and structured (perovskite-type) materials. The procedure can be used to comfortably explore the vast compositional space or as the first step in a multi-stage high-throughput screening process. Instead of being a roadblock to achieving new functionality, the combinatorial explosion for multi-component compounds provides fertile ground for the discovery of innovative materials.</p><!><p>To begin, one can map chemical space by enumerating the ways in which the constituent elements of the periodic table can combine. If we restrict ourselves to the first 103 elements (to the end of the actinide series), the combinations (i.e., C 103 n ) for two, three, and four components are 5,253, 176,851, and 4,421,275, respectively. For five components, the combinations exceed 87 million.</p><p>Physically, the situation is more complex. Elements can combine in different ratios, leading to variation in material stoichiometry, e.g., the binary combinations AB, AB 2 , A 2 B 3 , and A 3 B 4 . Given elements can also adopt multiple oxidation states, each with a unique chemical behavior, e.g., Sn(II)O, Sn(IV)O 2 , and Sn(II)Sn(IV)O 3 . For our enumeration of feasible compounds, we next consider the accessible oxidation states of each element in stoichiometry up to quaternary A w B x C y D z , where the integers w, x, y, and z % 8. This definition includes, for example, common ternary pyrochlore oxides (A 2 B 2 O 7 ) and quaternary double perovskites (A 2 BCO 6 ). Using the most common oxidation states extends the first 103 elements of the periodic table to 403 unique ions.</p><p>The number of combinations is now drastically increased, as shown in Table 1, such that four-component candidate materials exceed 10 12 . In order to reduce this composition space, we can introduce selection rules (filters) from chemical theory.</p><p>We note that the estimations discussed here represent a lower limit on the number of accessible materials. We consider regular inorganic compounds and exclude, for example, non-stoichiometry, organic systems, hybrid organic-inorganic materials, electrides, and intermetallics, for which additional considerations are required for predicting viability. [17][18][19] Chemical Filters Rule 1: Charge Neutrality Ions tend to combine into charge-neutral aggregates. The same thinking applies to both ionic solids and more covalently bonded semiconductors. Any periodic solid must be charge neutral; otherwise, it would have an infinite electrostatic potential. Balancing oxidation states and fulfilling the valence octet rule are equivalent, e.g., III-V semiconductors, such as GaAs, can be represented as Ga 3+ As 3À . Our implementation is inspired by the work of Pamplin 20 and Goodman 21 on the subject of multi-component semiconductors.</p><p>A charge-neutrality constraint significantly reduces the total number of candidate materials. The rule states that the formal charges (q) of the components sum to 0, i.e., wq A + xq B + yq C + zq D = 0.</p><p>(Equation 1)</p><p>Charge neutrality contracts the compositional space by at least an order of magnitude for binaries, ternaries, and quaternaries (Table 1).</p><p>Rule 2: Electronegativity Balance Further to assuming that all charge-neutral combinations of oxidation states are accessible, we can implement a second constraint based on the electronegativity of the component elements. The empirical electronegativity (c) scale represents the ''attraction'' of a particular atom for electrons. For a stable compound, the relation c cation < c anion should be obeyed, i.e., the most electronegative element carries the most negative charge. Here, we employ the Pauling electronegativity scale, which reduces the allowed compositions by a factor of between 4 and 10 for the different numbers of components (Table 1).</p><p>It is also instructive to consider existing materials databases (the ICSD and Materials Project). For binary compounds, we find fewer combinations from our estimates as implemented in SMACT (Semiconducting Materials from Analogy and Chemical Theory) than from the ICSD (Figure 1), which can largely be attributed to our exclusion of intermetallics and polymorphs. In the Materials Project, multiple entries for a single crystal structure and chemical composition are removed, and the number of compositions are in close agreement. For ternaries and quaternaries, the compositions passing both charge and electronegativity tests continue to rise exponentially, whereas the number in existing databases remains relatively constant. The increased complexity of ternary and quaternary systems means that their synthesis, characterization, and reporting are more challenging than for binary systems. Nevertheless, the large differences between the numbers of potential and reported materials suggest that wide areas of unexplored compositional space may contain stable and useful materials.</p><p>a q, charge neutralitiy; X, electronegativity balance.</p><p>The numbers reported in this section are vast, and using modern electronic-structure techniques to perform quantitative screening for application is unimaginable. Exploration of the hitherto neglected compositional space will require further guidelines.</p><p>In the following sections, we demonstrate how additional descriptors can be applied for identifing materials for specific applications.</p><!><p>Several useful properties can be estimated from knowledge of the chemical composition alone, and here we explore the application of some of these approaches.</p><!><p>The concept of atomic electronegativity has been successfully extended to solids, where the geometric mean becomes the single-value descriptor, i.e.,</p><p>This descriptor represents a mid-gap energy between the filled (valence band) and empty (conduction band) electronic states. This corresponds to the electronic chemical potential (Fermi level) at 0 K. 22 Butler and Ginley 23 found a linear correlation between the solid electronegativity and the electrochemical flat-band potentials for a range of semiconductors. This was subsequently extended to a wider dataset including metal oxides, chalcogenides, and halides. 24 The method provides a rapid procedure for the estimation of absolute electron energies for multi-component materials. It is now commonly used in the computational screening of new materials for electrochemical applications. [25][26][27][28] Descriptor 2: Electronic Structure Many tight-binding model Hamiltonians exist for semiconductors and dielectrics. 8 One recent approach is based on the atomic solid-state energy (SSE) scale, 29 which provides information on valence and conduction bands on the basis of the frontier orbitals of the constituent ions. Whereas the Mulliken definition of electronegativity is an average of the ionization potential (IP) and electron affinity (EA) of an atom, the SSE reflects the IP of an anion (filled electronic states) and EA of a cation (empty electronic states). The energies of 40 elements were fitted from a test set of 69 closed-shell binary inorganic semiconductors, 29 which has recently been extended to 94 elements. 30 According to the tabulated SSE scale, the band gap (E g ) can be estimated as</p><p>For multi-component systems, the limiting values (cation with highest EA and anion with lowest IP) are used. The SSE has a root-mean-square deviation of 0.66 eV against the measured band gaps of 35 ternary semiconductors (see Table S1). This simple method allows for rapid screening of band gaps and absolute bandedge alignment.</p><p>Both methods (Equations 2 and 3) have been implemented for arbitrary compositions on the basis of tabulated atomic data in the SMACT package. Because no crystal-structure information is included at this level, the results are qualitative, and the models do not distinguish, for example, between polymorphs.</p><!><p>We now use the compositional space and chemical descriptors defined above to search for potential materials for solar fuel generation via photoelectrochemical water splitting.</p><p>The properties that are required for viable photoelectrodes include (1) a band gap in the visible range of the electromagnetic spectrum to absorb a significant fraction of sunlight and (2) upper valence and lower conduction bands bridging the water oxidation and reduction potentials, enabling the redox reaction. We set an optimal band-gap range between 1.5 and 2.5 eV. Although the free energy for water dissociation is 1.2 eV, the combination of loss mechanisms found in practical devices could require a band gap as large as 2.2 eV. 31,32 Metal oxides combine many attractive properties for water splitting (e.g., stability and cost), but they usually have band gaps too large to absorb a significant fraction of sunlight. The formation of multi-anion compounds offers a route to modifying the electronic structure. We consider ternary metal chalcohalides (i.e., A x B y C z ), with B = [O,S,Se,Te] and C = [F,Cl,Br,I]. We restrict the A cations to those with an SSE higher than the water reduction potential (approximately À4.5 V in relation to the vacuum at pH = 0). The conditions of charge neutrality and electronegativity are used for performing an initial screening that yields 52,094 combinations. With the additional band-gap criterion, the combinations are reduced to 7,676, and the pool of cations is reduced from 25 to 7 with A = [B,Ti,V,Zn,Ga,Cd,Sn]. We further rule out any boron-containing combinations at this stage, because these are known to form discrete molecular units (e.g., BClSe).</p><p>Finally, we screen compositions according to the environmental sustainability of the elements. We use the Herfindahl-Hirschman Index (HHI R ), which has been developed in the context of thermoelectric applications, for elemental reserves. 33 This index includes factors such as geopolitical influence over materials supply and price.</p><p>The HHI R for a given composition can be obtained as the weighted average over the constituent elements. At this stage, because stoichiometry is variable, we consider the mean HHI R for each A 1 B 1 C 1 combination.</p><p>The band-edge positions of the 20 candidates with the smallest HHI R values are presented in Figure 2. The HHI R has the effect of eliminating all combinations containing Ga, Te, and Br (although relatively abundant, most of the world's Br is produced from the Dead Sea, making it geopolitically sensitive, as reflected in a high HHI R ). There are no entries in the ICSD for most of the candidates that we identified; however, reports can be found for Cd 2 O 6 I 2 , Sn 2 SI 2 , and Zn 6 S 5 Cl 2 . [34][35][36] Both Cd 2 O 6 I 2 and Sn 2 SI 2 feature in the Materials Project and have band gaps of 3.3 and 1.6 eV, respectively, calculated within density functional theory (DFT). These compare with the SSE band gaps of 2.5 and 2.0 eV. The third compound, Zn 6 S 5 Cl 2 , is reported to have an optical gap of 2.7 eV 36 , which compares with the SSE band gap of 2.4 eV.</p><p>Only one oxygen-containing compound survived the band-gap screening criterion; the values for metal oxyhalides are generally too large. For O y I z , the iodide forms the upper valence band (low binding energy of I 5p), whereas it is the oxide (O 2p) for other halides. However, the sensitivity of the oxide ion to its crystal environment is well documented, 27,37 and consequently its SSE carries the greatest uncertainty. 29 This is one aspect where knowledge of the local structure (electrostatic potential) could significantly improve the accuracy of the results.</p><p>We must connect composition to crystal structure in order to make more accurate property predictions. Global optimization of crystal structures from first principles is a formidable task, although great progress is being made in this area. 38 We instead adopt an approach based on analogy with known structures through chemical substitutions, as developed by Hautier et al. 39 It uses data-mined probability functions, as implemented in the Materials Project.</p><p>To demonstrate the translation from composition to material, we performed crystalstructure mining for the four combinations with the lowest HHI R . The 88 predicted structures were then subjected to a full DFT lattice optimization procedure and ranked by total internal energy. Finally, accurate band gaps were predicted for the lowest-energy structures by hybrid DFT (HSE06 electron exchange and correlation 40,41 ). The compound Sn 5 S 4 Cl 2 has an indirect band gap of 1.6 eV and a direct gap of 1.8 eV, which lies within the target range. The band gaps of the other three lowest-energy compounds were calculated to be between 3.0 and 3.4 eV. Full details of the workflow (Figure S1) and band gaps (Table S2) can be found in the Supplemental Information.</p><p>The newly identified compound, Sn(II) 5 S 4 Cl 2 , adopts a structure formed from two distinct Sn centered polyhedra: (1) a distorted octahedron with equatorial S and apical Cl ions and (2) a distorted tetrahedron with 4 S ions and a stereochemically active Sn lone pair (Figure S2). The polyhedra form interlocking chains in three dimensions. The electronic density of states reveals an upper valence band composed of hybridized Sn s -Cl p orbitals; such Sn s-based valence bands are considered promising indicators for hole mobility. 42 The lower conduction band is composed mainly of overlapping Sn p orbitals. The chemical structure and bonding characteristics suggest that this material should have favorable carrier transport, crucial for optoelectronic applications.</p><p>Crystal Structure: Perovskites One of the most successful approaches to discovering new materials is structural analogy. The concept is to take a crystal structure with a known chemistry and to replace elements within the structure to tune the physical properties. In the simplest case, this involves direct isovalent substitution, e.g., Zn(II)S / Cd(II)S. Structural analogy can be extended to aliovalent cross-substitution (also termed cation mutation), e.g., Zn(II)S / Cu(I)Ga(III)S 2 . A systematic methodology was outlined more than 40 years ago in a paper by Pamplin 20 for enumerating charge-neutral tetrahedral semiconductors.</p><p>The challenge of going beyond tetrahedral semiconductors is predicting crystal structure. The radius of ions within a lattice has a long history as a geometric descriptor of structural stability. A key example is the application of radius ratio rules by Goldschmidt 43 to predict the propensity of a ternary ABC 3 combination to form the perovskite structure:</p><p>where t is the tolerance factor and r is the ionic radius. Values of t > 1 imply a relatively large A site favoring a hexagonal structure, 0.9 < t < 1 predicts a cubic structure, and 0.7 < t < 0.9 means that the A site is small, preferring an orthorhombic structure. For t < 0.7, other (non-perovskite) structures are predicted. These rules have recently been extended to describe structure-property relationships in hybrid organic-inorganic perovskites. 11,12 In this section, we apply our screening procedure to include knowledge of the crystal structure and estimate the size of the perovskite materials space. We start by enumerating the elemental combinations. We then reduce the set by requiring an octahedral coordination environment for the B site, as contained in the Shannon dataset, 44 and require a combination of oxidation states that are charge neutral. This list is then assessed in terms of t, as defined by the Shannon ionic radii. 44 We consider single-anion compositions based on C = [O,S,Se,F,Cl,Br,I]. The chargeneutrality and octahedral B-site constraints reduce the 176,851 elemental combinations to 41,725. The tolerance-factor constraint, 0.7 < t < 1.0, further reduces this to 26,567. For potential applications in the energy sector, we can consider candidates with HHI R smaller than that of CdTe (a commercial thin-film photovoltaic material), resulting in a final population of 13,415.</p><p>For each anion, an orthorhombic perovskite structure is the most common prediction, and hexagonal is the most rare (Figure 3). The fraction of cubic perovskite structures remains roughly constant within the respective halide and oxide or chalcogenide series; however, it is more dominant for the halides. The presence of Br or I makes a material less sustainable (higher HHI R ); otherwise, there is little to differentiate the anions.</p><p>Far more oxide and chalcogenide perovskites are predicted than halides. The higher anion charge allows for three distinct cation combinations (I-V, II-IV, and III-III), whereas halides have only I-II. In addition, a greater radius compatibility is found for the group VI anions. We find that the number of plausible perovskite structures increases with the anion radius; however, the lower crustal abundance for heavier elements reduces the number that meet the sustainability criterion.</p><p>A search of the Materials Project over the same anion space reveals 920 materials, a small fraction of those predicted from SMACT (26,567). The search includes all standard perovskite space groups. 45 For oxide perovskites, 8.26% of those identified from SMACT are found in the Materials Project; for sulfides, this falls to 0.45% and to 0.12% for selenides. To some extent, the greater number of oxide perovskites discovered reflects the greater research activity in this field; however, synthesis of chalogenide perovskites has been reported, [46][47][48] and there is interest in these materials for technological applications. 49,50 Of the ABC 3 materials reported in the Materials Project, 48% of oxides, 35% of sulfides, and 20% of selenides are in perovskite space groups.</p><p>Why are there so few chalcogenide perovskites? The tolerance-factor arguments that work well for metal oxides may not hold for chalcogenide perovskites. Oxygen forms more ionic compounds because of its higher electronegativity and lower polarizability than those of S, Se, and Te. When covalent bonding becomes prevalent, it is known to result in deviations from tolerance-factor behavior. An example is the case of NaSbO 3 , for which t = 0.92 is commensurate with the formation of cubic perovskite but which forms the non-perovskite ilmenite structure. Goodenough and Kafalas 51 explained this deviation as a result of strong s bonding between Sb and O. This procedure demonstrates the power of searching through materials on the basis of structural analogy. Only a small fraction of possible perovskite materials have been synthesized. Although some may not represent thermodynamic ground states, they could be accessible through kinetic control of crystal growth or templated on a substrate. Many interesting chalcogenide perovskites are waiting to be discovered. The final pool of 13,415 feasible compositions is within the grasp of explicit computation by quantum mechanical methods, albeit as part of an ambitious project. Indeed, high-throughput screening of 5,400 multi-anion cubic perovskite structures via DFT has been reported 25,52 and revealed 32 promising new materials for watersplitting applications.</p><!><p>We have demonstrated the utility of chemical theory in quantifying the magnitude of the compositional space for multi-component inorganic materials. Even after the application of chemical filters, the space for four-component materials exceeds 10 10 combinations. We further estimate that the five-component space exceeds 10 13 combinations. There are many applications in which materials with even higher-order compositions have been developed, e.g., in high-temperature superconductors, where six to seven component materials are common. The number of potential materials is not infinite, but it is immense. The scale of the combinatorial explosion emphasizes the need for effective material-design procedures that employ existing chemical and physical knowledge in a targeted manner. Stochastic sampling of this chemical space is unlikely to be effective in yielding materials with specific functionality. We have presented a procedure that uses simple descriptors to support materials exploration, discovery, and design. All element counts and plots presented in this paper were created with custom codes based on SMACT and written in the Python 2.7 programming language. Elemental data are collated from multiple sources (see Table 2) and made HHI elemental Herfindahl-Hirschman Index calculated from geological and geopolitical data 33 Ionization potential NIST Atomic Spectra Database 59 Pauling electronegativity updated values of electronegativity on Pauling's scale were compiled in the CRC Handbook; 53 for elements 95 (Am) and above, Pauling's recommended value of 1.3 was used; 60 the value for krypton (3.0) was derived from the bond energy of KrF 2 and reported in a scientific paper 61 SSE ''solid-state energy'' model of semiconductors and dielectrics 29,30 SSE (Pauling) extended estimates of solid-state energy from the correlation between known values and Pauling electronegativity 30 Where possible, values recommended by the National Institute of Standards and Technology (NIST) were used.</p><!><p>Chem 1, 617-627, October 13, 2016 625 algorithmically accessible in a unified object-orientated interface. Example routines that check element and oxidation-state combinations against the conditions of charge neutrality and electronegativity are provided.</p><p>Scripts that generate the results and plots reported in this paper are available with the SMACT codes. A number of tutorials working through the combinatorial explosion are provided at https://github.com/WMD-group/SMACT_practical.</p><p>The codes, collectively named Semiconducting Materials by Analogy and Chemical Theory, are inspired by the pen-and-paper procedure reported by Pamplin in 1964. 20</p>

Chem Cell

Circular dichroism and UV resonance Raman study of the impact of alcohols on the Gibbs free energy landscape of an \xce\xb1-helical peptide\xe2\x80\xa0

We used CD and UV resonance Raman spectroscopy to study the impact of alcohols on the conformational equilibria and relative Gibbs free energy landscapes along the Ramanchandran \xce\xa8-coordinate of a mainly poly-ala peptide, AP of sequence AAAAA(AAARA)3A. 2,2,2-trifluroethanol (TFE) most stabilizes the \xce\xb1-helical-like conformations, followed by ethanol, methanol and pure water. The \xcf\x80-bulge conformation is stabilized more than the \xce\xb1-helix, while the 310-helix is destabilized due to the alcohol increased hydrophobicity. Turns are also stabilized by alcohols. We also found that while TFE induces more \xce\xb1-helices, it favors multiple, shorter helix segments.

circular_dichroism_and_uv_resonance_raman_study_of_the_impact_of_alcohols_on_the_gibbs_free_energy_l

<!>Experimental Details<!>CD measurements<!>UVRR measurements<!>Impact of alcohols on the Gibbs free energies of helices<!>TFE induce multi helix segments<!>Conclusions<!>

<p>Protein (peptide) folding depends both on its primary sequence and its solvent environment. Addition of alcohol to aqueous solution changes the hydration of protein (peptide). The resulting conformational changes can be used as a valuable tool for probing protein (peptide) – water interactions.1–7 It is important to realize that despite intensive investigations over the years, the mechanism(s) by which alcohols perturb protein conformation is still poorly understand. 8–18</p><p>In this work, we used CD and UV resonance Raman (UVRR) spectroscopy to study the impact of alcohols on the conformational equilibria and relative Gibbs free energy landscapes along the Ramanchandran Ψ-coordinate of a mainly poly-ala peptide, AP of sequence AAAAA(AAARA)3A. We find that the α-helix and π-bulge conformations are most stabilized by 2,2,2-trifluroethanol (TFE), followed by ethanol, methanol and pure water. Turn conformations are also stabilized. However, 310-helices are destabilized. We also find that TFE induces an increased abundance of α-helices. However, the average α-helix length is decreased.</p><!><p>The 21-residue peptide AP of sequence AAAAA(AAARA)3A was purchased from AnaSpec Inc. (>95% purity). Absolute methanol was purchased from J. T. Baker. Absolute ethanol was purchased from Pharmco. 2,2,2-trifluroethanol (TFE, 99.8% purity) was purchased from Acros. The pH7 solution samples contain 1 mg/ml concentration of AP and 0.05 M NaClO4.</p><p>The CD spectra were measured by using a Jasco-715 spectropolarimeter, by using a 0.02 cm path length cuvette. We co-added five individual CD spectra.</p><p>The UV resonance Raman (UVRR) apparatus was described in detail by Bykov et al. 19 Briefly, 204 nm UV light (1 mW average power, 100 μm diameter spot) was obtained by mixing the 3rd harmonic with the fundamental (816 nm wavelength, 1 kHz repetition rate, 0.6 W average power, 25–40 ns pulse width) of a tunable Ti:Sapphire laser system from Photonics Industries. The sample was circulated in a free surface, temperature-controlled stream. A 180° sampling backscattering geometry was used. The collected light was dispersed by a double monochromator onto a back thinned CCD camera (Princeton Instruments Spec 10 System, 1.5 cm−1 resolution with 100 μm slit width). We used 5 min accumulation times, and four accumulations were co-added. The 732 cm−1 and 1379.5 cm−1 bands of Teflon were utilized to calibrate the frequency. The frequencies are reproducible to less than 1 cm−1. Raman spectra were normalized to the peak height of the 932 cm−1 ClO4− band. No Raman saturation occurs at these low excitation powers.</p><!><p>Fig. 1 shows the temperature dependence of the CD spectra of AP in pure water. The low temperature CD spectra show two troughs at 222 nm and 206 nm which are characteristic of α-helix conformations.20 As the temperature increases the ellipticity at 222 nm, θ222 becomes less negative indicating α-helix melting. The isosbestic point at 202 nm indicates that the melting behavior appears spectroscopically as a "two-state" process. Previous work by our group demonstrated that the AP α-helix conformation melts to a dominantly PPII-like conformation. 21</p><p>Fig. 2a and 2b show the temperature dependence of the mean residue ellipticity at 222 nm, θ222 of AP in pure water and in the presence of different alcohols. Alcohols increase the α-helix content. At 20°C and 25% alcohol by volume, TFE most stabilizes the α-helix, followed closely by ethanol and then methanol, consistent with previous studies 8,12,18. At 50% (v/v) alcohol, ethanol is the most α-helix stabilizing, followed by TFE and then methanol. As the alcohol concentration increases from 25% to 50%, θ222 decreases in methanol and ethanol but changes little in TFE (Fig. 2c). Previous studies also showed that TFE does not appear in CD measurements to induce additional α-helix concentrations above 25% (v/v). 11</p><!><p>204 nm UV Raman spectra (UVRS) of AP in pure water (Fig. 3) show mainly the amide RR bands. In contrast, 204 nm UVRS of AP in 50% methanol (Fig. 3) show methanol Raman bands which we numerically removed in the Fig. 4 UVRS. The resulting spectra show the temperature dependence of the 204 nm UVRS of AP in 50% methanol. As the temperature increases, the AmI band upshifts from 1652 cm−1 to 1658 cm−1 while the AmII band downshifts from 1556 cm−1 to 1552 cm−1. 22 Previous work 23 indicates that water hydrogen bonding to the peptide bond (PB) C=O site increases the C=O bond length and, thus, downshifts the AmI band, while water hydrogen bonding to the PB N-H upshifts the AmII band. The AmI (AmII) band in pure water (Fig. 4) is upshifted (downshifted) relative to that in 50% methanol, indicating less C=O (N-H) hydrogen bonding in alcohol solution. 23 The Cα-H doublet (~1372 cm−1 and ~1393 cm−1) frequency does not shift as the temperature increases but its intensity increases. The Cα-H doublet intensity only slightly increases from 2°C to 20°C, indicating little α-helix melting. 24 Significant intensity changes observed from 20°C to 40°C indicates extensive α-helix melting. The AmIII3 band downshifts from ~1264 cm−1 at 2°C to ~1259 cm−1 at 40°C while its intensity increases. 22 UVRS of AP in other alcohols (not shown) show very similar α-helix melting behaviors.</p><p>To calculate the α-helical fractions, we subtracted appropriate amounts of the temperature dependent PPII-like conformation basis spectra 22 from the measured and digitally smoothed UVRS of AP to minimize the Cα-H region intensity in the difference spectra. The basis spectra intensities subtracted are directly proportional to the concentrations of the PPII-like conformation at each temperature. The resulting difference spectra appear to be mainly α-helix-like. Fig. 5 shows UVRR calculated fractions of α-helix-like conformation of AP in pure water and in 50% (v/v) alcohol. The α-helical-like conformations are dramatically stabilized in alcohol, and melt little as the temperature increases. TFE stabilizes the α-helical-like conformations the most, followed by ethanol, and then methanol, as previously observed. 8,12,18 The α-helix-like conformation melting curves in ethanol and in methanol are essentially identical. These conclusions obviously differ from the CD conclusions.</p><p>Fig. 6 shows the calculated α-helix-like spectra of AP in pure water and in 50% (v/v) methanol. The AmIII3 band in pure water shows a peak at ~1258 cm−1 with a shoulder at ~1280 cm−1 and another shoulder at ~1240 cm−1. Previous work showed that an AmIII3 band at ~1258 cm−1 indicates the pure α-helical conformations, while a band at ~1280 cm−1 indicates a π-helix (bulge) while a band at ~1240 cm−1 indicates a 310-helix. 25 The AmIII3 band in pure water narrows at higher temperature as previously observed,26,27 indicating decreased concentrations of 310-helix and π-bulge conformations relative to the pure α-helix concentration as the temperature increases. The AmIII3 band in 50% methanol shows a shoulder at ~1258 cm−1 and another shoulder at ~1280 cm−1 while the ~1240 cm−1 component is missing, indicating a lack of 310-helices. All helical spectra show an AmIII3 band at ~1200 cm−1, indicating turn structures. 26 Calculated α-helix-like spectra in other alcohols (not shown) are essentially identical to those in methanol, indicating similar ensembles of helical conformations.</p><p>We calculated the Gibbs free energy landscapes of AP (Fig. 7) along the Ψ-folding coordinate from the UVRR by using the methodology of Mikhonin et al. 19,26,28 The energy landscape (Fig. 7) is bumpy within the α-helix-like basin. Within this basin the pure α-helix conformation (Ψ ~ −45°) is always lowest in energy, followed by the π-bulge conformation. The 310-helix conformation (Ψ ~ −20°) lies at a slightly higher relative energy in pure water, but at much higher energies in alcohols. As the temperature increases the α-helix basin Gibbs free energy in pure water increases indicating that the α-helix is destabilized relative to the PPII-like conformation. The relative α-helix basin energies change very little with temperature in 50% alcohols. For all temperatures, the lowest α-helix Gibbs free energies occur in 50% TFE, followed by ethanol, methanol and finally pure water. The same trend is seen with the π-bulge energies. The alcohol induced π-bulge energy decrease is larger than that of the α-helix. Turn conformations are stabilized by alcohols, consistent with previous observations that alcohols stabilize turns over PPII-like conformations.29 In contrast, 310-helix conformations are dramatically destabilized by alcohols.</p><!><p>Numerous studies indicate that alcohols induce α-helix formation in proportion to the bulkiness of their alcohol hydrocarbon group. 8,12,18,30 This is confirmed by our UVRR results that the α-helix has the lowest Gibbs free energy in 50% TFE, followed by ethanol and methanol.</p><p>Alcohol molecules displace water in the peptide hydration shell which increases the hydrophobicity of the peptide-solvent interface, which should enhance intramolecular hydrogen bonding which should increase the α-helical content 31,32. Previous studies 33 indicate that 310-helices allow greater solvent access to the peptide bonds and thus are favored as the solvent hydrophilicity increases. In contrast, the α-helix and π-helix are more favored as the solvent hydrophobicity increases. It is also known that the 310-helix is favored in the peptide terminal regions where solvent exposure is greatest. 34</p><!><p>Our UVRR measurements that indicate that 50% TFE most stabilizes α-helical-like conformations, appears to conflict with the CD measurements that 50% TFE does not significantly stabilize α-helical conformations more than 25% TFE. Previous studies22,35–37 showed that UVRR calculated α-helical conformation concentrations are higher than those calculated from CD 36 because the magnitude of the molar ellipticity per peptide bond (PB) decreases dramatically as the number of PB within an α-helix decreases. 11,38,39 In contrast, Raman is more linear; each peptide bond independently contributes to the Raman intensity 36,40 (except for the AmI band of the α-helical conformation where strong coupling between AmI vibrations exist 41). Thus, we can explain the spectroscopic results by proposing that TFE induces the most α-helical PBs but also breaks long helices into short helices (See Appendix I). Recent studies have showed that TFE binds strongly to peptides, 42,43 while ethanol does not directly bind. 10</p><p>To quantify the dependence of the CD molar ellipticity per PB of an α-helix, θn on the number of PBs within the helix, n, we fitted our experimental data to the empirical equation proposed by Chen et al, 39 (See Appendix II);</p><p>This allows us to relate the observed θ222 values to the UV Raman calculated helical fractions (See Appendix III). The Fig. 8 calculated θ222 in 50% TFE is modeled to be less negative than that in 50% ethanol at low temperatures. (Calculated θ222 are slightly more negative than those measured in Fig. 2b because the NaClO4 used as an internal standard in the UVRR measurements as an internal intensity standard but not included in the CD measurements stabilizes the α-helix conformation.27)</p><!><p>CD and UVRR measurements indicate that TFE most stabilizes the α-helix, followed by ethanol, methanol and pure water. We determined the Gibbs free energy landscape from the UVRR spectra and found that the alcohol induced π-bulge energy decrease is larger than that of the α-helix, while the 310-helix energy increases due to the alcohol increased hydrophobicity. Turns are stabilized by alcohols as well. We also found that while TFE induces more α helices, it favors multiple, shorter helical segments.</p><!><p>Temperature dependence of the CD spectra of 1 mg/ml AP in pure water.</p><p>(a) θ222 of AP in 25% (v/v) alcohol; (b) θ222 of AP in 50% (v/v) alcohol; (c) Δθ222 (θ222 in 50% alcohol minus θ222 at 25% alcohol).</p><p>204 nm excited UVRS of AP in pure water (solid line); AP in 50% methanol (dashed line) at 10°C. The UVRS of AP in pure water was scaled to facilitate comparison.</p><p>Temperature dependence of 204 nm excited UVRS of AP in 50% methanol and UVRS of AP in pure water at 2°C (dashed line); The methanol contribution was subtracted. The UVRS of AP was scaled to facilitate comparison.</p><p>Raman calculated AP α-helical-like fractions (primarily α- and 310 and π-helix(bulge)) of AP in different solutions.</p><p>Calculated α-helix-like spectra of AP in pure water (solid line) and in 50% (v/v) methanol (dashed line). Calculated α-helix-like difference spectra were normalized to the intensity of the AmIII1 band.</p><p>Calculated Gibbs free energy landscape of AP along the Ramachandran Ψ angle coordinate. in pure water; in 50% methanol; in 50% ethanol; in 50% TFE;. The PPII-like conformation is the reference state.</p><p>Calculated θ222 of AP in pure water and in 50% (v/v) alcohols.</p><p>fαCD vs. fαRaman of AP.</p><p>Linear fit of previously measured θ222 and fαRaman of AP in pure water.27</p>

PubMed Author Manuscript

Online coupling of digital microfluidic devices with mass spectrometry detection using an eductor with electrospray ionization

MS detection coupled with digital microfluidic (DMF) devices has only been demonstrated in an off-line manner using matrix assisted laser desorption ionization. In this work, an eductor is demonstrated which facilitated online coupling of DMF with electrospray ionization MS detection. The eductor consisted of a transfer capillary, a standard ESI needle, and a tapered gas nozzle. As a pulse of N2 was applied to the nozzle, a pressure differential was induced at the outlet of the ESI needle that pulled droplets from the DMF, past the ESI needle, and into the flow of gas exiting the nozzle, allowing detection by MS. Operating position, ionization potential, and N2 pressure were optimized, with the optimum ionization potential and N2 pressure found to be 3206 V and 80 psi, respectively. Online MS detection was demonstrated from both open and closed DMF devices using 2.5 \xce\xbcL and 630 nL aqueous droplets, respectively. Relative quantitation by DMF-MS was demonstrated by mixing droplets of caffeine with droplets of theophylline on an open DMF device, and comparing the peak area ratio obtained to an on-chip generated calibration curve. This eductor-based method for transferring droplets has the potential for rapid, versatile, and high throughput microfluidic analyses.

online_coupling_of_digital_microfluidic_devices_with_mass_spectrometry_detection_using_an_eductor_wi

Introduction<!>Materials<!>Eductor fabrication and operation<!>DMF Device Fabrication<!>DMF device operation<!>Mass spectrometry parameters<!>Optimization and characterization experiments<!>Results<!>Design and principles of operation<!>Performance characterizations and optimization<!>DMF-MS analysis<!>Conclusion

<p>Digital microfluidics (DMF) is a lab-on-a-chip format where discreet droplets are manipulated on an array of patterned electrodes to accomplish complex tasks. DMF devices operate by electrowetting, in which the wetting of a hydrophobic dielectric surface can be reversibly affected by application of a potential between electrodes embedded below the surface and a counter electrode in contact with the fluid. Precise manipulation of the wetting via actuation of electrode arrays allows sub-microliter droplets to be dispensed from larger volume reservoirs and enables merging, mixing, and splitting of such droplets to accomplish a multitude of analyses.</p><p>Many of the benefits of DMF devices result from the small, discreet droplet volumes and the versatility of analyses that can be performed in the digital regime. The utility of discreet droplets in chemical analysis has been demonstrated in both flow-based systems1 and in DMF format2–4 as isolated reaction vessels5,6 and isolated sample storage vessels.7 Sampling in the form of droplets allows for increased temporal8,9 and spatial10 resolution, while the small volume of these droplets maintains high concentrations in sample-limited analyses11 and reagent-limited reactions.12 Performing chemical analysis in the digital regime involves combining discreet packets (droplets) of information (analytes and reagents) with a finite number of operations (droplet generation, merging, mixing and splitting) to accomplish a wide variety of analytical tasks. In this way, digital microfluidic devices are analogous to digital electronics in that they offer similar versatility from a limited number of basic tools and operations.</p><p>While the use of small volume droplets lends benefits to DMF analyses, it also poses a challenge to many traditional detection methods. As with other microfluidic formats, this challenge can be addressed by employing fluorescence detection,13,14 however this detection scheme limits the types of analyses and the amount of information that can be attained. Mass spectrometry (MS) offers a more information-rich approach to detection, and while multiple instances of offline coupling of DMF with matrix assisted laser desorption ionization (MALDI) have been reported,15,16 reports of electrospray ionization (ESI) sources for DMF devices are fewer.17 A challenge to integrating the two methods is that the droplets on a DMF device are unconfined and at ambient pressure, which makes it difficult to introduce them to ESI without a pressure-assisted mechanism. Previous methods have interfaced segmented droplets from microfluidic devices, but these methods utilized pressure-induced transport to deliver these droplets to the MS.18,19</p><p>Another challenge with online coupling is that the typical operating voltages for DMF and ESI are dissimilar, with DMF devices operating with AC voltages20, 21 and ESI with high DC voltages. Ambient desorption ionization methods such as desorption electrospray ionization (DESI)22 and direct analysis in real time (DART)23 may be amenable to online detection from DMF devices, however these methods would not be effective for ionizing droplets in closed DMF devices and would not easily allow analysis of the entire droplet volume. Additionally, integration of ESI sources with DMF devices would allow online detection, improving the versatility and throughput of DMF analyses.</p><p>A technology capable of transferring droplets from the DMF device to the ion source is needed to decouple AC and DC voltage operation while allowing online MS detection from DMF devices. An eductor is a device that uses the flow of one fluid to entrain the flow of another. Eductors operate by the Venturi effect as described by the Bernoulli principle, and are a mature technology appearing in a wide range of applications,24–26 however this common method of fluid control is rarely employed on microfluidic devices. In this work, an eductor with an integrated ion source is presented to address the challenges of integrating DMF with online MS detection. A fused silica transfer capillary was used to couple the DMF device to a standard ESI needle. The ESI needle was aligned inside of a gas nozzle so that as N2 flowed through the nozzle, a drop in pressure resulted at the outlet of the ESI needle by the Venturi effect. This pressure differential from the inlet of the transfer capillary to the outlet of the ESI needle pulled sub-microliter droplets from the surface of the DMF device, past the ESI needle, and ejected into the flow of N2 to the MS inlet. A multivariate approach was used to optimize eductor operating position, applied ionization potential, and applied N2 pressure. To demonstrate the versatility of coupling DMF with online MS analysis, droplets of caffeine were merged with droplets of an internal standard and peak areas of the resulting selected ion current (SIC) were used for quantification. This method of integration is simple and should be applicable to a large number of DMF analyses.</p><!><p>Caffeine, theophylline and Met-Arg-Phe-Ala peptide (MRFA) were purchased from Sigma-Aldrich (St. Louis, MO). Acetic acid was purchased from EMD Chemicals Inc. (Gibbstown, NJ). SU8-3035 was purchased from Microchem Corporation (Newton, MA). Teflon AF solution was purchased from DuPont (Wilmington, DE). EGC-1700 was purchased from 3M (St. Paul, MN).</p><!><p>High density polyethylene tubing with an inner diameter of 3 mm was heat drawn to an outlet diameter of 1.5 mm to form the nozzle of the eductor. A regulator controlled the pressure from the N2 tank. Timing of N2 delivery to the nozzle was controlled by 9 computer-controlled solenoid valves (model A00SC232P, Parker Hannifin Corp., Cleveland, OH) with the outlets connected together. The transfer line from the DMF device was a 10 cm × 150 μm o.d. × 100 μm i.d. fused silica capillary (Polymicro Technologies, Phoenix, AZ) while a standard ESI needle (P/N 00950-00951, Thermo Fisher Scientific Inc., Waltham, MA) was used. Ionization potential was applied by a high voltage DC power supply (model 6AA12-P4, Ultravolt, Ronkonkoma, NY) controlled by software written in Labview 8.5 (National Instruments, Austin, TX).</p><!><p>Electrode patterns were fabricated on chrome photomask blanks (Telic Co., Valencia, CA) by photolithography and chrome etching. The electrode pattern consisted of a 5 mm × 5 mm reservoir electrode segmented into two parts, and six working electrodes for droplet dispensing and delivery to the transfer capillary. Electrodes were connected to electrical contact pads at the edge of the device designed to interface with a card edge connector (model 3666-0000, 3M, St. Paul, MN). To improve the uniformity of dielectric coatings, patterned electrodes were subjected to two minutes of plasma oxidation (model PDC-32G, Harrick Plasma, Ithaca, NY) prior to spin coating SU8-3035 at 7000 rpm for 40 s. The dielectric thickness was 19.7 ± 0.9 μm as measured by a profilometer (P-15, KLA-Tencor, Milpitas, CA). Coated devices were baked for 5 min at 65 °C followed by 5 min at 95 °C before being exposed to 25 s of UV radiation at an intensity of 20 mW cm−2 using a collimated UV source (OAI, San Jose, CA). The devices were left overnight to complete the curing of SU8. Cured devices were spin coated with a 1% solution of Teflon AF 1600 at 2500 rpm for 20 s then baked for 5 min at 75 °C to evaporate the solvent. The coated surface was then quickly passed over a gas burner several times (< 1 s per pass) to reduce surface roughness.</p><p>Grounding electrodes for open DMF devices were fabricated by dip coating 30 gauge tin plated copper wire in EGC-1700 electronics coating. The grounding electrodes were placed approximately 100 μm to the side of the working electrodes which allowed continuous contact with the droplets. Cover slides for closed DMF devices consisted of 25 mm × 75 mm ITO-coated glass slides (P/N 576352, Sigma-Aldrich). The ITO surface was coated with Teflon AF by the same procedure described above, except a final baking step of 15 min at 330 °C was performed rather than passing the devices over a gas flame. Closed devices were assembled by spacing the ITO cover slides 175 μm from the DMF surface using a gasket made from two layers of blue low tack tape (Semiconductor Equipment Corporation, Moorpark, CA).</p><!><p>Droplet actuation was achieved by applying 175 – 225 Vrms at 1 kHz to the patterned electrodes while the 30 gauge tin plated copper wire or ITO cover slide was grounded. The control voltage was generated by passing the output of a function generator (model 4003A, B&K Precision Corporation, Yorba Linda, CA) through a high voltage amplifier (model 601C, TREK, Inc., Medina, NY). Voltage application was controlled using computer-controlled 12V relays (CK1601A, Carl's Electronics, Oakland, CA) interfaced to the DMF device with a card edge connector. Droplet volumes in closed DMF devices were determined from digital images of droplet generation using ImageJ software.27</p><!><p>Mass spectra were collected using a Finnigan LCQ Duo (Thermo Fisher Scientific Inc., Waltham, MA). Unless otherwise noted, selected ion monitoring (SIM) was performed over a 10 m/z range centered at the indicated ion of interest. Selected ion currents (SIC) were taken from the SIM data using a 1 m/z window centered on the ion of interest. For measurement of caffeine:theophylline ratios, the mass analyzer was scanned from 175 – 200 m/z, and the SIC of each compound using a 1 m/z window was used for each compound. The ratio of the areas of these SIC traces was then taken. The MS instrument was operated with a capillary temperature of 200 °C and a 10 ms ion injection time. Ionization voltages ranged from 0 to +6 kV DC, and were applied using a computer-controlled high voltage power supply as mentioned previously.</p><!><p>Design Expert 7 software (StatEase, Minneapolis, MN) was used to develop the experimental design and to perform all statistical analyses. Four experimental factors were varied over three levels each, as described in Table 1. A Box-Behnken experimental design was used to reduce the 64 possible combinations to 29 experiments (including 5 replicates of the median condition, x = 5 mm, y = 37.5 mm, P = 60 psi, and V = 3000 V). Each of the four factors was then fit to a polynomial model with the general form: (1)R=βi+x+y+V+P+xy+xV+xP+yV+yP+VP+x2+y2+V2+P2+xyV+xyP+xVP+yVP+x2y+x2V+x2P+xy2+xV2+xP2+y2V+y2P+yV2+yP2+V2P+VP2+x3+y3+V3+P3+Ei where R was the response (average peak area or peak area RSD), βi was the model coefficient, Ei was the residual error, x was the position off-axis relative to the MS inlet, y was the distance from the MS inlet, V was the ionization potential, and P was the applied N2 pressure. Analysis of variance (ANOVA) was used to partition the total variation in the data into the variations due to the four experimental factors and the variation due to random error. These components were used to calculate an F-value, which was compared to a tabulated F-distribution to generate a p-value. The p-value described the probability that a variation resulted from error rather than from an experimental factor. When p < 0.05, the effect of that factor was considered to be significant. Model reduction, which pooled non-significant terms (p > 0.10) with the residual error by backward elimination regression, was applied to find the best fit for each response. The final reduced model for each response was described by the following equations: (2)Log(peakarea)=β1+x+V+P (3)RSDofpeakarea=β2+x+y+V+P+VP+y2</p><p>ANOVA tables for each of the reduced models above are given in Tables S1 and S2 in the supporting information.</p><p>Unless otherwise stated, all signals were quantified by integrating the area under the SIC traces, and the analyte was 2.5 μL aqueous droplets of 10 μg mL−1 MRFA (m/z = 524.3) acidified with 1% acetic acid.</p><p>Semi-quantitative analysis was performed with 2.5 μL aqueous droplets containing caffeine concentrations of 5 – 250 μg mL−1, with theophylline (m/z = 181.1, [M+H]+) as an internal standard at a final concentration of 100 μg mL−1.</p><!><p>Online MS detection from DMF devices is an attractive option for high throughput and parallel analyses with information rich detection. Coupling DMF to ESI-MS is challenging because it is difficult to introduce a pressure-assisted mechanism to droplets that are at ambient pressure and not confined by fluidic channels. Additionally, the voltage regimes of DMF (typically hundreds of volts AC) and ESI (typically kilovolts DC) are difficult to integrate into a single electronic system. To address these challenges, we have developed a microfluidic eductor that transfers DMF droplets to an ESI source and subsequently into an MS instrument for detection.</p><!><p>The eductor, illustrated in Figure 1A, consisted of a transfer capillary, a standard ESI needle, and a tapered gas nozzle. The transfer capillary coupled the DMF device to the ESI needle, with the outlet of the transfer capillary recessed 2 mm from the outlet of the ESI needle. The ESI needle was aligned coaxially inside the gas nozzle, with the outlet of the needle recessed 1 mm from the outlet of the gas nozzle. With this configuration it was possible to introduce sub-microliter droplets into the mass spectrometer by applying a 15 s pulse of N2 to the nozzle. As the gas pulse passed the ESI needle, a drop in pressure was induced at the outlet of the ESI needle, which pulled droplets from the DMF device, through the transfer capillary, past the ESI needle, and into the flow of gas exiting the nozzle.</p><p>The device was easily coupled to DMF devices of both closed and open configurations, provided that the gap spacing of the closed DMF devices was greater than the 150 μm outer diameter of the transfer capillary. Video demonstrations of the eductor operating with both open and closed DMF devices can be found in the supporting information. Droplet volumes of 630 ± 90 nL were successfully transferred from closed DMF devices, whereas open DMF devices were loaded with 2.5 μL droplets by pipette. In either case, droplets from the DMF device resulted in peaks in the SIC that were rectangular in shape with the duration of the signal proportional to the volume of the droplet being transferred (Figure 1B). The RSD of the ion current magnitude from a single peak was higher than 20%, but the areas of the SIC produced from droplets of identical composition and volume were less than 10% RSD.</p><p>MS detection of small molecules, peptides, and proteins were all possible with this system. The present studies focused on detection of small molecules and peptides because biomolecule adsorption on the surface of the DMF device, a well known phenomenon,28 hindered the analysis of proteins (data not shown). This carryover does not limit the efficacy of the system, however, since methods exist to address protein carryover on DMF devices.29</p><!><p>The eductor was positioned in front of the MS inlet on a two axis positioning stage, and a multivariate approach was used to characterize the performance of the eductor as a function of four factors: x-position (off-axis position relative to the MS inlet), y-position (distance from the MS inlet), applied ionization potential, and applied N2 pressure. Table 1 shows the levels over which each of these factors was tested. A Box-Bhenken experimental design, similar to those described previously,30, 31 was used to reduce the total number of experiments needed. In each experiment, the average SIC peak area from 5 replicate droplets and the RSD of the replicate peak areas were the measured responses. ANOVA was used to identify statistically significant relationships between the factors and these responses.</p><p>The peak area response was fit to a model (p < 0.0001) where the log of peak area varied linearly with respect to x-position (p < 0.0001) and ionization voltage (p = 0.0320). Neither y-position nor applied N2 pressure affected peak area in a statistically significant manner. Figure 2 illustrates the relationship between the log of peak area and both x-position and applied ionization potential. Not surprisingly, peak area increased with increasing ionization potential and decreased as the eductor was moved off-axis with respect to the MS inlet. The maximum experimentally observed peak area occurred at x = 0 mm and at an ionization potential of 5000 V. The factors found to be insignificant in this model, y-position and N2 pressure, both play a role in the desolvation of ions prior to MS detection. The lack of a statistically significant relationship between these factors and peak area suggests that either sufficient desolvation occurred even at low N2 pressure and at short distances from the MS inlet, or that desolvation did not play a significant role in changing the SIC peak areas. Further studies will be required to completely understand the role these factors have on ion desolvation in this system.</p><p>Peak area reproducibility followed a more complex trend, with the RSD of SIC peak areas fitting a quadratic model (p = 0.0030). In this model, significant relationships were found for both applied N2 pressure (p = 0.0063) and the square of y-position (p = 0.0027). The first order y-position term was not statistically significant (p = 0.837) but was retained in the model to maintain hierarchy with the y2 term. Figure 3 shows the relationship between peak area RSD and both y-position and applied N2 pressure. The dependence of RSD on y2 is evident by the parabolic shape of the response surface, with the minimum RSD found at a y-position of 37.5 mm at each value of N2 pressure. Peak area RSD decreased with increasing N2 pressure, with the highest experimentally observed peak area reproducibility (5% RSD) at the highest N2 pressure (80 psi). As discussed previously, N2 pressure and y-position were insignificant factors when attempting to increase the average peak area, suggesting that droplet desolvation did not play a significant role in peak area. But, due to their significance in this study, the shape and mechanics of plume generation and ion desolvation appear to strongly influence the reproducibility of the SIC peak areas. Previous reports have looked at plume shape and mechanics in similar ionization sources,32 but we felt a peak area precision of 5% was sufficient. Relationships of borderline significance were x-position (p = 0.0794) and the product of N2 pressure and ionization potential (p = 0.0903). Ionization potential was not found to be statistically significant (p = 0.232), but was retained in the model to preserve hierarchy. Preliminary studies indicated that modulating N2 pressure was also an effective method for affecting the rate of droplet transfer from the DMF device to the MS detector, with decreased N2 pressure leading to slower transfer speeds (data not shown). While this may be useful in tuning an analysis to particularly small or large volume droplets, it should be noted that doing so would have an impact on peak area reproducibility.</p><p>The performance characterizations described above were used to identify optimum operating conditions. Two equally weighted optimization goals were defined: 1) maximizing the SIC peak area, and 2) minimizing peak area RSD. A desirability function scored how closely the response met the optimization goals, with a desirability score of 1 indicating that the response was in perfect agreement with the optimization goals.33 Figure 4 shows a spatial plot of the desirability score as a function of x- and y-position at the identified optimal N2 pressure and ionization potential. A maximum desirability score of 0.919 is predicted when the eductor is positioned on-axis with the MS inlet, at a distance of 37.5 mm, with an ionization potential of 3206 V and an N2 pressure of 80.0 psi. At these conditions, the experimentally observed average peak area was 26308 arbitrary units with an RSD of 5%, which agreed with the results predicted by the model of an average peak area of 26440 arbitrary units and an RSD of 3% (Figure 4B).</p><!><p>Preliminary studies suggested that peak area in the SIC was an effective means for quantifying signal in these studies, and thus motivated the investigation of peak area in the device characterizations described above. To validate the use of SIC peak area in DMF-MS analyses, a semi-quantitative DMF-MS analysis was performed.</p><p>The ability to rapidly mix droplets makes DMF devices a promising platform for relative quantitation by MS because analyte-containing droplets can be combined with internal standards prior to analysis. Relative quantitation of caffeine from aqueous droplets on a DMF device is illustrated in Figure 5. Droplets containing premixed solutions of caffeine with theophylline internal standard were transferred from the DMF device to the MS by the eductor. Ratios of the caffeine/theophylline peak areas (Figure 5A, squares) gave a calibration curve with a linear range of 5 – 250 μg mL−1 (12.5 – 625 ng per droplet) and a best fit line of y = 0.0159 x + 0.0237 (R2 = 0.999). Under these conditions, caffeine was detected regularly at concentrations of 1 μg/mL (2.5 ng per droplet), but 10-fold lower concentrations were not. Shown in the inset of Figure 5A are examples of the SIC traces obtained at two points within the calibration curve. The blue trace is the SIC of caffeine (m/z = 195) and the black trace is from theophylline (m/z = 181). Aqueous droplets spiked with caffeine were combined on the DMF device with droplets containing theophylline (Figure 5B) and allowed to mix by diffusion. The resulting relative peak areas agreed well with the calibration curve (Figure 5A, diamonds), and validated the use of relative peak areas as a means of quantifying DMF droplet contents by online MS detection.</p><!><p>Coupling of DMF devices to ESI-MS allows the advantages of digital analyses with the information-rich detection offered by MS. The eductor-integrated ionization method presented here facilitates online MS detection from open or closed DMF devices in two ways. First, the eductor imparts a motive force to the droplets to deliver them to the ESI needle addressing the challenge associated with ambient and unconfined droplets. Ambient ionization mass spectrometry methods, such as DESI or DART, could be used to analyze droplets from open DMF devices, but it would be a challenge to analyze droplets from closed DMF devices. Secondly, the eductor is designed to remove droplets from the DMF device, which allows the high DC ionization potential to be decoupled from the voltage scheme of the DMF device permitting MS analysis while movement of other droplets on the DMF can continue uninterrupted. Droplet removal has the additional benefits of allowing detection from the entire droplet volume, and of clearing droplets from the device to allow high throughput or highly paralleled analyses.</p><p>DMF devices achieve complex tasks from a limited number of operations, which makes this technology ideally suited for highly versatile analysis systems. Online MS detection from DMF devices complements this versatility well, since it will allow optimization, calibration, and analytical measurements to be performed within the same experiment, without taking the DMF device offline.</p>

PubMed Author Manuscript

Carbon nanopipette electrodes for dopamine detection in Drosophila

Small, robust, sensitive electrodes are desired for in vivo neurotransmitter measurements. Carbon nanopipettes have been previously manufactured and used for single cell drug delivery and electrophysiological measurements. Here, a modified fabrication procedure was developed to produce batches of solid carbon nanopipette electrodes (CNPEs) with ~250 nm diameter tips, and controllable lengths of exposed carbon, ranging from 5 \xce\xbcm to 175 \xce\xbcm. The electrochemical properties of CNPEs were characterized with fast-scan cyclic voltammetry (FSCV) for the first time. CNPEs were used to detect the electroactive neurotransmitters dopamine, serotonin, and octopamine. CNPEs were significantly more sensitive for serotonin detection than traditional carbon fiber microelectrodes (CFMEs). Similar to CFMEs, CNPEs have a linear response for dopamine concentrations ranging from 0.1 to 10 \xce\xbcM and a LOD of 25 \xc2\xb1 5 nM. Recordings with CNPEs were stable for over 3 hours when the applied triangle waveform was scanned between \xe2\x88\x920.4 and 1.3 V vs. Ag/AgCl/Cl\xe2\x88\x92 at 400 V/s. CNPEs were used to detect endogenous dopamine release in Drosophila larvae using optogenetics, which verified the utility of CNPEs for in vivo neuroscience studies. CNPEs are advantageous because they are an order of magnitude smaller in diameter than typical CFMEs and have a sharp, tunable geometry that facilitates penetration and implantation for localized measurements in distinct regions of small organisms, such as the Drosophila brain.

carbon_nanopipette_electrodes_for_dopamine_detection_in_drosophila

<!>Solutions and Chemicals<!>Carbon Nanopipette Electrode fabrication<!>Scanning electron microscopy<!>Instrumentation and Electrochemistry<!>Endogenous Dopamine Evoked by CsChrimson Channelrhodopsin Stimulation<!>Statistics<!>Results and Discussion<!>Fabrication of Carbon Nanopipette Electrodes<!>Comparison of CNPEs and CFMEs<!>CNPE Stability Over Time<!>CNPE characterization<!>Measurements of Endogenous Dopamine in Drosophila Evoked by CsChrimson Stimulation<!>Conclusions

<p>Carbon-fiber microelectrodes (CFMEs) are traditionally used with fast-scan cyclic voltammetry (FSCV) to study rapid neurotransmitter changes in vivo.1 They allow real-time detection of catecholamines with high sensitivity and selectivity. Traditional CFMEs are 7 μm in diameter2; however, smaller electrodes would be useful for neurochemical studies in small organisms such as Drosophila melanogaster (the fruit fly). The larval fly central nervous system is extremely small, only about 100 μm wide and the brain is about 8 nL in volume.3 Individual neuropil, or brain regions, of the adult fly are only a few microns in diameter.4,5 Drosophila is a convenient model organism because it has homologous neurotransmitters with mammals and is easy and fast for genetic manipulation. CFMEs have been used to make electrochemical measurements of exogenously applied dopamine in the adult fly mushroom body.6 In addition, endogenous, stimulated dopamine changes have been measured in a single fruit fly larva.3 Drosophila have glial sheaths surrounding their neuropil that can be tough to penetrate. In larvae, a cut surface is made to insert the electrode3,7,8,9 and in adults, collagenase has been applied to chemically digest the tissue.6,10 Therefore, studying the release of endogenous neurotransmitters with better spatial resolution requires a small, dagger-like electrode that can penetrate through the tough glial sheath barrier with minimal tissue damage. Although CNPs have not been previously tested with intact brain tissue, they have been used to penetrate individual mammalian cells while retaining cell viability.11,12,13 Hence, it is reasonable to expect that the CNPs can penetrate the brain tissue without causing significant damage.</p><p>Over the past few decades, nanoelectrodes have been developed for electrochemical applications. Carbon electrodes are preferred for neurotransmitter applications because of their low cost, wide potential window, and good adsorption properties.14 To make smaller electrodes, carbon fibers can be either flame etched or electrochemically etched to sub-micron tips.15–18 Recently, flame-etched carbon fibers encased in nanopipettes have been used to make measurements at single synapses, but these electrodes must be individually fabricated.19 Carbon nanomaterials, such as nanotubes, can also be used as smaller electrodes. Carbon nanofiber microelectrodes have been developed for neurotransmitter detection, but they are on a larger chip and not easily implantable.20 Small carbon paste electrodes have been made for scanning electrochemical microscopy studies, but are not easy to batch fabricate.21 Tiny, nanometer-sized electrodes have been made using a single-walled carbon nanotube either sticking out22 or on a silicon wafer.23 Alternatively, some methods completely insulate an etched carbon fiber except for the very tip leaving an effective diameter of a few nanometers.11,24 However, insulation is difficult and a single carbon nanotube or nanometer-sized fiber is not robust enough to be implanted into tissue.</p><p>For in vivo measurements in Drosophila, we desire a sharp, carbon nanoelectrode with high sensitivity to detect nanomolar concentrations that can be easily batch fabricated. Carbon nanopipette electrodes (CNPEs) are nanometer sized electrodes, which have been previously used for electrophysiological measurements and delivering fluids into cells.25,26,35 CNPEs are fabricated by selectively depositing a carbon layer on the inside of a pulled-quartz capillary. The capillary is then chemically etched to expose the carbon tip. CNPEs are batch-fabricated in a furnace and are rigid because of the quartz insulation. While many of our past designs consisted of hollow pipettes, allowing for drug delivery to cells, extending the carbon deposition time can lead to a sealed, solid tip with a 50–400 nm diameter. Recently, CNPEs with recessed tips have been evaluated as nanosamplers and scanning electrochemical microscopy tips.27 Here, solid-tipped, cylindrical CNPEs and fast-scan cyclic voltammetry (FSCV) are coupled for the first time.</p><p>The objective of this study was to characterize the electrochemical properties of CNPEs using FSCV for the detection of electroactive neurotransmitters and test their suitability for measurements in Drosophila. We use three parameters to define the truncated cone geometry of the CNPE tip: the tip diameter, exposed length along the pipette axis, and cone angle. Tip diameter affects invasiveness, cone angle affects sharpness and rigidity, and the exposed length controls the electrode interfacial surface area. The CNPEs used here were approximately 250 nm in diameter at the tip, with a cone angle of 1.6°, and exposed carbon length ranging from 5 μm to 175 μm, controlled with etch time. CNPEs were successfully used to detect dopamine, serotonin, and octopamine. Dopamine current was stably detected with CNPEs with an optimized triangular waveform of −0.4 V to 1.3 V at a scan rate of 400 V/s and a frequency of 10 Hz. The current was linear with dopamine concentration up to 10 μM. CNPEs are sharp and robust enough to successfully penetrate into a Drosophila larva central nervous system without breaking and endogenous, stimulated dopamine release could be measured. CNPEs coupled with FSCV will facilitate fast, real-time measurements of neurotransmitters in specific brain regions of the Drosophila.</p><!><p>All reagents were purchased from Fisher Scientific (Fair Lawn, NJ) unless otherwise specified. Dopamine hydrochloride, serotonin hydrochloride, and octopamine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Each neurotransmitter was dissolved in 0.1M HClO4 for a 10mM stock solution and diluted daily in phosphate buffered saline (PBS) for testing. The PBS was 131.25 mM NaCl, 3.0 mM KCl, 10.0 mM NaH2PO4 monohydrate, 1.2 mM MgCl2 hexahydrate, 2.0 mM Na2SO4 anhydrous, and 1.2 mM CaCl2 dihydrate with the pH adjusted to 7.4. Sodium chloride was purchased from VWR International LLC (West Chester, PA), sodium phosphate from Ricca Chemical Company (Arlington, TX) and calcium chloride from Sigma-Aldrich. All aqueous solutions were made with deionized water (Milli-Q Biocel, Millipore, Billerica, MA).</p><!><p>CNPEs were fabricated with 1 mm outer diameter, 0.7 mm inner diameter, filamented quartz capillaries of 10 cm length (Sutter Instrument Co., Novato, CA) or 1mm outer diameter, 0.8mm inner diameter non-filamented quartz capillaries of 10 cm length (VitroCom, Mountain Lakes, NJ). Pipettes were pulled using a Sutter P-2000 laser-based pipette puller with the parameters: HEAT 800, FIL 4, VEL 60, DEL 128, and PULL 100 (1×0.7mm filamented) or HEAT 750, FIL 4, VEL 50, DEL 150, PULL 55 (1×0.8mm non-filamented). Chemical vapor deposition (CVD) was performed on the pipettes in a 3-zone horizontal tube furnace (Carbolite HVS, Hope Valley, UK) with a 1.3″ inner diameter quartz tube at 900 °C, with flow conditions of 400 sccm methane and 600 sccm argon, for a 3 hour duration. During deposition, the pipette tips were oriented against the flow of the gas, i.e., the tip pointed upstream. Pipettes were cooled under argon flow to prevent the oxidation of the carbon at elevated temperatures. The carbon deposited selectively inside the pipette, not on the outer surface due to the gas confinement effect described by Singhal et al.28 No catalyst was used.</p><p>The carbon-coated pipettes were etched in 5:1 buffered hydrofluoric acid (Transene Co. Inc., Danvers, MA) for 10 to 15 minutes, followed by a 10-minute rinse in deionized water. A friction grip was used to hold pipettes, and a manual manipulator was used to lower the tips into beakers of either HF or water. Short CNPEs for use in the Drosophila larval ventral nerve cord were prepared with an etch time of 60 seconds. The pipettes were inspected under an optical microscope (Olympus Corp. BX-51) and imaged with a SEM (FEI Quanta 600 ESEM, Hillsboro, OR). The tip outer diameter ranged from 50 to 400 nm (average of 250 nm) and the exposed carbon tip length depended on the etch time and tolerances of the pipette puller, but typically was 125 to 175 μm for etch times of 10 to 15 minutes and 5 to 10 μm for a 1 minute etch. For additional details on CNP fabrication we refer readers to the following references.12,28,29</p><p>To ensure the pipettes were properly sealed, CNPEs were connected to the headstage of a HEKA EPC 10 patch-clamp amplifier using a standard 1.0mm HEKA pipette holder. The pipettes were also connected to a pressure-injection pump (Eppendorf Femtojet, Happauge, NY). The CNPE tip was submerged in phosphate buffered saline (HyClone, PBS1X) and a 0.5 mm diameter silver chloride wire (WPI Inc., Sarasota, FL) was used as a counter/reference electrode in solution in a 2-electrode configuration. The digital lock-in module of the PATCHMASTER software was used to measure the equivalent capacitance of the CNPE interface with a 10mV, 1kHz sinusoidal potential, as the pressure within the pipette was adjusted between 0 and 300 kPa. The tip was first checked for bubbles, which would indicate a broken tip, and then the capacitance was monitored with changing pressure. The capacitance is proportional to the electrode interfacial area, and if it is stable with varying pressure it indicates that there is minimal capillary rise and that the tip is well-sealed. CNPEs that were not well sealed were discarded.</p><!><p>Scanning electron microscopy was performed in a FEI Quanta 600 ESEM (FEI, Hillsboro, Oregon) in secondary electron mode. CNPEs were adhered to a standard sample mount with carbon tape such that the CNPE axis was orthogonal to the electron beam. A short working distance (5mm) and low accelerating voltage (2 keV) were used in high-vacuum mode to attain enhanced surface detail and to minimize charging effects.30 The Environmental SEM provides a large sample chamber that allows CNPEs to be mounted without breaking or modification.</p><!><p>Fast-scan cyclic voltammograms were collected using a Chem-Clamp potentiostat (Dagan, Minneapolis, MN). TarHeel CV software (gift of Mark Wightman, University of North Carolina) was used for data collection and analysis. The hardware and data acquisition were the same as previously described.31 A triangular waveform was applied to the electrode. The electrode was scanned at a scan rate of 400 V/s from −0.4 V to 1.3 V and back at a frequency of 10 Hz unless otherwise noted. A Ag/AgCl wire was used as a reference electrode. The flow injection apparatus with a six-port, stainless steel HPLC loop injector used is the same as previously described.32 Electrodes were tested with a 5 s injection time. Because carbon is deposited on the entire length of the CNPEs, a direct electrical connection was made between a silver wire and the inner carbon lining of the CNPE at its distal end in the Universal Pipette Holder (HB180, Dagan Corp., Minneapolis, MN). No backfill solution was used. The holder was connected to a 1 MΩ headstage (Dagan Corp., Minneapolis, MN).</p><!><p>Virgin females with UAS-CsChrimson inserted in attp1833 (a gift of Vivek Jayaraman) were crossed with th-GAL4 flies (a gift of Jay Hirsh). Resulting heterozygous larvae were shielded from light and raised on standard cornmeal food mixed 250:1 with 100 mM all-trans-retinal. A small amount of moistened Red Star yeast (Red Star, Wilwaukii, WI) was placed on top of the food to promote egg laying. The central nervous system of a third instar wandering larva was dissected in the buffer. Isolated ventral nerve cords were prepared and recorded from as previously described.34 The electrode was allowed to equilibrate in the tissue for 15 minutes prior to data collection. A baseline recording was taken for 10 seconds prior to stimulation. Red-orange light from a 617 nm fiber-coupled high-power LED with a 200 μm core optical cable (ThorLabs, Newton, NJ) was used to stimulate the CsChrimson ion channel. The light was modulated with Transistor-Transistor Logic (TTL) inputs to a T-cube LED controller (ThorLabs, Newton, NJ), which was connected to the breakout box. TTL input was driven by electrical pulses controlled by the TarHeel CV software.</p><!><p>Statistics were performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). Data are reported as the mean ± standard error of the mean (SEM) for n number of different electrodes. Significance was determined by unpaired t-tests and defined as p ≤ 0.05.</p><!><p>The first goal of this study was to electrochemically characterize CNPEs using FSCV. FSCV allows measurements of rapid changes in neurotransmitter concentrations. CNPEs have traditionally been manufactured with open tips25, but that is not suitable for rapid electrochemistry as sample would wick up into the pipette. Here, the fabrication was modified slightly to grow enough carbon to make an electrode with a solid tip ~250 nm in diameter. The cone angle was 1.6° and the exposed carbon length, controlled with etch time, ranged from 5 μm to 175 μm. The second goal was to test the suitability of CNPEs for dopamine measurements in Drosophila larvae, which have a very small central nervous system. This nanoscale electrode would allow for high spatial resolution measurements.</p><!><p>The carbon nanopipette electrode (CNPE) consists of a pulled-glass/quartz pipette coated with a layer of pyrolytic carbon along its entire inner surface to a thickness sufficient to seal the pipette's narrow opening (Figure 1). Figure 1A shows the fabrication process. First, a quartz capillary was pulled into a fine-tipped nanopipette (Figure 1A (i)). Next, carbon was deposited by CVD until the tip was sealed with carbon (Figure 1A (ii)). Further up, the carbon-coated pipette was still hollow, which facilitates electrical connection via contact with a metal wire. Subsequent to the carbon deposition, the quartz/glass at the tip of the CNPEs was etched in buffered hydrofluoric acid to expose a desired length of a tapered carbon cylinder (Figure 1A (iii)). The exposed length was controlled by the etching time. For the pipette geometry used here, this corresponds to an exposed length of 12.5–17.5 μm/minute as measured with an optical microscope and confirmed with SEM. Figure 1B shows an example CNPE tip with ~170 μm exposed carbon. The interface between the exposed carbon and the quartz insulation is clearly visible. Figure 1C is an enlarged image of the quartz/carbon interface. The tip diameter of the individual CNPEs used in this work was measured with SEM and the range of tip sizes was 50–400nm (Figure 1D), with an average of 250 nm. Since the glass/quartz template controls the outer dimensions of the deposited carbon, it is likely that the primary source of tip variability stems from the pipette puller parameters. Even with the above variability, this fabrication method consistently yields sub-micron sized tips, an order of magnitude smaller than traditional CFMEs. All electrodes were tested for tip closure prior to use.</p><!><p>Dopamine was chosen to analyze with CNPEs because it is an important neurotransmitter, easily oxidized, and adsorbs to carbon surfaces.35 Dopamine plays an important role in reward, addiction, and motor behaviors.36 Figure 2 shows examples of background-subtracted cyclic voltammograms (A and B) as well as normalized current vs time plots (C and D) for dopamine at two different waveforms for a CNPE (dashed line) and a CFME (solid line). The first waveform (referred to as the 1.0 V waveform) scanned from −0.4 to 1.0 V vs. Ag/AgCl/Cl− at a scan rate of 400 V/s and a frequency of 10 Hz (A and C). The second waveform (referred to as the 1.3 V waveform) was the same as the 1.0 V waveform except the switching potential was 1.3 V (B and D). Figures 2B and D show larger currents for both CNPEs and CFMEs with the 1.3 V potential limit than the ones with the 1.0 V potential limit, as expected due to oxidation of carbon.37 The CFME had more current for dopamine than the CNPE for both waveforms. However, the peak oxidation voltage was lower for CNPEs (Figures 2A and B). CNPEs and CFMEs have a similar time response at the 1.3 V waveform, although CNPEs were slightly slower with the 1.0 V waveform (Figures 2C and D).</p><p>Table 1 shows average peak oxidative currents (ip,a), background currents, and the difference between the oxidative and reductive peak potentials (ΔEP) for CNPEs (150 μm in length, ~250 nm diameter) and CFMEs (50–75 μm in length, 7 μm diameter) at the 1.0V and 1.3V waveforms. The average peak oxidative current (ip,a) for 1 μM dopamine is about 30% lower for CNPEs than CFMEs at both waveforms; however, the difference was not significant (unpaired t-test, 1.0 V waveform, p=0.1273; 1.3 V waveform, p=0.2353). The background currents were obtained in the absence of dopamine and compared using the maximum values during the forward scans. Background currents for CNPEs were higher than CFMEs, although not significantly (unpaired t-test, 1.0 V waveform, p=0.2484; 1.3 V waveform, p=0.3730). Background current is proportional to electrode surface area. The 7 μm diameter, 62 μm long CFME has a surface area of 1410 μm2, while the 150 μm long CNPE with a cone angle of 1.6° and a tip diameter of 250 nm has a surface area of 1970 μm2. Thus, the CNPEs are about 1.4 times larger than CFMEs, which is consistent with background current ratios of 1.5 and 1.4 documented in Table 1 for the 1.0V and 1.3V waveforms, respectively. For in vivo measurements, the exposed carbon length was successfully reduced below 10 μm (8.2 ± 1.4 μm) by decreasing the quartz etch duration time. This size was appropriate for producing sufficient current magnitude from a highly localized region.</p><p>A lower oxidation current correlated with lower background charging current was expected; however, this was not true for the CNPEs of the sizes used in our experiments. The ratio of background current to peak oxidative current is significantly higher for CNPEs than CFMEs at both waveforms signifying CNPEs are less sensitive per unit area than CFMEs. At the 1.0 V waveform, CNPEs have a background current to peak oxidative current of 75 ± 10; whereas, the CFME ratio is 29 ± 4 (unpaired t-test, p<0.005, CNPE n=8, CFME n=6). For the 1.3 V waveform, CNPEs have a background current to peak oxidative current of 42 ± 5, and the CFME ratio is 21 ± 4 (unpaired t-test, p<0.005, CNPE n=11, CFME n=6). These differences in sensitivity per unit area may due to different types of carbon in CFMEs vs CNPEs or capacitive coupling with the thin quartz near the CNPE tip.</p><p>ΔEp is the difference between the oxidative and reductive peak potentials. The average ΔEp for CNPEs is significantly lower than CFMEs for the two waveforms (Table 1, unpaired t-test p<0.0001). As in the case of the CFMEs, there is no significant difference in ΔEp for CNPEs for the 1.0 V and 1.3 V waveforms (p=0.4010). The decrease in ΔEp for CNPEs compared to CFMEs implies decreased overpotential for dopamine. However, one would expect to observe higher sensitivity with lower overpotential, and the CNPEs had less sensitivity per unit area. Alternatively, the IR drop may be different for the different materials. The lower ΔEp might also be due to differences in diffusion and adsorptive behavior due to different carbon types and geometries between the CNPEs and CFMEs. The CNPE carbon is amorphous with graphitic islands, and has surface functional groups that depend on deposition conditions.38 In contrast, the CFMEs are predominantly graphitic. The lower ΔEp could be due to different functional groups or the amount of edge plane graphite sites on CNPEs, which play a role in electron transfer and adsorption reactivity.14</p><p>CNPEs were also used to detect serotonin and octopamine, which are other electroactive signaling molecules in the Drosophila central nervous system. Figure 3 shows examples of background-subtracted cyclic voltammograms with inserts of peak oxidation current over time for octopamine (A and B) and serotonin (C and D). The standard waveform (−0.4 to 1.3 V at 400 V/s) was applied at 10 Hz to CNPEs for octopamine detection (Fig. 3A) and serotonin detection (Fig. 3C). In addition, specialized waveforms previously developed for these neurotransmitters were applied, a positive waveform for octopamine detection39 (0.1 to 1.0 V and back at 600 V/s, Figure 3B) and the serotonin waveform for serotonin detection40 (0.1 to 1.0 to −0.1 to 0.1 at 1000 V/s, Figure 3D). Both analytes exhibited less electrode fouling when using their respective specialized waveforms, which is indicated by a faster return of the current to baseline after removal of the analyte from the flow cell. For octopamine, a strong secondary peak is observed using the positive waveform at CNPEs (Fig 3B), whereas less secondary peak was observed when using the positive waveform with CFMEs39. This could indicate adsorption of the secondary oxidation product of octopamine to the surface of CNPE despite the positive holding potential.</p><p>The average peak oxidation current of 1 μM serotonin was 43 ± 11 nA (n=4) for CFMEs and 33 ± 2 nA (n=4) for CNPEs. The average peak oxidation current of 1 μM octopamine was 10 ± 3 nA (n=4) for CFMEs and 2.8 ± 0.2 nA (n=4) for CNPEs. The ratio of background charging current to peak oxidative current for serotonin is significantly lower for CNPEs (2.5 ± 0.4, n=4) than CFMEs (11 ± 2, n=4) (unpaired t-test, p=0.0117). However, the ratio of background charging current to peak oxidative current for octopamine it is not significantly different between CNPEs (52 ± 15, n=4) and CFMEs (31 ± 6, n=4) (unpaired t-test, p=0.2489). Therefore, CNPEs have higher sensitivity for serotonin than CFMEs, while having the same sensitivity for octopamine.</p><!><p>Electrode stability is important for in vivo experiments, which can last hours.41 To test stability, the 1.0 V waveform was applied continuously to the CNPE in buffer and the response to a five-second injection of 1 μM dopamine was measured every hour. Current was normalized for each electrode to the first response to dopamine to take into account differences in individual electrodes. Figure 4 shows that at the 1.0 V waveform, the CNPE sensitivity dropped to 32% of the original current after 3 hours. CFMEs are stable over the same time.42 The left inset shows example cyclic voltammograms taken at the first injection of dopamine and after three hours for the 1.0 V waveform. The oxidative and reductive peak voltages shifted outward, signifying slower electron transfer kinetics accompanied the decrease in sensitivity. We hypothesize that the surface of the electrode is fouled, which would reduce the sensitivity and slow the transfer kinetics; however, dopamine diffuses to the electrode and some current is still measured from electron tunneling.</p><p>This problem of electrode surface fouling is overcome by electrochemically renewing the surface. Scanning to 1.3 V allows for the regeneration of a fresh carbon surface and maintains electrode sensitivity.37 For the stability experiments using the 1.3 V waveform, the CNPEs were allowed to stabilize with the waveform applied for 30 minutes before taking the initial measurement due to the oxidation of the electrode surface. If not allowed to stabilize, the signal actually increases during this time due to increased surface area from carbon-carbon bonds breaking and increased adsorption due to carbon functional groups.31 The peak oxidative current was constant over three hours (Figure 4) with the 1.3 V waveform, indicating that CNPEs are stable at this waveform and suitable for longer in vivo studies. This is confirmed by the right inset CVs which show the sensitivity and the electron transfer kinetics remained the same after three hours. Three hours is longer than a typical Drosophila experiment, and some electrodes were used for much longer or in multiple larvae and showed no degradation in signal. From this stability experiment, we determined that the 1.3 V waveform was most appropriate and we used this waveform for the remaining studies.</p><!><p>Figure 5 shows that the CNPE peak oxidation current for 1 μM dopamine is proportional to the scan rate, v (R2=0.984, n=4). The frequency was varied to keep equal time between scans. Current is normalized to the maximum value per electrode to minimize effects due to varying surface areas of different electrodes. For a diffusion-controlled process we anticipate a v1/2 proportionality with peak current, arising from the diffusive time scale in the transport equation. For an adsorption-controlled process we expect a proportionality with scan rate, which arises upon the inclusion of adsorption kinetics via a Langmuir or linearized Langmuir isotherm.43 This plot indicates that the kinetics are more adsorption-controlled than diffusion-controlled (plot of i vs v1/2 yields an R2=0.956, n=4), similar to carbon-fiber microelectrodes.35 Figure 5B shows peak currents for various dopamine concentrations (100 nM to 10 μM). Current is linear with concentration up to 10 μM. The average LOD, calculated from the 100 nM data, was 25 ± 5 nM (n=3).</p><!><p>To test the use of CNPEs to detect endogenous dopamine in Drosophila, dopamine release was stimulated with the red light sensitive cation channel CsChrimson and detected using a CNPE. CsChrimson is a channel that is more red-shifted than the traditional Channelrhodopsin-2 which has been used in optogenetics.33 Upon red light stimulation, the CsChrimson channels open and cations enter the cell, depolarizing the neuron and causing an action potential. A th-GAL4 driver was used to express UAS-CsChrimson in only the dopaminergic cells of the heterozygous crossed flies.33 The CNPE does not penetrate cells but measures extracellular changes in dopamine that occur due to volume transmission.</p><p>CNPEs with lengths of 125–175 um would be suitable for measurements in mammalian tissues. However, because the Drosophila larval VNC is so small (only 200 μm in length), smaller CNPEs were needed. CNPEs with short exposed tips 8.2 ± 1.4 μm in length, were characterized and the average current for 1 μM DA at these electrodes is 0.39 ± 0.08 nA. However, the noise is also small and the S/N values are still good (37 ± 4). Figure 6 shows the cyclic voltammogram for dopamine (Fig. 6A) when a short, 7 μm long CNPE (Fig. 6B) was used to detect stimulated release. The cyclic voltammogram has the characteristic oxidation and reduction peaks for dopamine. A false color plot of the data (Fig. 6C) shows the dopamine release during a 5 second long red light stimulation. Consecutive voltammograms are plotted over time on the x-axis, the y-axis is applied voltage, and current is shown in false color. Green/purple is dopamine oxidation and blue/yellow is dopamine reduction. The concentration versus time plot is made using an in vitro calibration to convert maximum peak oxidation current to dopamine concentration. Dopamine is cleared from the extracellular space by dopamine transporters8 and the concentration begins to decrease after the stimulation is finished. Using this short CNPE, endogenous dopamine was successfully detected, verifying that CNPEs are suitable for dopamine measurements in Drosophila tissue.</p><p>The batch fabrication of robust, nanosized electrodes suitable for in vivo studies is difficult and most methods to fabricate smaller electrodes have involved etching a single electrode by hand. The CNPEs developed here are batch fabricated and have the robustness to be implanted in tissue. The sensitivity per unit area for CNPEs with FSCV is slightly less than traditional CFMEs for dopamine, but CNPEs are able to measure endogenous dopamine in Drosophila larvae. Reducing the length to 5 to 10 μm makes these CNPEs useful for high spatial resolution measurements in Drosophila. The Mirkin group has recently made disk electrodes from CNPEs with recessed tips for use as scanning electrochemical microscopy tips.27 Direct fabrication or mechanical polishing of CNPEs to disk electrodes that are flat and not recessed would allow future measurements from discrete regions and at single neuronal cells. The ability to measure endogenous dopamine release in Drosophila will allow for studies on how genetics or behavior affects neurotransmission regulation. CNPEs could also be applied to study other neurotransmitters in vivo such as serotonin and octopamine in the future.</p><!><p>We fabricated solid-tipped CNPEs that allow high spatial resolution measurements of dopamine. CNPEs are batch fabricated and the electrode geometry can be easily modified via puller parameters, deposition conditions, and etch duration, to produce electrodes of desired tip size, taper, and exposed surface area. The nanoscopic tip provides for highly localized measurements, and its sharp conical shape makes it ideal for implantation into small regions such as the dopaminergic centers of the Drosophila brain. CNPEs were characterized for the first time with FSCV and their electrochemical signals for dopamine were suitable for in vivo measurements in Drosophila. CNPEs have fast electron transfer kinetics, stability, and good sensitivity. For dopamine, they are less sensitive per unit area compared to CFMEs, but still have sufficient signal for in vivo measurements. Interestingly, CNPEs showed improved sensitivity for serotonin compared to CFMEs. Coupled with FSCV, CNPEs could be used to measure real-time dopamine changes in specific regions of the adult fly, where the neuropil are only a few microns in diameter. Future studies in specific brain regions will give a better understanding of neurotransmission underlying discrete physiological processes.</p>

PubMed Author Manuscript

Tamoxifen plus tegafur-uracil (TUFT) versus tamoxifen plus Adriamycin (doxorubicin) and cyclophosphamide (ACT) as adjuvant therapy to treat node-positive premenopausal breast cancer (PreMBC): results of Japan Clinical Oncology Group Study 9404

PurposeA prospective randomized clinical trial was conducted to evaluate the efficacy of tamoxifen plus doxorubicin and cyclophosphamide compared to tamoxifen plus tegafur-uracil as an adjuvant therapy to treat node-positive premenopausal breast cancer (PreMBC).MethodsEligibility criteria included pathologically node-positive (n = 1–9) preMBC with curative resection, in stages I–IIIA. Patients were randomized to receive either tamoxifen 20 mg/day plus tegafur-uracil 400 mg/day (TU) for 2 years or six courses of a 28-day cycle of doxorubicin 40 mg/m2 plus cyclophosphamide 500 mg/m2 on day 1 along with tamoxifen (ACT) given for 2 years as adjuvant therapy. Primary endpoint was overall survival (OS), and secondary endpoint was recurrence-free survival (RFS).ResultsIn total, 169 patients were recruited (TU arm 87, ACT arm 82) between October 1994 and September 1999. The HR for OS was 0.76 (95 % CI 0.35, 1.66, log-rank p = 0.49) and that for RFS was 0.77 (95 % CI 0.44, 1.36, log-rank p = 0.37), with ACT resulting in a better HR. The 5-year OS was 79.7 % for patients in the TU arm and 83 % for those in the ACT arm. The 5-year RFS was 66.1 % for patients in the TU arm and 70.6 % for those in the ACT arm. A higher proportion of patients in the ACT arm experienced grade 3 leucopenia (0 % in the TU arm, 4 % in the ACT arm).ConclusionsThere were no significant differences in the efficacy of TU and ACT as adjuvant therapy.

tamoxifen_plus_tegafur-uracil_(tuft)_versus_tamoxifen_plus_adriamycin_(doxorubicin)_and_cyclophospha

Introduction<!>Eligibility and excluding criteria<!>Planned treatment schedules<!>Patient assessment<!>Study endpoint<!>Statistical analysis plan<!>Interim analysis and monitoring plan<!>Results<!><!>Discussion<!>

<p>Progression-free survival and overall survival have been improved according to the development of postoperative adjuvant therapy using drugs based on clinical trials. Prior to the 1980s, cyclophosphamide, methotrexate, and fluorouracil (CMF) therapy was the standard therapy, but development of Adriamycin in the 1990s indicated that Adriamycin might surpass CMF in terms of prolonging prognosis. Prior to the 1990s, oral anticancer agents became the standard therapy since they were thought to cause fewer adverse events in Japan.</p><p>Combined administration of oral fluoropyrimidine plus tamoxifen for 2 years postoperatively was reported to result in a high 5-year survival of 91 % for patients with Stage II breast cancer and 78 % for those with Stage III breast cancer [1, 2], and combined administration of oral fluoropyrimidine plus tamoxifen was reported to diminish QOL less [1]. The criteria for determination of estrogen receptor (ER) status at the time differed from the current criteria, and tamoxifen was supposed to be less efficacious in ER-negative patients. However, tamoxifen was administered regardless of the patient's ER status in general. Moreover, the form of administration was typically in combination with an anticancer agent including chemotherapy and hormone therapy. This study was planned within this context.</p><p>Current postoperative drug therapy to treat breast cancer is often chosen depending on the breast cancer subtype, which is determined based on panels for markers such as ER, HER2, and Ki67 [2]. This selection is based on predicted drug efficacy. The fact that lymph node metastasis is a prognostic factor was true when this trial began and it remains true today. When numerous lymph node metastases are noted, standard therapy is the administration of anthracycline and taxane, regardless of the cancer subtype. This study sought to assess the superiority of Adriamycin and cyclophosphamide (AC) + tamoxifen (ACT regimen) over oral tegafur-uracil (UFT) + tamoxifen (TU regimen), which was the standard therapy in Japan when the trial began, as a postoperative adjuvant therapy to treat premenopausal breast cancer in patients who were histopathologically confirmed to have lymph node metastasis. This trial also sought to determine whether all patients with node-positive breast cancer needed to be administered anthracycline or whether administration of oral fluoropyrimidine was sufficient.</p><!><p>Premenopausal female patients over the age of 15 with Stage I–IIIa breast cancer were eligible for this study. All patients had to have undergone curative mastectomy with axillary node dissection, and a histological examination had to reveal involvement of 1–9 axillary nodes. Other eligibility criteria were a World Health Organization (WHO) performance status of 0–1, adequate bone marrow and liver and kidney function, and no evidence of metastasis. Patients who received previous systemic treatment for breast cancer were excluded. The informed consent of each patient was obtained before study participation.</p><!><p>All patients randomized to TU or ACT regimen. For patients in the TU arm, tamoxifen (20 mg/day) and UFT (400 mg/day) were administered for a maximum of 2 years in all patients. For patients in the ACT arm, Adriamycin (40 mg/m2 intravenously) and cyclophosphamide (500 mg/m2 intravenously) were administered on day 1 every 28 days. This cycle was repeated six times. Tamoxifen (20 mg/day) was administered for a maximum of 2 years in all patients, regardless of hormonal receptor status.</p><p>Randomization was done using the minimization method, and the arms were balanced with regard to ER and progesterone receptor (PR) status (either one positive (>10 %) versus both negative and unknown), HER2 status (positive versus negative or unknown), number of metastatic nodes (1–3 versus 4–9), and institution.</p><!><p>Initial workup included medical history, tumor assessment, physical examination, routine hematology and chemistry test, chest radiography, liver ultrasonography, and a bone scan. Hematology and chemistry tests, tumor marker measurements, and urinalysis were repeated monthly. To check for distant metastasis, a chest radiography and liver ultrasonography were performed every 6 months, a bone scan was performed every year, and bilateral mammography was performed every 2 years. Hematological disorders and toxicity were evaluated according to the Toxicity Grading Criteria of the Japan Clinical Oncology Group (JCOG) [3] and were recorded on case report forms.</p><!><p>The primary endpoint of this study was overall survival (OS), and the secondary endpoint was recurrence-free survival (RFS). OS was defined as the time from randomization to death from any cause, and it was censored as of the date of final follow-up. RFS was defined as the time from randomization to either the first incidence of recurrence or death from any cause, and it was censored as of the date of final follow-up. OS and RFS were evaluated according to hormone receptor status (either ER- or PR-positive versus both ER- and PR-negative or unknown) in subgroup analyses. In addition, the safety of treatment was evaluated.</p><!><p>If patients treated with ACT had a significantly longer OS than patients treated with TU, then ACT would be recommended as the new standard treatment. The estimated 5-year OS of these patients is commonly 64–88 % [4–6]. A total of 342 patients were needed to detect a prolongation of the 5-year OS from 75 % for patients in the TU arm to 85 % for patients in ACT arm with an 80 % power and a two-sided alpha of 5 %. Considering some patients potentially lost to follow-up, the sample size was set at 400 patients in total. The planned study period was originally 2 years for recruitment and an additional 5 years for follow-up. Due to the slow recruitment, the protocol was revised to extend the recruitment period, and the sample size was revised to 330 patients with a recruitment period of 5 years. OS was analyzed for all randomized patients and RFS for randomized patients excluding a patient with bone metastasis at the registration. OS and RFS were estimated using the Kaplan–Meier method, and curves were compared using a log-rank test. Hazard ratios of treatment effects were estimated by a Cox regression model. All analyses were based on intent to treat. All statistical analyses were performed using SAS release 8.2 (SAS Institute, Cary, NC).</p><!><p>An interim analysis was to be performed when half of the total number of patients was enrolled. The JCOG Data and Safety Monitoring Committee (DSMC) independently reviewed the interim analysis report, and premature termination of the trial could be considered at that stage. In-house interim monitoring was performed by the JCOG Data Center to ensure data submission, patient eligibility, protocol compliance, safety, and on-schedule study progress. The monitoring reports were submitted to and reviewed by the DSMC every 6 months.</p><!><p>This study began in 1994. At an interim analysis on June 1999, patient recruitment was so slow that the DSMC recommended terminating patient recruitment or continuing but changing the primary endpoint to RFS. Furthermore, a consensus meeting in St. Gallen in 1997 deemed that administering tamoxifen to hormone receptor-negative patients was ethically unacceptable [7]. Therefore, recruitment of patients was terminated pursuant to suggestions from the JCOG DSMC.</p><!><p>Trial profile of Japan Clinical Oncology Group study, JCOG 9404</p><p>Patient characteristics</p><p>Kaplan–Meier curves of overall survival (a) and relapse-free survival (b) for node-positive breast cancer patients treated with tamoxifen plus tegafur-uracil or tamoxifen with anthracycline and cyclophosphamide</p><p>Kaplan–Meier curves of overall survival (a) and relapse-free survival (b) for node-positive breast cancer patients treated with tamoxifen plus tegafur-uracil or tamoxifen with anthracycline and cyclophosphamide according to estrogen receptor (ER) and progesterone receptor (PgR) status</p><p>Hematological (A) and non-hematological (B) toxicities</p><!><p>The decision to administer postoperative adjuvant drug therapy, which seeks to inhibit the recurrence of breast cancer, is often currently made based on the primary tumor's subtype. Breast cancer is essentially categorized into four subtypes depending on the expression of ER, PgR, HER2, and Ki67 [2]. Endocrine drugs are given to patients with ER- and/or PgR-positive luminal tumors. Trastuzumab (a molecular-targeted agent) and an anticancer agent are both administered to HER2-positive patients. These strategies are tailor-made target therapies according to the prediction of efficacy of drugs. In addition to endocrine drugs, anticancer agents are often administered to patients with breast cancer expressing a high level of Ki67 [8, 9]. The individual determination of whether or not a tumor is sensitive to a drug is difficult, and despite this, anticancer agents are administered. Including anticancer agents is considered acceptable when patients have numerous lymph node metastases (irrespective of tumor subtype), if their cancer is ER- and/or PgR-positive and expressing a low level of Ki67. The validity and evaluation of Ki-67 are not definitive [10]. Both anthracycline and taxane are often administered sequentially for these patients despite the possibility that efficacy of these drugs is low. These classifications of breast cancer and administration of taxane and molecular drugs were widely in use after the current trial began.</p><p>At the beginning of this study, tamoxifen was administered as the standard therapy even if the patient was ER-negative. In light of current evidence, there is no doubt that tamoxifen has little efficacy in treating ER-negative breast cancer [11], though there are also no data indicating that the efficacy of anticancer agents will diminish if used in combination with tamoxifen. Thus, the results of this trial simply compared taking UFT for 2 years to taking AC to treat node-positive premenopausal breast cancer. Previous meta-analyses clearly revealed data indicating that AC therapy is more effective at preventing recurrence than CMF [12–16], but AC therapy has not been compared to oral fluoropyrimidine. The results of this trial indicated no difference between oral fluoropyrimidine and AC therapy in terms of prolonging survival in patients overall. AC therapy resulted in a longer recurrence-free survival (RFS) in only ER-negative patients. These results do not have a meaning for recent breast cancer treatment strategy, because of the insufficiency of patients recruitment and old adjuvant treatment design. However, this finding suggests that AC therapy has limited efficacy when treating node-positive breast cancer by administering tamoxifen as a postoperative adjuvant therapy to treat ER-positive breast cancer. This finding also suggests that administration of oral fluoropyrimidine alone may be sufficient in some cases. In fact, OS and PFS were similar between ACT and TU arm with ER-positive breast cancer. A potent anticancer agent, like anthracycline, may not be needed to treat ER-positive breast cancer even if it has lymph node metastasis.</p><p>The question of whether UFT is needed or if tamoxifen alone is sufficient remains. Results of the JCOG9401 study [17], which examined patients with postmenopausal breast cancer with lymph node metastasis during the same period as the current trial, may offer an answer. The study compared tamoxifen alone and ACT therapy to treat patients with node-positive breast cancer, and results indicated that ER-positive patients had a 5-year RFS of 59.3 % when given tamoxifen alone versus 76.9 % when given ACT therapy and a 5-year OS of 87.1 % when given tamoxifen alone versus 90 % when given ACT therapy. Patients in this trial who were given UFT+tamoxifen had a 5-year RFS of 74.5 % and a 5-year OS of 89 %. There was possibility of prognostic benefit of additional UFT for ER-positive node-positive patients. Thus, comparison of TU therapy to tamoxifen alone is needed. In Japan, a prospective clinical trial on adding S-1 to treat patients with ER-positive breast cancer after completion of standard chemotherapy is currently enrolling subjects (UMIN000003969).</p><p>No major differences were noted in ER-negative patients in either arm of this trial. That said, ER-negative patients had a 5-year OS and a 5-year RFS that was about 30 % shorter than the 5-year OS and 5-year RFS of ER-positive patients. Trastuzumab tends to be administered to patients with ER-negative breast cancer if they are HER2-positive [18], and taxane tends to be administered along with anthracycline if they are HER2-negative [14]. The regimens in this trial were inadequate to evaluate the appropriate adjuvant drugs for ER-negative patients with node metastases.</p><p>In terms of adverse events, a hematological event in the form of a grade 3 decline in the white blood cell count was noted only in patients in the ACT arm. In terms of non-hematological events, abnormal liver function was noted in patients in the TU arm and nausea was often noted in patients in the ACT arm. Results of this trial revealed numerous adverse events in patients in the ACT arm as a whole. Since the current dose of AC is higher than that used in this trial, UFT may be less damaging. However, results suggested that sufficient caution in abnormal liver function is necessary to use UFT for long time as adjuvant therapy. The current trial did not administer both endocrine therapy and chemotherapy concurrently. Previous data on such chemoendocrine therapy have highlighted the enhancement of adverse events and an increase in thrombosis in particular [19–21]. Neither group of patients in this trial had thrombosis/embolism. Existing data are from the USA and Europe, where thrombosis is more prevalent. These conditions may pose far less of a problem in Japan because of their different physique. Chemoendocrine therapy is ruled out based on current data from Europe and the USA, but there may be leeway for therapy selection depending on the patient.</p><p>This trial prospectively studied the usefulness of ACT therapy to treat patients with node-positive premenopausal breast cancer. This trial began prior to 2000, and modern standard adjuvant therapy was established during collecting patients for this trial. There were some issues with trial design and trial enrollment since the standard therapy changed substantially during trial enrollment. However, the times changed from an era of actively administering anticancer agents to every patient with breast cancer with lymph node metastasis to an era of selecting therapy by predicting drug efficacy. Postoperative adjuvant therapy with oral FU was the standard therapy in this trial, and a new appreciation for the efficacy of that therapy is developing. In this trial, ACT did not significantly prolong survival compared to TUFT, especially in ER-positive patients. Without a doubt, these findings pose clinical questions that should be answered when formulating a treatment strategy for postoperative adjuvant therapy. Further studies via prospective trials (which include those currently underway) are needed.</p><!><p>On behalf of the JCOG Breast Cancer Study Group.</p>

PubMed Open Access

Interactions between shape-persistent macromolecules as probed by AFM

Water-soluble shape-persistent cyclodextrin (CD) polymers with amino-functionalized end groups were prepared starting from diacetylene-modified cyclodextrin monomers by a combined Glaser coupling/click chemistry approach. Structural perfection of the neutral CD polymers and inclusion complex formation with ditopic and monotopic guest molecules were proven by MALDI-TOF and UV-vis measurements. Small-angle neutron and X-ray (SANS/SAXS) scattering experiments confirm the stiffness of the polymer chains with an apparent contour length of about 130 Å. Surface modification of planar silicon wafers as well as AFM tips was realized by covalent bound formation between the terminal amino groups of the CD polymer and a reactive isothiocyanate-silane monolayer. Atomic force measurements of CD polymer decorated surfaces show enhanced supramolecular interaction energies which can be attributed to multiple inclusion complexes based on the rigidity of the polymer backbone and the regular configuration of the CD moieties. Depending on the geometrical configuration of attachment anisotropic adhesion characteristics of the polymer system can be distinguished between a peeling and a shearing mechanism.

interactions_between_shape-persistent_macromolecules_as_probed_by_afm

Introduction<!>Synthesis of the shape-persistent CD polymer<!>SANS and SAXS measurements of the CD polymer<!>Complexation of monotopic and ditopic guests<!>Probing multivalent interactions by AFM<!>Conclusion

<p>Shape-persistence is an important key feature in self-organisation strategies of supramolecular building blocks resulting in high structural perfection of the obtained molecular assemblies [1], such as shape persistent macrocycles, cage compounds or rotaxanes [2][3][4]. Especially shape-persistent polymers are of significant scientific interest as their defined structural characteristics offer various applications as sensor materials, biomimetic filaments or organic electronics [5][6][7]. Furthermore, compared to polymers with flexible chains, shape persistent macromolecules with high structural rigidity are able to form stable aggregates based on multiple supramolecular interactions, which can be detected and quantified without the presence of side effects, such as self-passivation or coiling processes. Dendrimers, nanoparticles and shape-persistent polymers had been previously discussed as scaffolds for the design of multiple ligands of high affinity [8]. Nevertheless, well-defined model systems in which the influence of rigidity and regularity on cooperativity of binding was systematically investigated have not been reported so far. Rigid linear polymers have been considered as suitable scaffolds for the design of supramolecular systems showing multiple interactions. A high rigidity of the macromolecule is maintained by rigid, linear repeat units, such as trans-ethenylene, ethynylene, or p-phenylene moieties. The observed persistence lengths of polyconjugated polymers ranged from 6 to 16 nm, depending on the side groups and the method of determination [9][10][11].</p><p>Among many supramolecular interactions, such as hydrogen bonding, π-π-interactions or hydrophobic host-guest interactions [12][13][14][15][16], the interactions of cyclodextrins (CDs) with hydrophobic guest molecules are of special interest, since CDs are readily available bio-based materials and interactions take place under physiological conditions [17]. CDs are ideal candidates for the investigation of multivalent interactions as they combine high affinities with a versatile integrability in macromolecular systems [18]. CDs have already been employed for the construction of supramolecular polymers [19][20][21], supramolecular hydrogels [22,23], molecular printboards [24,25] or multivalent interfaces [26][27][28] with tunable chemical and physical properties. Herein, for the first time, we present studies concerning the synthesis of shape-persistent CD polymers to investigate multivalent binding with ditopic guest molecules on the molecular level (Figure 1). The ditopic guest (shown in red colour) should act as a connector between opposing CD moieties.</p><p>Only a few examples of shape-persistent CD polymers have been reported so far, including CD-modified conjugated oligomers and polymers composed of rigid phenylene ethynylene (PPE) structure units which are able to form self-inclusion complexes with tunable electrochemical properties [29][30][31][32][33][34][35]. The synthesis of PPE, in which two β-CD rings were attached to every second phenylene group, was described by Ogoshi et al. [36] using a Sonogashira-Hagiwara coupling. We preferred a poly-phenylene-butadiynylene backbone, synthesized by a Glaser-Eglington coupling, since the repeating unit is long enough (l = 0.944 nm) to allow the connection of one CD moiety at each phenylene unit. Based on the stiffness of the polymer chain self-passivation of CD polymer modified surfaces is reduced to a minimum. Furthermore, the ethynyl end groups are easily functionalized by click chemistry.</p><p>Isothermal titration calorimetry (ITC), fluorescence spectroscopy, quartz crystal microbalance (QCM), surface plasmon resonance (SPR) and atomic force microscopy (AFM) have been employed to quantify the strength of the multivalent interactions [8]. Because binding affinities can be very high for multivalent supramolecular systems, the constituents are commonly used in low equilibrium concentrations. Since AFM even allows the investigation of single molecules, such as DNA [37,38] or molecular self-assembling based on "Dip-Pen" nanolithography [39], it was chosen as the most reliable technique to probe highly cooperative recognition processes.</p><p>The investigation of cooperativity of multiple host-guest interactions using AFM has been reported by several groups [40][41][42][43][44][45]. Huskens and co-workers measured the supramolecular interactions between a β-CD-modified planar surface and mono-, diand trivalent adamantane guest molecules attached to an AFM tip and found enhancement factors up to 2, depending on the force loading rate [46]. We have previously explored the adhesion characteristics of dense CD layers on an AFM tip and a planar silicon surface connected by various ditopic linker molecules. In this system we were able to switch adhesion and friction by applying external stimuli onto the responsive ditopic linkers [47][48][49]. In contrast to previous work our molecular toolkit, based on ditopic connector molecules, allows the independent determination of unspecific interactions between CD polymers at tip and planar surface as well as the specific interactions to ditopic connector molecules. In the following, we describe the first example of multivalent interaction of ditopic guest molecules with shape-persistent CD polymers covalently attached to an AFM tip and a planar surface. Nano force measurements between CD and CD polymer, CD polymer and CD, and CD and CD at the tip and the planar surface, respectively, exerted by the adamantane ditopic connector molecules were systematically investigated. All four configurations are schematically depicted in Figure 2.</p><!><p>Our synthetic approach for the preparation of modified poly(phenylene butadiynylene)s bearing one CD molecule per repeat unit started from 2,5-dibromo-4-methylbenzoic acid (2) [50,51], which was esterified to 3 with tert-butanol catalyzed by H 2 SO 4 (Scheme 1). The TMS-protected diacetylene derivative 4 was prepared by Sonogashira reaction of 3 with trimethylsilylacetylene. Subsequent deprotection of the TMS groups using tetra-n-butylammonium fluoride and saponification of the tert-butyl ester with trifluoroacetic acid resulted in the corresponding benzoic acid 6. The latter was coupled to 6-monoamino-6-deoxy-β-CD [52] using N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) applying a procedure known for terephthalic acid [53]. The resulting product, monomer 7, was easily isolated due to its low solubility in water which was attributed to self-inclusion between hydrophobic phenyl moieties and β-CD rings leading to daisy chains [54].</p><p>The polymerization of 7 was performed through Glaser coupling in pyridine catalyzed by Cu(I)/Cu(II). After removal of low molecular weight material by ultrafiltration polymer 8 was isolated as a light orange solid in 91% yield. Polyrotaxane formation, which might prevent the accessibility of the CD-moieties located on the polymer backbone, was avoided by the presence of pyridine as a non-polar solvent. Both NOESY NMR experiments and circular dichroism (results not shown) do not indicate any significant interaction of the CDs and the aromatic backbone. Compared to monomer 7, peak broadening and the disappearance of the 1 H NMR signals of the acetylene protons at 4.54 and 4.36 ppm indicate the formation of polymer 8. The presence of the conjugated backbone was confirmed by UV-vis and fluorescence measurements in water. Compared to 7, a characteristic bathochromic shift could be observed both in the absorption and emission spectra of polymer 8 (Figure 3) showing the presence of the extended polyconjugated π-system.</p><p>Quantitative information about the molecular weight distribution of 8 was obtained by MALDI-TOF measurements using an ionic liquid matrix (HABA/TMG 2 ) [55]. A representative MALDI spectrum, shown in Figure 4, exhibits a wide range of broad signals starting from the signal of the dimer at m/z 2,621.33 Da detected as [M + Na] + and ending at the 38mer at m/z 48,196.23 Da for a S/N ratio ≥3, with an average 1297.4 mass units shift corresponding to one additional repeating unit. Among each discrete envelope, one to three supplementary ions, have been detected with a constant 165.2 mass unit shift, revealing the presence of small quantities of the repeat unit originating from unmodified benzoic acid derivative 6, e.g., at 2,621.33 and 2,786.52 Da (Figure 4). The MS analysis reveals the high structural perfection of the polymer 8 where at most one CD entity per polymer molecule is missing. Integration of the relative distribution of the most intense ions of each population allowed to estimate both the number average molecular weight, M n , and the mass average molecular weight, M w , of 8,765.77 Da, and 22,023.56 Da, respectively. These values result in a polydispersity index PDI = M w /M n of 2.59 typical for normal distributions. From the value of M w an average contour length L = 17 nm of the macromolecule was calculated. A more detailed analysis of the MS data is provided in Supporting Information File 1.</p><!><p>Structural characteristics of the CD polymer 8 have been investigated by small-angle neutron and X-ray scattering experiments (SANS/SAXS). SANS data (KWS-1, JCNS at Heinz Maier-Leibnitz Zentrum [56]) for a polymer concentration range from 0.005 to 0.03 g/cm 3 are presented in Figure 5. SANS intensities are normalized to polymer concentration and therefore scattering intensities depend on polymer chain mass (or mass of chain aggregates), square of scattering contrast, conformation of polymer chain, and interaction between the Table 1: Structural parameters of polymer 8 (apparent radius of gyration, scattering at zero angle, radius of gyration of polymer cross-section, scattering at zero angle of polymer cross-section, apparent contour length obtained from the ratio between I(0) and I CS (0), and calculated apparent mass of polymer 8, obtained from the length of monomer unit chains (aggregates). There are only minor differences in scattering for concentrations up to 0.02 g/cm 3 indicating no significant aggregation between polymer chains with increasing concentration which would lead to highly ordered polymer species. The decrease of scattering intensity for the highest concentration of 0.03 g/cm 3 can be attributed to interaction of polymer chains. The SAXS curve measured at 0.03 g/mL shows a similar shape as the neutron data (Supporting Information File 1, Figure S1). The low-q range of scattering data has been analyzed with a Debye function. The apparent radius of gyration R g,app and the scattering at "zero angle", I(0), were obtained by fitting the scattering data for q < 0.02 Å −1 [57]:</p><p>(</p><p>where x= q 2 R g,app 2 . The scattering intensity is given by (2) where the apparent molar weight, M app , is connected with the real molar weight, M, via a structure factor S(0) (interaction among polymer chains) as M × S(0) = M app and Δρ m is the difference in neutron scattering length density between polymer and solvent normalized to the density of polymer. The local structure of the polymer cylindrical cross-section was extracted by applying indirect Fourier transformation (IFT) [58] to the experimental data from the high-q range. Detailed information applying this method is presented in Supporting Information File 1. The resulting parameters for the concentration dependence of I(0), scattering at "zero angle" of a cylindrical crosssection of polymer I CS (0), radius of gyration R g,app , and radius of gyration of a cylindrical cross-section R g,CS are presented in The flexibility of chains of polymer 8 was determined by means of a Holtzer plot [59]. Detailed information and the corresponding data are presented in Supporting Information File 1. The absence of a characteristic inflection point, where the scattering intensity changes from q −1 as for rigid cylinder to q −2 (or to q −5/3 when self-avoidance is important) as for flexible chains, indicates that polymer chains are short and rigid, i.e., that the persistence length is of the same order as the contour length of the polymer.</p><p>The SAXS data has been analyzed by models representing the expected shape of polymers. It was assumed that there is no interaction between aggregates, which means that the scattering intensities depend only on the size and shape of the aggregates [60]. Details are shown in Supporting Information File 1.</p><p>The scattering data could be described (Figure 5 above and Figure S1 in Supporting Information File 1) by a population of rigid cylinders of length 110 ± 5 Å and radius of cross-section of 12 ± 2 Å. Neglecting the interaction between polymer chains in the model leads to the slightly lower length values.</p><!><p>In contrast to monomer 7, polymer 8 was soluble in water up to a concentration of 0.15 mM (based on the repeating unit). This allows the investigation of the complexation of ditopic and monotopic guests, 9 and 10, respectively. The solubility of the host polymer 8 as a function of the concentration of both guests 9 and 10 (Scheme 2) was determined by UV−vis spectroscopy using the extinction coefficient ε of 8 (14,800 M −1 cm −1 ) at 425 nm. A more detailed description of the solubility measurements is presented in Supporting Information File 1.</p><p>Addition of hydrophilic guest 10 caused an increase in solubility of host polymer 8 in water (Figure 6). The surprisingly steep initial slope of the phase solubility diagram, m = 1.4 (repeating unit/guest) could be well represented by a model where every second CD moiety has to be complexed by the hydrophilic guest to significantly improve the solubility in water. Binding constants of about 40,000 M −1 , which were in the same range as literature values for the incorporation of adamantane derivatives into β-CD, [61] were obtained using ITC measurements considering a two-step sequential complexation with guest 10. Further information is provided in Supporting Information File 1. Incomplete complexation with cationic guest molecules is indicated by a significant lower binding constant of 670 M −1 for the second binding complexation step, which is strongly inhibited as a result of the electronic repulsion of charged guest molecules in close proximity to each other. In contrast, a pronounced reduction of the solubility of CD polymer 8 was observed in the presence of ditopic guest 9, which was attributed to the interconnection of polymer chains through the complexation of the ditopic guest. The very low concentration of connector 9 necessary for the almost complete precipitation of the host polymer 8 can be explained by the high integrability of the host-guest system based on the shape-persistence of the polyconjugated polymer backbone of 8. cyanate groups, which smoothly react with amines forming stable thiourea links [48]. Monolayers of β-CD or β-CDpolymer were obtained by attachment of monoamino β-CD or amino-modified CD polymer 12, synthesized from polymer 8 (Scheme 3) through Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with the triethylene glycol linker 11 (N 3 -TEG-NH 2 ) which had been prepared in a five-step procedure [62,63].</p><!><p>The adhesive forces of 12, due to supramolecular interactions with ditopic guest 9, between a planar silicon surface and an AFM tip both modified with the CD polymer 12 or 6-monoamino-6-deoxy-β-CD were systematically investigated by AFM. While adhesion was very weak in pure water, significant adhesion took place over a wide range of distances in a 10 μM solution of ditopic guest 9 (Figure 7a-d). For comparison, we also investigated the adhesion forces between CD and 12, 12 and CD, and CD and CD at the tip and the planar surface, respectively, caused by the adamantane connector 9.</p><p>Adhesive forces were recorded as function of the tip-surface distance upon retracting of the tip from the surface for all four configurations. The pull-off force required to detach the tip from the surface in the presence of connector molecules was of the order of 500 pN for the CD-CD configuration and about 1 nN for all configurations involving CD polymers (12). These values are significantly higher than the pull-off forces of about 250 pN measured in control experiments for all configurations. The graphical summary in Figure 7a suggests that the pull-off forces for the 12-12 configuration are slightly higher than for the 12-CD and for the CD-12 configuration.</p><p>While the pull-off force is similar, the overall appearance of the force curves differs for the three polymer configurations. The interaction distance varies significantly for the different configurations. The CD-CD configuration has the shortest and the polymer-polymer configuration the longest range of interactions. The interaction range can be quantified by the tip-surface distance at which the last rupture occurs, referred to as maximum rupture length. The histograms of the maximum rupture length for all four configurations are presented in Figure 7.</p><p>For the CD-CD configuration, the most probable maximum rupture length of 5 nm corresponds to the combined height of the monolayers on tip and surface, each of about 2.5 nm. The typical rupture length for the CD-12 configuration is 10 nm, while it is 29 nm for the 12-CD configuration. The difference in maximum rupture length indicates a difference in the detachment mechanism. In the CD-12 configuration, the polymers bind to the sloped facets of the asperity of the AFM tip. Upon pulling, the polymers are sheared from the tip apex by rupturing all bonds simultaneously leading to one large rupture peak at a small tip-surface distance. For the 12-CD configuration, a force plateau observed in the force-distance curve in Figure 7c reveals the peeling of a polymer chain from the CD-coated surface resulting in a rupture length similar to the length of the polymer chains.</p><p>For the 12-12 configuration, many additional small detachment events lead to a broadening of the pull-off curve and reveal the rupture of bonds for tip-surface distances as large as 110 nm in Figure 7d. The broad distribution of rupture length, which extends to roughly the double of that of the 12-CD configura- tion, indicates that individual long polymer chains interlock, explaining also the characteristic stretching events in the forcedistance curve. The most probable maximum rupture length for the 12-12 system is 38 nm, which is double of the average polymer length of 17 nm predicted from the MALDI-TOF and SANS/SAXS results. The agreement confirms the picture that the maximum rupture length reflects the final detachment of supramolecular bonds at the end of stretched polymer chains attached to AFM tip and surface.</p><p>The higher sensitivity of our AFM set up compared to the MALDI-TOF instrument allowed us to even detect single rup- ture events at a distance up to 250 nm, which proved that some individual chains had a length of at least 125 nm. Compared to MALDI-TOF measurements in which the small number of high molecular weight polymer chains are hardly detectable, AFM experiments overemphasize the few longest polymer chains probing the interactions of the regularly spaced CDs in CD polymer 12 and ditopic connector molecules. Due to this observation AFM is an excellent detection tool for analysing cooperative effects in ordered supramolecular systems.</p><p>The differences between the four configurations of functionalization can be further quantified by integration of the force curves, resulting in the work of separation which has been employed before as a suitable parameter for the quantification of polymer detachment [45]. In line with the characteristic shape of the example force curves, the work of separation increased significantly in the order CD-CD, CD-12, 12-CD and 12-12 configuration (Figure 8). The relative increase in the work of adhesion from control experiments to measurements of the specific interactions caused by the connector molecule 9 was even higher than the respective increase in pull-off force due to the very short range of the non-specific adhesive interactions.</p><p>The significant difference in the interaction range and thus in the work of separation between CD-12 and 12-CD configuration can be explained by the asymmetry between curved tip and flat surface and the resulting difference in the detachment mechanism. Polymers attached to the surface bind to the side faces of the tip with its nanometer-scale apex radius. Upon retraction, the force acts along the polymer and shears the polymer off the tip, with all bonds rupturing more or less simultaneously. In contrast, polymers attached to the tip bind to the flat surface such that upon retraction the polymer is peeled from the surface by the orthogonal force, one bond breaking after another. The different detachment scenarios are depicted in the schematic drawings in Figure 2. The shearing configuration (CD-12) leads to simultaneous rupture of all bonds, while the peeling configuration (12-CD) involves bending of the polymer and consecutive rupture. The strongest adhesion is offered by the supramolecular interlocking of polymers attached to tip and surface. Supramolecular interconnection between two CD polymer 12 molecules through the ditopic guest 9 is expected to be superior to the one between CD polymer 12 and CD because of the higher regularity of the CD spacing at the polymer compared to the spacing within the CD monolayer. We conclude that the regularity of the CD polymer 12 allows to establish a much higher number of supramolecular bonds with the connector 9 giving rise to about a fivefold enhancement of the work of separation.</p><p>Many force curves exhibit a well-defined last rupture event. A representative example is shown in Figure 9a, where the force drops from around 63 pN to zero at a distance of 110 nm. The distribution of rupture forces for the last rupture events, shown in Figure 9b, has a clear maximum at 63 pN, determined by a Gaussian fit to the distribution, and a weak second maximum at about double this value.</p><p>We conclude that 63 ± 10 pN is the rupture force for a single bond between our supramolecular polymers 12 established by the ditopic guest 9. The value agrees with rupture force measured for adamantane-CD complexes with CD molecules in the surface layers when the stiffness of the AFM cantilever is taken into account [64].</p><p>Force curves like those shown in Figure 7 can be repeated on the same spot of one sample many times with very similar results. The repeatability confirms the reversibility of the underlying interactions. It is difficult to estimate the number of supramolecular bonds contributing to pull-off forces of 1 nN in to the one for the CD-CD system previously described [48]. This spike force may be enhanced by the multivalency effects discussed above, but its strength indicates that more than one polymer molecule might be involved.</p><!><p>In conclusion, regular water-soluble shape-persistent CD polymers based on poly(phenylene butadiynylene) were prepared by a straightforward Glaser coupling/click chemistry approach, which can be attached to planar silicon surfaces as well as AFM tips. Structural perfection of the resulted polymers was con-firmed by MALDI-TOF measurements revealing the presence of high molecular weight materials with up to 38 repeat units. High integrability of the scaffold was proven by UV-vis supported solubility measurements upon addition of ditopic adamantane connectors. Small-angle neutron scattering and X-ray experiments reveal the presence of stiff cylindrical polymer chains with contour lengths of about 13-16 nm, which corresponds to the values obtained by MALDI and AFM measurements. Hard substrates with the shape-persistent polymers and interconnected by ditopic guest molecules require about five times higher separation energies than those functionalized with conventional CD monolayers. This significant enhancement of adhesion can be attributed to a strong cooperative effect favored by the rigidity of the polymer backbone and the regular spacing of the CD moieties. The range of adhesive interactions could be extended from 5 to 38 nm, which will also allow the interconnection of surfaces with higher roughness. The stiff polymers exhibit a clear contrast between shearing and peeling mechanisms, depending on the geometrical configuration of attachment. The distribution of the maximum rupture lengths in the force microscopy experiments confirms the molecular weight distribution of the CD polymers estimated by MALDI-TOF and the average contour length determined by SANS/SAXS. In addition, force microscopy experiments emphasize the longest polymer chains and their maximum length.</p>

Beilstein

Crystal‐Size‐Induced Band Gap Tuning in Perovskite Films

AbstractA comprehensive picture explaining the effect of the crystal size in metal halide perovskite films on their opto‐electronic characteristics is currently lacking. We report that perovskite nanocrystallites exhibit a wider band gap due to concurrent quantum confinement and size dependent structural effects, with the latter being remarkably distinct and attributed to the perturbation from the surface of the nanocrystallites affecting the structure of their core. This phenomenon might assist in the photo‐induced charge separation within the perovskite in devices employing mesoporous layers as they restrict the size of nanocrystallites present in them. We demonstrate that the crystal size effect is widely applicable as it is ubiquitous in different compositions and deposition methods employed in the fabrication of state‐of‐the‐art perovskite solar cells. This effect is a convenient and effective way to tune the band gap of perovskites.

crystal‐size‐induced_band_gap_tuning_in_perovskite_films

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

<p>A. Ummadisingu, S. Meloni, A. Mattoni, W. Tress, M. Grätzel, Angew. Chem. Int. Ed. 2021, 60, 21368.</p><!><p>Perovskite materials have recently emerged as excellent candidates for opto‐electronic applications, photovoltaics in particular. Several perovskite deposition methods have been developed in the recent past for the fabrication of prototype perovskite solar cells. The most commonly used ones are the sequential deposition method [1] and the anti‐solvent method. [2] The former involves two steps, the deposition of the lead iodide (PbI2) as a film and the dipping of the film in a methylammonium iodide (CH3NH3I, MAI) solution to form the perovskite, methylammonium lead iodide (CH3NH3PbI3, MAPbI3) in‐situ. [1] The latter involves the deposition of a perovskite precursor solution containing the reactants needed to form the perovskite, followed by dripping of an anti‐solvent on top and heating to form the perovskite thin film. [2]</p><p>Perovskite solar cells have also been developed in a variety of architectures, including the mesoscopic, [2] planar [3] and inverted ones. [4] The state‐of‐the‐art mesoscopic cells [2] are based on a mesoporous layer of TiO2 particles, which serves as the electron transport layer. Such cells consist of a capping layer of perovskite crystals on top of the mesoscopic TiO2 layer, which is itself infiltrated with perovskite. In contrast, planar architectures consist only of a compact perovskite layer between electron and hole transport layer, [5] with the simpler architecture presenting its own advantages.</p><p>Reports [6] describe spectroscopic investigations looking into the correlation between perovskite microstructure and key opto‐electronic properties. They identified that properties such as the optical absorption,[ 6b , 6c ] the photoluminescence [6c] (PL) and the electron‐hole interaction, [6a] which are important for photovoltaics, are dependent on whether the perovskite lies in the mesoscopic or capping layer. Specifically, the main differentiating aspect of the microstructure in these two cases: the crystal size, has been of interest in the past. While several aspects commonly associated with the crystal size such as lattice strain have been proposed as possible explanations for the variations in opto‐electronic properties observed when crystal size changes, the underlying cause remains elusive.[ 6a , 6b ] In order to effectively design and fabricate solar cells to achieve the highest performances, knowledge about the influence of crystal size on some of the relevant material properties is essential. Thus, a fundamental explanation behind this phenomenon needs to be identified and this is the focus of this study.</p><p>Herein, we aim to address the above‐mentioned aspects by coupling experimental observations with insights from classical and first principle atomistic simulations. First, we investigate the steady state PL from methylammonium lead iodide perovskite (CH3NH3PbI3) samples at different stages of conversion from lead iodide (PbI2) in the sequential deposition reaction [1] and observe a blue‐shift of the PL spectra and an unusual asymmetry in the emission weighted towards higher energies at the start of the reaction. Then, from classical molecular dynamics (MD) and density functional theory (DFT) calculations, we identify that the band gap narrows with increase in perovskite nanocrystallite size, independent of quantum confinement (QC). This is because PbI6 octahedra in smaller nanocrystallites are more tilted and Pb atoms are more off‐centered. We associate this finding with the presence of smaller sized nanocrystallites at the start of the reaction, which manifests itself as a blue‐shift and an asymmetry in the emission at the early stages of the reaction.</p><p>Next, using cross‐sectional confocal laser scanning fluorescence microscopy (CLSM), we show that the CH3NH3PbI3 perovskite in the mesoporous layer has a blue‐shifted emission compared to the capping layer. We also examine perovskite samples of the nominal composition Cs0.05MA0.16FA0.79Pb(I0.83Br0.17)3 made using the anti‐solvent method and show that the crystal size effect is present in these as well, demonstrating the broad implications of our work. Examining samples made on mesoscopic scaffolds of different particle sizes, which restrict the size of perovskite crystallites in them, we clearly show that crystal size can be used to tune opto‐electronic properties such as the band gap. Finally, we discuss the direct implications of our observations for the performance of mesoporous layer‐based devices and propose the use of crystal‐size‐control achieved via tailoring of the mesoporous scaffold for effective photovoltaic device design.</p><!><p>The sequential deposition method used to deposit perovskite thin films is interesting for kinetic studies as it allows us to accurately follow and monitor the progress of the perovskite formation. The concentration of the MAI solution can be kept low enough to practically halt the reaction. This allows us to control the progress of the reaction, thus monitoring the changes in the film, such as the growth of perovskite nanocrystallites, as the reaction progresses. This makes the sequential deposition method an apt choice for this part of the study rather than the anti‐solvent method. Furthermore, the sequential deposition has been demonstrated as a viable option for the large‐scale production of perovskite thin films for opto‐electronics through scaling up of the process. [7] This exemplifies the great potential of this method and the need to study it further.</p><p>Following the sequential deposition method here, PbI2 films are deposited onto a mesoporous Al2O3 scaffold and then dipped in a MAI solution in 2‐propanol to form a CH3NH3PbI3 film (see the Supporting Information, Materials and Methods). The path of this reaction has been reported in our previous study. [8] Al2O3 scaffolds rather than TiO2 were used for these measurements to prevent quenching of the PL signal. To begin with, we look at the steady‐state PL spectra of samples made using sequential deposition where the reaction was arrested at different points of conversion from PbI2 to CH3NH3PbI3 (see ex‐situ measurements in the Supporting Information for details).</p><p>The emission spectra of these PbI2 samples dipped in MAI solution for different periods (dipping time), containing different quantities of CH3NH3PbI3 and unconverted PbI2, are presented in Figure 1 a and b. We assign the emission between 700 and 800 nm to CH3NH3PbI3 at room temperature based on the literature. [6c] Interestingly, we observe that the CH3NH3PbI3 emission spectra unexpectedly shift to higher wavelengths as dipping time increases, i.e., as the conversion reaction from PbI2 to CH3NH3PbI3 progresses. Through further analysis of these spectra (discussed in the SI), we identify that a reduction of the full width at half maximum (FWHM) (Figure 1 d) and the high energy tail in the emission takes place with time. The emission from unreacted PbI2 is expected to be between 500 and 550 nm and therefore does not interfere with the emission detected from the perovskite. [8]</p><!><p>Photoluminescence spectra of samples of different dipping times in sequential deposition. a),b) Normalized emission spectra of samples of gradually increasing dipping times of 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 25, 50, 200 and 400 s. c) Maxima position and d) FWHM obtained from each of the spectra shown in (a) and (b).</p><!><p>Complementary measurements (see SI, Figure S1) demonstrate that the PL shift is also detected in in‐situ measurements where the sequential deposition reaction have not been arrested at different points in time. Through another investigation (Figure S2) of samples with crystals in the capping layer of increasing sizes deposited on identical mesoporous scaffolds, we observe that smaller crystals in the capping layer also exhibit blue‐shifted emission compared to larger crystals. The phenomenon we report is therefore not limited to perovskite nanocrystallites in the mesoporous layer, but also applies to crystals in the capping layer. This finding is also consistent with the literature investigating mesoscopic and planar films deposited separately.[ 6a , 6c ]</p><p>According to the literature, the formation of the perovskite from the PbI2 takes place via the intercalation of MAI into the PbI2 followed by structural reorganization of the crystal lattice. In this stage, perovskite formation in the capping layer proceeds through an increase in the size of the perovskite nanocrystallites in the PbI2 precursor with dipping time. [8]</p><p>In this work we consider two hypotheses: i) that the red‐shifting with dipping time is due to either the reduction of QC or ii) to structural changes with the growth of the perovskite nuclei. Previous reports have considered QC mainly focusing on the particle‐in‐a‐box effect, [9] which widens the band gap in smaller nuclei. Here we also consider the effect of enhanced exciton binding associated to the confinement, which has the opposite effect on the band gap.</p><p>The reduction in QC alone cannot explain the red‐shift of the PL peak with dipping time. In fact, we still observe a significant shift when nuclei largely exceed Bohr exciton radius of the perovskite (a 0=2.2 nm), [10] as indicated by the detection of the X‐ray diffraction (XRD) signal for dipping time as short as 27 s, implying that the film contains nuclei larger than 10 nm (Figure S3), the approximate detection limit of our XRD measurements. This aspect is explored further at a later point.</p><p>Reports[ 6c , 11 ] have pointed to the possibility that the perovskite band gap is size dependent, with smaller crystals exhibiting larger band gaps as a consequence of lattice strain. To begin with, Grancini et al. [6c] posited that an ordered arrangement of the organic cations within the inorganic cage in larger crystallites grown on a flat substrate results in a reduction in the strain felt on the Pb‐I cage when compared to smaller ones grown in the mesoporous scaffold. This in turn red‐shifted the optical absorption onset. However, as we show later, we observe no change in the orientation of cations with grain size. Drawing from the conclusions of the above report [6c] among others, D'Innocenzo et al. [11] in turn suggested that the reduction in the band gap for larger crystals is possibly due to a change in the stress in the Pb−I bond. A precise identification of the nature of this stress and a quantification of the effect on the band gap is still lacking.</p><p>Surface effects, limited to the context of those resulting in structural strains and doping [6b] or dislocations, [12] have been suggested as the possible origin of the red‐shift in the emission with crystal size. Nevertheless, the surface‐induced structural effects in perovskite crystals have not yet been fully studied or understood. Here, we perform simulations that will identify the origin of the red‐shift and the reduction in the asymmetry of the emission observed with increasing dipping time, paying close attention to surface‐induced effects.</p><p>To identify the origin of the change in emission (red‐shift and decrease in the FWHM) with dipping time, we perform combined classical MD and DFT simulations of clusters of increasing size modeling perovskite nanocrystallites forming and growing in the PbI2 precursor. Classical MD allows us to analyze relatively large nanocrystallites investigating the effect of their size on the structure. The classical model potential for hybrid perovskites (MYP) developed by Mattoni et al. [13] is used here. MYP has been shown to give excellent results in predicting structural and dynamical bulk [14] and non‐bulk properties of halide perovskites, [15] including surfaces[ 15a , 16 ] and grain boundaries. [17] See Materials and Methods for additional details and references.</p><p>The growing nanocrystallites of CH3NH3PbI3 are modeled as cubic‐like clusters ranging from ca. 3 to ca. 10 nm (Figure 2 a). This shape is consistent with the TEM images of nanocrystals in the literature [18] and classical MD and DFT results showing that the (100) surface is the most stable one.[ 15a , 19 ] Additional details on the model chosen to represent CH3NH3PbI3 nanocrystals are discussed in the Materials and Methods section, where all the computational details are also provided. This model takes into account both local (e.g., surface defects) and global (e.g., Laplace pressure) effects of the nanocrystallite surface. For reference, we also consider a CH3NH3PbI3 (infinite periodic) bulk sample.</p><!><p>Results of atomistic simulations on CH3NH3PbI3 nanocrystallites of increasing size. a) Snapshots of the 3.2, 4.4 and 10.1 nm nanocrystallites taken from the classical MD. b) Pb‐I‐Pb angular distribution function, P(α) c) off‐centering distribution, P(r Pb). The maximum of P(r Pb) is at r off≈0.2 nm, consistent with literature data on the CsSnBr3 analogue. [21] d) Estimated wavelength of the PL peak as a function of the nanocrystallite size due to Pb‐I‐Pb distortions, off‐centering and quantum confinement. Theoretical values have been rigidly shifted to meet the bulk value. e) Energy of the valence band maximum (VBM) and conduction band minimum (CBM) as a function of the nanocrystallite size due to Pb‐I‐Pb distortion. Data are relative to the smallest nanocrystallite, δE=E(l)−E(3.2 nm). For reference, we also report data for an ideal (infinite) CH3NH3PbI3 crystal. The colors of the symbols in (d) and (e) denote the sizes of the nanocrystallites according to the colors in the legend in (b). We remark that the difference between the band gap of a ca. 10 nm nanocrystallite and the bulk crystal due to surface‐induced structural effects corresponds to a shift in emission of ca. 13 nm, while the difference due to QC is ca. 9 nm. We also look at the apparent abnormal trend of the predicted PL maximum of the "tilting" curve (in (d)) and VBM curve (in (e)) between clusters of 5.1 and 5.8 nm. This is likely a result of our classical MD+DFT simulation strategy that, while allowing to treat large clusters, is affected by statistical fluctuations, resulting in (random) errors like in experiments. More details are available in the SI.</p><!><p>Four structural characteristics affecting the electronic structure of perovskite, the Pb‐I bond length [20] (Figure S4), the Pb‐I‐Pb angle (Figure 2 b), the off‐centering of Pb atoms in the PbI6 octahedron [21] (Figure 2 c) and the reorientation of the cation [6c] (Figure S5) are considered. The Pb‐I bond length does not show any significant dependence or clear trend with nanocrystallite size (as shown in Figure S4). Similarly, the distribution of the orientation of cations is not affected by the nanocrystallite size (Figure S5). The Pb‐I‐Pb angle (α) is a convenient measure of the tilting of PbI6 octahedra, [22] the former is easier to compute than the latter in MD, in which PbI6 octahedra are dynamically distorted. A visual representation of the angle α between adjacent PbI6 octahedra is shown in Figure 3 a. P(α) is characterized by an intense peak at ca. 170° corresponding to the tilted quasi‐cubic structure of halide perovskites (Figure 2 b). We also find Pb‐I‐Pb angles close to 90° but these are associated with defects forming at the surfaces, edges and corners of nanocrystallites.</p><!><p>Visual representation of PbI6 octahedra in CH3NH3PbI3 nanocrystallites showing the two structural characteristics affecting the electronic structure of perovskite. a) Definition of the Pb‐I‐Pb angle α. α is a proxy of the tilting angle. In configurations extracted from MD simulations, in which PbI6 octahedra are dynamically distorted, α is easier to compute than the tilting angle. b) Off‐centering is defined as the distance between the barycenter of the iodine atoms of a PbI6 octahedron and the corresponding lead atom.</p><!><p>The P(α) becomes sharper and shifts towards higher angles with increase in nanocrystallite size (Figure 2 b). The P(α) is broader near the surface of the nanocrystallite than in the bulk and the reduced fraction of stoichiometric units in the former region with respect to the latter in larger nanocrystallites explains the sharpening of α with their size. This can be explained considering that the larger core, exhibiting a more bulk‐like crystalline order, has a templating effect on the periphery, making the structure of this region more regular (Figure S6). Since the crystal size increases with dipping time, we associate the effects of the nanocrystallite size on the Pb‐I‐Pb distribution with dipping time.</p><p>The off‐centering distribution of Pb from the nominal center of the PbI6 octahedron (Figure 3 b), measured by the average of the iodine positions, P(r Pb), is shown in Figure 2 c for nanocrystallites of various sizes. We see that the maximum of P(r Pb) shifts to slightly shorter distances and the distribution becomes sharper with nanocrystallite size. Similar to the α case, P(r Pb) is broader at the periphery of the nanocrystallite than in the core and since the core/periphery ratio increases with nanocrystallite size, we observe a templating effect in this case as well.</p><p>To understand the effect of changes of the Pb‐I‐Pb angle and Pb off‐centering on the PL spectrum as opposed to just QC, we perform DFT calculations of a bulk system using the α max and r Pb max values determined in the above‐described MD simulations (Figure S7).</p><p>Previous DFT calculations on small nanocrystallites have investigated the dependence of the band gap on their size [23] without distinguishing between structural effects and QC. We find that a narrower band gap is associated with a larger α max, i.e., less tilted structures, and lower r Pb max, i.e., lower Pb off‐centering. Thus, simulations predict that surface‐induced structural effects produce a shift in the PL spectra of ca. 13 nm towards longer wavelengths between a ca. 10 nm nanocrystallite and the bulk (Figure 2 d) as an effect of the increase in α max and a corresponding shift of ca. 8 nm due to the reduction of r Pb max, in good agreement with the experimental results from the steady state PL measurements presented earlier (Figure 1 c).</p><p>The narrowing of the band gap with increasing α max and decreasing r Pb max is due to the character of the valence band maximum (VBM) and the conduction band minimum (CBM) of CH3NH3PbI3, which are made of Pb‐6s/I‐5p and Pb‐6p/I‐5s antibonding atomic orbitals. The overlap of both orbitals increases with a larger α max and a smaller r Pb max, and the VBM and CBM move towards the vacuum level. [24] However, the change in the antibonding overlap of the VBM is stronger and the band gap narrows with the nanocrystallite size (Figure 2 e).</p><p>The other effect observed in the experimental steady‐state PL spectra, the decrease in the FWHM (Figure 1 d) and asymmetry, can also be attributed to the growth of perovskite crystallites. In the early stages of the reaction small perovskite crystals are formed, which grow larger at longer dipping times through the phenomenon of Ostwald ripening. [8] Since the emission wavelength converges to the bulk value for larger crystallites, the shorter wavelength contribution of smaller crystallites disappears from the PL spectra, reducing the FWHM and asymmetry. We also observe an intrinsic sharpening and reduction of asymmetry in P(α) and P(r Pb) with nanocrystallite size (Figure 2 b and c), which is expected to result in a corresponding sharpening and symmetrization of the PL spectra with dipping time.</p><p>For a comprehensive image of the system, we look into QC effects for nanocrystallites of the same size considered above. The blue‐shift of the PL spectrum due to QC is the result of two terms of opposite sign: Firstly, the particle‐in‐a‐box effect, which widens the band gap and this widening is proportional to the inverse of the square size of the nanocrystallite and of the reduced mass of the exciton. Secondly, the enhanced exciton binding associated with the confinement of the corresponding electron and hole, which reduces the band gap and is proportional to the inverse of the size of the crystal [25] (see Materials and Methods for details). This second contribution, which reduces the length scale of QC effect, has been often neglected in the literature. [9] Here, we use the reduced mass of the exciton obtained from DFT data available in the literature, [26] which is consistent with experimental values (0.15 m0). [10] For very small nanocrystallites, the QC effect dominates. However, this effect vanishes rapidly thereafter and for larger nanocrystallites of ca. 10 nm, the surface‐induced structural effects (bending of the Pb‐I‐Pb angle and off‐centering of Pb) are larger than the QC, with the predicted shift in PL maxima obtained as ca. 13 and ca. 8 nm for the former vs. ca. 9 nm for the latter as shown in Figure 2 d. We see that QC becomes negligible for nanocrystallites of 15 nm (see Figure 2 d).</p><p>Summarizing the calculations, we show that the band gap narrows with the increase of nanocrystallite size due to surface‐induced structural effects, in particular a reduction in the tilting of PbI6 octahedra and off‐centering of Pb (as visually depicted in Figure 3 b). Thus, DFT calculations predict that the emission maxima red‐shifts with increase in the nanocrystallite size and hence with dipping time. However, for large nanocrystallites these effects become negligible, which is consistent with the observation that there is no change in the emission maxima between 200 and 400 s (Figure 1 c). We remark that due to these concomitant structural effects, QC alone is insufficient to explain the dependence of PL spectra on crystal size.</p><p>Our observations are in keeping with the "soft" nature of perovskite crystals, which has been demonstrated clearly by a number of recent studies. These revealed the unique feature of perovskites that tuning of the surface can affect the property evolution within the interior of perovskite crystals. Xue et al. [27] have shown the occurrence of surface‐induced secondary grain growth in perovskite thin films, while we have recently reported on the long‐term stabilization of α‐FAPbI3 films by covering them with an overlayer of two‐dimensional perovskite. [28] The soft nature of perovskites is responsible for the failure of the Fröhlich polaron model of electron‐lattice coupling to explain charge carrier transport, [29] the formation and/or ordering of polar nanodomains surrounding charge carriers, [30] and the simultaneous presence of charge carriers with different lifetimes. [31] We believe that the soft nature of perovskites, which can react to "random" stress associated with different kinds of grain boundaries in polycrystalline films presenting non‐uniform strain, might be responsible for macroscopic inhomogeneity of optical (e.g., PL) characteristics of samples. [32] Similarly, stress/strain induced by the interface with the substrate or layers underneath the perovskite, e.g., mesoscopic TiO2, might affect its local opto‐electronic properties.</p><p>We employ hyperspectral cross‐sectional CLSM to look at fully formed methylammonium lead iodide perovskite films on an Al2O3 mesoscopic scaffold to study the emission from the mesoporous and capping layers individually. The cross‐sectional imaging technique has been described in our previous work [8] and in the Materials and Methods section. Figure 4 b shows the normalized emission spectra for perovskite in the mesoporous and the capping layers, where each data point was obtained from the images constituting Figure 4 a by isolating both the layers and averaging over the respective areas through image processing for each image (See Materials and Methods for details about sampling). We observe that there is a blue‐shift in the emission profile of the perovskite in the mesoporous layer. This observation is consistent with steady state PL measurements reported in the literature where perovskite infiltrated into a mesoscopic layer and deposited as a flat layer were compared. Perovskite infiltrated into the mesoporous scaffold consists of smaller crystals compared to the capping layer. [6c] Therefore, based on our simulations, perovskite in mesoporous layers is expected to exhibit a blue‐shifted emission and our experimental findings confirm the same.</p><!><p>Study of the emission from the mesoporous layer and the capping layer in a partial cell. a) Cross‐sectional CLSM imaging of CH3NH3PbI3 sample consisting of a capping layer and the mesoporous perovskite infiltrated into an Al2O3 mesoporous scaffold. Pseudo‐color cross‐sectional image of fully converted sample. 32 images showing the emission between 640 and 800 nm in steps of 5 nm each have been summed. Color scale assignment to the emission intensity in the image shown. Scale bar, 2.5 μm. b) Normalized emission from the perovskite in the capping layer and the mesoporous layer obtained from the images that make up (a). The emission from the mesoporous layer is blue‐shifted compared to the capping layer. c) A view of a perovskite solar cell without a back contact. The emissions from the perovskite in the mesoporous layer and the capping layer in the partial cell are shown, d) energy band diagram depicting the perovskite levels in the mesoporous and capping layers.</p><!><p>TiO2 mesoporous layers are employed in the fabrication of perovskite solar cells with mesoporous architectures. [2] We characterize both the 18 NRT TiO2 and 30 NRD TiO2 mesoporous layers, those commonly used in solar cells, using scanning electron microscopy (Figure S8) and N2 gas adsorption measurements (Table S1). Using the Brunauer–Emmett–Teller (BET) method, [33] we find that the average pore diameter of the mesoporous matrix is 26 and 31 nm for the 18 NRT and 30 NRD TiO2 mesoporous films, respectively. From the Barrett–Joyner–Halenda (BJH) method, [33] the average pore diameter was found to be 22 and 27 nm for the 18 NRT and 30 NRD TiO2 mesoporous films, respectively.</p><p>The mesoporous scaffold restricts the size of perovskite crystals that can grow in its pores, as mentioned in the literature. [34] Therefore, these perovskite crystals formed in‐situ in these mesoporous layers are likely to have a crystal size distribution with average diameters similar to those estimated for the average pore diameters of these mesoporous layers. The larger values for the average diameters when compared to the Bohr exciton radius again indicate that the QC regime has been surpassed for a large fraction of the crystals in the mesoporous layers as well. Furthermore, similar to the Al2O3 case imaged earlier (which consisted of mesoporous particles of comparable size), we expect the blue‐shift due to surface‐induced structural changes described earlier to take place for perovskite in TiO2 mesoporous layers as well. We expect this blue‐shifting to have no drawback on re‐absorption (see Sec. 6 in SI).</p><p>It is interesting to look at the schematic of a partial solar cell shown in Figure 4 c and the corresponding schematic of the energy band diagrams shown in Figure 4 d (see also Figure S9). Due to the predominance of small crystals in the mesoporous layer, the VBM is expected to be at more negative values compared to the capping layer (Figure 4 d) as identified thorough the above simulations. This offset in the VBMs of the perovskite in these layers has implications for the performance of mesoscopic solar cells as it can affect charge transport in the device. We posit that it provides additional selectivity for hole transport as photo‐generated holes in the mesoporous layer can be transported into the capping layer (as shown in Figure 4 d) where they come in contact with the hole transport material, while hole transport in the opposite direction is not favorable. Moreover, the reduction in the population of photo‐generated holes in the mesoporous layer compared to electrons lowers the possibility of recombination taking place there. The nearly unchanged CBM position in the perovskite (as shown in Figure 2 e) is an advantage as the alignment with the CBM of the TiO2 electron transport layer [5] is maintained.</p><p>We also consider the disadvantage of mesoscopic architectures, the numerous grain boundaries in the perovskite in the mesoporous layer are likely to be potential recombination centers, which may result in performance losses, due to non‐radiative recombination lowering the open‐circuit voltage. [35] However, our previous study [2] with mesoporous‐based solar cells (composed of a Rb‐incorporated quadruple‐cation perovskite on ca. 100 nm thick TiO2 mesoporous layer) demonstrated open‐circuit voltages approaching theoretically achievable values, pointing to low losses due to non‐radiative recombination. While extremely thick mesoporous layers might adversely affect photovoltaic performance due to large losses from non‐radiative recombination, the use of thin mesoporous scaffolds still exploits the advantages discussed above.</p><p>Based on our findings, we propose a novel strategy wherein the mesoporous scaffold itself can be used to tune the VBM and CBM in devices and employed in designing new architectures for perovskite photovoltaics, especially band gap graded ones. This is an alternative to current design strategies such as compositional tuning where the perovskite composition would be graded through the depth of the device and would practically require an extremely well‐controlled reactant evaporation process for the deposition of the graded perovskite layer. Using mesoporous scaffolds of increasing sizes, we suggest utilizing the crystal‐size‐induced structural effects in each layer to tune the VBM and CBM (shown schematically in Figure S9 with details in the SI), to achieve a fully graded band gap with just one absorber material, fabricated using a convenient solution‐based deposition process. Experimental results relevant to this discussion on the opto‐electronic properties of perovskite in mesoporous scaffolds of different sizes are presented later.</p><p>Our results are particularly relevant for cells that already consist of multiple mesoporous layers such as those reported by Mei et al. [36] This architecture is well suited to produce a graded band gap solar cell using several different mesoporous layers. Moreover, many of the highest performing prototype solar cells make use of mesoporous layers. These include those reported by Saliba et al. [2] at 21.6 %, Tavakoli et al. [37] at about 22 % and Min et al. over 23 %, [38] which all employ 100–150 nm thick TiO2 mesoscopic layers. The same holds for perovskite devices showing the present certified record efficiency of 25.2 %. [39] The perovskite in these mesoporous layers will likely be subject to the size effects explored in our study. Furthermore, the potential of mesoscopic TiO2 based perovskite solar cells fabricated with high efficiencies recently through inkjet printing has been demonstrated by Huckaba et al. [40] which shows the scalability of this technology.</p><p>To explore the broader implications of our work, we examine perovskite samples of the nominal composition Cs0.05MA0.16FA0.79Pb(I0.83Br0.17)3, henceforth referred to as the triple‐cation composition, which is used in a number of high‐performing solar cells[ 37 , 41 ] (Figure S10). This perovskite is predominantly composed mainly of formamidinium ions (FA, HC(NH2)2 +) in place of methylammonium ions (MA, CH3NH3 +). Moreover, the perovskite is deposited using the anti‐solvent method as described in the literature, [41] which is the other deposition method widely used to deposit perovskite films for high‐performing devices such as those reported in these references.[ 37 , 41 ] We perform hyperspectral cross‐sectional CLSM to study the emission from the perovskite in both the mesoporous layer and the capping layer in partial solar cells, in a manner similar to that done for the methylammonium lead iodide sample in Figure 4. We image two triple‐cation perovskite samples, one deposited on a mesoscopic scaffold made of 17 nm diameter Al2O3 particles (Figure S10a and b), while the other is made of 95 nm diameter Al2O3 particles (Figure S10c and d). The perovskite is infiltrated into the mesoporous layer and also forms an additional capping layer on top in both samples (Figure S11, discussed more later).</p><p>For both these samples, the normalized emission obtained from the images (Figure S10b and d) shows that the emission maxima of the perovskite in the mesoporous layer is blue‐shifted compared to the capping layer, similar to observations made for methylammonium lead iodide in Figure 4. Moreover, we observe longer tails in the emissions towards the blue (high energy, associated with larger band gaps) for the perovskite in the mesoporous layers compared to the capping layers, which we attribute to the size restriction of nanocrystallites in the mesoporous layers. This is comparable to our previous investigation of methylammonium lead iodide samples made with short dipping times in the sequential deposition method, where small sized nanocrystallites are expected to form. These findings are therefore consistent with our experiments and theoretical results discussed earlier in this report. In more general terms, we demonstrate that the crystal size effect we have described above for methylammonium lead iodide is also clearly observed for this complex triple‐cation composition deposited via the anti‐solvent method.</p><p>Comparing the samples with each other (Figure S10b and d), we notice that perovskite nanocrystallites in both the mesoporous layers have different band gaps and so do the capping layers. We see that the CLSM spectra from the perovskite in the mesoporous and capping layers from the 95 nm sample (Figure S10d) exhibit emission maxima at longer wavelengths, associated with smaller band gaps, when compared to the corresponding spectra from the 17 nm sample (Figure S10b). We take a closer look at the morphology of the perovskite in the mesoporous and capping layers for both samples using scanning electron microscopy (SEM) (Figure S11). The size of perovskite crystals in the 95 nm sample is larger in both the capping and mesoporous layers compared to the 17 nm sample in these SEM images. There are two implications for these observations:</p><p>Firstly, the smaller band gap of the perovskite in the mesoporous layer associated with the lower energy emission from the 95 nm mesoporous layer sample, compared to the one from the 17 nm sample, supports our previous hypothesis. For crystallites of larger size obtained within the 95 nm mesoporous scaffold, which is well beyond the QC regime (ca. 20 times the Bohr exciton diameter: d0 of methylammonium lead iodide perovskite, [10] ca. 15 times d0 of FAPbBr3 and ca. 7 times d0 of FAPbI3, [42] which we take as a reference for the unknown value of the Bohr exciton radius of mixed perovskites), the crystal size effect is present and it is an effect distinct from QC. For very small nanocrystallites, this crystal size effect occurs in addition to and alongside QC. Therefore, comparing the CLSM results for the 17 nm and 95 nm mesoporous scaffold samples, with a corresponding change in the perovskite nanocrystallite size in the mesoporous layer as the mesoporous scaffolds restrict the size, we confirm the trend of the emission peak with size for larger crystallites, well beyond the Bohr exciton radius, previously discussed for methylammonium lead iodide on the basis of experiments and simulations. Furthermore, the presence of very large crystals (Figure S11) with the emission maxima at longer wavelength (Figure S10) in the capping layer for the 95 nm mesoporous layer‐based sample compared to the capping layer of the 17 nm sample, is consistent with our previous conclusion in Figure S2 for methylammonium lead iodide demonstrating that the crystal size effect is present for quite large crystals in the capping layer as well and not limited to nanocrystallites in the mesoporous layer.</p><p>Secondly, these findings demonstrating the difference in opto‐electronic properties in terms of the band gap for perovskite in mesoporous scaffolds of different sizes, have practical implications as discussed in the previous section. These include improving the charge extraction in state‐of‐the‐art mesoscopic layer‐based solar cells and in the design of novel graded band gap devices constructed using mesoscopic layers to tune the band gap. Our work highlights the immense potential for designing perovskite opto‐electronic devices that exploit the phenomenon of crystal‐size‐induced band gap tuning conveniently via the use of tailored mesoscopic scaffolds.</p><p>In summary, the crystal size phenomenon we discussed is widely generalizable and we demonstrate that it influences various perovskites and deposition methods of current technological interest as well. The comparison of samples made with mesoporous layers of different particle sizes proves a) the effect of crystal size restriction on the opto‐electronic properties of the material and b) the practical use of this phenomenon in devices currently employing mesoscopic layers, as well as their potential for use in new designs.</p><!><p>Our study demonstrates that methylammonium lead iodide perovskite films exhibit a blue‐shift in the PL spectra and the presence of an asymmetry in the emission during formation in the sequential deposition method, attributed to small perovskite nanocrystallites which are present at the early stages of the reaction. The asymmetry weighted towards higher energies is shown to be associated with the surface‐induced structural effects in small perovskite nanocrystallites, namely PbI6 octahedra tilting and Pb off‐centering. We distinguish this phenomenon from QC effects as changes in the perovskite band gap are observed for nanocrystallites of size greatly exceeding the Bohr exciton radius. We show that the perovskite in the mesoporous layer has a larger band gap compared to the capping layer due to the presence of smaller crystals in the former. Our results have broad implications for solar cells employing mesoporous architectures, as an intrinsic feature of the perovskite band gap for mesoporous layers, has been identified.</p><p>The wide scope of the crystal size effect is also demonstrated in our investigation of samples of the complex triple‐cation perovskite composition, prepared through the anti‐solvent method on mesoporous scaffolds of different particle sizes. This investigation confirms the ubiquitous nature and extensive applicability of the crystal size effect studied in our work in both: different perovskite compositions and various thin film deposition methods which are of current interest in the field. We show that band gap tuning can be conveniently achieved through control of the crystal size via the use of tailored mesoscopic scaffolds in perovskite solar cells. Overall, our work systematically unravels the phenomenon of crystal‐size‐induced band gap tuning in perovskites and highlights its immense potential for the design of highly efficient opto‐electronic devices in the near future.</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>Supporting Information</p><p>Click here for additional data file.</p>

PubMed Open Access